Unveiling Unique Expression Patterns of D20S16 Satellite DNA in Human Embryonic Development

Unveiling Unique Expression Patterns of D20S16 Satellite DNA in Human Embryonic Development | 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 Research Article Unveiling Unique Expression Patterns of D20S16 Satellite DNA in Human Embryonic Development Yajie Hu, Kenji Mizuguchi, Kousuke Hashimoto This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5191409/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 Background: Satellite DNA plays a crucial role in maintaining chromosomal stability and gene regulation. However, its specific functions during early embryonic development are not fully understood. In this study, we investigate the expression patterns and regulatory mechanisms of D20S16 satellite DNA during critical stages of human embryogenesis by integrating the complete human genome reference (T2T-CHM13) with RNA-seq data. Results: Our comprehensive analysis reveals that D20S16 exhibits high expression levels in early developmental stages, followed by a significant decline as development progresses. We identified 20 distinct D20S16 elements in the human genome and found that only two elements, located on chromosome 20, are transcriptionally active during embryogenesis. Comparative analysis with macaque data further demonstrates that D20S16 is minimally expressed in macaque embryos, which have shorter and fewer repeat units compared to humans. Conclusion: These findings suggest that D20S16 plays a unique regulatory role in human embryonic development, with its expression being potentially linked to specific chromosomal locations. This study provides new insights into the role of satellite DNA in early development and sets the foundation for future research into its function and evolutionary significance. RNA-seq T2T-CHM13 Comparative genomics Repeat units HMM Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Embryonic development, a pivotal stage in mammalian life, begins with the fusion of sperm and egg. This marks the start of a major transformation involving chromatin structure and transcriptional activity. The process starts with a totipotent fertilized egg and proceeds through cleavage, morula, and blastocyst stages. This succession of early embryonic events essentially represents a process of cell proliferation with large-scale epigenetic remodelling, involving various biological processes that require precise transcriptional regulation, epigenetic reprogramming, and orderly metabolic changes [ 1 ]. Recent advances in RNA sequencing technologies have deepened our understanding of transcriptional changes in embryos at different stages [ 2 , 3 ]. Epigenetic reprogramming during gametogenesis and embryonic development has also been progressively revealed [ 4 – 6 ]. However, despite these advancements, knowledge of satellite DNA expression during human embryonic development remains limited. A substantial portion of the genome is non-coding DNA, including tandem repeat sequences such as satellite DNA that occupy many eukaryotic chromosomes. These sequences are predominantly located in (peri)centromeric and (sub)telomeric regions of chromosomes. Initially, owing to their non-coding nature and lack of sequence conservation across closely related species, satellite DNAs were primarily considered “junk DNA” [ 7 ]. However, contemporary research has revealed that satellite DNA is crucial in maintaining chromosomal segregation and genetic stability [ 8 – 13 ]. Therefore, a thorough investigation into the role of satellite DNA in embryonic development is essential for unveiling the intricate mysteries of embryogenesis. In the past, the study of satellite DNA was particularly challenging owing to their short repetitive sequences. In addition, the incompleteness of the human GRCh38 reference genome, with missing or incorrect data in over 5% of sequences [ 14 – 16 ], compounded the difficulty. These missing or incorrect parts were located mainly where satellite DNA is prevalent, notably in telomeric and centromeric regions. However, with newer DNA sequencing technologies, such as PacBio HiFi and Oxford Nanopore’s ultra-long-read sequencing [ 17 ], the Telomere-to-Telomere (T2T) Consortium successfully filled these regions and released the first complete human genome reference, CHM13 [ 18 ]. Furthermore, it has completed the sequence information for the Y chromosome, unveiling v. 2.0 This breakthrough provides invaluable data for our research into satellite DNA. This article presents our key findings on the expression profiles of satellite DNAs such as the highly expressed D20S16 during human embryonic development, contributing to our understanding of their potential functions and implications. Results 1 Stage-Specific Expression Patterns of D20S16 Satellite DNA, comprising highly repetitive non-coding sequences, includes 33 distinct types in the human genome as defined by RepeatMasker ( http://repeatmasker.org ). We analysed an RNA-Seq dataset [ 19 ] (SRA ID SRP062850) representing a comprehensive set of transcriptomes of human oocytes and preimplantation embryonic development. This dataset includes samples from seven stages of human oogenesis and early embryogenesis, processed with a transposase-based library method to sequence total RNA without 3′ bias [ 19 ]. Through analysis, we have discovered that satellite DNAs exhibit stage-specific expression patterns during embryonic development (Fig. 1 A). D20S16, along with GSATII and TAR1, shows high expression before the cleavage (CL) stage of embryonic development, followed by a noticeable decrease in later stages (Fig. 1 B). Conversely, other satellite DNAs, such as ACRO1 [ 20 ] and BSR/Beta, display increasing expression after the CL stage. To validate these findings and enhance the robustness of our observations, we used an additional dataset [ 21 ] (SRA ID SRP061636), which encompasses a comprehensive set of transcriptomes, covering both polyA + and polyA − mRNAs. The results corroborated our initial observations and provided further insights into the timing of D20S16 expression decline. Specifically, while the first RNA-Seq analysis showed a marked decrease in D20S16 expression after the CL stage, the second dataset showed that this decline begins after the 8-cell (8C) stage, which is part of the cleavage stage (Fig. S1 ). Furthermore, the 2-cell (2C), 4-cell (4C), and 8-cell (8C) stages included in this dataset provide more detailed insights into the expression dynamics of satellite DNA during this critical developmental phase. These results demonstrate that satellite DNAs, such as D20S16, exhibit distinct expression patterns during embryonic development. 2 Discovery of Twenty Distinct Yet Continuous D20S16 Elements in the Human Genome After identifying the stage-specific expression patterns of D20S16, the next challenge was to determine which of the many D20S16 copies in the genome were actually being transcribed. To address this, we first needed to clarify the exact number and locations of D20S16 copies in the human genome. Using RepeatMasker annotations, we initially identified 123 copies of D20S16 dispersed throughout the human genome (Fig. S2), 38 on chromosome (chr.) 20 and 30 on chr. 3, and the rest distributed across chrs. 2 and 5 to 9. On chr. 20, D20S16 spans 61,231 base pairs (bp), occupying 46.8% of the total sequence, a much larger region than on other chromosomes (Fig. 2 A). We found a marked tendency to cluster in specific regions (Fig. 2 B): specifically, 38 copies on chr. 20 are entirely concentrated into 5 clusters around the 50-Mb region, each separated by very short distances (Fig. 2 C). Upon closer examination using the Integrative Genomics Viewer (IGV) [ 22 ], we realized that these clustered D20S16 were actually a single long D20S16 element, rather than multiple short copies. (Fig. 2 D). These segments were mistakenly annotated as MLT2B4 due to sequence similarities between parts of D20S16 and MLT2B4 (Fig. 2 E). MLT2B4 is categorized as a long terminal repeat (LTR) within the endogenous retrovirus (ERV) family. While the length of MLT2B4 consensus sequence is 557 bp, most observed instances were approximately 100 bp (Fig. 2 D). Sequence comparison revealed that a part of MLT2B4 (272–368 bp) has similarity to D20S16 (Fig. 2 E), which led to these mis-annotations. Expression data displayed continuous transcription from a broad region of D20S16, also indicating that the D20S16 copies, segmented by “MLT2B4” short sequences, constitute a single larger D20S16 element rather than a mixture of D20S16 and MLT2B4 copies. We thus merged 123 individual D20S16 copies fragmented by MLT2B4 into 20 elements (Fig. 2 F; Table S1 ). This process significantly altered the sequence lengths, transforming multiple short copies into longer substantial elements (Fig. S3). The expression analysis of the 20 refined elements revealed that two adjacent elements on chr. 20 (chr20 #4 and chr20 #5) are predominantly expressed in total of GV-CL (Fig. 2 G). These two elements accounted for the vast majority of D20S16 expression in embryo. In contrast, elements on chrs 3, 5 and 7 exhibited minimal expression and no expression was detected from other chromosomes, as confirmed by the second dataset (Fig. S4). 3 Identifying D20S16 Repeat Units via Manual Extraction and Hidden Markov Models To elucidate the reasons behind the specific high expression of D20S16 from chr20#4 and chr20#5, we aimed to explore the internal sequence composition of these elements. D20S16 is composed of short repetitive units, each 49 bp long [ 23 ] (Fig. 3 A). However, whether the sequences of the repetitive units vary among different copies remains unclear. We tried to extract these repetitive units from the chr20#4 and #5 elements using publicly available tools such as RepeatModeler2 [ 24 ] and RepEx [ 25 ], but failed owing to their high diversity and short sequence lengths. We then manually investigated and identified a highly conserved 5-nucleotide motif, “CAGCT”, in the repetitive units. Consequently, we resorted to manual extraction, using “CAGCT” as a reference point for cutting and aligning multiple sequences from all 20 refined elements. We further corrected and adjusted sequences with minor variations, including “CAGCC” or “CATGT”. After 2 or 3 iterations of this process, we extracted 2611 repetitive units from the 20 elements, and generated a single multiple sequence alignment (Fig. 3 B). Using the multiple sequence alignment, we used Hidden Markov Models (HMMs) [ 26 ] to capture repetitive units potentially missed during manual extraction. We built an HMM model using the hmmbuild function in HMMER (v. 3.3.2) and searched for repetitive units in the human genome using the hmmsearch function (E-value < 0.01). Finally, we identified 2809 repeat units (Fig. 3 C) with an average length of 53 bp. Of these, 483 were located in chr20#2, 332 in chr20#5, and 96 in chr20#4. Despite their relatively smaller number, the repeat units in chr20#4 had remarkably high expression levels. Compared with the RepeatMasker data (Fig. 3 D), our approach added 8528 bases and removed 2811 bases with errors (Fig. 3 E), refining the D20S16 profile and establishing a new HMM model for further research. 4 Identifying four types of D20S16 on the basis of the variable region Following the extraction of repeat units, we conducted a comprehensive sequence conservation analysis of each element and generated sequence logos (Fig. S5), which revealed the coexistence of conserved and variable sequences. The 5′ and 3′ regions of all D20S16 repeat units are conserved, with the variable region located in the middle of the units (starting from 25 bp). We categorized them into 4 types on the basis of their sequence length: that characterized by a conserved “CATCAG” sequence is 47 bp; that by “CAA-A–G” is 49 bp; that by “CAACACCAG” is 50 bp; and that by “CAGCAGCACC-G” is 53 bp (Fig. 4 A). Specifically, the 47-bp group comprises 7 elements found on chrs. 2, 3, and 5 (1003 units); the 49-bp group comprises 5 elements from chrs. 8 and 20 (1195 units); the 50-bp group comprises 5 elements across chrs. 2, 3, 5, 8, and 9 (339 units); and the 53-bp group comprises 3 elements, including the prominent chr20#4 (272 units). For each of these sequence groups, phylogenetic trees were constructed to further explore their expression in relation to the variable regions. Our analysis of the phylogenetic tree of the 53-bp group (Fig. 4 B) revealed that, with a few exceptions, the units of chr20#4 clustered together, suggesting that this region is a result of tandem repeat duplication that might have increased the unit numbers during evolution. In addition, we examined the relationship between expression levels and sequence similarity. Intriguingly, despite high sequence homogeneity, expression was exclusive to chr20#4 (gray bars in Fig. 4 B). This pattern was also observed in the 50-bp group containing chr20#5 (Fig. S6). These findings prompted us to reassess the mechanisms governing D20S16 expression. From our observations, expression seems to be more intricately associated with specific chromosomal locations than with sequence variation. However, the precise mechanisms and functions warrant further investigation. 5 Specific Expression of D20S16 in Human Embryonic Development To broaden our understanding of the role of satellite DNA in embryonic development, particularly D20S16, we explored its expression patterns during macaque embryonic development. We selected rheMac10 (Rhe10) as the reference macaque genome [ 27 ] and used the associated RNA-Seq dataset (SRA ID: SRP089891) [ 28 ] for analysis, which includes single oocytes/embryos or multiple biological replicates at each developmental stage. In addition, it pools samples of 5 to 23 oocytes or embryos collected from 1 to 10 female macaques at each developmental stage. We compared the expression patterns of D20S16 between humans and macaques during embryonic development. In contrast to the high expression levels observed in humans, D20S16 expression was barely detectable in macaque early development (Fig. 5 A). Even during the cleavage stages (2C-8C), where D20S16 is strongly expressed in humans, its expression remained minimal in macaques (Fig. 5 B). This clear difference highlights the pronounced disparity in D20S16 expression between the two species. To investigate whether these differences in expression are related to variations in the D20S16 sequence, we compared the genomic regions containing D20S16 in humans and macaques. As shown in Fig. 2 C, the 50 Mb region of the human chromosome 20 contains five D20S16 elements, of which two were transcriptionally active. By using BLAST [ 29 ], we identified a syntenic region in the macaque genome at 83 Mb on chromosome 10, corresponding to this locus in the human genome (Fig. 5 C). We then precisely aligned the five D20S16 elements between the two genome sequences. As a result, we found that the macaque genome also contains five elements; however, in all cases, their lengths are shorter than those in the human elements (Fig. 5 D). Notably, the #4 element, which is highly expressed in humans, was only half the length in macaques. This length difference indicates that the number of repeat units in macaques is lower than in humans, potentially contributing to the observed differences in expression levels. By applying the same workflow, we extracted the consensus sequence from the corresponding region in Rhe10 and found that the variable region of chr10 #4 shares the sequence ‘CAGCAGCACC-G’ with chr20 #4 in CHM13 (Fig. 5 E). Sequence alignment between chr20 #4 in CHM13 and chr10 #4 in Rhe10 revealed gaps (Fig. 5 F), suggesting that either humans gained additional repeat units or macaques lost them during evolution. Discussion This study explored the expression of satellite DNAs throughout human embryonic development, providing insights into potential regulatory roles during embryonic development. Notably, the behaviour of D20S16, GSATII, and TAR1 may indicate that they play significant roles in early developmental stages, possibly orchestrating key transitions and gene expression profiles. Similarly, the observed expression patterns of ACRO1 and BSR/Beta might align with their potential roles in critical phases of embryonic development, such as cell division and differentiation processes. The transient expression peaks of ALR/Alpha and (GAATG) n could be indicative of their importance during specific windows of embryonic development, although the direct correlation to specific biological functions remains speculative. Among these, D20S16 emerged as particularly noteworthy owing to its pronounced expression levels and distinct trend, driving a comprehensive investigation into its copy number, chromosomal positioning, and consensus sequences. This analysis enhanced our understanding of satellite DNA and introduced a new perspective: that chromosomal positioning might contribute to the regulatory expression of satellite DNA. Moreover, the elevated expression of D20S16 in pathological contexts, such as breast cancer [ 30 ], highlights its broader biological relevance and potential as a target for therapeutic strategies. While previous studies [ 31 ] focused on expression analysis, our research also examined the genomic regions of individual elements where this expression occurs. By using a combination of manual curation and HMM methodologies, we constructed HMM profiles for D20S16 subtypes. This allowed us to identify the variable and conserved regions within D20S16. Although this lays a solid foundation for further research, we must acknowledge that the subjectivity inherent in manual extraction and the assumptions underlying the HMM may introduce unconfirmed variations. Notably, our study did not find a D20S16 expression pattern in the macaque model analogous to that in humans. While this reinforces the hypothesis that D20S16 exhibits a unique expression pattern in human embryonic development, it also highlights a significant limitation of our research, namely the incompleteness of the macaque genome data. Future research should consider gathering more comprehensive macaque genome data, akin to the T2T-CHM13, or use genome data from other closely related species to better understand the evolutionary and functional differences of satellite DNA in primates. Future research should focus on the experimental validation of the function of D20S16, mainly through the precise manipulation of its expression in model organisms by using gene editing technologies, such as the CRISPR-Cas9 system. This could reveal its specific effect on embryonic development. If feasible, creating transgenic mice carrying the human D20S16 sequence through gene knock-in experiments could provide insights into its role throughout embryonic development. In addition, analysing the dynamic expression of D20S16 by using real-time quantitative PCR (qRT-PCR) to obtain quantitative expression data at different developmental stages would be beneficial. Future studies should investigate the interactions between D20S16, other genes, and signalling pathways during embryogenesis, as well as its specific roles in cell division and differentiation. In summary, our study offers new insights into the expression patterns of D20S16 in human embryonic development. These findings could pave the way for new fields of research into the function of satellite DNA and potentially provide new understandings and therapeutic strategies for diseases related to embryonic development. Conclusion This study has enhanced our understanding of the function and expression patterns of the D20S16 satellite DNA during human embryonic development. Our analyses indicate that D20S16 exhibits a distinct pattern of high expression in the early stages of embryogenesis and suggest that its expression may be influenced by its specific chromosomal location. These insights are significant for understanding the gene regulatory networks during early embryonic development. To address the challenges in the analysis of satellite DNA sequences, this study used a novel approach combining manual curation with the HMM to identify distinct types of D20S16. This method has not only increased the accuracy of sequence identification, but will also aid in more precisely revealing the function of D20S16 in future model organism research. Despite challenges posed by the incomplete macaque genome data, our work indicates the potential of D20S16 as a satellite DNA with high expression specificity in humans. Methods Collecting transcriptome and genome data We used the fasterq-dump v. 3.0.3 tool to retrieve RNA-sequencing (RNA-Seq) data derived from 658 cells and 667 developing human embryos (GEO Accession GSE85632) in FASTQ file format from the Sequence Read Archive (SRA) database, and to retrieve RNA-Seq data of human (GSE71318) and macaque (GSE86938) supplementary cells of developing embryos. We used the T2T-CHM13 v. 1.1 human genome reference and the rhcMac10 macaque genome reference and their corresponding gene annotations in GTF format from the UCSC Genome Browser ( https://genome.ucsc.edu/ ). Sequence information for satellite DNA was sourced from the RepeatMasker database ( https://www.repeatmasker.org/ ). Profiling Expression of Satellite DNA For read mapping, we first pre-processed the data in FastQC v. 0.11.9 to perform quality analysis with the command ‘ fastqc -t 2 -q ’. We then used trim_galore v. 0.6.7 (‘ trim_galore -q 20 --nextera --paired ’) to remove base sequences with quality scores < 20 and to trim Nextera primers. To remove rRNA reads, we processed the dataset in rRNAdust v. 1.06. The paired fastq files were then concatenated in seqkit [ 32 ] v. 2.2.0 (‘ seqkit pair − 1 file1 -2 file2 ’). Index generation and read mapping were conducted in STAR [ 33 ] v. 2.7.10a. The resulting read mappings were then normalized to the volume of RNA-Seq data in the dataset, and counts per million (CPM) values were computed across all samples. Finally, to visually represent the expression of satellite DNA during embryonic development, heatmaps and line graphs were generated and presented in R software. Merging D20S16 Copies Owing to the sequence similarity between MLT2B4 and D20S16, which complicates the identification of D20S16 sequences, we merged copy regions that were separated because of this similarity. First, we measured the distance between adjacent copies on each chromosome, using regular expression . This process entailed calculating the distance by subtracting the end position of a given copy from the start position of the next copy on the same chromosome. Typically, the distances between proximate copies ranged from 10 to 200 bp. Copies that were separated by more than 1000 bp were considered sufficiently distant to be classified as distinct elements. We used Mafft [ 34 ] v. 7.515 for comprehensive sequence comparison and further examination of these regions, and visualized the results in the Integrative Genomics Viewer [ 22 ]. Extraction of Repeat Unit in D20S16 Upon acquiring the data from the merged region, we initiated extraction by using a Regex command in awk to isolate the conserved region “CAGCT” as the reference locus. Then we used Mafft for multi-sequence alignment ( mafft --thread 12 --globalpair --maxiterate 1000 ) of the segmented regions to scrutinize the initial cutting results. During this phase, any inaccuracies due to non-conservative sequences were identified and rectified. This cycle of cutting, aligning, and correcting was performed two or three times to enhance the precision of the extracted sequences. Through these iterations, a more comprehensive multiple sequence comparison file was compiled. Following this, HMMER [ 35 ] v. 3.3.2 was used to construct a model (hmmbuild) and search for sequences similar to the identified unit across the entire genome (hmmsearch). Finally, to visually represent the consensus sequence, we generated sequence logos in weblogo [ 36 ], using custom Python scripts. This graphical representation provides a clear and concise visualization of the sequence conservation and variability within the repeat unit of D20S16. Phylogenetic tree construction The consensus sequence was generated from all units in Mafft for multiple sequence comparison. The specific sequences spanning positions 26–37 within the consensus sequence were precisely extracted through the use of custom Python scripts. This targeted approach allowed for a focused analysis of the region of interest. Thereafter, the extracted sequences were used to construct a phylogenetic tree in PhyML [ 37 ] v. 3.3.20220408. Abbreviations RNA-Seq RNA Sequencing HMM Hidden Markov Model CPM Counts Per Million GRCh38 Genome Reference Consortium Human Build 38 T2T-CHM13 Telomere-to-Telomere CHM13 (Complete Human Genome) IGV Integrative Genomics Viewer LTR Long Terminal Repeat ERV Endogenous Retrovirus GTF Gene Transfer Format FASTQ FASTQ (Sequence file format) SRA Sequence Read Archive HMMER Hidden Markov Model Software qRT-PCR Quantitative Real-Time Polymerase Chain Reaction CRISPR-Cas9 Gene Editing Technology BLAST Basic Local Alignment Search Tool Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Competing interests The authors declare no competing interests Funding Not applicable Author Contribution HK and HY are the primary authors of this manuscript. They were responsible for the analysis of the RNA-seq data, conducted the bioinformatics analysis, and generated the visual representations and figures. Both authors contributed significantly to the interpretation of the data and drafting of the manuscript. In addition, HK led the experimental design, while HY managed the data processing and statistical analysis.MK provided valuable guidance throughout the project, offering critical feedback on the study's methodology and theoretical framework. He also contributed to the refinement and editing of the manuscript, ensuring clarity and coherence. All authors read and approved the final manuscript. Acknowledgements We thank the scientists for providing the RNA-seq data and make the the T2T-CHM13 reference sequence available. Their contributions have enabled us to gain insights into the expression patterns of satellite DNA during embryonic development. Data Availability We are currently making revisions in preparation for the upload. The data availability information will be updated once the upload is successful. References Jukam D, Shariati SAM, Skotheim JM. 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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-5191409","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":370006015,"identity":"7e8fb7ac-c9ad-45c9-957b-0346269f00f4","order_by":0,"name":"Yajie Hu","email":"","orcid":"","institution":"Osaka University","correspondingAuthor":false,"prefix":"","firstName":"Yajie","middleName":"","lastName":"Hu","suffix":""},{"id":370006019,"identity":"6d8c93a0-1bf6-4a9b-b255-f64a26be0e9b","order_by":1,"name":"Kenji Mizuguchi","email":"","orcid":"","institution":"Osaka University","correspondingAuthor":false,"prefix":"","firstName":"Kenji","middleName":"","lastName":"Mizuguchi","suffix":""},{"id":370006023,"identity":"5ea2d2a3-d114-4824-af7f-4297e41b73c4","order_by":2,"name":"Kousuke Hashimoto","email":"data:image/png;base64,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","orcid":"","institution":"Osaka University","correspondingAuthor":true,"prefix":"","firstName":"Kousuke","middleName":"","lastName":"Hashimoto","suffix":""}],"badges":[],"createdAt":"2024-10-02 07:53:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5191409/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5191409/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68242521,"identity":"ae409f0d-6da4-4f70-b4c8-3427b00839be","added_by":"auto","created_at":"2024-11-05 08:25:13","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":151200,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSatellite DNA expression in embryonic development.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Heatmap illustrating expression patterns of 33 satellite DNAs across eight stages of human embryonic development: Germinal Vesicle (GV), Meiosis I (MI), Meiosis II (MII), Pronuclei (PN), Cleavage (CL), Morula (MO), Inner Cell Mass (IC), and Trophectoderm (TP). CPM = counts per million. To visualize gene expression levels, we normalized data as log\u003csub\u003e2\u003c/sub\u003e(CPM+1). (\u003cstrong\u003eB\u003c/strong\u003e) D20S16 expression is high before CL (cleavage) stage and decreases after.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5191409/v1/86f58069d6e57476b03b15df.jpeg"},{"id":68242516,"identity":"5b10bb79-851a-4dc7-bd16-abb781852283","added_by":"auto","created_at":"2024-11-05 08:25:11","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":439105,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eD20S16 distribution in the genome. (A) \u003c/strong\u003eThe proportion of D20S16 sequence length varies across different chromosomes, with the largest on chr20, followed by chr3, and the smallest on chr9. \u003cstrong\u003e(B)\u003c/strong\u003e The distribution of D20S16 copies across different chromosomes. Gray bar shows the length of each chromosome. Dark blue points mean each copy site. Boxed point means 38 copies clustered at the 50-Mb region on chr. 20.\u003cstrong\u003e (C) \u003c/strong\u003eMap outlining the specific locations of D20S16 sequences on chr. 20 from 49.571 to 50.286 Mb, numbered from #1.\u003cstrong\u003e (D) \u003c/strong\u003eComparative genomic map displaying the relative positions of D20S16 and MLT2B4 on the T2T-CHM13 chr. 20. Insertion of MLT2B4 into D20S16 leads to a RepeatMasker annotation error and misidentification of D20S16 regions.\u003cstrong\u003e (E) \u003c/strong\u003eAlignment comparison highlighting sequence similarities between MLT2B4 and D20S16: part of MLT2B4 (272–368 bp) has similarity to D20S16. \u003cstrong\u003e(F) \u003c/strong\u003eThe process of integrating individual copies of D20S16 into composite elements, we categorized the distances between the copies on each chromosome, using the copies with larger intervals as demarcation points and combining copies with smaller intervals into a single, continuous D20S16 region. \u003cstrong\u003e(G)\u003c/strong\u003e D20S16 copy expression levels on different chromosomal elements. Expression in chr20#4 and #5 is much higher than in the other elements.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5191409/v1/3d00e3a8dbec16962dbadab1.jpeg"},{"id":68242520,"identity":"884c0b3f-4712-4ca3-b050-34d887c50632","added_by":"auto","created_at":"2024-11-05 08:25:13","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":301570,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe composition of D20S16, along with the revised workflow and results. (A)\u003c/strong\u003e Schematic representation of the D20S16 locus, showcasing a single-copy sequence composed of sequential-dynamic-variability repeat units (49 bp). \u003cstrong\u003e(B)\u003c/strong\u003e Diagram outlining the step-by-step protocol for isolating D20S16 repeat units. We manually investigated and identified a conserved 5-amino-acid motif, “CAGCT”, in the repetitive units. Consequently, we resorted to manual extraction, using “CAGCT” as a reference point for cutting and aligning multiple sequences for all 20 refined elements. We further corrected and adjusted sequences with minor variations, including “CAGCC” or “CATGT”. After 2 or 3 iterations of this process, we used Hidden Markov Models (HMMs) to capture repetitive units potentially missed during manual extraction. \u003cstrong\u003e(C) \u003c/strong\u003eBar graph quantifying repeat units identified within each D20S16 element: 483 were located in chr20#2, 332 in chr20#5, and 96 in chr20#4.\u003cstrong\u003e (D)\u003c/strong\u003e Describes the differences in base lengths between the RepeatMasker data (raw data) versus newly extracted data (new data). \u003cstrong\u003e(E) \u003c/strong\u003eA comparison of the old and new D20S16 using chr20 #4 as an example revealed the addition of regions previously misannotated, as well as the removal of redundant insertions with errors.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5191409/v1/7ee759b02bd93ef974fa9587.jpeg"},{"id":68242484,"identity":"e8fa9cba-738b-4ee5-ac7f-078b4e25a43c","added_by":"auto","created_at":"2024-11-05 08:24:58","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":602903,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe consensus sequence and phylogenetic tree of D20S16. (A) \u003c/strong\u003eSequence logos illustrating the consensus sequence and variable regions for D20S16 units from the 47-, 49-, 50- and 53-bp groups. Larger letters mean they’re more conservative in their position.\u003cstrong\u003e(B) \u003c/strong\u003ePhylogenetic tree consisting of three elements (chr6 #1, chr7 #1, chr20 #4) of the 53-bp group. Gray bars show expression level.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5191409/v1/3cae599edc6e0925c3ad3f92.jpeg"},{"id":68242515,"identity":"b14af0ee-cb3e-4072-9491-e08bcbacbab5","added_by":"auto","created_at":"2024-11-05 08:25:08","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":292841,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression and Sequence Analysis of D20S16 in Macaque Embryonic Development. (A) \u003c/strong\u003eHeatmap detailing the expression levels of 22 satellite DNA sequences across eight stages of macaque embryonic development: mature blastocyst stage oocytes (GVO), mature oocytes (MOT), 1-cell embryos at the prokaryotic stage (1C), 2-cell embryos (2C), 4-cell embryos (4C), 8-cell embryos (8C), morula (MOR), blastocysts (BL). CPM = counts per million. To visualize gene expression levels, we normalized data as log\u003csub\u003e2\u003c/sub\u003e(CPM+1). \u003cstrong\u003e(B)\u003c/strong\u003e The negligible developmental expression pattern of D20S16 during macaque embryogenesis. \u003cstrong\u003e(C)\u003c/strong\u003e Homologous Regions of D20S16 in Rhe10 (macaque) and CHM13 (human), Sequence Length, and Expression Levels of Various Elements. \u003cstrong\u003e(D) \u003c/strong\u003eComparison of sequence lengths of D20S16 in 5 elements between CHM13 and Rhe10. \u003cstrong\u003e(E)\u003c/strong\u003e Consensus sequence of D20S16 in 5 elements on chr10, and comparison of #4 with other elements. \u003cstrong\u003e(F)\u003c/strong\u003e Sequence Alignment of CHM13 chr20 #4 with Rhe10 chr10 #4.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5191409/v1/39640e16d57a5da87fd7bbde.jpeg"},{"id":75557299,"identity":"e5111ac3-cf31-44cd-bbb7-924463e6ed29","added_by":"auto","created_at":"2025-02-05 21:46:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2586203,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5191409/v1/af9f2eb6-7b5e-4106-bba6-2e1257a6b209.pdf"},{"id":68242514,"identity":"955f1aa0-532b-40c0-b3e1-d27f031c86e5","added_by":"auto","created_at":"2024-11-05 08:25:07","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":983253,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-5191409/v1/edb2b8ec3e419740c806c9ee.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Unveiling Unique Expression Patterns of D20S16 Satellite DNA in Human Embryonic Development","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEmbryonic development, a pivotal stage in mammalian life, begins with the fusion of sperm and egg. This marks the start of a major transformation involving chromatin structure and transcriptional activity. The process starts with a totipotent fertilized egg and proceeds through cleavage, morula, and blastocyst stages. This succession of early embryonic events essentially represents a process of cell proliferation with large-scale epigenetic remodelling, involving various biological processes that require precise transcriptional regulation, epigenetic reprogramming, and orderly metabolic changes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent advances in RNA sequencing technologies have deepened our understanding of transcriptional changes in embryos at different stages [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Epigenetic reprogramming during gametogenesis and embryonic development has also been progressively revealed [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, despite these advancements, knowledge of satellite DNA expression during human embryonic development remains limited.\u003c/p\u003e \u003cp\u003eA substantial portion of the genome is non-coding DNA, including tandem repeat sequences such as satellite DNA that occupy many eukaryotic chromosomes. These sequences are predominantly located in (peri)centromeric and (sub)telomeric regions of chromosomes. Initially, owing to their non-coding nature and lack of sequence conservation across closely related species, satellite DNAs were primarily considered \u0026ldquo;junk DNA\u0026rdquo; [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, contemporary research has revealed that satellite DNA is crucial in maintaining chromosomal segregation and genetic stability [\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Therefore, a thorough investigation into the role of satellite DNA in embryonic development is essential for unveiling the intricate mysteries of embryogenesis.\u003c/p\u003e \u003cp\u003eIn the past, the study of satellite DNA was particularly challenging owing to their short repetitive sequences. In addition, the incompleteness of the human GRCh38 reference genome, with missing or incorrect data in over 5% of sequences [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], compounded the difficulty. These missing or incorrect parts were located mainly where satellite DNA is prevalent, notably in telomeric and centromeric regions. However, with newer DNA sequencing technologies, such as PacBio HiFi and Oxford Nanopore\u0026rsquo;s ultra-long-read sequencing [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], the Telomere-to-Telomere (T2T) Consortium successfully filled these regions and released the first complete human genome reference, CHM13 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Furthermore, it has completed the sequence information for the Y chromosome, unveiling v. 2.0 This breakthrough provides invaluable data for our research into satellite DNA.\u003c/p\u003e \u003cp\u003eThis article presents our key findings on the expression profiles of satellite DNAs such as the highly expressed D20S16 during human embryonic development, contributing to our understanding of their potential functions and implications.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1 Stage-Specific Expression Patterns of D20S16\u003c/h2\u003e \u003cp\u003eSatellite DNA, comprising highly repetitive non-coding sequences, includes 33 distinct types in the human genome as defined by RepeatMasker (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://repeatmasker.org\u003c/span\u003e\u003cspan address=\"http://repeatmasker.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). We analysed an RNA-Seq dataset [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] (SRA ID SRP062850) representing a comprehensive set of transcriptomes of human oocytes and preimplantation embryonic development. This dataset includes samples from seven stages of human oogenesis and early embryogenesis, processed with a transposase-based library method to sequence total RNA without 3\u0026prime; bias [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Through analysis, we have discovered that satellite DNAs exhibit stage-specific expression patterns during embryonic development (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). D20S16, along with GSATII and TAR1, shows high expression before the cleavage (CL) stage of embryonic development, followed by a noticeable decrease in later stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Conversely, other satellite DNAs, such as ACRO1 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and BSR/Beta, display increasing expression after the CL stage.\u003c/p\u003e \u003cp\u003eTo validate these findings and enhance the robustness of our observations, we used an additional dataset [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] (SRA ID SRP061636), which encompasses a comprehensive set of transcriptomes, covering both polyA\u0026thinsp;+\u0026thinsp;and polyA\u0026thinsp;\u0026minus;\u0026thinsp;mRNAs. The results corroborated our initial observations and provided further insights into the timing of D20S16 expression decline. Specifically, while the first RNA-Seq analysis showed a marked decrease in D20S16 expression after the CL stage, the second dataset showed that this decline begins after the 8-cell (8C) stage, which is part of the cleavage stage (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Furthermore, the 2-cell (2C), 4-cell (4C), and 8-cell (8C) stages included in this dataset provide more detailed insights into the expression dynamics of satellite DNA during this critical developmental phase. These results demonstrate that satellite DNAs, such as D20S16, exhibit distinct expression patterns during embryonic development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2 Discovery of Twenty Distinct Yet Continuous D20S16 Elements in the Human Genome\u003c/h3\u003e\n\u003cp\u003eAfter identifying the stage-specific expression patterns of D20S16, the next challenge was to determine which of the many D20S16 copies in the genome were actually being transcribed. To address this, we first needed to clarify the exact number and locations of D20S16 copies in the human genome. Using RepeatMasker annotations, we initially identified 123 copies of D20S16 dispersed throughout the human genome (Fig. S2), 38 on chromosome (chr.) 20 and 30 on chr. 3, and the rest distributed across chrs. 2 and 5 to 9. On chr. 20, D20S16 spans 61,231 base pairs (bp), occupying 46.8% of the total sequence, a much larger region than on other chromosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). We found a marked tendency to cluster in specific regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB): specifically, 38 copies on chr. 20 are entirely concentrated into 5 clusters around the 50-Mb region, each separated by very short distances (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eUpon closer examination using the Integrative Genomics Viewer (IGV) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], we realized that these clustered D20S16 were actually a single long D20S16 element, rather than multiple short copies. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These segments were mistakenly annotated as MLT2B4 due to sequence similarities between parts of D20S16 and MLT2B4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). MLT2B4 is categorized as a long terminal repeat (LTR) within the endogenous retrovirus (ERV) family. While the length of MLT2B4 consensus sequence is 557 bp, most observed instances were approximately 100 bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Sequence comparison revealed that a part of MLT2B4 (272\u0026ndash;368 bp) has similarity to D20S16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), which led to these mis-annotations. Expression data displayed continuous transcription from a broad region of D20S16, also indicating that the D20S16 copies, segmented by \u0026ldquo;MLT2B4\u0026rdquo; short sequences, constitute a single larger D20S16 element rather than a mixture of D20S16 and MLT2B4 copies. We thus merged 123 individual D20S16 copies fragmented by MLT2B4 into 20 elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). This process significantly altered the sequence lengths, transforming multiple short copies into longer substantial elements (Fig. S3).\u003c/p\u003e \u003cp\u003eThe expression analysis of the 20 refined elements revealed that two adjacent elements on chr. 20 (chr20 #4 and chr20 #5) are predominantly expressed in total of GV-CL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). These two elements accounted for the vast majority of D20S16 expression in embryo. In contrast, elements on chrs 3, 5 and 7 exhibited minimal expression and no expression was detected from other chromosomes, as confirmed by the second dataset (Fig. S4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003e3 Identifying D20S16 Repeat Units via Manual Extraction and Hidden Markov Models\u003c/h3\u003e\n\u003cp\u003eTo elucidate the reasons behind the specific high expression of D20S16 from chr20#4 and chr20#5, we aimed to explore the internal sequence composition of these elements. D20S16 is composed of short repetitive units, each 49 bp long [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). However, whether the sequences of the repetitive units vary among different copies remains unclear. We tried to extract these repetitive units from the chr20#4 and #5 elements using publicly available tools such as RepeatModeler2 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and RepEx [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], but failed owing to their high diversity and short sequence lengths. We then manually investigated and identified a highly conserved 5-nucleotide motif, \u0026ldquo;CAGCT\u0026rdquo;, in the repetitive units. Consequently, we resorted to manual extraction, using \u0026ldquo;CAGCT\u0026rdquo; as a reference point for cutting and aligning multiple sequences from all 20 refined elements. We further corrected and adjusted sequences with minor variations, including \u0026ldquo;CAGCC\u0026rdquo; or \u0026ldquo;CATGT\u0026rdquo;. After 2 or 3 iterations of this process, we extracted 2611 repetitive units from the 20 elements, and generated a single multiple sequence alignment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eUsing the multiple sequence alignment, we used Hidden Markov Models (HMMs) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] to capture repetitive units potentially missed during manual extraction. We built an HMM model using the \u003cem\u003ehmmbuild\u003c/em\u003e function in HMMER (v. 3.3.2) and searched for repetitive units in the human genome using the \u003cem\u003ehmmsearch\u003c/em\u003e function (E-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003eFinally, we identified 2809 repeat units (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) with an average length of 53 bp. Of these, 483 were located in chr20#2, 332 in chr20#5, and 96 in chr20#4. Despite their relatively smaller number, the repeat units in chr20#4 had remarkably high expression levels. Compared with the RepeatMasker data (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), our approach added 8528 bases and removed 2811 bases with errors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), refining the D20S16 profile and establishing a new HMM model for further research.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003e4 Identifying four types of D20S16 on the basis of the variable region\u003c/h3\u003e\n\u003cp\u003eFollowing the extraction of repeat units, we conducted a comprehensive sequence conservation analysis of each element and generated sequence logos (Fig. S5), which revealed the coexistence of conserved and variable sequences. The 5\u0026prime; and 3\u0026prime; regions of all D20S16 repeat units are conserved, with the variable region located in the middle of the units (starting from 25 bp). We categorized them into 4 types on the basis of their sequence length: that characterized by a conserved \u0026ldquo;CATCAG\u0026rdquo; sequence is 47 bp; that by \u0026ldquo;CAA-A\u0026ndash;G\u0026rdquo; is 49 bp; that by \u0026ldquo;CAACACCAG\u0026rdquo; is 50 bp; and that by \u0026ldquo;CAGCAGCACC-G\u0026rdquo; is 53 bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eSpecifically, the 47-bp group comprises 7 elements found on chrs. 2, 3, and 5 (1003 units); the 49-bp group comprises 5 elements from chrs. 8 and 20 (1195 units); the 50-bp group comprises 5 elements across chrs. 2, 3, 5, 8, and 9 (339 units); and the 53-bp group comprises 3 elements, including the prominent chr20#4 (272 units). For each of these sequence groups, phylogenetic trees were constructed to further explore their expression in relation to the variable regions.\u003c/p\u003e \u003cp\u003eOur analysis of the phylogenetic tree of the 53-bp group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) revealed that, with a few exceptions, the units of chr20#4 clustered together, suggesting that this region is a result of tandem repeat duplication that might have increased the unit numbers during evolution.\u003c/p\u003e \u003cp\u003eIn addition, we examined the relationship between expression levels and sequence similarity. Intriguingly, despite high sequence homogeneity, expression was exclusive to chr20#4 (gray bars in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This pattern was also observed in the 50-bp group containing chr20#5 (Fig. S6). These findings prompted us to reassess the mechanisms governing D20S16 expression. From our observations, expression seems to be more intricately associated with specific chromosomal locations than with sequence variation. However, the precise mechanisms and functions warrant further investigation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003e5 Specific Expression of D20S16 in Human Embryonic Development\u003c/h3\u003e\n\u003cp\u003eTo broaden our understanding of the role of satellite DNA in embryonic development, particularly D20S16, we explored its expression patterns during macaque embryonic development. We selected rheMac10 (Rhe10) as the reference macaque genome [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and used the associated RNA-Seq dataset (SRA ID: SRP089891) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] for analysis, which includes single oocytes/embryos or multiple biological replicates at each developmental stage. In addition, it pools samples of 5 to 23 oocytes or embryos collected from 1 to 10 female macaques at each developmental stage.\u003c/p\u003e \u003cp\u003eWe compared the expression patterns of D20S16 between humans and macaques during embryonic development. In contrast to the high expression levels observed in humans, D20S16 expression was barely detectable in macaque early development (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Even during the cleavage stages (2C-8C), where D20S16 is strongly expressed in humans, its expression remained minimal in macaques (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). This clear difference highlights the pronounced disparity in D20S16 expression between the two species.\u003c/p\u003e \u003cp\u003eTo investigate whether these differences in expression are related to variations in the D20S16 sequence, we compared the genomic regions containing D20S16 in humans and macaques. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, the 50 Mb region of the human chromosome 20 contains five D20S16 elements, of which two were transcriptionally active. By using BLAST [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], we identified a syntenic region in the macaque genome at 83 Mb on chromosome 10, corresponding to this locus in the human genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). We then precisely aligned the five D20S16 elements between the two genome sequences. As a result, we found that the macaque genome also contains five elements; however, in all cases, their lengths are shorter than those in the human elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Notably, the #4 element, which is highly expressed in humans, was only half the length in macaques. This length difference indicates that the number of repeat units in macaques is lower than in humans, potentially contributing to the observed differences in expression levels.\u003c/p\u003e \u003cp\u003eBy applying the same workflow, we extracted the consensus sequence from the corresponding region in Rhe10 and found that the variable region of chr10 #4 shares the sequence \u0026lsquo;CAGCAGCACC-G\u0026rsquo; with chr20 #4 in CHM13 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Sequence alignment between chr20 #4 in CHM13 and chr10 #4 in Rhe10 revealed gaps (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), suggesting that either humans gained additional repeat units or macaques lost them during evolution.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study explored the expression of satellite DNAs throughout human embryonic development, providing insights into potential regulatory roles during embryonic development. Notably, the behaviour of D20S16, GSATII, and TAR1 may indicate that they play significant roles in early developmental stages, possibly orchestrating key transitions and gene expression profiles. Similarly, the observed expression patterns of ACRO1 and BSR/Beta might align with their potential roles in critical phases of embryonic development, such as cell division and differentiation processes. The transient expression peaks of ALR/Alpha and (GAATG)\u003cem\u003en\u003c/em\u003e could be indicative of their importance during specific windows of embryonic development, although the direct correlation to specific biological functions remains speculative.\u003c/p\u003e \u003cp\u003eAmong these, D20S16 emerged as particularly noteworthy owing to its pronounced expression levels and distinct trend, driving a comprehensive investigation into its copy number, chromosomal positioning, and consensus sequences. This analysis enhanced our understanding of satellite DNA and introduced a new perspective: that chromosomal positioning might contribute to the regulatory expression of satellite DNA. Moreover, the elevated expression of D20S16 in pathological contexts, such as breast cancer [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], highlights its broader biological relevance and potential as a target for therapeutic strategies.\u003c/p\u003e \u003cp\u003eWhile previous studies [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] focused on expression analysis, our research also examined the genomic regions of individual elements where this expression occurs. By using a combination of manual curation and HMM methodologies, we constructed HMM profiles for D20S16 subtypes. This allowed us to identify the variable and conserved regions within D20S16. Although this lays a solid foundation for further research, we must acknowledge that the subjectivity inherent in manual extraction and the assumptions underlying the HMM may introduce unconfirmed variations.\u003c/p\u003e \u003cp\u003eNotably, our study did not find a D20S16 expression pattern in the macaque model analogous to that in humans. While this reinforces the hypothesis that D20S16 exhibits a unique expression pattern in human embryonic development, it also highlights a significant limitation of our research, namely the incompleteness of the macaque genome data. Future research should consider gathering more comprehensive macaque genome data, akin to the T2T-CHM13, or use genome data from other closely related species to better understand the evolutionary and functional differences of satellite DNA in primates.\u003c/p\u003e \u003cp\u003eFuture research should focus on the experimental validation of the function of D20S16, mainly through the precise manipulation of its expression in model organisms by using gene editing technologies, such as the CRISPR-Cas9 system. This could reveal its specific effect on embryonic development. If feasible, creating transgenic mice carrying the human D20S16 sequence through gene knock-in experiments could provide insights into its role throughout embryonic development. In addition, analysing the dynamic expression of D20S16 by using real-time quantitative PCR (qRT-PCR) to obtain quantitative expression data at different developmental stages would be beneficial. Future studies should investigate the interactions between D20S16, other genes, and signalling pathways during embryogenesis, as well as its specific roles in cell division and differentiation.\u003c/p\u003e \u003cp\u003eIn summary, our study offers new insights into the expression patterns of D20S16 in human embryonic development. These findings could pave the way for new fields of research into the function of satellite DNA and potentially provide new understandings and therapeutic strategies for diseases related to embryonic development.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study has enhanced our understanding of the function and expression patterns of the D20S16 satellite DNA during human embryonic development. Our analyses indicate that D20S16 exhibits a distinct pattern of high expression in the early stages of embryogenesis and suggest that its expression may be influenced by its specific chromosomal location. These insights are significant for understanding the gene regulatory networks during early embryonic development.\u003c/p\u003e \u003cp\u003eTo address the challenges in the analysis of satellite DNA sequences, this study used a novel approach combining manual curation with the HMM to identify distinct types of D20S16. This method has not only increased the accuracy of sequence identification, but will also aid in more precisely revealing the function of D20S16 in future model organism research. Despite challenges posed by the incomplete macaque genome data, our work indicates the potential of D20S16 as a satellite DNA with high expression specificity in humans.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCollecting transcriptome and genome data\u003c/h2\u003e \u003cp\u003eWe used the fasterq-dump v. 3.0.3 tool to retrieve RNA-sequencing (RNA-Seq) data derived from 658 cells and 667 developing human embryos (GEO Accession GSE85632) in FASTQ file format from the Sequence Read Archive (SRA) database, and to retrieve RNA-Seq data of human (GSE71318) and macaque (GSE86938) supplementary cells of developing embryos. We used the T2T-CHM13 v. 1.1 human genome reference and the rhcMac10 macaque genome reference and their corresponding gene annotations in GTF format from the UCSC Genome Browser (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://genome.ucsc.edu/\u003c/span\u003e\u003cspan address=\"https://genome.ucsc.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Sequence information for satellite DNA was sourced from the RepeatMasker database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.repeatmasker.org/\u003c/span\u003e\u003cspan address=\"https://www.repeatmasker.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eProfiling Expression of Satellite DNA\u003c/h2\u003e \u003cp\u003eFor read mapping, we first pre-processed the data in FastQC v. 0.11.9 to perform quality analysis with the command \u0026lsquo;\u003cem\u003efastqc -t 2 -q\u003c/em\u003e\u0026rsquo;. We then used trim_galore v. 0.6.7 (\u0026lsquo;\u003cem\u003etrim_galore -q 20 --nextera --paired\u003c/em\u003e\u0026rsquo;) to remove base sequences with quality scores\u0026thinsp;\u0026lt;\u0026thinsp;20 and to trim Nextera primers. To remove rRNA reads, we processed the dataset in rRNAdust v. 1.06. The paired fastq files were then concatenated in seqkit [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] v. 2.2.0 (\u0026lsquo;\u003cem\u003eseqkit pair \u0026minus;\u0026thinsp;1 file1 -2 file2\u003c/em\u003e\u0026rsquo;). Index generation and read mapping were conducted in STAR [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] v. 2.7.10a. The resulting read mappings were then normalized to the volume of RNA-Seq data in the dataset, and counts per million (CPM) values were computed across all samples. Finally, to visually represent the expression of satellite DNA during embryonic development, heatmaps and line graphs were generated and presented in R software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMerging D20S16 Copies\u003c/h2\u003e \u003cp\u003eOwing to the sequence similarity between MLT2B4 and D20S16, which complicates the identification of D20S16 sequences, we merged copy regions that were separated because of this similarity. First, we measured the distance between adjacent copies on each chromosome, using \u003cem\u003eregular expression\u003c/em\u003e. This process entailed calculating the distance by subtracting the end position of a given copy from the start position of the next copy on the same chromosome. Typically, the distances between proximate copies ranged from 10 to 200 bp. Copies that were separated by more than 1000 bp were considered sufficiently distant to be classified as distinct elements. We used Mafft [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] v. 7.515 for comprehensive sequence comparison and further examination of these regions, and visualized the results in the Integrative Genomics Viewer [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eExtraction of Repeat Unit in D20S16\u003c/h2\u003e \u003cp\u003eUpon acquiring the data from the merged region, we initiated extraction by using a Regex command in \u003cem\u003eawk\u003c/em\u003e to isolate the conserved region \u0026ldquo;CAGCT\u0026rdquo; as the reference locus. Then we used Mafft for multi-sequence alignment (\u003cem\u003emafft --thread 12 --globalpair --maxiterate 1000\u003c/em\u003e) of the segmented regions to scrutinize the initial cutting results. During this phase, any inaccuracies due to non-conservative sequences were identified and rectified. This cycle of cutting, aligning, and correcting was performed two or three times to enhance the precision of the extracted sequences. Through these iterations, a more comprehensive multiple sequence comparison file was compiled. Following this, HMMER [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] v. 3.3.2 was used to construct a model (hmmbuild) and search for sequences similar to the identified unit across the entire genome (hmmsearch). Finally, to visually represent the consensus sequence, we generated sequence logos in weblogo [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], using custom Python scripts. This graphical representation provides a clear and concise visualization of the sequence conservation and variability within the repeat unit of D20S16.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic tree construction\u003c/h2\u003e \u003cp\u003eThe consensus sequence was generated from all units in Mafft for multiple sequence comparison. The specific sequences spanning positions 26\u0026ndash;37 within the consensus sequence were precisely extracted through the use of custom Python scripts. This targeted approach allowed for a focused analysis of the region of interest. Thereafter, the extracted sequences were used to construct a phylogenetic tree in PhyML [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] v. 3.3.20220408.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eRNA-Seq\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;RNA Sequencing\u003c/p\u003e\n\u003cp\u003eHMM\u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Hidden Markov Model\u003c/p\u003e\n\u003cp\u003eCPM\u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Counts Per Million\u003c/p\u003e\n\u003cp\u003eGRCh38\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Genome Reference Consortium Human Build 38\u003c/p\u003e\n\u003cp\u003eT2T-CHM13\u0026nbsp; \u0026nbsp;Telomere-to-Telomere CHM13 (Complete Human Genome)\u003c/p\u003e\n\u003cp\u003eIGV\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Integrative Genomics Viewer\u003c/p\u003e\n\u003cp\u003eLTR\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Long Terminal Repeat\u003c/p\u003e\n\u003cp\u003eERV\u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Endogenous Retrovirus\u003c/p\u003e\n\u003cp\u003eGTF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Gene Transfer Format\u003c/p\u003e\n\u003cp\u003eFASTQ\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;FASTQ (Sequence file format)\u003c/p\u003e\n\u003cp\u003eSRA\u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Sequence Read Archive\u003c/p\u003e\n\u003cp\u003eHMMER\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Hidden Markov Model Software\u003c/p\u003e\n\u003cp\u003eqRT-PCR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Quantitative Real-Time Polymerase Chain Reaction\u003c/p\u003e\n\u003cp\u003eCRISPR-Cas9\u0026nbsp;Gene Editing Technology\u003c/p\u003e\n\u003cp\u003eBLAST \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Basic Local Alignment Search Tool\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eNot applicable\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eHK and HY are the primary authors of this manuscript. They were responsible for the analysis of the RNA-seq data, conducted the bioinformatics analysis, and generated the visual representations and figures. Both authors contributed significantly to the interpretation of the data and drafting of the manuscript. In addition, HK led the experimental design, while HY managed the data processing and statistical analysis.MK provided valuable guidance throughout the project, offering critical feedback on the study's methodology and theoretical framework. He also contributed to the refinement and editing of the manuscript, ensuring clarity and coherence. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe thank the scientists for providing the RNA-seq data and make the the T2T-CHM13 reference sequence available. Their contributions have enabled us to gain insights into the expression patterns of satellite DNA during embryonic development.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eWe are currently making revisions in preparation for the upload. The data availability information will be updated once the upload is successful.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJukam D, Shariati SAM, Skotheim JM. Zygotic Genome Activation in Vertebrates. Dev Cell. 2017;42:316\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang F, Barbacioru C, Nordman E, Bao S, Lee C, Wang X, Tuch BB, Heard E, Lao K, Surani MA. Deterministic and stochastic allele specific gene expression in single mouse blastomeres. PLoS ONE. 2011;6:e21208.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan L, Yang M, Guo H, et al. 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Syst Biol. 2010;59:307\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"RNA-seq, T2T-CHM13, Comparative genomics, Repeat units, HMM","lastPublishedDoi":"10.21203/rs.3.rs-5191409/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5191409/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Satellite DNA plays a crucial role in maintaining chromosomal stability and gene regulation. However, its specific functions during early embryonic development are not fully understood. In this study, we investigate the expression patterns and regulatory mechanisms of D20S16 satellite DNA during critical stages of human embryogenesis by integrating the complete human genome reference (T2T-CHM13) with RNA-seq data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Our comprehensive analysis reveals that D20S16 exhibits high expression levels in early developmental stages, followed by a significant decline as development progresses. We identified 20 distinct D20S16 elements in the human genome and found that only two elements, located on chromosome 20, are transcriptionally active during embryogenesis. Comparative analysis with macaque data further demonstrates that D20S16 is minimally expressed in macaque embryos, which have shorter and fewer repeat units compared to humans.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e These findings suggest that D20S16 plays a unique regulatory role in human embryonic development, with its expression being potentially linked to specific chromosomal locations. This study provides new insights into the role of satellite DNA in early development and sets the foundation for future research into its function and evolutionary significance.\u003c/p\u003e","manuscriptTitle":"Unveiling Unique Expression Patterns of D20S16 Satellite DNA in Human Embryonic Development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-05 08:23:58","doi":"10.21203/rs.3.rs-5191409/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":"836c6690-4fb2-4bbb-8477-5acd1f6a97b2","owner":[],"postedDate":"November 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-16T10:23:13+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-05 08:23:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5191409","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5191409","identity":"rs-5191409","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}