Replication Timing Uncovers a Two-Compartment Nuclear Architecture of Interphase Euchromatin in Maize

Replication Timing Uncovers a Two-Compartment Nuclear Architecture of Interphase Euchromatin in Maize | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Replication Timing Uncovers a Two-Compartment Nuclear Architecture of Interphase Euchromatin in Maize Hafiza Sara Akram, Emily E. Wear, Leigh Mickelson-Young, Zachary M. Turpin, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6116464/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Genome replication is temporally regulated during S phase, with specific genomic regions replicating at defined times in a process known as Replication Timing (RT). Based on 3D cytology in replicating nuclei, we previously proposed a “mini-domain chromatin fiber RT model” for maize euchromatin that suggested it is subdivided into early-S and middle-S compartments distinguished by chromatin condensation and RT. However, whether this compartmentalization reflects a general nuclear architecture that persists throughout the cell cycle was unclear. To test this model, we conducted two orthogonal assays—Hi-C for genome-wide interaction data and 3D FISH for direct visualization of chromatin organization. Hi-C eigenvalues and insulation scores revealed distinct patterns of early-S regions having negative insulation scores with long-range contacts while middle-S regions showed the opposite. Early-S regions also correlated more strongly with epigenomic signatures of open, transcriptionally active chromatin than middle-S regions. 3D oligo FISH painting confirmed that early-S and middle-S regions occupy adjacent but largely non-overlapping nucleoplasmic spaces during all interphase stages, including G1. Our findings redefine the maize euchromatin “A” compartment as having two distinct subcompartments—Early-S and Middle-S—and underscore the importance of replication timing as a defining feature of chromatin architecture and genome organization. Biological sciences/Cell biology Biological sciences/Genetics Biological sciences/Genetics/Genomics/Epigenomics Replication timing (RT) chromatin nuclear architecture Hi-C 3D FISH 5-ethynyl deoxyuridine (EdU) DAPI Early-S RT Middle-S RT maize Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 INTRODUCTION The eukaryotic cell cycle consists of a widely conserved series of events, including cell growth, DNA replication, and division into two daughter cells. DNA replication occurs during the S phase of the cell cycle, and the specific time within S phase when each genomic region replicates can be measured and annotated as Replication Timing (RT) 1 – 3 . The fact that some cells or tissues have different RT profiles reveals the existence of an underlying RT program coupled to development or differentiation 4 , 5 . The RT program is generally recognized as ensuring the faithful reproduction and transmission not only of the nucleotide sequence but also of the chromatin state, contributing to the propagation of epigenomic cell type identity and functions 6 . Most eukaryotic RT knowledge comes from yeast and mammals, where complex temporal programs have been described and extensively studied 7 , 8 . However, the regulatory mechanisms that govern temporal RT programs remain understudied in plants. Early studies of plants concluded that genome replication is essentially biphasic, with early-replicating and late-replicating regions 9 , 10 . Later studies greatly improved the resolution of the replication timing profile by using nucleotide analogs such as 5-bromo-2′-deoxyuridine (BrdU) 11 and 5-ethynyl-2'-deoxyuridine (EdU) for pulse-labeling replicating DNA. EdU has become the analog of choice because it does not require acid or heat denaturation for detection and, instead, is directly conjugated with click chemistry to add a fluorescent dye used for both fluorescence activated nuclei sorting (FANS) and microscopy 12 . These EdU-labeled nuclei can be separated by flow cytometry from unlabeled G1 and G2 nuclei and further divided by increasing DNA content into subpopulations representing sequential stages of S phase. Three separated populations, early-S, middle-S, and late-S, have been used for microscopic analysis and for sequencing of replicated DNA (Repli-seq) 13 . The first genome-wide RT maps in plants were produced for Arabidopsis thaliana and maize ( Zea mays L.) using these techniques 2 , 14 . The comparative analysis of early, middle and late replication in Arabidopsis revealed that early-S and middle-S are quite similar to each other and distinct from late-S. Early and middle replicating domains are enriched with open chromatin as defined by nuclease sensitivity and histone acetylation markers with early-replicating regions being gene-rich, while late-replicating domains are characterized by an enrichment of transposons and repressive epigenetic markers 11 , 14 – 16 . Although Arabidopsis has provided a foundational understanding of plant replication timing, its small genome size and high gene density are unusual among plant genomes. Plants with larger genome sizes, such as maize (2 n = 2X = 20) with a 1C value of 2.3 Gb, have expanded intergenic content, which generally tends to replicate primarily during middle-S and exists in a slightly more compacted state compared to the early-S replicating chromatin 17 , 18 . The maize genome, which is about two thirds the size of the human genome and contains a complex array of transposons, is more typical of higher plants. This fact, together with the fact that maize has been the subject of detailed genetic and cytogenetic studies for more than 100 years 19 , 20 , makes maize an attractive system for exploring the control of replication in plants with complex genomes and chromatin architecture. Furthermore, as one of the world’s most agriculturally significant crops, insights into the replication process in maize are relevant not only for fundamental research but also for strategies to enhance crop performance and resilience. Our group has developed a powerful experimental system to characterize replication in actively growing, intact maize root tip meristems that enables examination of replication patterns, genome features, and associated chromatin structure parameters at different stages of the cell cycle 13 , 21 . Although heterochromatic regions in maize replicate late, and many genes replicate early as they do in most other eukaryotic systems, the spatiotemporal patterns of early and middle S-phase replication in maize differ significantly from those in mammalian and yeast systems. In mammals, DNA synthesis occurs at different nuclear regions throughout S phase, classified as 5 sequential stages referred to as patterns 1–5 22 or Types I-V 23 , in which the first, third, and fifth stages represent the earliest, the middle most, and latest stages of S phase, respectively. Despite the different naming conventions, there is agreement on the nuclear distribution of replication in mammalian nuclei, which starts with broad distribution in euchromatic portions of the nucleoplasm at early S phase, then in perinuclear and perinucleolar regions during middle S phase, followed by heterochromatic portions of the nucleoplasm at late S phase 6 , 22 – 25 . Much of the mammalian replication timing studies compare Early to Late replication timing, and genomic regions where they switch, with relatively few studies focusing directly on middle S 4 , 26 , 27 . In maize, DNA synthesis is thoroughly dispersed throughout the nucleus during both early and middle S phase but becomes more punctate and clustered in late S phase. Analysis of DNA RT in maize shows that early S-phase replicative labeling primarily colocalizes with regions of relatively weak DAPI fluorescence, indicating a low DNA density, while middle S-phase EdU labeling aligns with regions of euchromatin showing stronger DAPI staining. Based on these findings, we proposed a "minidomain chromatin fiber RT model”, suggesting that maize euchromatin exists as an interspersed mixture of two subcompartments, each characterized by distinct replication timing and chromatin morphology 18 . To test the minidomain chromatin fiber RT model, we employed a combination of proximity ligation (Hi-C) and quantitative in situ hybridization (3D-FISH) techniques in this study. Here we provide new evidence for this model throughout the cell cycle, recontextualizing our view of the euchromatin global "A" compartment into two spatially and epigenetically distinct compartments. METHODS Plant materials: Experiments were done using maize ( Zea mays L., inbred B73) root tips or earshoots. For root tips, as described in a previous protocol 28 , seeds were surface sterilized and imbibed overnight with constant stirring and aeration. Seeds were surface-sterilized again with 0.5% sodium hypochlorite, 0.05% Tween-20 solution, and then rinsed with water. Seeds were germinated in boxes with paper towels moistened with sterile water at 28°C for 3 days under continuous light (Feit Electric OneSync LED light system, 3000K, 300–400 lux). The material for biological replicates were grown independently and harvested on different days. For earshoots, field-grown maize plants were harvested between 9-11am on sunny days, and earshoots ranging in size from 0.5-1 cm were flash frozen in liquid nitrogen, and pooled (15–20 earshoots) for storage at -80°C as described in previous paper 29 . Root tip and earshoot nuclei for HiC: For Hi-C on seedling root nuclei, the samples were not subjected to EdU labeling or the click reaction, but otherwise isolated from formaldehyde fixed maize root tip for FACS as previously described 13 , 30 . Nuclei were stained with DAPI and sorted to collect between 1.25–1.53 million nuclei as input (Fig. 1 h) for each biological replicate of the Hi-C assay. For Hi-C on field-grown earshoot nuclei, we used the method of Savadel et al., (2021) 29 to isolate nuclei. The frozen tissues were ground in liquid nitrogen to a fine powder, and the nuclei were formaldehyde crosslinked. The fixation reaction was stopped by addition of 0.1 vol (~ 1 mL) of 2.5 M glycine. The tissue was then mechanically disrupted with a Polytron (3 x 10 seconds at ~ 1/5 maximum speed) to liberate nuclei. Nuclei were pelleted by centrifugation at 2000 xg for 15 min at 4ºC and resuspended in a 15 mL buffer. Total earshoot nuclei were used for the Hi-C assay. Preparation and sequencing of Hi-C libraries from maize root tip and ear shoot: The Hi-C libraries were generated essentially following the protocol first described for maize leaf by Dong et al.(2017) and later detailed in a methods chapter by Dong and Zhong (2020) 31 , 32 . We made some minor modifications to accommodate the library kits currently utilized by the Molecular Cloning Facility (Dept. Biol. Sci, Florida State University, Tallahassee, FL) and reduced washing steps to avoid nuclei loss as described in the wet bench full protocol (Supplementary File 1). Among the key steps were permeabilization of nuclei with 0.5% (w:v) SDS for 5 min at 65°C, DNA digestion with Dpn-II restriction enzyme, overhang repair with Klenow and biotinylated nucleotides, and ligation with T4 DNA ligase. Ligated DNA was biotin affinity purified, crosslinks reversed, and sheared to 300–500 bp to yield DNA fragments for library construction using NEBNext Adapters for Illumina. These libraries were sequenced using Illumina Hi-Seq 2500 paired-end 150-bp chemistry (Translational Science Lab at College of Medicine, Florida State University, Tallahassee, FL) to obtain library sequences at a depth ranging from 127M to 309M (Supplementary Table 1) per replicate, with four replicates for root tips and three for earshoots. Raw sequences were processed to remove adapters by the sequencing facility, providing library Fastq files for HiC analysis. Nuclei isolation for MMOA-seq For MMOA nuclei preparation was done using the previously published protocol (Bass et al. 2014; Wear et al. 2016). Briefly, maize seedling roots were labeled in 25 µM EdU for 20 min, rinsed and placed in 100 µM thymidine to stop labeling. The terminal 1-mm maize root tip segments were excised, fixed and lysed for the isolation of nuclei for MMOA-seq. Incorporated EdU was then conjugated to Alexa Fluor 488 (AF-488) using a Click-iT EdU Alexa Fluor 488 imaging kit (Life Technologies). The nuclei were then counterstained with DAPI using a cell lysis buffer containing 2 µg/mL DAPI and 40 µg/mL Ribonuclease A and filtered through a 20-µm nylon mesh filter (Partec). Nuclei were flow sorted on a FACS Aria III flow cytometer (BD Biosciences) equipped with UV (355 nm) and blue (488 nm) lasers. G1 nuclei were used for MMOA-seq library preparation. MMOA-seq library preparation and Sequencing MMOA-seq libraries were prepared using nuclei in 500-µL aliquots per replicate. Briefly, 60 µL of nuclei were distributed to each of four 1.5 mL screw cap tubes. 10x MNase working dilutions (25 U/mL, 12.5 U/mL, and 6.25 U/mL) were prepared from a 20,000 U/mL stock. 6.7 µL of each working dilution (or MDB) were added to each of the 60 µL nuclei aliquots and immediately vortexed and briefly centrifuged. Complete digestion reactions were transferred to a 37ºC shaking water bath and incubated for 15 min. Reactions were promptly stopped after 15 min by addition of 5 µL of 0.5 M EGTA (pH 8.0). Digested chromatin was then decrosslinked overnight at 65ºC in 1% SDS, 150 mM NaCl, 20 µg/mL Proteinase K. DNA was purified by 25:24:1 Phenol:Chloroform:Isoamyl alcohol (pH 8.0). Nucleic acids were precipitated and the pellets were washed with ice cold 70% ethanol and air dried before being redissolved in 100 µL of 10 mM Tris EDTA buffer. For each library, 75 ng of selected digests (1.25 and 2.5 U/mL MNase) were combined and diluted to 50 µL with 0.1X TE. Sequencing libraries were made according to the NEBNext Ultra II DNA Library Prep Kit for Illumina with the following modifications to the size selection protocol for retention of small fragments (< 200 bp): the first bead addition used 50 µL of beads, the second used 100 µL of beads. A total of 8 cycles of PCR were completed at the barcoding step. Libraries were quantified by Qubit dsDNA HS fluorimetry and KAPA qPCR. DNA fragment size distribution was analyzed on an Agilent High Sensitivity D1000 Screentape assay. An equimolar pool of all 24 MOA-seq libraries was prepared and sequenced using 50-bp paired-end reads on a Novaseq S1 flow cell in the FSU College of Medicine Translational Science Lab. HiC-Pro analysis of Hi-C data: HiC-Pro 33 is a custom bioinformatic software for the analysis of Hi-C datasets. The pipeline performs sequential steps, including short read mapping, detection of valid ligation fragments, and various quality controls. The output consists of inter- and intra-chromosomal contact maps at various resolutions. The genome was divided into non-overlapping bins of equal size at different resolutions (1 kb, 5 kb,10 kb, 50 kb, 250 kb, 500 kb, and 1mb) and contacts were scored in each bin. The frequency of interaction between bins was represented by bi-dimensional heatmaps (“contact matrices''), containing both inter- and intra-chromosomal contacts. Contact matrices were visualized using Juicebox 34 , and used to calculate a Pearson correlation matrix for observed/expected intra- and inter-chromosomal interactions. Insulation index and Eigenvector analysis: To systematically identify folding structures at the local scale, we applied several techniques, such as Principal Component Analysis (PCA), the Insulation Index method and Virtual 4C analysis. We ran PCA to calculate the first eigenvector (principal component) of each contact matrix at 50-kb resolution to identify two distinct A/B genomic compartments as described in Lieberman-Aiden et al 35 . The Insulation Index analysis 36 , 37 was calculated as the cumulative frequency of interactions in all the 50-kb bins within the 1 mb window. We calculated the log2 of the observed/expected interaction frequencies using the median scores for the expected values. Valleys/minima (negative values) indicate loci of reduced frequency of interaction with flanking regions, whereas local maxima (positive values) reveal folding structures. Virtual 4C analysis 38 was centered over “viewpoints” of interest (“baits”), chosen in our case among replication segments of different classes, Early-S or Middle-S 2 . We scored all the valid interactions between each viewpoint and the rest of the chromosome, and compared the frequency of Early to Early and Middle to Middle versus Early to Middle interactions. Statistical analysis: For virtual 4C arc plots, we used plotGardner v1.0.17 39 package for the R statistical suite 40 for the visualization of genomic regions. Two similar sized early and middle RT segments were selected as 4C baits and then their corresponding 10-kb bins were extracted. Each 10-kb bin was annotated with its RT class, and all bins in contact with the bait bins were retrieved. Hexbin plots were also drawn using the R package ggplot2 v4.1.2 41 with Hi-C EV versus different chromatin marks and Early-S, Middle-S RT versus chromatin marks at 50-kb resolution. Root tip nuclei for 3D-FISH: Maize seedlings were grown and nuclei were prepared and sorted as described above. For 3D quantitative FISH with the mixed population nuclei, we combined 30 uL each of the flow-sorted EdU-labeled nuclei gates for G1 (1.5M/mL, 16%), early-S (1.9M/mL, 21%), middle-S (1.5M/mL, 16%), late-S (0.36M/mL, 4%), and G2 (3.9M/mL, 41%). In this mix, the S-phase nuclei were ~ 41% of the total. For 3D-FISH with G1 nuclei, only flow-sorted G1 nuclei were used (Fig. 8 c, inset panel). Probes labeling for 3D-FISH: Oligonucleotide probe libraries were designed by DAICEL Arbor Biosciences using three sets of sequences as inputs, early-S, middle-S, and late-S RT segments. These annotation segments were based on the Repli-seq data from Emily et. al, (2017) 2 , realigned to B73v5 using the RepliScan pipeline 42 to produce a DNA RT class annotation file, RT_class_ALL_27800_b73v5_9colBed_vhsf521e.BED (Supplementary File-2). From this annotation file, we derived coordinates for three sets of genomic regions (Early, Middle, or Late) within the first 105 Mbp on chromosome 5, which corresponds to the short arm. These were used as input to obtain three libraries of oligos as T m -matched ~ 45-mers, high-density, and uniformly spaced at high density across the chromosome arm. The resulting sets of oligos included a total of 22,460, 27,390, or 4,640 oligos from the early-S, middle-S, or late-S regions, respectively, and the coordinates of the uniquely named oligos are provided as bed files (Supplementary File-3). A series of experiments were conducted to convert these dsDNA libraries into fluorescently labeled single-stranded DNA (ssDNA) libraries (Supplementary File-4). Briefly, the dsDNA library underwent PCR amplification to obtain a DNA yield sufficient for in-vitro transcription. The purified DNA was processed using the Qiagen QIAquick PCR Purification Kit and quantified using a spectrophotometer (NanoDrop). After DNA purification, in-vitro transcription was carried out using the MEGAshortscript TM T7 Kit (Thermo Fisher), using 480 ng/µL DNA as template per reaction. Following transcription, RNA purification was performed using the Macherey-Nagel RNA Clean-Up Mini Kit. RNA was quantified by NanoDrop to verify that the concentration was above 1 µg/µL, as required by the reverse transcription PCR (RT PCR) step. Single-stranded DNA (ssDNA) was generated through a reverse transcription reaction using SuperScript IV Reverse Transcriptase, with the addition of 52 µg of RNA to obtain sufficient yield of ssDNA. At this stage, ssDNA probes were labeled with three different fluorophores: Alexa fluor 488 (ALP-a488, green), Alexa fluor 546 (ALP-a546, red), and Alexa fluor 647 (ALP-a647N, far red). The process resulted in RNA-DNA hybrids and some unincorporated primers. Exonuclease-I was used to digest unincorporated primers, and RNase was used to digest the RNA from RNA-DNA hybrids. After RNA and primer digestion, labeled ssDNA was purified using the Zymo Quick-RNA purification kit and quantified. Fluorescent in-situ hybridization: The FISH protocol was adapted from Bass et al. 1997 43 that used the polyacrylamide embedded technique for 3D FISH analysis. Fixed EdU-labeled nuclei were embedded in a thin layer of optically clear 3X acrylamide mix on a glass slide. Then wash buffers and prehybridization buffers were exchanged by 200-µL droppers. EdU-labeled nuclei remained intact throughout the procedures of washing and equilibrating. A buffer containing RT class-labeled probes, 50% formamide, and 2X SSC was added and incubated at 37°C for 30 min for prehybridization. After prehybridization, incubation slides were placed on a hot plate at 65°C for 30 min for genomic DNA denaturation. After denaturation, the slides were placed back in the 37°C incubator overnight for hybridization. The following day, the slides underwent serial washing with buffers of increasing stringency. Finally, slides were mounted with mounting media and sealed with Sally Hanson nail hardener. Deltavision microscopy and Image analysis: Following the FISH experiment, 3D images were captured with 0.2-micron projections in multiple wavelength iterative deconvolution microscope. The raw data was subsequently subjected to 3D iterative deconvolution and chromatic aberration correction (CAC). For analysis purposes after 3D data collection, individual nuclei with probe signals were cropped to allow quantitative signal colocalization analysis using the inbuilt solid object builder polygons method. For these analyses, the control regions were non-FISH euchromatin areas selected from the same nuclei. Quantitative segmentation data analysis was performed on at least 50 nuclei for each Early and Middle probes labeled experiment. All 3D images were stored on the image repository omero.bio.fsu.edu, allowing for additional segmentation analysis. RESULTS Repli-seq scheme and minidomain chromatin fiber RT model The maize ( Zea mays L.) genome consists of 10 metacentric chromosomes, ranging from 150 to 300 Mb in size, and has been annotated with regard to DNA replication timing (RT) using previously published Repli-seq data 2 and the RepliScan pipeline to segment the genome into discrete RT classes 42 . For this study, the Repli-seq data was remapped to the B73 AGPv5 assembly and segmented using RepliScan. The DNA RT profiles for sequences that replicate at early-S, middle-S, or late-S are visualized in a genome browser as shown in Fig. 1 for maize chromosome 5. We selected chromosome 5 as a representative chromosome, based on the criterion that it reflected the whole genome RT class frequencies previously defined by Repli-seq 2 . The (RT) profile along the entire length of maize chromosome 5 (Fig. 1 a) illustrates regional enrichments for early and late replication, corresponding to euchromatic, gene-rich areas and heterochromatic, gene-poor sections, respectively. The early-S RT profiles show the highest coverage in the distal regions of the chromosome arms where gene density is higher 44 . In contrast, late-S RT profiles show the highest coverage near the center of the chromosome at the centromeric and pericentromeric regions, which are enriched in repetitive sequences and transposons. The middle-S RT profiles show a unique pattern of being relatively evenly distributed across most of the chromosome. A distinct non-centromeric block of late-S replicating chromatin is also seen at the heterochromatic knob, embedded in the long arm of chromosome 5. To gain further insight into the relationships among the different RT classes, we inspected a 2-Mb non-heterochromatin region on chromosome 5 to specifically focus on the distribution of early-S versus middle-S (Fig. 1 b, blue and green) RT classes. These are shown in relation to genes and chromatin accessibility data 45 , reflecting the known stronger coupling of early-S replicating regions with genes and open chromatin compared to middle-S replicating regions. This 2-Mb region illustrates a striking pattern of alternating early-S and middle-S segments, which switch back and forth repeatedly over these large genomic areas. Based on cytological analysis of early and middle replication patterns, a model of euchromatin organization was proposed as the "minidomain chromatin fiber RT model" 18 . According to this model, maize euchromatin, which is not uniformly stained in cytological preparations, exists in two interspersed subcompartments corresponding to early-S and middle-S replicating regions (Fig. 1 c-e). These early-S chromatin regions are relatively weakly stained with DAPI, whereas the middle-S chromatin regions exhibit stronger DAPI staining (Fig. 1 e). Given that these two distinct chromatin types were defined in S phase, we set out to test the idea that they reflect a general nuclear architecture in maize present throughout the interphase. This idea was first tested using high-throughput conformation capture (Hi-C) analysis with nuclei from the entire mitotic cell cycle (Fig. 1 f-h). Hi-C data reveals covariance and correlation with early and middle replication timing in maize euchromatin We used formaldehyde-fixed root tip nuclei from 3-day-old seedlings (Fig. 1 f-h) to prepare Hi-C libraries (Supplemental Fig. 1) in biological replicates of 1.25 million nuclei each. For comparison, we also made Hi-C libraries from immature earshoot nuclei. The analysis of the resulting Hi-C libraries provided genome coverage and valid pair contact frequencies comparable to that previously reported for maize bundle sheath cells 32 . This confirmed that the contact patterns remained intact following the flow-sorted nuclei isolation and library preparation steps (Supplementary Table 1). To compare nuclear architecture to DNA replication timing, we plotted Hi-C contact matrices from selected regions of chromosome 5 as shown in Fig. 2 . At the whole chromosome scale, we see the typical maize Hi-C chromosome structure in which euchromatin and centromere-associated/pericentromeric heterochromatin are evident (Fig. 2 a), reflecting the global A (black) and B (grey) compartments, respectively 32 , 35 . To more specifically examine early-S versus middle-S, we looked at three different 2-Mb regions (Fig. 2 b-d) on the short arm of chromosome 5. These 2-Mb heatmap matrices show the Hi-C contacts along with early, middle, and late RT segments (top, left) in blue, green, and red, respectively. We observed the expected strong diagonal signal, indicating frequent interactions among adjacent loci. Beyond the diagonal, interaction hotspots were observed (examples highlighted in blue and pink circles). Blue circles highlight contacts between two non-adjacent early RT regions, while pink circles mark interactions between early and middle RT regions. These represent long-range cis-interactions within these representative 2-Mb regions. Notably, early-S replicating segments appeared to display more long-reaching interactions with other early-S replicating segments compared to adjacent middle-S replicating segments. Some of these early-to-early contacts extend beyond 1 Mb, skipping multiple intervening RT segments. To compare more directly the HiC contact matrices to RT classes, we looked at these same 2-Mb regions by plotting RT profiles and segments along with the chromosome-wide eigenvector (EV) and insulation score (IS) analyses for all four biological replicates (Figs. 2 e–g). The insulation score of a genomic region measures its frequency of interaction with the neighboring regions. A low or negative IS indicates a scarcity of local interactions relative to longer-range contacts 36 . Strikingly, most of the points at which the EV and IS values switched from positive to negative mirrored the boundaries between the DNA replication timing segments. For instance, the light blue-shaded regions, representing early-replicating segments, showed positive EV and negative IS. The low insulation scores of early-S RT regions denote a greater tendency for long-range contacts, consistent with the hotspots of contacts highlighted in the contact matrices (blue circles, Fig. 2 b-d). Relatedly, the green-shaded region, indicating middle-S RT segments, corresponded to negative EVs and positive ISs, suggesting a higher insulation score with more confined interactions 36 . Finally, the red-shaded areas denoted late-S RT regions with corresponding negative EV and positive IS. These patterns reflect the fact that Hi-C is capturing smaller-scale nuclear architectural features beyond that of the global A and B compartments. Notably, the annotations for RT and HiC mirror each other in distinguishing early-S and mid-S euchromatin regions. To determine if these relationships between nuclear architecture and replication timing hold true for the rest of the genome, we carried out a correlation analysis between RT and Hi-C. For this we combined the chromosome-wide HiC data (EV and IS) from all ten chromosomes. The early-S Repli-seq coverage values showed a strong positive correlation ( r = + 0.81) with the eigenvectors, whereas the middle-S Repli-seq coverage showed a slight negative correlation ( r = -0.15). The late-S Repli-seq coverage values showed a strong negative correlation ( r = -0.67) with the eigenvectors (Fig. 2 h). When doing a similar genome-wide correlation with IS, we observed a strong negative correlation with early-S Repli-seq coverage ( r = -0.59), a weak positive correlation with middle-S coverage ( r = + 0.18), and a strong positive correlation with late-S- Repli-seq coverage ( r = + 0.41) (Fig. 2 i). For both eigenvector and insulation scores, the strongest but opposite correlations were observed for early-S compared to late-S chromatin. The middle-S chromatin showed the same direction of correlation as that of the late-S chromatin, but with much smaller correlation values. Together, these data show that genomic regions distinguished by replication timing also correlate with distinct Hi-C-defined chromatin and genomic organization across the whole genome. We compared our Hi-C data for root tips to the eigenvector and IS data from earshoots analyzed in parallel (Supplementary Table 1) and bundle sheath cells from a published study 32 . Pairwise comparisons between tissues (supplementary Fig. 2) revealed strong genome-wide similarity between root tip and earshoot Hi-C profiles ( r = 0.85 for both EV and IS), both of which are actively growing tissues. We also observed slightly weaker but significant correlations between root tip and bundle sheath Hi-C profiles ( r = 0.73 for EV and 0.66 for IS), possibly reflecting a more cell-type independent global conservation of genome organization. We also investigated whether any of the ten maize chromosomes showed more or less tissue-specific variation compared to the whole genome (Supplementary Fig. 3a & 3b). Interestingly, we found that the root tip and ear shoot Hi-C data showed a strong positive correlation across all chromosomes with a notable exception for chromosome 6, which contains the nucleolar organizing region near the end of the short arm. The correlation coefficient for chromosome 6 was r = 0.62, compared to all the others which ranged from r = + 0.78 to + 0.95 (Supplementary Fig. 3a). We found a similar situation for root tip versus bundle sheath, with chromosome 6 showing the lowest correlation (Supplementary Fig. 3b). In addition, most of the other chromosomes showed relatively weaker correlations with bundle sheath data, likely due to variations in Hi-C library preparation and/or tissue type. Using a stratum-adjusted correlation coefficient (SCC) analysis 46 , we also found that root tip to earshoot similarity was higher than that of either one compared to bundle sheath (Supplementary Fig. 4). Given the strong correlations between replication timing and Hi-C defined nuclear architecture in root tip, we wondered if the RT classification alone from root tip could be predictive of HiC nuclear architecture in other tissues. For this, we did correlation analysis between root tip Repli-seq and the Hi-C EV data of the other two tissues, for which we do not have replication timing data (summarized in Fig. 3 ). The root tip Repli-seq-defined genome structure closely mirrored the Hi-C Eigenvector (Fig. 3 a-b), which can be seen locally (Supplementary Fig. 5b-c) and globally (Fig. 3 c). These analyses demonstrated that replication timing in root tip cells predict chromatin structure across other tissues, including earshoot and bundle sheath, suggesting a robust association between replication timing and chromatin organization across the plant. HiC contacts are enriched within RT classes Given the high similarity among the root tip biological replicates (Supplementary Fig. 4), Hi-C contacts from all root tip replicates were pooled for subsequent analyses. We used the pooled data to examine the degree to which moderate to long range contacts were occurring between regions with the same replication timing assignments, i.e. within-class contacts, as predicted by the 2-compartment model for euchromatin (Fig. 4 ). We assigned replication timing (RT) class annotations to each of the two members of the valid pair contacts and calculated the frequencies of contacts with matching and differing pairs for the RT classes, for Early, Middle, or Late regions. The analysis revealed that of all the valid pair contacts (Fig. 4 a), Early-Early accounted for 28% (2.93 million), Middle-Middle for 24% (2.47 million), and Late-Late for 16% (1.65 million). Contacts between two different RT classes were less frequent, with 16% Early-Middle, 13% Middle-Late, and only 3% Early-Late. These findings indicate the "same-class" categories are the most frequent, even after normalizing to account for their relative abundance (Fig. 4 b). To examine more closely the same-RT-class contacts, we conducted a virtual 4C analysis of two early and two middle RT genomic regions, each approximately 150 Kb, located on chromosome 5 (Fig. 4 c). This approach allowed for the identification of long-range interactions centered around a specific genomic region of interest, known as the “bait” region, or virtual bait in this case. Two early and two middle RT segments were designated as baits, and their same-class contacts were plotted along the entire chromosome 5 (Fig. 4 d). All four baits showed long distance contacts, including to regions on the other arm of the chromosome, but the early-early contacts extended further, and even beyond the distal knob on the long arm of chromosome 5. This difference can not be explained by the location of the baits, because they come from the same local region (Fig. 4 c), which represents less than 1% of the 226 Mb chromosome 5. We observed consistent results when we decomposed the contact matrices for all of chromosome 5 into middle-versus-middle (Fig. 4 e) and early-versus-early RT contacts only (Fig. 4 f). The per-bait analysis also showed that the same class contacts were the predominant type (Supplementary Fig. 6). The least frequent contacts were between early or middle baits and late-S regions for all four segments analyzed (Supplementary Fig. 6a-d). We next investigated the persistence of these same-class contact tendencies as a function of distance relative to the linear genome. The results demonstrated the expected distance-dependent decay of all contact classes, with early-early RT class contacts showing the highest frequency at any given distance (Supplementary Fig. 6e). This enrichment was not a result of the relative abundance of the RT classes per se, as shown using a shuffled-position RT control (Supplementary Fig. 6f). Overall, the contact class pair enrichments and the virtual 4C bait results showed that same-class contacts are more frequent than cross-class contacts, and that early RT segments exhibit more of the longest-range interactions than the middle RT segments. HiC-based chromatin architecture aligns with epigenomic and transcriptional features To test how our Hi-C data correlated to genomic and genetic features, we checked the correlation of our Hi-C eigenvectors with epigenetic and transcriptional features known to be associated with chromatin structure (Fig. 5 ). We first examined how the Hi-C data correlated with histone post-translational modifications using ChIP-seq data from B73 maize root tips 2 , 47 , 48 . We previously anticipated that modifications associated with active genes would be positively correlated with early-S replication timing regions within euchromatin, whereas repressive marks would not be or less so 21 . Indeed, we found significant correlations between our root tip Hi-C eigenvector (EV) data analyzed at 50-kb resolution on chromosome 5 and the active gene-associated marks (Fig. 5 a-d). Among the marks with positive correlations, H3K27ac and H3K4me3 had the strongest correlations with r = + 0.73 and + 0.65. The values for the histone marks and the eigenvector varied along the length of the chromosome, showing a general trend of higher values in the chromosome arms for all of the marks tested, except for H3K9me2 (Supplementary Figs. 7a-d). Consistent with our findings, regions enriched for H3K56ac (EV r = + 0.45) and H3K4me3 (EV r = + 0.65) were previously classified as early-S replicating regions, which also shows enrichment for genes with high median expression levels 2 . Regions enriched for H3K27me3, on the other hand, exhibited a weakly positive association with Hi-C EVs (Fig. 5 e, r = + 0.13). Interestingly, the H3K27me3 mark has been associated with facultative heterochromatin 49 , 50 and also found to be enriched in middle-S replicating regions and genes with lower median expression levels 2 . The only histone mark we examined that showed a negative correlation with our root tip EV was H3K9me2 (Fig. 5 f, r = -0.20), which is consistent with its known association with constitutive heterochromatin 51 , 52 . We also investigated the correlation between Hi-C EVs and transcriptional activity estimates from the RAMPAGE transcription start site mapping data and it showed a positive correlation (Fig. 5 g, r = + 0.53) 48 . Finally, we compared Hi-C EV with chromatin accessibility using micrococcal nuclease (MNase) sensitivity profiling datasets from maize root tip nuclei. The read coverage levels from a light MNase digestion 45 showed a positive correlation of r = + 0.48 with Hi-C EVs (Fig. 5 h). Likewise, the open chromatin defined by sequencing small fragments from light MNase digests in purified G1 nuclei showed a positive correlation of r = + 0.42 with Hi-C EVs (Fig. 5 i). These were calculated for chromosome 5 because it was selected for additional cytological analysis, but similar trends were observed genome wide as shown in Fig. 6 for correlations between these same epigenomic features and both HiC and replication timing. 3D cytological evidence for the spatial bifurcation of euchromatin into early-S and middle-S chromatin regions We next tested the 2-compartment euchromatin model using a chromosome painting strategy based on probes from different replication timing classes (Fig. 7 ). For this independent strategy, we continued to use the representative chromosome 5, one that reflected the whole genome RT class frequencies previously defined by Repli-seq 2 . We found that the RT class frequencies for the short arm of chromosome 5 (chr5S) closely matched those of the whole genome (Fig. 7 a). For instance, Early-S segments were 30% of chr5S and 30% of the whole genome, whereas middle-S segments were 38% and 35% of chr5S and the genome, respectively. Therefore, we designed RT-class-specific oligo FISH painting probes at comparable densities (Fig. 7 b) spanning the entire chr5S. For the oligo FISH probes, we developed three sets of oligo painting libraries: Early (E), Middle (M) and Late (L) across our three RT class segments which collectively account for 82% of chr5S. Probe distribution along the chr5S arm aligns with the RT classes. Distal regions are enriched for early-S segments and probes (blue tracks, while centromere proximal regions are enriched for late-S segments and probes (red tracks). Middle-S segments and probes (green tracks) were evenly distributed across the short arm (Fig. 7 c). The interspersed pattern of early S and middle S segments and probes is better seen by focusing on a 2-MB region from the middle of chr5S (Fig. 7 d). For the RT class oligo FISH painting experiment (Fig. 8 ), early and middle probes were labeled with different fluorophores and hybridized to a mixed population of nuclei from the entire cell cycle, made by pooling G1, E, M, L and G2 nuclei (Supplementary Fig. 8d). Representative images from 3D deconvolution image datasets are shown as 1-µm Z-stack projections, a five-optical-section projection spanning the FISH probe signal regions. For quantitative analysis, we selected nuclei in which two similar sized paint signals were observed for diploid chromosome 5 (Fig. 8 a, circles). In these nuclei, non-specific background staining of the nucleolus (labeled "n") was commonly seen, but the maize NOR is not on chromosome 5 and this non-specific signal was disregarded. To quantify the DAPI in the FISH-marked regions, we manually segmented the FISH paint signals to create volumetric polygon objects to obtain DAPI density (average photon counts per cubic micron). As a control, random non-FISH euchromatin regions were selected, avoiding the nucleolus and bright knobs. In this way, we could obtain the DAPI values for chromatin in painted RT class probe regions relative to bulk euchromatin control regions (Fig. 8 b). By plotting the log2 ratio of DAPI density in the FISH versus control regions, we showed that the average DAPI concentration in Early-S chromatin (mean of -0.26) was less than the control regions, whereas the middle-S FISH chromatin (mean of + 0.21) was higher. The RT painted and control region ratios did not overlap (Fig. 8 C, box plot). Given that observation, along with the fact that we sampled nuclei from a mixture of G1, S, and G2 in which S phase was only ~ 41% of the total, we concluded that this relationship between replication timing and DAPI density holds true for nuclei across the cell cycle, not just in S phase (Fig. 8 c). To more definitively establish the spatial bifurcation of euchromatin outside of S phase, we performed dual probe FISH experiments using early and middle probes on purified G1 nuclei as summarized in Fig. 9 . Representative images of dual-labeled G1 nuclei (Fig. 9 a) showed spatial separation of early-S and middle-S probe regions corresponding to the same unreplicated chromosome. We repeatedly observed paired but spatially separated signals for the early-S versus middle-S probes, in contrast to their intermixed, linear arrangement along chromosome (Fig. 1 b). By segmenting the FISH signal regions, we could directly compare DAPI concentration in early-S chromatin (green outlines) relative to middle-S (red outlines) chromatin from the same chromosome, avoiding any uncertainty in selecting "control" regions from other parts of the nucleus. We quantified the DAPI intensity for each chromosome-based pair of FISH signals (Fig. 9 b), providing a log2 ratio that captures the relative chromatin compaction of the two FISH-delineated areas. We found that the average DAPI concentration ratios for all the Early-Middle FISH region pairs quantified was 0.86 (sd 0.07) in the G1 nuclei (Fig. 9 c), clearly establishing that RT defined regions are spatially separated and differentially condensed, even outside of S phase. These results validate the mini-domain RT chromatin model, demonstrating that maize euchromatin exists as an interspersed mixture of two compartments distinguishable by condensation state and replication timing. Early-S FISH probes localize preferentially in regions with low DAPI signals, whereas middle-S FISH probes coincide with regions of higher DAPI intensity. 3D dual-label RT FISH painting in G1 nuclei provided compelling evidence, consistent with Hi-C data, that the two euchromatin compartments are integral features of nuclear organization. DISCUSSION This study sheds light on the close relationship between DNA replication timing (RT) and chromatin organization in the maize genome, focusing particularly on nuclei in the terminal 1 mm of actively growing root tips and their euchromatin compartments. By using two orthogonal methods for defining nuclear architecture, Hi-C, and 3D FISH, we demonstrated how replication timing reveals a spatial bifurcation of maize euchromatin (the global A) into separate early-S and middle-S compartments. These findings support our earlier minidomain chromatin fiber RT model 18 , 21 and extend it into G1 (Fig. 9 a-c), outside of S-phase. Other studies in eukaryotic systems point to the fact that simple global A and B compartments are widely conserved, but are insufficient to understand the complexity of structural and functional organization of chromatin. Mammalian chromatin, for instance, has been divided into a variable number of states or subcompartments, ranging from three to eight or more, based on various approaches including epigenomic marks, modularity-based analyses, trans contacts, hierarchical clustering of domains, and data from TSA-seq, DamID, and Hi-C 53 – 57 . Similarly, plant chromatin, primarily from Arabidopsis studies, has also been shown to possess multiple chromatin states defined by distinct features 14 , 58 – 60 . These studies tend to agree that the global A compartment can be split into two subcompartments, A1 and A2, which replicate in that order in plants and animals 32 , 53 , 56 . We speculate that the maize early-S and middle-S chromatin described here may reflect an A1 and A2 type of subdivision, which a single assay, replication time profiling, can be used to map. Consistent with this, a recent light and EM cytological analysis of the very large genome plant, Nigella damascena , also revealed that early-S versus middle-S replication partitioned into more open versus less open chromatin, respectively 17 . Evidence for the nonrandom spatial distribution of early, middle, and late RT domains is supported by genomic RT profiling with Repli-seq 2 and 3D quantitative microscopy. Within S-phase, using replicative labeling, we know that early replication predominantly occurs in distal, gene-rich euchromatic regions with open chromatin features, whereas middle replication corresponds to denser euchromatin associated with repetitive elements and less active genes 18 . The interspersed early and middle RT domains in the 2-Mb segment of chromosome 5 (Fig. 1 b) reflect their alternating nature and associated chromatin states. Here we document the robust covariance between chromatin interactions and RT annotations. Early RT domains exhibit higher contact frequencies with other early regions, even across long genomic distances, as evidenced by the eigenvector (EV) and insulation score (IS) analyses (Figs. 2 & 4 ). Middle RT domains exhibit a lesser tendency for long reaching interaction and show a more condensed chromatin structure. The "same-RT-class" interactions may reflect different functions. For example, early-early interactions might be largely based on transcriptional activity, while middle-middle interactions - occurring in regions with less active genes and abundant retroelements - might reflect a chromatin packaging function. In general, it is known that the chromatin regions that replicate at different time points in S phase, such as early versus late, also show distinct enrichments for epigenomic features in plants and animals 2 , 11 , 61 . For instance, early-S replicating regions generally exhibit open and accessible chromatin, enrichment for active chromatin marks such as histone acetylation, and relatively higher levels of transcriptional activity. On the other hand, late-S replicating regions generally exhibit the opposing features such as closed and repressive chromatin marks, including histone methylation on H3K27 or H3K9, and DNA methylation. In contrast to the established framework of a binary division of S-phase into early-S versus late-S stages, our study emphasizes a tripartite replication timing framework, with early, middle, and late S profiling. We focused on the early versus middle because they appear similar in their broad distribution within nucleoplasmic HiC-defined as global A compartment but are distinct in other ways. Interestingly, the Middle-S chromatin has features of both euchromatin and heterochromatin. Middle-S tends to lack the marks associated with the most active chromatin but is only weakly enriched for repressive histone marks. For instance, the gene-active chromatin histone acetylation mark H3K27ac had correlation values of r = + 0.72 for Early-S versus r = − 0.61 for Middle-S chromatin. Similarly, but in the opposite direction, the gene-repressive chromatin mark H3K9me2 had correlation values of r = − 0.19 for Early-S versus r = + 0.044 for Middle-S chromatin (Fig. 6 ) consistent with previous study 2 . In all cases, we saw that the Middle-S regions have chromatin mark correlations that can be described as (1) being in between those of Early-S and Late-S, (2) weaker overall, and (3) more similar to those of Late-S regions. In addition, transcriptional characteristics of genomic regions, including transcription start site mapping and transcriptomics, also tend to mirror the pattern of epigenomic marks with regard to gene activity. Specifically, analyzing expression levels for promoters using the RAMPAGE assay 48 , 62 , we observed a correlation of r = + 0.55 for Early-S chromatin and r = − 0.12 for Middle-S chromatin. Indeed, our epigenomic enrichment and Hi-C analyses provide concordant substantiation for the unique identity of Middle-S chromatin, which comprises a third of the maize genome. The FISH experiments also provided new and compelling cytological evidence for the bifurcation of the global A compartment into early and middle chromatin. Unlike our previous work, which was limited to replicative labeling within Early-S or Middle-S, our RT-FISH approach allowed us to examine the spatial location of the RT-defined sequences outside of S-phase and independent of replicative labeling. We carried out two related types of experiments. The first experiment used early and middle RT FISH probes separately on total nuclei and quantified the DAPI concentration in the FISH-defined regions relative to a similar sized chromatin control region with properties typical of bulk euchromatin (Fig. 8 ). The second experiment used both early and middle probe sets labelled with different fluorophores on G1 nuclei. This enabled us to simultaneously visualize the spatial separation and nuclear localization of both RT-FISH signals, as well as to quantify their relative DAPI densities, without the need for an external control region (Fig. 9 ). In every case, we observed signal partitioning validating the 2-compartment architecture, which we now know is present in G1 nuclei. This finding demonstrates that RT-related nuclear architecture exists prior to S phase, and is not simply a result of processes in S-phase itself. Given that Hi-C compartments and replication timing are tightly coupled in root tip, we reasoned that one could ask to what extent replication timing programs might be conserved in other tissues, using Hi-C structure as a proxy for replication timing. We showed that biologically vastly different organs, earshoot versus root tip, had strong correlations for Hi-C based compartmentalization genome-wide, with r = + 0.85 for both Eigenvalues and insulation scores (Supplementary Fig. 2). When correlating replication timing from root tip with Hi-C measures from earshoot or bundle sheath, the correlations were similar in strength and direction to those from within-tissue comparisons (Fig. 3 ). These findings provide evidence in support of the idea that the two compartment architecture of euchromatin (as depicted in Fig. 2 c-e) not only extends throughout the cell cycle in root tips but exists in other tissues as well. This observation raises the intriguing possibility that this pattern of nuclear architecture may reflect a broader organizational principle in other large-genome eukaryotes. Declarations AUTHOR CONTRIBUTIONS HWB, LH-B and WFT conceived the study. HSA, HWB, LC, LH-B and WFT designed the experiments. HSA prepared Hi-C libraries and performed 3D FISH experiments, collected and analyzed the data. ZMT prepared MOA-seq libraries. LC and HWB contributed to the analysis and interpretation of data. EEW and LM-Y provided maize root tip nuclei for the study. HSA and HWB drafted the manuscript and all authors critically revised and approved the final manuscript. COMPETING INTERESTS We do not have any competing interests. MATERIAL AND CORRESPONDENCE Material request from the corresponding author Hank W. Bass. ACKNOWLEDGEMENTS The authors would like to acknowledge help with early versions of the data analysis by Dr. Jawon Song and Joshua Urrutia at the Texas Advanced Computing Center. This work was supported by a grant from the National Science Foundation Plant Genome Research Program (NSF IOS 2025811 to LH-B, LC, WFT, and HWB), awards from Florida State University (Dean's Award for Doctoral Excellence to HSA; Ben and Karen Thrower award to HSA), and funds from North Carolina State University. DATA AVAILABILITY STATEMENT Raw Hi-C data, MOA-seq data, processed all valid pairs files, HiC files and bigwig files can be accessed at NCBI GEO under the accession number GSE287128. 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Genetics 200:1105–1116 Hufford MB et al (2021) De novo assembly, annotation, and comparative analysis of 26 diverse maize genomes. Science 373:655–662 Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryFile1HiCprotocol.docx HiC protocol SupplementaryFile2allRTsegments.txt Dataset 1 SupplementaryFile3EMLprobes.zip Dataset 2 SupplementaryFile4probeLabelProtocol.docx FISH probe labeling protocol SuppFilesAllSuppT1.pdf Supp Figures 1-8 and Supp Table 1 SuppFilesAllSuppT1V2.pdf Supplementary Information Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6116464","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":426871778,"identity":"9bf7b88c-7173-4445-a3e8-e83e1705caa3","order_by":0,"name":"Hafiza Sara Akram","email":"","orcid":"https://orcid.org/0000-0002-6375-0568","institution":"Department of Biological Science, Florida State University, Tallahassee, FL, USA, 32303","correspondingAuthor":false,"prefix":"","firstName":"Hafiza","middleName":"Sara","lastName":"Akram","suffix":""},{"id":426871779,"identity":"ea3212df-ecdd-4843-a714-a62dee3309c2","order_by":1,"name":"Emily E. 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Turpin","email":"","orcid":"https://orcid.org/0000-0002-6488-2503","institution":"Department of Biological Science, Florida State University, Tallahassee, FL, USA, 32303","correspondingAuthor":false,"prefix":"","firstName":"Zachary","middleName":"M.","lastName":"Turpin","suffix":""},{"id":426871782,"identity":"050a5d9c-74a0-44ad-8c12-eb48e4f33c74","order_by":4,"name":"Linda Hanley-Bowdoin","email":"","orcid":"https://orcid.org/0000-0001-7999-8595","institution":"North Carolina State University, Department of Plant and Microbial Biology; Raleigh, NC USA 27695","correspondingAuthor":false,"prefix":"","firstName":"Linda","middleName":"","lastName":"Hanley-Bowdoin","suffix":""},{"id":426871783,"identity":"060942ac-c623-4803-aaa6-6e69ba3fcc40","order_by":5,"name":"William F. Thompson","email":"","orcid":"https://orcid.org/0000-0001-5034-8337","institution":"North Carolina State University, Department of Plant and Microbial Biology; Raleigh, NC USA 27695","correspondingAuthor":false,"prefix":"","firstName":"William","middleName":"F.","lastName":"Thompson","suffix":""},{"id":426871784,"identity":"e972ce71-f88b-4922-a397-f1744a6b0e41","order_by":6,"name":"Lorenzo Concia","email":"","orcid":"https://orcid.org/0000-0002-7401-7214","institution":"University of Texas at Austin, Texas Advanced Computing Center, Austin, TX, USA, 78758","correspondingAuthor":false,"prefix":"","firstName":"Lorenzo","middleName":"","lastName":"Concia","suffix":""},{"id":426871777,"identity":"c80f8072-bac1-4fef-8417-41a45cf14314","order_by":7,"name":"Hank W. Bass","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYBACAxDxsUGCgYH5AIjJTJwWxpkgLWwJJGhh5m1gIEGLOfvxx59td1jIMbCxP5NgqLBObCCkxbInx0w694yEMQMbj5kEw5l0wloMDuSwMee2SSQ2yPewSTC2HSZCy/nnjz9bgrSAHMb4jxgtNxIMpBnBWhjMJBgbiNBiOeONmWQv0C9sbDzGFgnH0o0JajHnT3/84eeOOjl+NvaHNz7UWMsS1AIHbCAigWjlo2AUjIJRMArwAgBfJDaFIdjvmgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-0522-0881","institution":"Department of Biological Science, Florida State University, Tallahassee, FL, USA, 32303","correspondingAuthor":true,"prefix":"","firstName":"Hank","middleName":"W.","lastName":"Bass","suffix":""}],"badges":[],"createdAt":"2025-02-27 00:50:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6116464/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6116464/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78332959,"identity":"a5462a65-f1bc-4b30-b933-a9a4fd09d61a","added_by":"auto","created_at":"2025-03-12 07:29:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":581471,"visible":true,"origin":"","legend":"\u003cp\u003eRepli-Seq scheme and minidomain chromatin fiber RT model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e A schematic representation of euchromatin and heterochromatin distributed along chromosome 5, including a centromere (CEN) \u003ca href=\"https://paperpile.com/c/oAFTKb/uRUN+awuw\"\u003e\u003csup\u003e63,64\u003c/sup\u003e\u003c/a\u003e and\u0026nbsp; knob (K) on the long arm. Below, the Repli-seq is shown, with early RT (blue), middle RT (green), and late RT (red) spanning the entire chromosome. \u003cstrong\u003e(b)\u003c/strong\u003e UCSC Genomaize browser view zoomed into a 2-Mb non-heterochromatin region, showing RT segments defined by Repliscan, gene models, the replication timing profile, MNase light digest coverage and Root DNS-seq tracks. Digestion sensitivity is represented by dark color on density heatmap. \u003cstrong\u003e(c)\u003c/strong\u003e DAPI-stained image of maize interphase nuclei. The nucleolus is labeled “n”, and one of the knobs is highlighted as “K”. The yellow box indicates the euchromatin region magnified in the next panel. \u003cstrong\u003e(d)\u003c/strong\u003e A zoomed area of the euchromatin region with fiber-like structures. \u003cstrong\u003e(e)\u003c/strong\u003e Illustration depicting two euchromatin sub-compartments: Early-S (thin/gray) and Middle-S (thick/black). \u003cstrong\u003e(f-h)\u003c/strong\u003e Workflow: \u003cstrong\u003e(f)\u003c/strong\u003e 3-day-old seedlings; \u003cstrong\u003e(g)\u003c/strong\u003e excised 0-1 mm root tip for fixation and nuclei purification \u003cstrong\u003e(h)\u003c/strong\u003e nuclei stained with DAPI, analyzed using flow cytometry (DAPI fluorescence with emission filter 460 ± 50 nm). Total nuclei were collected for each biological replicate of Hi-C.\u003c/p\u003e","description":"","filename":"Figure1RepliSeqModel.png","url":"https://assets-eu.researchsquare.com/files/rs-6116464/v1/59c264e7063bb801be515030.png"},{"id":78331980,"identity":"77b5bc3f-a6e1-4de2-aeaf-0996c29bb3ff","added_by":"auto","created_at":"2025-03-12 07:21:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":878633,"visible":true,"origin":"","legend":"\u003cp\u003eHi-C on maize root tip nuclei.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Repli-seq and its correlation with chromatin structure (Hi-C) eigenvector analysis across chromosome 5, with the knob (K) and centromere (CEN) labeled. \u003cstrong\u003e(b-d)\u003c/strong\u003e Hi-C contact matrices for 2-Mb genomic regions (indicated by orange rectangles) from chromosome 5. RT classification segments (early in blue, middle in green and late in red) are shown on the top and left edges of the matrices. Color intensity represents the frequency of contacts between two loci. Blue and pink circles highlight examples of early-early and early-middle contacts, respectively. \u003cstrong\u003e(e-g)\u003c/strong\u003e UCSC Genomaize browser views of the same 2-Mb regions, showing the RT profile, chromosome-wide eigenvector (EV) analysis, and insulation score (IS) tracks for four root tip biological replicates. Light blue, green, and red shaded regions correspond to early, middle, and late RT segments, with their respective EV and IS patterns. \u003cstrong\u003e(h)\u003c/strong\u003e Correlation plots illustrate the correlation between root tip Hi-C eigenvector (y-axis) and root tip Repli-seq data (x-axis). \u003cstrong\u003e(i)\u003c/strong\u003e Correlation plots of root tip insulation score data (y-axis) and root tip Repli-seq data (x-axis) for RT classes: early, middle, and late. Hexagonal cell intensity indicates the number of data points, with density plots on the top and left showing independent distributions for each variable.\u003c/p\u003e","description":"","filename":"Figure2Chr5HiCmatrix.png","url":"https://assets-eu.researchsquare.com/files/rs-6116464/v1/106d6137ec984abe2b700734.png"},{"id":78331979,"identity":"ad5db19e-6786-4eae-93c9-23c4d811e0af","added_by":"auto","created_at":"2025-03-12 07:21:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":135016,"visible":true,"origin":"","legend":"\u003cp\u003eRoot Repli-seq versus Hi-C from different tissues\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Correlation plot showing root tip Repli-seq data on the x-axis and earshoot eigenvector (EV) analysis data on the y-axis. \u003cstrong\u003e(b)\u003c/strong\u003e Correlation plot showing root tip Repli-seq data on the x-axis and bundle sheath eigenvector (EV) analysis data on the y-axis. Density plots above and to the left of each plot represent the independent distributions of the variables. \u003cstrong\u003e(c)\u003c/strong\u003e Bar chart comparing replication timing (RT) classes (early: blue, middle: green, late: red) in root tip versus eigenvector in root tip, earshoot, and bundle sheath tissues.\u003c/p\u003e","description":"","filename":"Figure3HiCvsRT.png","url":"https://assets-eu.researchsquare.com/files/rs-6116464/v1/2b6134140ea0f47ef4f21d82.png"},{"id":78331462,"identity":"d43617ac-7378-4a7a-9132-4ec1107c9549","added_by":"auto","created_at":"2025-03-12 07:13:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":735493,"visible":true,"origin":"","legend":"\u003cp\u003eRT class contact enrichment and virtual 4C analysis\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Table showing the contacts for each replication timing (RT) class pair, number per million and percentages of valid Hi-C contact pairs. Contacts between different RT classes in both directions (e.g., early-middle or middle-early) are represented within the same cell. \u003cstrong\u003e(b)\u003c/strong\u003e Observed versus expected Hi-C valid contacts, with the same RT class contacts shown in blue (early-early), green (middle-middle), and red (late-late). Gray bars represent different RT class contacts. \u003cstrong\u003e(c)\u003c/strong\u003eUCSC Genomaize browser view of virtual 4C baits targeting \"Early\" and \"Middle\" RT segments, alongside the RT profile. \u003cstrong\u003e(d)\u003c/strong\u003e Blue and green arcs represent genomic contacts originating from early and middle RT segments, respectively, from each of the four bait regions and extending across chromosome 5. The RT profile is shown at the bottom of the plot. \u003cstrong\u003e(e) \u003c/strong\u003eHi-C contact matrix showing only middle-middle RT contacts for all of chromosome 5. \u003cstrong\u003e(f) \u003c/strong\u003eHi-C contact matrix showing only early-early RT contacts for chromosome 5. For panels e and f, the RT classification segments are displayed (MS green, ES blue) and the contract matrix plots are displayed with settings of 1-mb resolution and 40 color intensity. Contrasting regions illustrate long-distance contact frequency differences between M-M (dashed pink boxes) and E-E (solid pink boxes) in the contact matrices.\u003c/p\u003e","description":"","filename":"Figure4RTContactEnrichment.png","url":"https://assets-eu.researchsquare.com/files/rs-6116464/v1/b9d5af9f5909fe84e5d5baed.png"},{"id":78331982,"identity":"139301e1-aa30-4832-bdbb-6c1438a78240","added_by":"auto","created_at":"2025-03-12 07:21:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":144555,"visible":true,"origin":"","legend":"\u003cp\u003eHi-C correlation with other chromatin features\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a-f)\u003c/strong\u003e Correlation plots between root tip Hi-C data and histone post-translational modifications: \u003cstrong\u003e(a)\u003c/strong\u003e H3K27ac, \u003cstrong\u003e(b)\u003c/strong\u003e H3K4me3, \u003cstrong\u003e(c)\u003c/strong\u003eH3K56ac, \u003cstrong\u003e(d)\u003c/strong\u003e H3K4me1, \u003cstrong\u003e(e)\u003c/strong\u003e H3K27me3, and \u003cstrong\u003e(f)\u003c/strong\u003e H3K9me2. \u003cstrong\u003e(g)\u003c/strong\u003eCorrelation plots between root tip Hi-C data and transcriptional data: \u003cstrong\u003e(g)\u003c/strong\u003e RAMPAGE, \u003cstrong\u003e(h-i)\u003c/strong\u003e Correlation plots between root tip Hi-C data and chromatin accessibility: \u003cstrong\u003e(h)\u003c/strong\u003e differential nuclease sensitivity data and \u003cstrong\u003e(i)\u003c/strong\u003e G1 MOA light digestion.\u003c/p\u003e","description":"","filename":"Figure5EVcorrelations.png","url":"https://assets-eu.researchsquare.com/files/rs-6116464/v1/65cb30c590f4156b9251ab02.png"},{"id":78333376,"identity":"095287a7-97d5-4c45-9a12-4e10938622be","added_by":"auto","created_at":"2025-03-12 07:37:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1615180,"visible":true,"origin":"","legend":"\u003cp\u003eSummary genome-wide correlations of epigenomic features with HiC Eigenvectors and Repli-seq coverage.\u003c/p\u003e\n\u003cp\u003eCorrelation values between different chromatin features and Hi-C data, early RT, middle RT and late RT Repli-seq data. The H3K27ac, H3K4me3 and RAMPAGE data was taken from Cahn et al 2024 \u003ca href=\"https://paperpile.com/c/oAFTKb/MDCs\"\u003e\u003csup\u003e48\u003c/sup\u003e\u003c/a\u003e, H3K56ac, H3K4me1, H3K27me3 and H3K9me2 data from Wear et al 2017 \u003ca href=\"https://paperpile.com/c/oAFTKb/fPCO\"\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/a\u003e, DNS light data from Turpin et al 2018 \u003ca href=\"https://paperpile.com/c/oAFTKb/9Jxn\"\u003e\u003csup\u003e45\u003c/sup\u003e\u003c/a\u003e and MMOA-seq data from this paper.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure6v3.png","url":"https://assets-eu.researchsquare.com/files/rs-6116464/v1/c6e50e6d92c1b55dee281b32.png"},{"id":78331470,"identity":"900a8711-7722-425c-b18b-84ff66582b59","added_by":"auto","created_at":"2025-03-12 07:13:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":167574,"visible":true,"origin":"","legend":"\u003cp\u003eFISH probes designed for chromosome 5 short arm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Percentages of early, middle, and late replication timing (RT) segments are shown for comparison between the whole maize genome, chromosome 5, and the short arm of chromosome 5 (5S). The 5S region of chromosome 5 was selected as a representative genomic region for 3D FISH analysis due to its similar RT segment distribution. \u003cstrong\u003e(b)\u003c/strong\u003e Table displaying the total number of probes and their density. \u003cstrong\u003e(c, d)\u003c/strong\u003eUCSC Genomaize browser views of chromosome 5S and a 1-Mb zoomed-in region, showing RT segments and their corresponding 3D FISH probe oligo pools. Blue, green, and red tracks represent early, middle, and late RT segments and their respective RT FISH probes.\u003c/p\u003e","description":"","filename":"Figure7OligoFISHprobes.png","url":"https://assets-eu.researchsquare.com/files/rs-6116464/v1/6182eb3df6b9602ec8b51e44.png"},{"id":78331983,"identity":"765ef4a0-ebcb-4445-8ccd-896fdaf5de51","added_by":"auto","created_at":"2025-03-12 07:21:00","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":876475,"visible":true,"origin":"","legend":"\u003cp\u003e3D cytology of single labeled FISH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Cytology of total nuclei from root tip hybridized with Early-S and Middle-S FISH probes labeled with far-red fluorophores. Images were collected using 3D deconvolution microscopy, corrected for wavelength-dependent chromatic aberration, and displayed as grayscale (DAPI and FISH probes) or as color overlays (DAPI in red, FISH probes in green). Each row represents a single nucleus, showing intensity-averaged projections spanning 1 µm (5 Z-sections) that include the FISH signals. Total DNA was visualized in the DAPI channel, and FISH probe signals were captured as Alexa-647 fluorescence in the Cy5 channel. Two representative examples are shown for Early-S probes (1st two rows) and Middle-S probes (last two rows). The location of nucleoli is marked as \"n\". Regions where FISH probe signals occur are indicated by circles in grayscale images (red circles) or color overlays (blue circles). The two similarly sized signal clouds represent the chromosome 5 homologs. All scale bars = 5 µm. \u003cstrong\u003e(b)\u003c/strong\u003e Individual nuclei with FISH probe signals were cropped for quantitative colocalization analysis using the inbuilt solid-object builder polygons method. The DAPI density of FISH signal regions was compared with non-FISH euchromatin control regions from the same nucleus. \u003cstrong\u003e(c)\u003c/strong\u003e Box plot showing the log2 ratio of DAPI density in early probe/control and middle probe/control regions. This box plot has data from G1, S and G2 nuclei.\u003c/p\u003e","description":"","filename":"Figure8FISH1mixed.png","url":"https://assets-eu.researchsquare.com/files/rs-6116464/v1/392792a4bbe24696464664ea.png"},{"id":78331984,"identity":"b2cff040-a3fe-42a1-9486-830bf5777467","added_by":"auto","created_at":"2025-03-12 07:21:00","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1010866,"visible":true,"origin":"","legend":"\u003cp\u003e3D cytology of two color FISH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Cytology of G1 root tip nuclei hybridized with Early-S and Middle-S FISH probes labeled with green (Alexa Fluor 488) and far-red (Alexa Fluor 647) fluorophores respectively. Images were acquired using 3D deconvolution microscopy, corrected for wavelength-dependent chromatic aberration, and displayed as grayscale (DAPI, FITC, and Cy5) or as color overlays (DAPI in blue, Early-FISH in green, Middle-FISH in red). Each row represents a single nucleus, showing intensity-averaged projections spanning 1 µm (5 Z-sections) that include the FISH signals. Total DNA was visualized in the DAPI channel, and FISH probe signals were captured as A-488 fluorescence (FITC channel) and A-647 fluorescence (Cy5 channel). Four representative examples of dual-labeled G1 nuclei are shown. The location of nucleoli is marked as \"n\". Green and red illustrations on grayscale highlight Early-FISH (E-FISH) and Middle-FISH (M-FISH) signals and the regions on DAPI where these signals are present. \u003cstrong\u003e(b)\u003c/strong\u003eIndividual nuclei with FISH probe signals were cropped for quantitative colocalization analysis using the inbuilt solid-object builder polygons method. The DAPI concentration of Early-FISH and Middle-FISH signal regions was directly compared. \u003cstrong\u003e(c)\u003c/strong\u003e Box plot displaying the log2 ratio of DAPI concentration in Early-FISH versus Middle-FISH regions. Bivariate plot inset (asterisk) in the corner highlights the G1-gated nuclei population used for this FISH experiment.\u003c/p\u003e","description":"","filename":"Figure9FISH2G1.png","url":"https://assets-eu.researchsquare.com/files/rs-6116464/v1/8f7c511ba5d772d6f3cd1aa7.png"},{"id":89169997,"identity":"2c68d425-1646-48c9-bdcb-85203dfdc78f","added_by":"auto","created_at":"2025-08-15 18:38:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6949073,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6116464/v1/83fae62c-f011-4ed1-968b-68324a8b7bd0.pdf"},{"id":78332956,"identity":"b082e1e9-2a49-4c0c-a5d0-c59f739b1231","added_by":"auto","created_at":"2025-03-12 07:29:00","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":295187,"visible":true,"origin":"","legend":"HiC protocol","description":"","filename":"SupplementaryFile1HiCprotocol.docx","url":"https://assets-eu.researchsquare.com/files/rs-6116464/v1/a2a7f6ac295a35c853c06260.docx"},{"id":78332957,"identity":"05e5cc4f-1180-4588-8470-176254cae85f","added_by":"auto","created_at":"2025-03-12 07:29:00","extension":"txt","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1874160,"visible":true,"origin":"","legend":"Dataset 1","description":"","filename":"SupplementaryFile2allRTsegments.txt","url":"https://assets-eu.researchsquare.com/files/rs-6116464/v1/29a504bb3403cdc94862a8df.txt"},{"id":78331459,"identity":"930becf5-2fd6-4aaa-a998-fb76e407d15b","added_by":"auto","created_at":"2025-03-12 07:13:00","extension":"zip","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":482939,"visible":true,"origin":"","legend":"\u003cp\u003eDataset 2\u003c/p\u003e","description":"","filename":"SupplementaryFile3EMLprobes.zip","url":"https://assets-eu.researchsquare.com/files/rs-6116464/v1/21c1612093f6def76e777744.zip"},{"id":78331457,"identity":"10e456c8-78ae-4bc0-9da4-551d6beccfaa","added_by":"auto","created_at":"2025-03-12 07:13:00","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":17742,"visible":true,"origin":"","legend":"\u003cp\u003eFISH probe labeling protocol\u003c/p\u003e","description":"","filename":"SupplementaryFile4probeLabelProtocol.docx","url":"https://assets-eu.researchsquare.com/files/rs-6116464/v1/51295d34cd9d15626ec6b5cc.docx"},{"id":78332964,"identity":"0a3953bc-b764-420f-af73-5089359c0ad7","added_by":"auto","created_at":"2025-03-12 07:29:00","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":13806982,"visible":true,"origin":"","legend":"Supp Figures 1-8 and Supp Table 1","description":"","filename":"SuppFilesAllSuppT1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6116464/v1/4b8993ddaeacc247f4f5d734.pdf"},{"id":78331994,"identity":"3a8821fd-0d3e-48ee-9ff3-878f86806e6d","added_by":"auto","created_at":"2025-03-12 07:21:00","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":13806978,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SuppFilesAllSuppT1V2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6116464/v1/869f2c5e7d1459d2a62fa56e.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Replication Timing Uncovers a Two-Compartment Nuclear Architecture of Interphase Euchromatin in Maize","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe eukaryotic cell cycle consists of a widely conserved series of events, including cell growth, DNA replication, and division into two daughter cells. DNA replication occurs during the S phase of the cell cycle, and the specific time within S phase when each genomic region replicates can be measured and annotated as Replication Timing (RT) \u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The fact that some cells or tissues have different RT profiles reveals the existence of an underlying RT program coupled to development or differentiation \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The RT program is generally recognized as ensuring the faithful reproduction and transmission not only of the nucleotide sequence but also of the chromatin state, contributing to the propagation of epigenomic cell type identity and functions \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Most eukaryotic RT knowledge comes from yeast and mammals, where complex temporal programs have been described and extensively studied \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. However, the regulatory mechanisms that govern temporal RT programs remain understudied in plants.\u003c/p\u003e \u003cp\u003eEarly studies of plants concluded that genome replication is essentially biphasic, with early-replicating and late-replicating regions \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Later studies greatly improved the resolution of the replication timing profile by using nucleotide analogs such as 5-bromo-2\u0026prime;-deoxyuridine (BrdU) \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and 5-ethynyl-2'-deoxyuridine (EdU) for pulse-labeling replicating DNA. EdU has become the analog of choice because it does not require acid or heat denaturation for detection and, instead, is directly conjugated with click chemistry to add a fluorescent dye used for both fluorescence activated nuclei sorting (FANS) and microscopy \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. These EdU-labeled nuclei can be separated by flow cytometry from unlabeled G1 and G2 nuclei and further divided by increasing DNA content into subpopulations representing sequential stages of S phase. Three separated populations, early-S, middle-S, and late-S, have been used for microscopic analysis and for sequencing of replicated DNA (Repli-seq) \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The first genome-wide RT maps in plants were produced for \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and maize (\u003cem\u003eZea mays\u003c/em\u003e L.) using these techniques \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The comparative analysis of early, middle and late replication in Arabidopsis revealed that early-S and middle-S are quite similar to each other and distinct from late-S. Early and middle replicating domains are enriched with open chromatin as defined by nuclease sensitivity and histone acetylation markers with early-replicating regions being gene-rich, while late-replicating domains are characterized by an enrichment of transposons and repressive epigenetic markers \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Although \u003cem\u003eArabidopsis\u003c/em\u003e has provided a foundational understanding of plant replication timing, its small genome size and high gene density are unusual among plant genomes. Plants with larger genome sizes, such as maize (2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2X\u0026thinsp;=\u0026thinsp;20) with a 1C value of 2.3 Gb, have expanded intergenic content, which generally tends to replicate primarily during middle-S and exists in a slightly more compacted state compared to the early-S replicating chromatin \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe maize genome, which is about two thirds the size of the human genome and contains a complex array of transposons, is more typical of higher plants. This fact, together with the fact that maize has been the subject of detailed genetic and cytogenetic studies for more than 100 years \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, makes maize an attractive system for exploring the control of replication in plants with complex genomes and chromatin architecture. Furthermore, as one of the world\u0026rsquo;s most agriculturally significant crops, insights into the replication process in maize are relevant not only for fundamental research but also for strategies to enhance crop performance and resilience.\u003c/p\u003e \u003cp\u003eOur group has developed a powerful experimental system to characterize replication in actively growing, intact maize root tip meristems that enables examination of replication patterns, genome features, and associated chromatin structure parameters at different stages of the cell cycle \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Although heterochromatic regions in maize replicate late, and many genes replicate early as they do in most other eukaryotic systems, the spatiotemporal patterns of early and middle S-phase replication in maize differ significantly from those in mammalian and yeast systems. In mammals, DNA synthesis occurs at different nuclear regions throughout S phase, classified as 5 sequential stages referred to as patterns 1\u0026ndash;5 \u003csup\u003e22\u003c/sup\u003e or Types I-V \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, in which the first, third, and fifth stages represent the earliest, the middle most, and latest stages of S phase, respectively. Despite the different naming conventions, there is agreement on the nuclear distribution of replication in mammalian nuclei, which starts with broad distribution in euchromatic portions of the nucleoplasm at early S phase, then in perinuclear and perinucleolar regions during middle S phase, followed by heterochromatic portions of the nucleoplasm at late S phase \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Much of the mammalian replication timing studies compare Early to Late replication timing, and genomic regions where they switch, with relatively few studies focusing directly on middle S\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In maize, DNA synthesis is thoroughly dispersed throughout the nucleus during both early and middle S phase but becomes more punctate and clustered in late S phase. Analysis of DNA RT in maize shows that early S-phase replicative labeling primarily colocalizes with regions of relatively weak DAPI fluorescence, indicating a low DNA density, while middle S-phase EdU labeling aligns with regions of euchromatin showing stronger DAPI staining. Based on these findings, we proposed a \"minidomain chromatin fiber RT model\u0026rdquo;, suggesting that maize euchromatin exists as an interspersed mixture of two subcompartments, each characterized by distinct replication timing and chromatin morphology \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo test the minidomain chromatin fiber RT model, we employed a combination of proximity ligation (Hi-C) and quantitative in situ hybridization (3D-FISH) techniques in this study. Here we provide new evidence for this model throughout the cell cycle, recontextualizing our view of the euchromatin global \"A\" compartment into two spatially and epigenetically distinct compartments.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003ePlant materials:\u003c/p\u003e \u003cp\u003eExperiments were done using maize (\u003cem\u003eZea mays\u003c/em\u003e L., inbred B73) root tips or earshoots. For root tips, as described in a previous protocol \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, seeds were surface sterilized and imbibed overnight with constant stirring and aeration. Seeds were surface-sterilized again with 0.5% sodium hypochlorite, 0.05% Tween-20 solution, and then rinsed with water. Seeds were germinated in boxes with paper towels moistened with sterile water at 28\u0026deg;C for 3 days under continuous light (Feit Electric OneSync LED light system, 3000K, 300\u0026ndash;400 lux). The material for biological replicates were grown independently and harvested on different days. For earshoots, field-grown maize plants were harvested between 9-11am on sunny days, and earshoots ranging in size from 0.5-1 cm were flash frozen in liquid nitrogen, and pooled (15\u0026ndash;20 earshoots) for storage at -80\u0026deg;C as described in previous paper \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRoot tip and earshoot nuclei for HiC:\u003c/p\u003e \u003cp\u003eFor Hi-C on seedling root nuclei, the samples were not subjected to EdU labeling or the click reaction, but otherwise isolated from formaldehyde fixed maize root tip for FACS as previously described \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Nuclei were stained with DAPI and sorted to collect between 1.25\u0026ndash;1.53\u0026nbsp;million nuclei as input (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh) for each biological replicate of the Hi-C assay.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor Hi-C on field-grown earshoot nuclei, we used the method of Savadel et al., (2021) \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e to isolate nuclei. The frozen tissues were ground in liquid nitrogen to a fine powder, and the nuclei were formaldehyde crosslinked. The fixation reaction was stopped by addition of 0.1 vol (~\u0026thinsp;1 mL) of 2.5 M glycine. The tissue was then mechanically disrupted with a Polytron (3 x 10 seconds at ~\u0026thinsp;1/5 maximum speed) to liberate nuclei. Nuclei were pelleted by centrifugation at 2000 xg for 15 min at 4\u0026ordm;C and resuspended in a 15 mL buffer. Total earshoot nuclei were used for the Hi-C assay.\u003c/p\u003e \u003cp\u003ePreparation and sequencing of Hi-C libraries from maize root tip and ear shoot:\u003c/p\u003e \u003cp\u003eThe Hi-C libraries were generated essentially following the protocol first described for maize leaf by Dong et al.(2017) and later detailed in a methods chapter by Dong and Zhong (2020) \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. We made some minor modifications to accommodate the library kits currently utilized by the Molecular Cloning Facility (Dept. Biol. Sci, Florida State University, Tallahassee, FL) and reduced washing steps to avoid nuclei loss as described in the wet bench full protocol (Supplementary File 1). Among the key steps were permeabilization of nuclei with 0.5% (w:v) SDS for 5 min at 65\u0026deg;C, DNA digestion with \u003cem\u003eDpn-II\u003c/em\u003e restriction enzyme, overhang repair with Klenow and biotinylated nucleotides, and ligation with T4 DNA ligase. Ligated DNA was biotin affinity purified, crosslinks reversed, and sheared to 300\u0026ndash;500 bp to yield DNA fragments for library construction using NEBNext Adapters for Illumina. These libraries were sequenced using Illumina Hi-Seq 2500 paired-end 150-bp chemistry (Translational Science Lab at College of Medicine, Florida State University, Tallahassee, FL) to obtain library sequences at a depth ranging from 127M to 309M (Supplementary Table\u0026nbsp;1) per replicate, with four replicates for root tips and three for earshoots. Raw sequences were processed to remove adapters by the sequencing facility, providing library Fastq files for HiC analysis.\u003c/p\u003e \u003cp\u003eNuclei isolation for MMOA-seq\u003c/p\u003e \u003cp\u003eFor MMOA nuclei preparation was done using the previously published protocol (Bass et al. 2014; Wear et al. 2016). Briefly, maize seedling roots were labeled in 25 \u0026micro;M EdU for 20 min, rinsed and placed in 100 \u0026micro;M thymidine to stop labeling. The terminal 1-mm maize root tip segments were excised, fixed and lysed for the isolation of nuclei for MMOA-seq.\u0026nbsp;Incorporated EdU was then conjugated to Alexa Fluor 488 (AF-488) using a Click-iT EdU Alexa Fluor 488 imaging kit (Life Technologies). The nuclei were then counterstained with DAPI using a cell lysis buffer containing 2 \u0026micro;g/mL DAPI and 40 \u0026micro;g/mL Ribonuclease A and filtered through a 20-\u0026micro;m nylon mesh filter (Partec). Nuclei were flow sorted on a FACS Aria III flow cytometer (BD Biosciences) equipped with UV (355 nm) and blue (488 nm) lasers. G1 nuclei were used for MMOA-seq library preparation.\u003c/p\u003e \u003cp\u003eMMOA-seq library preparation and Sequencing\u003c/p\u003e \u003cp\u003eMMOA-seq libraries were prepared using nuclei in 500-\u0026micro;L aliquots per replicate. Briefly, 60 \u0026micro;L of nuclei were distributed to each of four 1.5 mL screw cap tubes. 10x MNase working dilutions (25 U/mL, 12.5 U/mL, and 6.25 U/mL) were prepared from a 20,000 U/mL stock. 6.7 \u0026micro;L of each working dilution (or MDB) were added to each of the 60 \u0026micro;L nuclei aliquots and immediately vortexed and briefly centrifuged. Complete digestion reactions were transferred to a 37\u0026ordm;C shaking water bath and incubated for 15 min. Reactions were promptly stopped after 15 min by addition of 5 \u0026micro;L of 0.5 M EGTA (pH 8.0). Digested chromatin was then decrosslinked overnight at 65\u0026ordm;C in 1% SDS, 150 mM NaCl, 20 \u0026micro;g/mL Proteinase K. DNA was purified by 25:24:1 Phenol:Chloroform:Isoamyl alcohol (pH 8.0). Nucleic acids were precipitated and the pellets were washed with ice cold 70% ethanol and air dried before being redissolved in 100 \u0026micro;L of 10 mM Tris EDTA buffer.\u003c/p\u003e \u003cp\u003eFor each library, 75 ng of selected digests (1.25 and 2.5 U/mL MNase) were combined and diluted to 50 \u0026micro;L with 0.1X TE. Sequencing libraries were made according to the NEBNext Ultra II DNA Library Prep Kit for Illumina with the following modifications to the size selection protocol for retention of small fragments (\u0026lt;\u0026thinsp;200 bp): the first bead addition used 50 \u0026micro;L of beads, the second used 100 \u0026micro;L of beads. A total of 8 cycles of PCR were completed at the barcoding step.\u003c/p\u003e \u003cp\u003eLibraries were quantified by Qubit dsDNA HS fluorimetry and KAPA qPCR. DNA fragment size distribution was analyzed on an Agilent High Sensitivity D1000 Screentape assay. An equimolar pool of all 24 MOA-seq libraries was prepared and sequenced using 50-bp paired-end reads on a Novaseq S1 flow cell in the FSU College of Medicine Translational Science Lab.\u003c/p\u003e \u003cp\u003eHiC-Pro analysis of Hi-C data:\u003c/p\u003e \u003cp\u003eHiC-Pro \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e is a custom bioinformatic software for the analysis of Hi-C datasets. The pipeline performs sequential steps, including short read mapping, detection of valid ligation fragments, and various quality controls. The output consists of inter- and intra-chromosomal contact maps at various resolutions.\u003c/p\u003e \u003cp\u003eThe genome was divided into non-overlapping bins of equal size at different resolutions (1 kb, 5 kb,10 kb, 50 kb, 250 kb, 500 kb, and 1mb) and contacts were scored in each bin. The frequency of interaction between bins was represented by bi-dimensional heatmaps (\u0026ldquo;contact matrices''), containing both inter- and intra-chromosomal contacts. Contact matrices were visualized using Juicebox \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, and used to calculate a Pearson correlation matrix for observed/expected intra- and inter-chromosomal interactions.\u003c/p\u003e \u003cp\u003eInsulation index and Eigenvector analysis:\u003c/p\u003e \u003cp\u003eTo systematically identify folding structures at the local scale, we applied several techniques, such as Principal Component Analysis (PCA), the Insulation Index method and Virtual 4C analysis. We ran PCA to calculate the first eigenvector (principal component) of each contact matrix at 50-kb resolution to identify two distinct A/B genomic compartments as described in Lieberman-Aiden et al \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. The Insulation Index analysis \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e was calculated as the cumulative frequency of interactions in all the 50-kb bins within the 1 mb window. We calculated the log2 of the observed/expected interaction frequencies using the median scores for the expected values. Valleys/minima (negative values) indicate loci of reduced frequency of interaction with flanking regions, whereas local maxima (positive values) reveal folding structures.\u003c/p\u003e \u003cp\u003eVirtual 4C analysis \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e was centered over \u0026ldquo;viewpoints\u0026rdquo; of interest (\u0026ldquo;baits\u0026rdquo;), chosen in our case among replication segments of different classes, Early-S or Middle-S \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. We scored all the valid interactions between each viewpoint and the rest of the chromosome, and compared the frequency of Early to Early and Middle to Middle versus Early to Middle interactions.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis:\u003c/h2\u003e \u003cp\u003eFor virtual 4C arc plots, we used plotGardner v1.0.17 \u003csup\u003e39\u003c/sup\u003e package for the R statistical suite \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e for the visualization of genomic regions. Two similar sized early and middle RT segments were selected as 4C baits and then their corresponding 10-kb bins were extracted. Each 10-kb bin was annotated with its RT class, and all bins in contact with the bait bins were retrieved. Hexbin plots were also drawn using the R package ggplot2 v4.1.2 \u003csup\u003e41\u003c/sup\u003e with Hi-C EV versus different chromatin marks and Early-S, Middle-S RT versus chromatin marks at 50-kb resolution.\u003c/p\u003e \u003cp\u003eRoot tip nuclei for 3D-FISH:\u003c/p\u003e \u003cp\u003eMaize seedlings were grown and nuclei were prepared and sorted as described above. For 3D quantitative FISH with the mixed population nuclei, we combined 30 uL each of the flow-sorted EdU-labeled nuclei gates for G1 (1.5M/mL, 16%), early-S (1.9M/mL, 21%), middle-S (1.5M/mL, 16%), late-S (0.36M/mL, 4%), and G2 (3.9M/mL, 41%). In this mix, the S-phase nuclei were ~\u0026thinsp;41% of the total. For 3D-FISH with G1 nuclei, only flow-sorted G1 nuclei were used (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e8\u003c/span\u003ec, inset panel).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eProbes labeling for 3D-FISH:\u003c/p\u003e \u003cp\u003eOligonucleotide probe libraries were designed by DAICEL Arbor Biosciences using three sets of sequences as inputs, early-S, middle-S, and late-S RT segments. These annotation segments were based on the Repli-seq data from Emily et. al, (2017) \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, realigned to B73v5 using the RepliScan pipeline \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e to produce a DNA RT class annotation file, RT_class_ALL_27800_b73v5_9colBed_vhsf521e.BED (Supplementary File-2). From this annotation file, we derived coordinates for three sets of genomic regions (Early, Middle, or Late) within the first 105 Mbp on chromosome 5, which corresponds to the short arm. These were used as input to obtain three libraries of oligos as \u003cem\u003eT\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e-matched\u0026thinsp;~\u0026thinsp;45-mers, high-density, and uniformly spaced at high density across the chromosome arm. The resulting sets of oligos included a total of 22,460, 27,390, or 4,640 oligos from the early-S, middle-S, or late-S regions, respectively, and the coordinates of the uniquely named oligos are provided as bed files (Supplementary File-3).\u003c/p\u003e \u003cp\u003eA series of experiments were conducted to convert these dsDNA libraries into fluorescently labeled single-stranded DNA (ssDNA) libraries (Supplementary File-4). Briefly, the dsDNA library underwent PCR amplification to obtain a DNA yield sufficient for \u003cem\u003ein-vitro\u003c/em\u003e transcription. The purified DNA was processed using the Qiagen QIAquick PCR Purification Kit and quantified using a spectrophotometer (NanoDrop). After DNA purification, \u003cem\u003ein-vitro\u003c/em\u003e transcription was carried out using the MEGAshortscript TM T7 Kit (Thermo Fisher), using 480 ng/\u0026micro;L DNA as template per reaction. Following transcription, RNA purification was performed using the Macherey-Nagel RNA Clean-Up Mini Kit. RNA was quantified by NanoDrop to verify that the concentration was above 1 \u0026micro;g/\u0026micro;L, as required by the reverse transcription PCR (RT PCR) step. Single-stranded DNA (ssDNA) was generated through a reverse transcription reaction using SuperScript IV Reverse Transcriptase, with the addition of 52 \u0026micro;g of RNA to obtain sufficient yield of ssDNA. At this stage, ssDNA probes were labeled with three different fluorophores: Alexa fluor 488 (ALP-a488, green), Alexa fluor 546 (ALP-a546, red), and Alexa fluor 647 (ALP-a647N, far red). The process resulted in RNA-DNA hybrids and some unincorporated primers. Exonuclease-I was used to digest unincorporated primers, and RNase was used to digest the RNA from RNA-DNA hybrids. After RNA and primer digestion, labeled ssDNA was purified using the Zymo Quick-RNA purification kit and quantified.\u003c/p\u003e \u003cp\u003eFluorescent in-situ hybridization:\u003c/p\u003e \u003cp\u003eThe FISH protocol was adapted from Bass et al. 1997 \u003csup\u003e43\u003c/sup\u003e that used the polyacrylamide embedded technique for 3D FISH analysis. Fixed EdU-labeled nuclei were embedded in a thin layer of optically clear 3X acrylamide mix on a glass slide. Then wash buffers and prehybridization buffers were exchanged by 200-\u0026micro;L droppers. EdU-labeled nuclei remained intact throughout the procedures of washing and equilibrating. A buffer containing RT class-labeled probes, 50% formamide, and 2X SSC was added and incubated at 37\u0026deg;C for 30 min for prehybridization. After prehybridization, incubation slides were placed on a hot plate at 65\u0026deg;C for 30 min for genomic DNA denaturation. After denaturation, the slides were placed back in the 37\u0026deg;C incubator overnight for hybridization. The following day, the slides underwent serial washing with buffers of increasing stringency. Finally, slides were mounted with mounting media and sealed with Sally Hanson nail hardener.\u003c/p\u003e \u003cp\u003eDeltavision microscopy and Image analysis:\u003c/p\u003e \u003cp\u003eFollowing the FISH experiment, 3D images were captured with 0.2-micron projections in multiple wavelength iterative deconvolution microscope. The raw data was subsequently subjected to 3D iterative deconvolution and chromatic aberration correction (CAC). For analysis purposes after 3D data collection, individual nuclei with probe signals were cropped to allow quantitative signal colocalization analysis using the inbuilt solid object builder polygons method. For these analyses, the control regions were non-FISH euchromatin areas selected from the same nuclei. Quantitative segmentation data analysis was performed on at least 50 nuclei for each Early and Middle probes labeled experiment. All 3D images were stored on the image repository omero.bio.fsu.edu, allowing for additional segmentation analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eRepli-seq scheme and minidomain chromatin fiber RT model\u003c/p\u003e \u003cp\u003eThe maize (\u003cem\u003eZea mays\u003c/em\u003e L.) genome consists of 10 metacentric chromosomes, ranging from 150 to 300 Mb in size, and has been annotated with regard to DNA replication timing (RT) using previously published Repli-seq data \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and the RepliScan pipeline to segment the genome into discrete RT classes \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. For this study, the Repli-seq data was remapped to the B73 AGPv5 assembly and segmented using RepliScan. The DNA RT profiles for sequences that replicate at early-S, middle-S, or late-S are visualized in a genome browser as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for maize chromosome 5. We selected chromosome 5 as a representative chromosome, based on the criterion that it reflected the whole genome RT class frequencies previously defined by Repli-seq \u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe (RT) profile along the entire length of maize chromosome 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) illustrates regional enrichments for early and late replication, corresponding to euchromatic, gene-rich areas and heterochromatic, gene-poor sections, respectively. The early-S RT profiles show the highest coverage in the distal regions of the chromosome arms where gene density is higher \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. In contrast, late-S RT profiles show the highest coverage near the center of the chromosome at the centromeric and pericentromeric regions, which are enriched in repetitive sequences and transposons. The middle-S RT profiles show a unique pattern of being relatively evenly distributed across most of the chromosome. A distinct non-centromeric block of late-S replicating chromatin is also seen at the heterochromatic knob, embedded in the long arm of chromosome 5.\u003c/p\u003e \u003cp\u003eTo gain further insight into the relationships among the different RT classes, we inspected a 2-Mb non-heterochromatin region on chromosome 5 to specifically focus on the distribution of early-S versus middle-S (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, blue and green) RT classes. These are shown in relation to genes and chromatin accessibility data \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, reflecting the known stronger coupling of early-S replicating regions with genes and open chromatin compared to middle-S replicating regions. This 2-Mb region illustrates a striking pattern of alternating early-S and middle-S segments, which switch back and forth repeatedly over these large genomic areas.\u003c/p\u003e \u003cp\u003eBased on cytological analysis of early and middle replication patterns, a model of euchromatin organization was proposed as the \"minidomain chromatin fiber RT model\" \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. According to this model, maize euchromatin, which is not uniformly stained in cytological preparations, exists in two interspersed subcompartments corresponding to early-S and middle-S replicating regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-e). These early-S chromatin regions are relatively weakly stained with DAPI, whereas the middle-S chromatin regions exhibit stronger DAPI staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eGiven that these two distinct chromatin types were defined in S phase, we set out to test the idea that they reflect a general nuclear architecture in maize present throughout the interphase. This idea was first tested using high-throughput conformation capture (Hi-C) analysis with nuclei from the entire mitotic cell cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef-h).\u003c/p\u003e \u003cp\u003eHi-C data reveals covariance and correlation with early and middle replication timing in maize euchromatin\u003c/p\u003e \u003cp\u003eWe used formaldehyde-fixed root tip nuclei from 3-day-old seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef-h) to prepare Hi-C libraries (Supplemental Fig.\u0026nbsp;1) in biological replicates of 1.25\u0026nbsp;million nuclei each. For comparison, we also made Hi-C libraries from immature earshoot nuclei. The analysis of the resulting Hi-C libraries provided genome coverage and valid pair contact frequencies comparable to that previously reported for maize bundle sheath cells \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. This confirmed that the contact patterns remained intact following the flow-sorted nuclei isolation and library preparation steps (Supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eTo compare nuclear architecture to DNA replication timing, we plotted Hi-C contact matrices from selected regions of chromosome 5 as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e. At the whole chromosome scale, we see the typical maize Hi-C chromosome structure in which euchromatin and centromere-associated/pericentromeric heterochromatin are evident (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), reflecting the global A (black) and B (grey) compartments, respectively \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo more specifically examine early-S versus middle-S, we looked at three different 2-Mb regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-d) on the short arm of chromosome 5. These 2-Mb heatmap matrices show the Hi-C contacts along with early, middle, and late RT segments (top, left) in blue, green, and red, respectively. We observed the expected strong diagonal signal, indicating frequent interactions among adjacent loci. Beyond the diagonal, interaction hotspots were observed (examples highlighted in blue and pink circles). Blue circles highlight contacts between two non-adjacent early RT regions, while pink circles mark interactions between early and middle RT regions. These represent long-range cis-interactions within these representative 2-Mb regions. Notably, early-S replicating segments appeared to display more long-reaching interactions with other early-S replicating segments compared to adjacent middle-S replicating segments. Some of these early-to-early contacts extend beyond 1 Mb, skipping multiple intervening RT segments.\u003c/p\u003e \u003cp\u003eTo compare more directly the HiC contact matrices to RT classes, we looked at these same 2-Mb regions by plotting RT profiles and segments along with the chromosome-wide eigenvector (EV) and insulation score (IS) analyses for all four biological replicates (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ee\u0026ndash;g). The insulation score of a genomic region measures its frequency of interaction with the neighboring regions. A low or negative IS indicates a scarcity of local interactions relative to longer-range contacts \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Strikingly, most of the points at which the EV and IS values switched from positive to negative mirrored the boundaries between the DNA replication timing segments. For instance, the light blue-shaded regions, representing early-replicating segments, showed positive EV and negative IS. The low insulation scores of early-S RT regions denote a greater tendency for long-range contacts, consistent with the hotspots of contacts highlighted in the contact matrices (blue circles, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-d). Relatedly, the green-shaded region, indicating middle-S RT segments, corresponded to negative EVs and positive ISs, suggesting a higher insulation score with more confined interactions \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Finally, the red-shaded areas denoted late-S RT regions with corresponding negative EV and positive IS. These patterns reflect the fact that Hi-C is capturing smaller-scale nuclear architectural features beyond that of the global A and B compartments. Notably, the annotations for RT and HiC mirror each other in distinguishing early-S and mid-S euchromatin regions.\u003c/p\u003e \u003cp\u003eTo determine if these relationships between nuclear architecture and replication timing hold true for the rest of the genome, we carried out a correlation analysis between RT and Hi-C. For this we combined the chromosome-wide HiC data (EV and IS) from all ten chromosomes. The early-S Repli-seq coverage values showed a strong positive correlation (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.81) with the eigenvectors, whereas the middle-S Repli-seq coverage showed a slight negative correlation (\u003cem\u003er\u003c/em\u003e = -0.15). The late-S Repli-seq coverage values showed a strong negative correlation (\u003cem\u003er\u003c/em\u003e = -0.67) with the eigenvectors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). When doing a similar genome-wide correlation with IS, we observed a strong negative correlation with early-S Repli-seq coverage (\u003cem\u003er\u003c/em\u003e = -0.59), a weak positive correlation with middle-S coverage (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.18), and a strong positive correlation with late-S- Repli-seq coverage (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.41) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). For both eigenvector and insulation scores, the strongest but opposite correlations were observed for early-S compared to late-S chromatin. The middle-S chromatin showed the same direction of correlation as that of the late-S chromatin, but with much smaller correlation values. Together, these data show that genomic regions distinguished by replication timing also correlate with distinct Hi-C-defined chromatin and genomic organization across the whole genome.\u003c/p\u003e \u003cp\u003eWe compared our Hi-C data for root tips to the eigenvector and IS data from earshoots analyzed in parallel (Supplementary Table\u0026nbsp;1) and bundle sheath cells from a published study \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Pairwise comparisons between tissues (supplementary Fig.\u0026nbsp;2) revealed strong genome-wide similarity between root tip and earshoot Hi-C profiles (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.85 for both EV and IS), both of which are actively growing tissues. We also observed slightly weaker but significant correlations between root tip and bundle sheath Hi-C profiles (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.73 for EV and 0.66 for IS), possibly reflecting a more cell-type independent global conservation of genome organization.\u003c/p\u003e \u003cp\u003eWe also investigated whether any of the ten maize chromosomes showed more or less tissue-specific variation compared to the whole genome (Supplementary Fig.\u0026nbsp;3a \u0026amp; 3b). Interestingly, we found that the root tip and ear shoot Hi-C data showed a strong positive correlation across all chromosomes with a notable exception for chromosome 6, which contains the nucleolar organizing region near the end of the short arm. The correlation coefficient for chromosome 6 was \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.62, compared to all the others which ranged from \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.78 to +\u0026thinsp;0.95 (Supplementary Fig.\u0026nbsp;3a). We found a similar situation for root tip versus bundle sheath, with chromosome 6 showing the lowest correlation (Supplementary Fig.\u0026nbsp;3b). In addition, most of the other chromosomes showed relatively weaker correlations with bundle sheath data, likely due to variations in Hi-C library preparation and/or tissue type. Using a stratum-adjusted correlation coefficient (SCC) analysis \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, we also found that root tip to earshoot similarity was higher than that of either one compared to bundle sheath (Supplementary Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003eGiven the strong correlations between replication timing and Hi-C defined nuclear architecture in root tip, we wondered if the RT classification alone from root tip could be predictive of HiC nuclear architecture in other tissues. For this, we did correlation analysis between root tip Repli-seq and the Hi-C EV data of the other two tissues, for which we do not have replication timing data (summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The root tip Repli-seq-defined genome structure closely mirrored the Hi-C Eigenvector (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-b), which can be seen locally (Supplementary Fig.\u0026nbsp;5b-c) and globally (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). These analyses demonstrated that replication timing in root tip cells predict chromatin structure across other tissues, including earshoot and bundle sheath, suggesting a robust association between replication timing and chromatin organization across the plant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHiC contacts are enriched within RT classes\u003c/p\u003e \u003cp\u003eGiven the high similarity among the root tip biological replicates (Supplementary Fig.\u0026nbsp;4), Hi-C contacts from all root tip replicates were pooled for subsequent analyses. We used the pooled data to examine the degree to which moderate to long range contacts were occurring between regions with the same replication timing assignments, i.e. within-class contacts, as predicted by the 2-compartment model for euchromatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We assigned replication timing (RT) class annotations to each of the two members of the valid pair contacts and calculated the frequencies of contacts with matching and differing pairs for the RT classes, for Early, Middle, or Late regions. The analysis revealed that of all the valid pair contacts (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), Early-Early accounted for 28% (2.93\u0026nbsp;million), Middle-Middle for 24% (2.47\u0026nbsp;million), and Late-Late for 16% (1.65\u0026nbsp;million). Contacts between two different RT classes were less frequent, with 16% Early-Middle, 13% Middle-Late, and only 3% Early-Late. These findings indicate the \"same-class\" categories are the most frequent, even after normalizing to account for their relative abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo examine more closely the same-RT-class contacts, we conducted a virtual 4C analysis of two early and two middle RT genomic regions, each approximately 150 Kb, located on chromosome 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). This approach allowed for the identification of long-range interactions centered around a specific genomic region of interest, known as the \u0026ldquo;bait\u0026rdquo; region, or virtual bait in this case. Two early and two middle RT segments were designated as baits, and their same-class contacts were plotted along the entire chromosome 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). All four baits showed long distance contacts, including to regions on the other arm of the chromosome, but the early-early contacts extended further, and even beyond the distal knob on the long arm of chromosome 5. This difference can not be explained by the location of the baits, because they come from the same local region (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), which represents less than 1% of the 226 Mb chromosome 5. We observed consistent results when we decomposed the contact matrices for all of chromosome 5 into middle-versus-middle (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ee) and early-versus-early RT contacts only (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eThe per-bait analysis also showed that the same class contacts were the predominant type (Supplementary Fig.\u0026nbsp;6). The least frequent contacts were between early or middle baits and late-S regions for all four segments analyzed (Supplementary Fig.\u0026nbsp;6a-d). We next investigated the persistence of these same-class contact tendencies as a function of distance relative to the linear genome. The results demonstrated the expected distance-dependent decay of all contact classes, with early-early RT class contacts showing the highest frequency at any given distance (Supplementary Fig.\u0026nbsp;6e). This enrichment was not a result of the relative abundance of the RT classes per se, as shown using a shuffled-position RT control (Supplementary Fig.\u0026nbsp;6f).\u003c/p\u003e \u003cp\u003eOverall, the contact class pair enrichments and the virtual 4C bait results showed that same-class contacts are more frequent than cross-class contacts, and that early RT segments exhibit more of the longest-range interactions than the middle RT segments.\u003c/p\u003e \u003cp\u003eHiC-based chromatin architecture aligns with epigenomic and transcriptional features\u003c/p\u003e \u003cp\u003eTo test how our Hi-C data correlated to genomic and genetic features, we checked the correlation of our Hi-C eigenvectors with epigenetic and transcriptional features known to be associated with chromatin structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). We first examined how the Hi-C data correlated with histone post-translational modifications using ChIP-seq data from B73 maize root tips \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe previously anticipated that modifications associated with active genes would be positively correlated with early-S replication timing regions within euchromatin, whereas repressive marks would not be or less so \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Indeed, we found significant correlations between our root tip Hi-C eigenvector (EV) data analyzed at 50-kb resolution on chromosome 5 and the active gene-associated marks (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-d). Among the marks with positive correlations, H3K27ac and H3K4me3 had the strongest correlations with \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.73 and +\u0026thinsp;0.65. The values for the histone marks and the eigenvector varied along the length of the chromosome, showing a general trend of higher values in the chromosome arms for all of the marks tested, except for H3K9me2 (Supplementary Figs.\u0026nbsp;7a-d). Consistent with our findings, regions enriched for H3K56ac (EV \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.45) and H3K4me3 (EV \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.65) were previously classified as early-S replicating regions, which also shows enrichment for genes with high median expression levels \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Regions enriched for H3K27me3, on the other hand, exhibited a weakly positive association with Hi-C EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ee, r\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.13). Interestingly, the H3K27me3 mark has been associated with facultative heterochromatin \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e and also found to be enriched in middle-S replicating regions and genes with lower median expression levels \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The only histone mark we examined that showed a negative correlation with our root tip EV was H3K9me2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ef, r = -0.20), which is consistent with its known association with constitutive heterochromatin \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe also investigated the correlation between Hi-C EVs and transcriptional activity estimates from the RAMPAGE transcription start site mapping data and it showed a positive correlation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eg, r\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.53) \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Finally, we compared Hi-C EV with chromatin accessibility using micrococcal nuclease (MNase) sensitivity profiling datasets from maize root tip nuclei. The read coverage levels from a light MNase digestion \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e showed a positive correlation of \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.48 with Hi-C EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eh). Likewise, the open chromatin defined by sequencing small fragments from light MNase digests in purified G1 nuclei showed a positive correlation of \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.42 with Hi-C EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ei). These were calculated for chromosome 5 because it was selected for additional cytological analysis, but similar trends were observed genome wide as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e for correlations between these same epigenomic features and both HiC and replication timing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3D cytological evidence for the spatial bifurcation of euchromatin into early-S and middle-S chromatin regions\u003c/p\u003e \u003cp\u003eWe next tested the 2-compartment euchromatin model using a chromosome painting strategy based on probes from different replication timing classes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e). For this independent strategy, we continued to use the representative chromosome 5, one that reflected the whole genome RT class frequencies previously defined by Repli-seq \u003csup\u003e2\u003c/sup\u003e. We found that the RT class frequencies for the short arm of chromosome 5 (chr5S) closely matched those of the whole genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). For instance, Early-S segments were 30% of chr5S and 30% of the whole genome, whereas middle-S segments were 38% and 35% of chr5S and the genome, respectively. Therefore, we designed RT-class-specific oligo FISH painting probes at comparable densities (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eb) spanning the entire chr5S.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor the oligo FISH probes, we developed three sets of oligo painting libraries: Early (E), Middle (M) and Late (L) across our three RT class segments which collectively account for 82% of chr5S. Probe distribution along the chr5S arm aligns with the RT classes. Distal regions are enriched for early-S segments and probes (blue tracks, while centromere proximal regions are enriched for late-S segments and probes (red tracks). Middle-S segments and probes (green tracks) were evenly distributed across the short arm (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). The interspersed pattern of early S and middle S segments and probes is better seen by focusing on a 2-MB region from the middle of chr5S (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eFor the RT class oligo FISH painting experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e8\u003c/span\u003e), early and middle probes were labeled with different fluorophores and hybridized to a mixed population of nuclei from the entire cell cycle, made by pooling G1, E, M, L and G2 nuclei (Supplementary Fig.\u0026nbsp;8d). Representative images from 3D deconvolution image datasets are shown as 1-\u0026micro;m Z-stack projections, a five-optical-section projection spanning the FISH probe signal regions. For quantitative analysis, we selected nuclei in which two similar sized paint signals were observed for diploid chromosome 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e8\u003c/span\u003ea, circles). In these nuclei, non-specific background staining of the nucleolus (labeled \"n\") was commonly seen, but the maize NOR is not on chromosome 5 and this non-specific signal was disregarded. To quantify the DAPI in the FISH-marked regions, we manually segmented the FISH paint signals to create volumetric polygon objects to obtain DAPI density (average photon counts per cubic micron). As a control, random non-FISH euchromatin regions were selected, avoiding the nucleolus and bright knobs. In this way, we could obtain the DAPI values for chromatin in painted RT class probe regions relative to bulk euchromatin control regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). By plotting the log2 ratio of DAPI density in the FISH versus control regions, we showed that the average DAPI concentration in Early-S chromatin (mean of -0.26) was less than the control regions, whereas the middle-S FISH chromatin (mean of +\u0026thinsp;0.21) was higher. The RT painted and control region ratios did not overlap (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, box plot). Given that observation, along with the fact that we sampled nuclei from a mixture of G1, S, and G2 in which S phase was only\u0026thinsp;~\u0026thinsp;41% of the total, we concluded that this relationship between replication timing and DAPI density holds true for nuclei across the cell cycle, not just in S phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e8\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eTo more definitively establish the spatial bifurcation of euchromatin outside of S phase, we performed dual probe FISH experiments using early and middle probes on purified G1 nuclei as summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. Representative images of dual-labeled G1 nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea) showed spatial separation of early-S and middle-S probe regions corresponding to the same unreplicated chromosome. We repeatedly observed paired but spatially separated signals for the early-S versus middle-S probes, in contrast to their intermixed, linear arrangement along chromosome (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). By segmenting the FISH signal regions, we could directly compare DAPI concentration in early-S chromatin (green outlines) relative to middle-S (red outlines) chromatin from the same chromosome, avoiding any uncertainty in selecting \"control\" regions from other parts of the nucleus. We quantified the DAPI intensity for each chromosome-based pair of FISH signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb), providing a log2 ratio that captures the relative chromatin compaction of the two FISH-delineated areas. We found that the average DAPI concentration ratios for all the Early-Middle FISH region pairs quantified was 0.86 (sd 0.07) in the G1 nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec), clearly establishing that RT defined regions are spatially separated and differentially condensed, even outside of S phase.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese results validate the mini-domain RT chromatin model, demonstrating that maize euchromatin exists as an interspersed mixture of two compartments distinguishable by condensation state and replication timing. Early-S FISH probes localize preferentially in regions with low DAPI signals, whereas middle-S FISH probes coincide with regions of higher DAPI intensity. 3D dual-label RT FISH painting in G1 nuclei provided compelling evidence, consistent with Hi-C data, that the two euchromatin compartments are integral features of nuclear organization.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study sheds light on the close relationship between DNA replication timing (RT) and chromatin organization in the maize genome, focusing particularly on nuclei in the terminal 1 mm of actively growing root tips and their euchromatin compartments. By using two orthogonal methods for defining nuclear architecture, Hi-C, and 3D FISH, we demonstrated how replication timing reveals a spatial bifurcation of maize euchromatin (the global A) into separate early-S and middle-S compartments. These findings support our earlier minidomain chromatin fiber RT model \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and extend it into G1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea-c), outside of S-phase.\u003c/p\u003e \u003cp\u003eOther studies in eukaryotic systems point to the fact that simple global A and B compartments are widely conserved, but are insufficient to understand the complexity of structural and functional organization of chromatin. Mammalian chromatin, for instance, has been divided into a variable number of states or subcompartments, ranging from three to eight or more, based on various approaches including epigenomic marks, modularity-based analyses, trans contacts, hierarchical clustering of domains, and data from TSA-seq, DamID, and Hi-C \u003csup\u003e\u003cspan additionalcitationids=\"CR54 CR55 CR56\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Similarly, plant chromatin, primarily from Arabidopsis studies, has also been shown to possess multiple chromatin states defined by distinct features \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThese studies tend to agree that the global A compartment can be split into two subcompartments, A1 and A2, which replicate in that order in plants and animals \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. We speculate that the maize early-S and middle-S chromatin described here may reflect an A1 and A2 type of subdivision, which a single assay, replication time profiling, can be used to map. Consistent with this, a recent light and EM cytological analysis of the very large genome plant, \u003cem\u003eNigella damascena\u003c/em\u003e, also revealed that early-S versus middle-S replication partitioned into more open versus less open chromatin, respectively \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEvidence for the nonrandom spatial distribution of early, middle, and late RT domains is supported by genomic RT profiling with Repli-seq \u003csup\u003e2\u003c/sup\u003e and 3D quantitative microscopy. Within S-phase, using replicative labeling, we know that early replication predominantly occurs in distal, gene-rich euchromatic regions with open chromatin features, whereas middle replication corresponds to denser euchromatin associated with repetitive elements and less active genes \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The interspersed early and middle RT domains in the 2-Mb segment of chromosome 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) reflect their alternating nature and associated chromatin states. Here we document the robust covariance between chromatin interactions and RT annotations. Early RT domains exhibit higher contact frequencies with other early regions, even across long genomic distances, as evidenced by the eigenvector (EV) and insulation score (IS) analyses (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Middle RT domains exhibit a lesser tendency for long reaching interaction and show a more condensed chromatin structure. The \"same-RT-class\" interactions may reflect different functions. For example, early-early interactions might be largely based on transcriptional activity, while middle-middle interactions - occurring in regions with less active genes and abundant retroelements - might reflect a chromatin packaging function.\u003c/p\u003e \u003cp\u003eIn general, it is known that the chromatin regions that replicate at different time points in S phase, such as early versus late, also show distinct enrichments for epigenomic features in plants and animals \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. For instance, early-S replicating regions generally exhibit open and accessible chromatin, enrichment for active chromatin marks such as histone acetylation, and relatively higher levels of transcriptional activity. On the other hand, late-S replicating regions generally exhibit the opposing features such as closed and repressive chromatin marks, including histone methylation on H3K27 or H3K9, and DNA methylation.\u003c/p\u003e \u003cp\u003eIn contrast to the established framework of a binary division of S-phase into early-S versus late-S stages, our study emphasizes a tripartite replication timing framework, with early, middle, and late S profiling. We focused on the early versus middle because they appear similar in their broad distribution within nucleoplasmic HiC-defined as global A compartment but are distinct in other ways. Interestingly, the Middle-S chromatin has features of both euchromatin and heterochromatin. Middle-S tends to lack the marks associated with the most active chromatin but is only weakly enriched for repressive histone marks. For instance, the gene-active chromatin histone acetylation mark H3K27ac had correlation values of \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.72 for Early-S versus r = \u0026minus;\u0026thinsp;0.61 for Middle-S chromatin. Similarly, but in the opposite direction, the gene-repressive chromatin mark H3K9me2 had correlation values of \u003cem\u003er\u003c/em\u003e = \u0026minus;\u0026thinsp;0.19 for Early-S versus r\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.044 for Middle-S chromatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e) consistent with previous study \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In all cases, we saw that the Middle-S regions have chromatin mark correlations that can be described as (1) being in between those of Early-S and Late-S, (2) weaker overall, and (3) more similar to those of Late-S regions. In addition, transcriptional characteristics of genomic regions, including transcription start site mapping and transcriptomics, also tend to mirror the pattern of epigenomic marks with regard to gene activity. Specifically, analyzing expression levels for promoters using the RAMPAGE assay \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e, we observed a correlation of \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.55 for Early-S chromatin and \u003cem\u003er\u003c/em\u003e = \u0026minus;\u0026thinsp;0.12 for Middle-S chromatin. Indeed, our epigenomic enrichment and Hi-C analyses provide concordant substantiation for the unique identity of Middle-S chromatin, which comprises a third of the maize genome.\u003c/p\u003e \u003cp\u003eThe FISH experiments also provided new and compelling cytological evidence for the bifurcation of the global A compartment into early and middle chromatin. Unlike our previous work, which was limited to replicative labeling within Early-S or Middle-S, our RT-FISH approach allowed us to examine the spatial location of the RT-defined sequences outside of S-phase and independent of replicative labeling. We carried out two related types of experiments. The first experiment used early and middle RT FISH probes separately on total nuclei and quantified the DAPI concentration in the FISH-defined regions relative to a similar sized chromatin control region with properties typical of bulk euchromatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The second experiment used both early and middle probe sets labelled with different fluorophores on G1 nuclei. This enabled us to simultaneously visualize the spatial separation and nuclear localization of both RT-FISH signals, as well as to quantify their relative DAPI densities, without the need for an external control region (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). In every case, we observed signal partitioning validating the 2-compartment architecture, which we now know is present in G1 nuclei. This finding demonstrates that RT-related nuclear architecture exists prior to S phase, and is not simply a result of processes in S-phase itself.\u003c/p\u003e \u003cp\u003eGiven that Hi-C compartments and replication timing are tightly coupled in root tip, we reasoned that one could ask to what extent replication timing programs might be conserved in other tissues, using Hi-C structure as a proxy for replication timing. We showed that biologically vastly different organs, earshoot versus root tip, had strong correlations for Hi-C based compartmentalization genome-wide, with \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.85 for both Eigenvalues and insulation scores (Supplementary Fig.\u0026nbsp;2). When correlating replication timing from root tip with Hi-C measures from earshoot or bundle sheath, the correlations were similar in strength and direction to those from within-tissue comparisons (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These findings provide evidence in support of the idea that the two compartment architecture of euchromatin (as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-e) not only extends throughout the cell cycle in root tips but exists in other tissues as well. This observation raises the intriguing possibility that this pattern of nuclear architecture may reflect a broader organizational principle in other large-genome eukaryotes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAUTHOR CONTRIBUTIONS\u003c/h2\u003e\n\u003cp\u003eHWB, LH-B and WFT conceived the study. HSA, HWB, LC, LH-B and WFT designed the experiments. HSA prepared Hi-C libraries and performed 3D FISH experiments, collected and analyzed the data. ZMT prepared MOA-seq libraries. LC and HWB contributed to the analysis and interpretation of data. EEW and LM-Y provided maize root tip nuclei for the study. HSA and HWB drafted the manuscript and all authors critically revised and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eCOMPETING INTERESTS\u003c/h2\u003e\n\u003cp\u003eWe do not have any competing interests.\u003c/p\u003e\n\u003ch2\u003eMATERIAL AND CORRESPONDENCE\u003c/h2\u003e\n\u003cp\u003eMaterial request from the corresponding author Hank W. Bass.\u003c/p\u003e\n\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e\n\u003cp\u003eThe authors would like to acknowledge help with early versions of the data analysis by Dr. Jawon Song and Joshua Urrutia at the Texas Advanced Computing Center. This work was supported by a grant from the National Science Foundation Plant Genome Research Program (NSF IOS 2025811 to LH-B, LC, WFT, and HWB), awards from Florida State University (Dean's Award for Doctoral Excellence to HSA; Ben and Karen Thrower award to HSA), and funds from North Carolina State University.\u003c/p\u003e\n\u003ch2\u003eDATA AVAILABILITY STATEMENT\u003c/h2\u003e\n\u003cp\u003eRaw Hi-C data, MOA-seq data, processed all valid pairs files, HiC files and bigwig files can be accessed at NCBI GEO under the accession number GSE287128. Scripts for Hi-C data analysis can be accessed at\u003c/p\u003e\n\u003cp\u003ehttps://github.com/Sarachaudry/HiC-contacts-with-RT-annotations\u003c/p\u003e\n\u003cp\u003ehttps://github.com/Sarachaudry/Correlation_Hexbin_plots\u003c/p\u003e\n\u003cp\u003ehttps://github.com/Sarachaudry/virtual-4C-interactions-with-Sushi-in-R\u003c/p\u003e\n\u003cp\u003eGEO accession GSE287128: (reviewer token azojyygqvfyhjcl)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRhind N, Gilbert DM (2013) DNA replication timing. Cold Spring Harb Perspect Biol 5:a010132\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWear EE et al (2017) Genomic analysis of the DNA replication timing program during mitotic S phase in maize (Zea mays) root tips. 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Science 373:655\u0026ndash;662\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Replication timing (RT), chromatin, nuclear architecture, Hi-C, 3D FISH, 5-ethynyl deoxyuridine (EdU), DAPI, Early-S RT, Middle-S RT, maize","lastPublishedDoi":"10.21203/rs.3.rs-6116464/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6116464/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGenome replication is temporally regulated during S phase, with specific genomic regions replicating at defined times in a process known as Replication Timing (RT). Based on 3D cytology in replicating nuclei, we previously proposed a \u0026ldquo;mini-domain chromatin fiber RT model\u0026rdquo; for maize euchromatin that suggested it is subdivided into early-S and middle-S compartments distinguished by chromatin condensation and RT. However, whether this compartmentalization reflects a general nuclear architecture that persists throughout the cell cycle was unclear. To test this model, we conducted two orthogonal assays\u0026mdash;Hi-C for genome-wide interaction data and 3D FISH for direct visualization of chromatin organization. Hi-C eigenvalues and insulation scores revealed distinct patterns of early-S regions having negative insulation scores with long-range contacts while middle-S regions showed the opposite. Early-S regions also correlated more strongly with epigenomic signatures of open, transcriptionally active chromatin than middle-S regions. 3D oligo FISH painting confirmed that early-S and middle-S regions occupy adjacent but largely non-overlapping nucleoplasmic spaces during all interphase stages, including G1. Our findings redefine the maize euchromatin \u0026ldquo;A\u0026rdquo; compartment as having two distinct subcompartments\u0026mdash;Early-S and Middle-S\u0026mdash;and underscore the importance of replication timing as a defining feature of chromatin architecture and genome organization.\u003c/p\u003e","manuscriptTitle":"Replication Timing Uncovers a Two-Compartment Nuclear Architecture of Interphase Euchromatin in Maize","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-12 07:04:54","doi":"10.21203/rs.3.rs-6116464/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ef7fa68b-56e4-4d05-b3af-9988d8c6166d","owner":[],"postedDate":"March 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":45484138,"name":"Biological sciences/Cell biology"},{"id":45484139,"name":"Biological sciences/Genetics"},{"id":45484140,"name":"Biological sciences/Genetics/Genomics/Epigenomics"}],"tags":[],"updatedAt":"2025-08-15T18:30:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-12 07:04:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6116464","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6116464","identity":"rs-6116464","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}