CROSS: A cross-species chromosome substitution platform for dissecting chromosome architecture and activity

CROSS: A cross-species chromosome substitution platform for dissecting chromosome architecture and activity | 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 CROSS: A cross-species chromosome substitution platform for dissecting chromosome architecture and activity Wei Li, Yihuan Mao, Ning Yang, Yulong Zhao, Libin Wang, Kai Xu, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8995396/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The functional integrity of a mammalian chromosome is shaped by its long-term, co-evolution with species-specific nuclear environment. How chromosomes co-adapt with their native environment to define 3D architecture and transcriptional activity remain poorly understood, largely due to a lack of experimental models capable of systematically dissecting this co-evolution relationship. Here, we report a cross-species chromosome substitution (CROSS) method, a robust genomic engineering method that enables the stable, scarless replacement of host chromosomes with evolutionarily divergent orthologs. By integrating microcell-mediated chromosome transfer with CRISPR/Cas9, we imported the intact 158-Mb rat X chromosome into mouse embryonic stem cells, and subsequently achieved targeted substitution of its endogenous mouse counterpart, maintaining stability and integrity. Using this model, we found that rat-specific LINE1 and RatSatRep2 repeats failed to adequately recruit host SETDB1 in mouse cells, leading to localized erosion of H3K9me3 heterochromatin. This further triggered 3D structural remodeling, characterized by the de novo formation of topologically associating domain (TAD) boundaries that aberrantly activated adjacent genes—including Rhox5 , the master regulator of the Rhox cluster—impairing cellular differentiation. Our method provides a powerful chromosome engineering platform for dissecting how genomic sequences and epigenetic mechanisms cooperate in regulating chromosome architecture and function, and for evaluating the structural and functional fidelity of large-scale synthetic or heterologous DNA across species. Biological sciences/Biotechnology Biological sciences/Evolution/Coevolution Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Main The 3D organization and function of mammalian genome are shaped by its long-term co-evolution with the nuclear environment. How this co-evolution dictates chromosome folding and transcriptional regulation, and ultimately governs cellular function is a central question. In simpler eukaryotes, large-scale genomic substitution has recently emerged as a transformative strategy for deciphering these sequence-dependent principles; notably, the integration of billion-year-diverged bacterial genomes into yeast demonstrated that foreign DNA can spontaneously adopt distinct chromatin archetypes even in the absence of host-sequence co-evolution 1 . However, while these "top-down" evolutionary perturbations have redefined our understanding of nuclear compartmentalization in yeast, achieving the stable, scarless replacement of an entire endogenous chromosome remains a formidable challenge in mammalian systems. Current "bottom-up" synthetic approaches, such as the SynNICE system 2 , provide powerful means to study de novo assembled DNA in vivo; yet, recapitulating the full structural complexity of a native mammalian chromosome—characterized by its dense landscape of repetitive elements 3 – 7 and multi-layered 3D architecture 8 – 10 —requires engineering methods capable of handling hundred-megabase-scale templates. To bridge this gap, we developed a robust cross-species chromosome substitution (CROSS) platform. This method employs an "add-then-delete" strategy, integrating microcell-mediated chromosome transfer (MMCT) 11 – 15 with CRISPR/Cas9 16,17 to achieve the high-fidelity replacement of an endogenous chromosome with a heterologous ortholog. We demonstrate the utility of this platform by substituting the mouse X chromosome with its 158-Mb rat ortholog in mouse embryonic stem cells (mESCs). The X chromosome serves as a high-stringency benchmark due to its requirement for chromosome-wide regulatory programs 18 – 20 and its high degree of synteny (gene-order) and gene-content conservation 4 , 18 (Fig. 1 a, b and Extended Data Fig. 1 a) contrasted by rapidly evolving non-coding sequences (Fig. 1 c). Our CROSS platform provided a "living stress test" to evaluate the structural and functional fidelity of large-scale exogenous DNA within a non-native nuclear environment. We showed that the substitution was stable and that the engineered mESCs maintained self-renewal and pluripotency. Beyond replacement, this system functioned as a discovery tool for identifying functional genomic elements sensitive to host regulatory machinery. By using the rat X chromosome as an evolutionary perturbation, we identified specific failures in host-dependent epigenetic recruitment. Species-specific LINE1 and RatSatRep2 repeats failed to adequately recruit the host methyltransferase SETDB1, leading to localized H3K9me3 erosion. This epigenetic incompatibility subsequently triggered local 3D structural remodeling, such as the de novo formation of topologically associating domain (TAD) boundaries, which were linked to the regulation of developmental loci including Rhox5 . By providing a streamlined workflow for whole-chromosome substitution, this study offers a powerful methodology for the functional annotation of non-coding sequences and establishes a critical framework for assessing the performance of large-scale heterologous or synthetic DNA in mammalian cells. Results A robust CROSS platform for scarless, intact, and cross-species chromosome substitution in mammalian cells To achieve X chromosome substitution, a two-step process is required: First, transfer of the rat Chr.X from donor cells (rat ESC-A9 hybrid cells) to recipient cells (male mouse ESCs); Second, loss of endogenous mouse Chr.X (Fig. 1 d). To enable the isolation of chromosome-transferred and subsequent chromosome-substituted clones, distinct selectable markers were introduced into male rat ESCs (hereafter referred to as rESCs) and male mouse ESCs (hereafter referred to as mESCs) by their X chromosomes through CRISPR/Cas9 system 16 , 17 , 21 , 22 (Fig. 1 d). The Chr.X of rESCs was labeled by puromycin resistant gene (Puro R ) and enhanced green fluorescent protein (EGFP) at the downstream of the X-linked housekeeping gene Pgk1 (Fig. 1 d, e and Extended Data Fig. 1 b). To generate donor cells, the above rESCs were fused with A9 cells, yielding rESC-A9 hybrids. For recipient cells, while X-linked Hypoxanthine Phosphoribosyltransferase ( Hprt) gene was simultaneously disrupted via targeted insertion of pEF1α-Thymidine kinase (TK)-DsRed-NeoR cassette into its coding sequence (Fig. 1 d, e and Extended Data Fig. 1 b). By using MMCT 11 , 13 , 14 , rat Chr.X can be randomly transfer from donor cells to recipient cells. mESC clones with rat Chr.X (X tra mESCs) were first isolated based on EGFP fluorescence and subsequently selected using HAT and puromycin (Fig. 1 d). Previous research has demonstrated that exogenous chromosomes exhibit instability within host cells and are susceptible to loss 23 . Under puromycin treatment, clones were capable of maintaining rat Chr.X (Extended Data Fig. 1 c). However, upon drug withdrawal, rat Chr.X underwent rapidly lost. After ten passages, proportion of GFP + cells dropped below 20% (Extended Data Fig. 1 c). Clones were further validated by polymerase chain reaction (PCR) amplification (Extended Data Fig. 1 d, e), combined with karyotypic analysis (Extended Data Fig. 1 f). To derive mESC lines with X chromosome substitution, we next subjected the X tra mESCs to ganciclovir (GCV) selection (Fig. 1 d). This strategy exploited the spontaneous loss of one X chromosome in XX ESCs, thereby enriching for clones that had lost the endogenous mouse Chr.X (which carries the TK negative selection marker) (Fig. 1 d, e). The resulting clones, designated X sub mESCs, were isolated (Fig. 1 f). PCR and karyotyping confirmed that these lines retained the rat Chr.X but lacked the mouse Chr.X (Fig. 1 g, h). The transferred rat Chr.X remained stable during extended culture of Xsub mESCs (Fig. 1 h and Extended Data Fig. 1 c). Clones with possibly intact Chr.X were selected for whole-genome sequencing. Results showed that the mouse Chr.X and rat autosomes were absent, whereas the rat Chr.X was present and intact, indicating successful replacement of the Chr.X (Fig. 1 i and Extended Data Fig. 1 g). X sub mESCs maintained morphology similar to ESCs (Fig. 1 f), expressed ESC marker genes (Fig. 1 j, k), and formed all three germ layers after teratoma differentiation (Fig. 1 l). Collectively, these findings demonstrated the successful construction of a male mouse ESC line harboring only rat Chr.X. High-fidelity conservation of 3D chromatin architecture on the substituted rat X chromosome We next explored the 3D organization of the rat Chr.X in X sub mESCs through high-throughput chromatin conformation capture (Hi-C). Hi-C contact maps indicated that the distal interactions of the rat Chr.X in X sub mESCs were slightly downregulated when compared to rESCs, whereas the proximal interactions were slightly enhanced (Fig. 2 a). Chromatin DNA folds at multiple scales, including compartment, TAD, chromatin loops, to build chromosomes 8 – 10 . Chromatin segregates into two compartments: compartment A (open, gene-active, and broadly euchromatic) and compartment B (closed, gene-inactive, and broadly heterochromatic) 10 . We first assessed Chr.X compartmentalization. The level of compartmentalization in both compartment A and compartment B decreased (Fig. 2 b, c). Next, compartment changes were analyzed by plotting the E1 values, which revealed a bidirectional shift involving both compaction and decompaction (Fig. 2 d-f). This analysis identified a pattern of global weakening, where the most definitive A and B compartments showed significant weaken strength (Fig. 2 d). Furthermore, compartment switching events were observed in specific genomic regions (Fig. 2 e). Correspondingly, 3D remodeling revealed that the chromatin underwent a moderate decompaction, resulting in a disorganized and more open architecture (Fig. 2 g). This was further confirmed by the increased ratio of the surface area (S) to the volume (V) (Fig. 2 h). TAD organization of rat Chr.X was subsequently analyzed. Profiling insulation scores across TAD boundaries (± 50 kb) confirmed that boundaries were characterized by local minima (characteristic troughs), forming distinct troughs (Fig. 2 i). Analysis of boundary dynamics revealed that gained boundaries exhibited a decrease in insulation score, whereas lost boundaries showed an increase; unchanged boundaries remained stable (Fig. 2 i). Representative genomic regions exhibiting both gained and lost TADs were identified (Fig. 2 j). The TAD landscape was largely stable, with 86% of boundaries remaining unchanged, while a small but detectable fraction of boundaries were gained (7.9%) (Fig. 2 k). In summary, the TAD architecture remained predominantly conserved, with a subset of novel TADs emerging. As a control, the 3D structure of Chr.1 in X sub mESCs showed slighter changes when compared to Chr.1 in mouse ESCs (Fig. 2 f, k; and Extended Data Fig. 2 a-f), reinforcing that the observed structural reorganization was more specific to the exogenous Chr.X. Taken together, no drastic alterations were observed in the overall 3D chromatin architecture of rat X chromosome in X sub mESCs. However, reduction in compartment strength, a weakened segregation between compartments A and B, and moderate decompaction were evident on Chr.X. At a finer scale, while the majority of TAD boundaries in Chr.X were unchanged, the emergence of de novo TADs at specific loci was noted. This TAD stability aligns with the established role of CTCF binding sites in defining TAD boundaries and loops 24 – 26 . As mentioned above, previous studies have demonstrated that heterochromatin is the primary factor driving the formation of chromosomal compartments 27 , 28 . Besides, it has been reported that Setdb1 knockdown/knockout resulted in H3K9me3 loss and gained ectopic binding of CTCF in mouse ESCs 29 , suggested a potential role of H3K9me3 in prevent genomic CTCF binding, a core protein for TAD boundary formation. Thus, we extended our analysis to investigate the related epigenetic landscape of Chr.X, including heterochromatic histone modifications, to figure out the mechanisms of alterations in 3D genome organization. Multi-omic characterization of H3K9me3 depletion and its association with local chromatin organization and transcriptional activity We characterized the epigenetic landscapes in X sub mESCs by analyzing multi-omics data from chromatin immunoprecipitation followed by sequencing (ChIP-Seq), Cleavage Under Targets and Tagmentation (CUT&Tag) assay, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) and RNA-Seq (Fig. 3 a and Extended Data Fig. 3 a, b). Global erosion of the constitutive heterochromatin mark H3K9me3—the most pronounced change among all these epigenetic marks—characterized the substituted chromosome, as revealed by ChIP-seq and confirmed by CUT&Tag assay (Fig. 3 a-c; Extended Data Fig. 3 a). Notably, the longest of these H3K9me3 loss regions was a 3.2-Mb region (Extended Data Fig. 3 c), indicating large-scale epigenetic reorganization. Meanwhile, we also observed focal increase in the facultative repressive mark H3K27me3 (Fig. 3 a, b)—a potential compensatory response to the loss of global heterochromatin 30 . Consistent with the observed changes of A-B compartments mentioned above, the active marks H3K4me3 and H3K27ac, along with chromatin accessibility, exhibited concordant and overlapping patterns of focal upregulation (Fig. 3 a, b). In contrast, all analyses showed less pronounced alterations on mouse autosomes (ranging from Chr.1 to Chr.19) in X sub mESCs when compared to autosomes in mouse ESCs (Fig. 3 b and Extended Data Fig. 3 b). Notably, decompacted regions across Chr.X were closely associated with regions exhibiting pronounced loss of H3K9me3 (Fig. 3 d). Similarly, de novo TAD boundaries formed on Chr.X in X sub mESCs were also associated with substantial H3K9me3 loss (Fig. 3 e, f). Multiple megabase-scale regions on Chr.X exhibited substantial H3K9me3 loss (Extended Data Fig. 3 c, d), and displayed changes in chromatin 3D architecture (compartments and TADs) (Extended Data Fig. 3 d). To summarize, our data demonstrated a profound epigenetic remodeling of Chr.X in X sub mESCs when compared to Chr.X in rat ESCs, marked specifically by H3K9me3-dependent heterochromatin loss, which possibly contributed to changes in 3D organization of rat Chr.X. Next, to assess the transcriptional impact of the chromosomal engineering, we performed pairwise correlation analysis of gene expression across samples. Importantly, X-linked gene expression in the X sub mESCs most closely resembled that of the male rat ESCs ( R = 0.911), exhibiting a higher correlation than X sub mESCs vs. male mouse ESCs ( R = 0.759) and mouse ESCs vs. rat ESCs ( R = 0.835) (Fig. 3 g and Extended Data Fig. 4 a). The expression profile of key ESC marker genes was largely maintained (Fig. 1 k). However, our data demonstrated a partial increase in transcriptional activity of rat Chr.X in X sub mESCs (Fig. 3 a, h). About 23% of the Chr.X-linked genes in X sub mESCs were dysregulated (13% upregulated and 10% downregulated) compared to rat ESCs (Fig. 3 h). This high degree of transcriptional conservation indicated that the rat Chr.X largely retained its native gene expression program with moderate disruption following its introduction into the mouse cellular environment. Meanwhile, 95% of the mouse autosome genes maintained their expression in the X sub mESCs (Fig. 3 h and Extended Data Fig. 4 b). And the correlation patterns for autosomal genes were consistent across all sample pairs (Extended Data Fig. 4 b). Then we assessed the X-chromosome-to-autosome (X: A) expression ratio to evaluate X chromosome dosage compensation. The X sub mESCs exhibited an X: A (X rat : A mouse ) ratio of coding genes comparable to male mouse ESCs, which was slightly higher than that of male rat ESCs (Fig. 3 i and Extended Data Fig. 4 c). This establishment of a mouse-like expression balance was mediated by a genome-wide upregulation of the rat Chr.X but not downregulation of autosomal genes, evidenced by an elevated X-derived RNA: total RNA ratio of X sub mESCs compared to rat ESCs (Extended Data Fig. 4 d) and the conservation of autosomal gene transcription mentioned above (Fig. 3 h and Extended Data Fig. 4 b). Besides, analysis of the Chr.X of X sub mESCs identified specific pathway enrichments in differentially expressed genes (DEGs) (Extended Data Fig. 4 e). GO analysis of autosomal DEGs indicated different pathways (Extended Data Fig. 4 e). Overall, while autosomes maintained a high degree of expression conservation, the Chr.X globally conserved its expression pattern but underwent specific upregulation, resetting the X:A ratio to a level comparable to that in male mESCs. To elucidate the mechanism underlying the transcriptional upregulation of rat X-linked genes in the mouse nuclear environment, we analyzed the chromatin landscape across gene bodies (± 3 kb from TSS to TES). We observed a pronounced global reduction of the heterochromatin mark H3K9me3, along with a focal increase in chromatin accessibility and H3K4me3 at transcription start sites (Extended Data Fig. 4 f). Previous study suggested that novel TAD boundaries were related to proximal gene upregulation 31 , 32 . Building on the above finding that the loss of H3K9me3 was associated with the formation of new TAD boundaries (Fig. 3 e, f), we asked whether this structural remodeling contributed to gene upregulation. Importantly, genes located near these de novo TAD boundaries exhibited a significant tendency for transcriptional activation (Fig. 3 j). Thus, our data supported a model in which H3K9me3 erosion facilitates the formation of new chromatin TAD boundaries, which promotes the upregulation of boundary-proximal genes. Taken together, our results delineated the epigenetic fate of the rat Chr.X within a mouse nuclear environment. the chromosome underwent a profound epigenetic remodeling, characterized by a global loss of H3K9me3 and a consequent collapse of its native 3D architecture. While overall gene expression was broadly conserved, it underwent specific upregulation on the Chr.X-linked genes, resetting the X:A ratio. Critically, we established that the formation of new TAD boundaries at sites of H3K9me3 depletion provided a mechanistic link, explaining the upregulation of adjacent genes. The central role of H3K9me3 erosion prompted a question: what leads to the dissolution of this repressive mark in a foreign nuclear environment? Defective SETDB1 recruitment at species-specific repeats as the molecular basis of heterochromatin erosion We first mapped the genomic regions (Fig. 4 a) and their top enriched motifs (Extended Data Fig. 5 a) with reduced H3K9me3 and revealed that H3K9me3 loss were predominantly localized to distal intergenic sequences. This finding, together with the established role of H3K9me3 in silencing repetitive DNA, TE composition within these depleted regions was further examined. Our findings revealed a significant enrichment of specific TE subfamilies, including LINE: LINE1 (L1), Unknown: RatSatRep2, LTR: ERVK, and LTR: ERV1, among the H3K9me3-lost regions on Chr.X (Fig. 4 b). Comparative genomic analysis of repetitive elements revealed that LINEs are highly abundant in both the rat and mouse genomes and are further enriched on their Chr.X (Fig. 4 b, c). Notably, the rat Chr.X exhibits a substantial proportion of rat-specific LINEs (Fig. 4 c). Previous studies have reported the evolutionary arms race between TEs and host defense mechanisms 33 – 38 . Thus, we hypothesized that the rat-specific TE sequences may no longer be efficiently recognized by the mouse heterochromatin machinery. To test this, the evolutionary conservation of those top TE types within H3K9me3-lost regions were analyzed. Importantly, rat-specific TEs constituted the majority of these analyzed TE types, with RatSatRep2 being exclusively rat-specific (100%) (Fig. 4 d). Consistent with this, species-specific LINEs displayed significantly sharper decreases in H3K9me3 levels than conserved LINEs (Fig. 4 e). And the completely species-specific RatSatRep2 elements also exhibited substantial H3K9me3 reduction (Fig. 4 e). Phylogenetic analysis of full-length L1 retrotransposons from mouse and rat further demonstrated species-specific clustering, with distinct clades corresponding to mouse and rat L1 sequences (Fig. 4 f). Within each species, L1s derived from the Chr.X did not form separate clusters but were intermixed with autosomal L1s (Fig. 4 f). Both the total number and the Chr.X-linked count of full-length L1s were higher in the rat genome compared to the mouse genome (Fig. 4 f). Further analysis revealed that those young L1s emerging after rat-mouse divergence showed high H3K9me3 in their native state and suffered from greater H3K9me3 loss in X sub mESCs (Fig. 4 g). This heterochromatin breakdown was accompanied by the aberrant transcriptional activation of the Chr.X-linked TEs (Extended Data Fig. 5 b). This indicated preferential epigenetic erosion of recently evolved repetitive elements. To further investigate the mechanism of this recognition failure, the key H3K9 methyltransferase SET Domain Bifurcated Histone Lysine Methyltransferase 1 (SETDB1) occupancy was examined by ChIP-seq. SETDB1 showed markedly reduced binding at H3K9me3-lost regions in Chr.X of X sub mESCs (Fig. 4 h), despite unchanged expression level of Setdb1 , Suv39h2 , Kdm4a , and Kdm4c (Extended Data Fig. 5 c). Within the H3K9me3-lost region, rat-specific L1s showed sharper decreases in SETDB1 levels than conserved L1s (Fig. 4 i). These results indicated impaired recruitment of the H3K9me3 silencing machinery at these regions. In summary, our data traced the origin of H3K9me3 erosion to an evolutionary incompatibility: the sequence divergence of rat-specific transposable elements compromises their targeting by the mouse heterochromatin machinery. This defect manifests through impaired SETDB1 recruitment, ultimately leading to large-scale heterochromatin loss of the rat X chromosome in a mouse cellular environment. CROSS-mediated discovery of functional TAD boundaries at the Rhox5 locus regulating cellular differentiation Next, the developmental potential of X sub mESCs was assessed using both in vivo and in vitro assays. Although X sub mESCs expressed core ESC marker genes (Fig. 1 j, k) and formed teratomas containing all three germ layers (Fig. 1 l), they exhibited profound defects in differentiation capacity (Fig. 5 a-d and Extended Data Fig. 6a-d). Diploid blastocyst injection and somatic cell nuclear transfer (SCNT) assays showed that X sub mESCs contributed only to pre-implantation embryos but not to post-implantation stages (Fig. 5 a-c and Extended Data Fig. 6a, b). Consistently, X sub -mESC-derived teratomas retained abundant KI67 + undifferentiated cells (Extended Data Fig. 6c), indicating impaired terminal differentiation. In vitro, X sub mESCs formed embryoid bodies (EBs) with delayed silencing of pluripotency genes (Fig. 5 d). Given the in vivo developmental arrest around implantation, we further examined the naive-to-primed transition using an ESCs to epiblast-like cells (EpiLCs) differentiation model in vitro, which revealed a significant delay in the downregulation of naive pluripotency genes (Extended Data Fig. 6d). To further investigate the potential contribution of the aforementioned epigenetic remodeling to the differentiation repair, we focused on genes near de novo TAD boundaries associated with H3K9me3 loss on Chr.X. We ranked these genes by expression level and selected the top 10 candidates (Fig. 5 e) for further functional validation. The majority of them were upregulated in the X sub mESCs compared to controls (Fig. 5 e). Among them, we focused on Rhox5 (reproductive homeobox 5), a gene thought to be related to survival of male germ cell and tumor progress 39 , 40 . This gene was highly expressed in our mouse ESCs but silenced upon differentiation (data not shown). Concomitantly, Rhox5 failed to silence properly during differentiation, maintaining aberrantly high expression in X sub mESC-derived- EpiLCs, teratomas and EBs (Fig. 5 f and Extended Data Fig. 6e). In X sub mESCs, the locus surrounding Rhox5 exhibited substantial epigenetic alterations: a prominent H3K9me3 domain immediately downstream was eroded, coinciding with the formation of a new TAD boundary, a local decrease in CTCF binding, and a gain of H3K4me3 (Fig. 5 g). We found that the newly formed TAD boundaries were enriched with rat-specific TEs, specifically L1VL4a, RatSatRep2, and RNIAP1bLTR (Fig. 5 g). To test whether Rhox5 dysregulation contribute to differentiation delay, we performed knockout (KO) of Rhox5 in X sub mESCs using CRISPR/Cas9 system (Fig. 5 h and Extended Data Fig. 6f). Rhox5 KO of X sub mESCs leaded to larger EBs with improved lineage gene expression and decreased ESC marker gene expression (Fig. 5 i and Extended Data Fig. 6g); improved naïve-to-primed differentiation in vitro (Fig. 5 j); and reduced KI67 + cells after teratoma differentiation (Extended Data Fig. 6h). Importantly, Rhox5 KO enabled development to E6.5 in SCNT assays (Fig. 5 k). However, embryos did not survive to later stages in both SCNT and diploid blastocyst injection assays (Extended Data Fig. 6i and data not shown), indicating that Rhox5 dysregulation was an important, but not the sole, contributor to the developmental failure. The case of Rhox5 demonstrated how the epigenetic instability of the rat Chr.X—initiated by TE-related H3K9me3 loss and 3D remodeling—propagates to disrupt key developmental genes. This connects a chromosome-scale epigenetic incompatibility to an observed developmental arrest, revealing of the important role of a single, X-linked gene during development. Our model thus provides a platform to identify X-linked dosage-sensitive genes involved in developmental regulation and investigate X chromosome dosage compensation evolution. Discussion In this study, we established CROSS as a high-fidelity platform for the intact and scarless substitution of hundred-megabase-scale chromosomes in mammalian cells. By replacing the mouse X chromosome with its rat ortholog, we demonstrated that the exogenous rat Chr.X is stably maintained in mouse ESCs in the absence of mouse Chr.X. The resulting mouse ESCs harboring only rat Chr.X (X sub mESCs) could be stably maintained in culture, expressed key pluripotency markers, and retained the capacity to differentiate into all three germ layers. This system directly tests how a chromosome’s native sequence governs its architecture and function within a heterologous nuclear environment. A key technical challenge in large-scale mammalian genome engineering is maintaining the integrity of the epigenetic and structural landscape. Our findings directly linked the evolutionary divergence of species-specific transposable elements (L1/RatSatRep2) to a failure in recruiting the histone methyltransferase SETDB1, resulting in pervasive H3K9me3 loss. This provides direct experimental support for the model that TEs act as genomic platforms for heterochromatin machinery, whose recognition is shaped by sequence-host co-evolution 41 – 44 . Notably, a recent study reported that synthetic megabase human DNA delivered into mouse embryos also lacked H3K9me3 enrichment 2 . This shared lack of heterochromatin in both cross-species systems suggests that H3K9me3 deposition on foreign DNA is not an automatic outcome of chromatinization and likely depends on co‑evolved, sequence‑specific recognition mechanisms. At the level of 3D organization, the rat X chromosome maintained broadly conserved architecture, consistent with previous reports suggesting the deterministic role of DNA sequence features like LINE/SINE distribution in establishing compartment identity 45 and CTCF-binding cite distribution in TAD boundary formation by CTCF/cohesin-mediated loop extrusion mechanism 24 – 26 . Nevertheless, However, heterochromatin erosion mediated localized structural changes, including reduced compartmentalization and the emergence of de novo TAD boundaries. These alterations were consistent with studies suggesting that Setdb1 knockdown/knockout results in H3K9me3 loss and ectopic binding of CTCF binding, and thereby influence TAD and chromatin loop landscapes 29 , 46 , 47 . Thus, we suggested that beyond the deterministic role of DNA sequence, the co-evolution of DNA and trans acting heterochromatin machinery dynamically shapes chromatin organization. Besides, our system offers a discovery tool to systematically identify functional non-coding elements that regulate epigenetic patterning and 3D genome folding. The characterization of the Rhox5 locus—where H3K9me3 loss and de novo TAD boundary formation correlate with differentiation defects—exemplifies the platform’s capacity to reveal functional dependencies between 3D organization and developmental phenotypes. Unlike traditional knockout screens, the "evolutionary perturbation" approach offered by CROSS allows for the identification of regulatory elements within their native chromosomal context, providing insights into the functional requirements of the non-coding genome. In parallel with very recent progress in high-fidelity chromosome transfer and engineering pipelines 48 , our work addressed a distinct and important question: the functional outcome of cross-species chromosomal replacement. While existing methods enable the physical transfer of intact chromosomes, our model demonstrated that epigenetic compatibility—especially at species-specific repeats—constitutes a newly identified, requirement for stable chromosomal function. In conclusion, the CROSS platform bridges the gap between small-scale gene editing and whole-genome synthesis. It provides a standardized workflow for probing genomic compatibility and offers a critical framework for the functional validation of large-scale synthetic DNA. As the field moves toward the de novo design of mammalian chromosomes, the principles of repeat-mediated epigenetic stability identified here will likely inform the engineering of functional and robust synthetic genomes. Methods Animal care and use All animal experiments were approved by the Institutional Animal Care and Use Committee of the Institute of Zoology, Chinese Academy of Sciences (IOZ, CAS). The C57BL/6, B6D2F1 and CD-1 mice and Dark Agouti (DA) rats were obtained from Beijing Vital River Laboratories. All the mice used were raised in the SPF environment of the Laboratory Animal Center of the IOZ, CAS. ESC derivation and culture Mouse and rat ESCs were derived from embryos at E3.5 and cultured on feeder cells in 2i/LIF medium 49 plus 5% knockout serum replacement. The 2i/LIF medium consists of N2B27 medium (DMEM/F12, Neurobasal, N2, B27, GlutaMAX, β-Mercaptoethanol, 2% Bovine Serum Albumin, 10 mg/mL Insulin, and Penicillin-Streptomycin) supplemented with 1 µM MEK inhibitor PD0325901 (Stemgent), 3 µM GSK3b inhibitor CHIR99021 (Stemgent), and 10 3 units/mL mLIF (Millipore) for mouse ESCs and X sub mESCs, or rLIF (Millipore) for rat ESCs. Culture of other cell types The A9 cells were purchased from the Cell Bank of the Chinese Academy of Sciences, and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. rES-A9 hybrid cells were also cultured in the same medium. Feeder cells, mouse embryonic fibroblasts inactivated by treatment with 10 µg/ml mitomycin C, were cultured in the above medium. Genetic engineering of chromosome donor cells and recipient cells To enable site-specific DNA cleavage and integration, we used the CRISPR-Cas9 system to generate double-strand breaks at designated genomic loci. Single-guide RNAs (sgRNAs) were designed using Cas-Designer ( http://www.rgenome.net/cas-designer/ ) and Cas-OFFinder ( http://www.rgenome.net/cas-offinder/ ). SgRNAs with a score higher than 60 and minimal predicted off-target activity in the genome were selected. Each sgRNA spacer sequences was cloned into a BsaI-digested pUC19-U6-sgRNA vector by ligation of annealed oligonucleotides. SgRNA spacer sequences were listed in Supplementary Table 4. Donor cell lines . To engineer the donor cell line, we inserted an pEF1α-EGFP-PuroR cassette downstream of the Pgk1 gene on the X chromosome of DA rat ESCs (Fig. 1 d, e). The engineered rat ESCs were further fused with A9 cell lines to produce rES-A9 hybrid cells (see Cell fusion section), which served as Chr.X donor cells in MMCT assay. Recipient cell lines . We used a male C57BL/6 mESC line that is competent for diploid aggregation and tetraploid complementation and carries a random ERT2-Cre-ERT2-DsRed integration. On this parental line, we knocked out the Hprt gene in X chromosome and inserted an pEF1α-TK-DsRed-NeoR cassette at the same locus in one step of this ESC line (Fig. 1 d, e). For each targeting experiment, we co-delivered a pCAG-SpCas9-EGFP plasmid, the relevant U6-sgRNA plasmid, and the appropriate homology-directed repair (HDR) donor plasmid. The transfection was carried out using the electroporation transfection instrument Neon transfection system (Invitrogen). 48–72 hours after electroporation, GFP- or DsRed-positive cells were sorted by flow cytometry, respectively. Following drug selection, single-cell clones were isolated, expanded, and genotyped to confirm correct modifications. Cell fusion Cell fusion was performed using Polyethylene Glycol 1500 (PEG 1500, ROCHE). The A cells and B cells to be fused were digested, terminated, and washed with PBS. Then, the cells were resuspended in PBS and counted. In brief, taking 10 8 rat ESCs and 2 × 10 7 A9 cells as an example. The mixed cell suspension was centrifuged: at a speed of 1000–1500 rpm for 2–3 minutes. Remove the supernatant completely. Place the centrifuge tube in a 37 °C water bath to maintain the fusion process at 37 °C. Slowly and continuously add 1 mL of 50% PEG 1500 solution preheated to 37 °C to the centrifuge tube within 1 minute, and continue to stir. Continue stirring the cells for 1–2 minutes. Slowly and continuously add 1 mL of preheated KnockOut™ DMEM (Gibco) medium at 37 °C to the centrifuge tube and continue to stir. Add 3 mL of preheated KnockOut™ DMEM medium at 37 °C slowly and continuously, and continue to stir. Slowly add 10 mL of preheated KnockOut™ DMEM medium at 37 °C, and the cell suspension will be incubated at 37 °C for 5 minutes. Centrifuge, discard the supernatant. Resuspend the cell clumps in fresh culture medium, and after 24 hours of culture, add the drug sieve culture medium. Microcell-mediated chromosome transfer (MMCT) The MMCT procedures were primarily adapted from previously established protocols 12 , 50 with modifications. In brief, we first screened several candidate cell lines (rat ESC-A9 hybrids harboring rat Chr.X) for micronucleation efficiency and selected efficient donors for subsequent MMCT experiments. Donor cell lines were treated with 0.04–0.05 µg/mL colcemid for 48 h to form micronuclei. After trypsinization and washing, cells were resuspended in KnockOut™ DMEM containing 10 µg/mL cytochalasin B and subjected to density gradient centrifugation in Percoll: KnockOut™ DMEM (1: 1) at 16,000 × g for 60–75 min. The microcell enriched fraction was collected, washed, and sequentially filtered through 8 µm and 5 µm membranes to isolate microcells containing one to several chromosomes. After additional washing and counting, purified microcells were fused with recipient cells using PEG 1500 as described in the Cell Fusion section. Screening and identification of chromosome-transfer cell lines (X tra mESC lines) 48 hours after MMCT, cells dual positive for GFP and RFP were enriched by fluorescence activated cell sorting (FACS). Puromycin and HAT were then added to the culture medium for selection. After 1–2 weeks, the clones were picked and expanded. Genomic DNA was extracted from each single clone cell line, and successful transfer of the rat X chromosome and the absence of contaminating other chromosomes (including rat autosomes) were verified by genotyping, karyotyping, and whole-genome sequencing. The primer sequences used for genotyping were listed in Supplementary Tables 1 and 3. Screening and identification of chromosome-substituted cell lines (X sub mESC lines) To obtain subclones that had lost the endogenous mouse X chromosome (and its linked HSV-TK negative selection marker) from the established X tra mESC lines, cells were treated with ganciclovir (GCV). GCV-resistant clones were picked and expanded. Initial genotypic screening for loss of the mouse X chromosome was conducted using locus-species PCR (primers listed in Supplementary Table 2). Finally, putative positive clones were then subjected to karyotype analysis, and the complete replacement of the mouse X chromosome by the rat X chromosome was confirmed by whole-genome sequencing. Karyotyping analysis Treat the cells with 0.05 µg/mL colcemid for 2 to 6 hours. After trypsin digestion, wash the cells with phosphate buffered saline (PBS). Resuspend the cells in 0.075 M KCl hypotonic solution and incubate at 37°C for 30 minutes. Then, fix the cells on a 3:1 (volume ratio) methanol-acetic acid fixative at 4°C for 30 minutes. The cell suspension is dropped onto a pre-cooled glass slide under the influence of gravity. The cells were stained using Giemsa Stain. Immunofluorescence staining Immunofluorescence staining was performed as previously described 51 .Fix the cells with 4% paraformaldehyde for 10 minutes, then permeabilize and block the cells with 0.5% Triton X-100 containing 2% BSA for 1 hour. Add the primary antibodies at 4°C for overnight incubation. Incubate with the secondary antibody at room temperature for 1 hour. DNA was stained with Hoechst 33342 for 10 minutes. Imaging was performed using a two-photon confocal microscope (Leica, TCS Sp8). Antibodies: anti-OCT4 (ab19857, Abcam); anti-NANOG (ab80892, Abcam); anti-SSEA1 (ab16285, Abcam); anti-KI67 (ab16667, Abcam). Formation of teratomas Teratoma assay was performed as previously published 51 . Briefly, 0.05% pancreatic trypsin was used to digest and collect ESCs. Then 10 7 cells were then subcutaneously injected into both sides of the abdomen of SCID mice. After 21 days or one month, the tumors were excised and subjected to histological analysis through fixation and H&E staining or immunofluorescence staining. Formation of Embryoid bodies (EBs) First, the ESCs are digested into single cells using 0.05% pancreatic trypsin. Subsequently, the ESCs were suspended and cultured in the N2B27 medium. After approximately 8 days, the differentiated EB spheres were picked out. Oocyte collection Oocyte collection was performed as previously described 52 . Eight-week-old female mice were super-ovulated by consecutive injection of 7.5 IU human chorionic gonadotropin (hCG) and 7.5 IU pregnant mare's serum gonadotropin (PMSG). MII oocytes were harvested from ovarian oviduct 13–15 hours after the injections. Based on the instructions, derived oocytes were cultured in M16 medium after being washed with M2 medium. Hyaluronidase was used to remove the cumulus cells. Oocytes were then cultured in M2 medium at 37°C with 5% CO 2 . Somatic cell nuclear transfer (SCNT) SCNT was conducted follow protocols of the published article 53 . In brief, inject ESCs into enucleated oocytes to form embryos. The embryos are incubated in M16 medium for 1 hour, then activated in the CZB medium containing 5 mg/mL Cytochalasin B (Abcam) and 10 mM SrCl 2 (Sigma) for 5 to 6 hours. Subsequently, the embryos are transferred to M16 medium and cultured for 18 to 22 hours at 5% CO 2 and 37°C. Then they are transferred to the KSOM solution and cultured for 3.5 days at 5% CO 2 and 37°C. Diploid blastocyst injection This assay was carried out following the published article 54 .3.5 days after the mating of superovulated female CD-1 mice with male CD-1 mice, blastocysts were taken from their uteri. The ESCs were digested with trypsin and 12 to 15 cells were microinjected into each blastocyst. After culturing for 1 to 4 hours, the treated embryos were transferred to the oviducts of pseudo-pregnant CD-1 mice at 0.5 days post-conception. Identification of the chimeras were based on GFP and coat color. Quantitative real-time PCR Total RNA was extracted using TRIzol® Reagent (Life Technologies) and further purified with the PureLink® RNA Mini Kit (Invitrogen). Genomic DNA was removed by treatment with DNase I (TIANGEN). Subsequently, the RNA was reverse-transcribed into cDNA using the ReverTra Ace® qPCR RT Master Mix with gDNA Remover Kit. The real-time fluorescent quantitative PCR system contained SYBR qPCR Mix and 50 ⋅ ROX reference dye. The fluorescence quantitative PCR experiment was performed using the QuantStudio 6 Pro Real-Time PCR System (Thermo Fisher Scientific). The reference gene was Gapdh . The primer sequences used were listed in Supplementary Table 5. CRISPR/Cas9-mediated deletion of Rhox5 The design and construction of CRISPR/Cas9 system was described in the above Genetic engineering of chromosome donor cells and recipient cells section. Briefly, we co-delivered a pCAG-SpCas9-EGFP plasmid and the relevant U6-sgRNA plasmid into X sub mESCs to knockout Rhox5 gene. SgRNA spacer sequences were listed in Supplementary Table 4. Reference Genome Construction Reference genomes for Mus musculus (mm39) and Rattus norvegicus (rn7) were downloaded from the Ensembl database (Release 106). A combined mm39-rn7 reference genome was generated by merging the sequences and annotations from both assemblies to facilitate integrated cross-species analysis. Whole genome sequencing Genomic DNA was extracted using the E.Z.N.A.® MicroElute Genomic DNA Kit (Omega Bio-tek) according to the manufacturer’s instructions. DNA quality was assessed by agarose gel electrophoresis and quantified using a Qubit® 3.0 Fluorometer. Library construction and high-throughput sequencing were performed on Illumina NovaSeq 6000 platform by Annoroad Gene Technology (Beijing) Co., Ltd. Raw reads underwent quality assessment using FastQC (v0.11.5) ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). Adapter sequences and low-quality bases were subsequently trimmed using trim_galore (v0.6.6) ( https://github.com/FelixKrueger/ TrimGalore). The cleaned reads were aligned to the above reference genome (mm39-rn7) using BWA-MEM (v0.7.17) 55 with default parameters, followed by duplicate marking and removal with GATK (v4.1.8) 56 . The resulting BAM files were used for copy number variation (CNV) analysis with SCCNV (v1.0.2) 57 , generating visualizations of genomic segments’ copy number states. Hi-C library construction and data analysis. Hi-C libraries were constructed using standard protocol by Annoroad and were sequenced on Illumina platforms. Hi-C data were processed using HiC-Pro (v3.1.0) 58 to generate raw contact matrices at 10-kb and 100-kb genomic bin resolutions. Systematic biases in the contact matrices were corrected using the ‘hicCorrectMatrix’ function from HiCExplorer (v3.6) 59 . The corrected 100 kb matrix was utilized for global structural assessment. Contact matrix visualization and saddle plots were generated with cooltools (v0.7.1) 60 , and three-dimensional (3D) genome structures were inferred using miniMDS 61 . Chromatin compartment (A/B) analysis was performed using cscoreTool (v1.1) 62 . A compartment switch was defined as a genomic bin where the absolute difference in compartment score between samples exceeded 0.3. Topologically Associating Domains (TADs) were identified at 10-kb resolution by calculating the insulation score with cooltools 60 . Dynamic TAD boundaries were classified as: Gain boundary , a genomic locus identified as a TAD boundary exclusively in the X sub mESC group; Lost boundary , a genomic locus identified as a TAD boundary exclusively in the control (ctrl) group; Maintained boundary , a TAD boundary called in both groups. In all analyses, the ctrl was rat ESCs when assessing the rat X chromosome, and mouse ESCs when assessing autosomes. TADs were visualized using pyGenomeTracks 63 , and insulation scores distribution plot were generated with deepTools 64 . ChIP-seq library preparation Chromatin immunoprecipitation was performed using the SimpleChIP® Plus Sonication Chromatin IP Kit (Cell Signaling Technology, 56383) following the manufacturer’s protocol. Briefly, cells were cross-linked with formaldehyde, lysed, and chromatin was fragmented by sonication. Immunoprecipitation was carried out with target-specific antibodies. Antibodies: anti-H3K9me3 (ab8898, Abcam), anti-H3K27me3 (9733s, Cell Signaling technology), anti-H3K27ac (ab4729, Abcam), anti-H3K4me3 (ab8580, Abcam), anti-CTCF (3418S, Cell Signaling technology), anti-SETDB1 (11231-1-AP, Proteintech). After reversal of cross-links, DNA was purified using the kit’s spin columns. Libraries were constructed and sequenced by Annoroad on Illumina NovaSeq platform. ATAC-seq library preparation ATAC‑seq libraries were prepared by Annoroad using their standard protocol, based on hyperactive Tn5 transposase 65 . In brief, 50,000 viable cells were used for transposase-mediated tagmentation, followed by PCR amplification with indexed primers. ChIP-seq and ATAC-seq data analysis ChIP-seq and ATAC-seq libraries were sequenced on the Illumina NovaSeq platform by Annoroad. ChIP-seq and ATAC-seq datasets were processed through an identical pipeline. Following quality control with FastQC (v0.11.5), adapter sequences were removed using trim_galore (v0.6.6). The processed reads were aligned to the reference genome with Bowtie2 (v2.3.5) 66 under the parameters “--sensitive --end-to-end”, and PCR duplicates were eliminated using a custom script. Read coverage was normalized to Reads Per Kilobase per Million mapped reads (RPKM) using the ‘bamCoverage’ utility from deepTools (v3.5.1) 64 . The bigwig files were visualized using pyGenomeTracks (v3.6) 63 . The mean read count distribution plot was generated using deeptools. Peak calling was executed with MACS2 (v2.1.2) 67 with the parameters “-p 0.01 --nomodel --nolambda”. All peaks were segmented into 1-kb windows using the sliding window method. These regions were categorized as follows: gain regions , peaks present in the X sub mESC group but absent in the control group (ctrl), exhibiting an RPM fold-change (X sub mESC/ctrl) ≥ 4; lost regions , peaks present in the ctrl group but absent in the X sub mESC group, exhibiting an RPM fold-change (ctrl/ X sub mESC) ≥ 4; maintained regions , peaks called in both X sub mESC and ctrl group. In all analyses, the ctrl was rat ESCs when assessing the rat X chromosome, and mouse ESCs when assessing autosomes. Motif enrichment de novo motif discovery within specific peaks was performed using the MEME suite (v5.5.2) 68 . CUT&Tag library preparation and data processing CUT&Tag libraries were prepared using the Hyperactive Universal CUT&Tag Assay Kit for Illumina Pro (Vazyme, TD904) following the manufacturer’s instructions. Cells were bound to ConA beads, permeabilized with digitonin, and incubated with primary antibodies (anti-H3K9me3, ab8898, Abcam) and secondary antibodies. Protein A/G-Tn5 transposase was then targeted to antibody-bound regions for tagmentation. DNA was extracted, amplified with indexed primers, and purified using DNA Clean Beads. Libraries were quantified and sequenced on the Illumina NovaSeq platform. After removing the adapter sequences with trim_galore, CUT&Tag reads were mapped to the reference genome (mm39-rn7) using bowtie2 66 with the parameters “--local --very-sensitive --no-mixed --no-discordant”. The redundant reads were removed using our own script. The mean read count distribution plot was generated using deeptools 64 . RNA-Seq library construction and data analysis Total RNA was extracted as described in the Quantitative real-time PCR section. Libraries were quantified and sequenced on Illumina platforms. Raw RNA-seq reads underwent quality assessment using FastQC. Adapter sequences and low-quality bases were trimmed with trim_galore. The resulting high-quality reads were aligned to the composite mm39-rn7 genome using STAR (v2.7.1a) 69 . Uniquely mapped reads were isolated using a custom script for subsequent quantification. Transcript abundance was estimated with StringTie (v2.0) 70 and normalized to Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Differential gene expression analysis was conducted using the DESeq2 package (v1.38.0) 71 in R. Genes demonstrating an absolute log2 fold change > 1 with p-value < 0.05 were designated as differentially expressed. Functional enrichment analysis for Gene Ontology (GO) terms was performed on these gene sets using the DAVID 72 bioinformatics database. X:A ratio: StringTie 70 -derived gene expression tables were merged into a unified matrix, retaining annotated protein-coding genes on autosomes and the X chromosome; genes in the top 5% of maximal expression were excluded. Two gene sets were used: all expressed protein-coding genes and one-to-one mouse–rat homologous protein-coding genes obtained from Ensembl BioMart 73 . X:A ratio was assessed using both expression-based and read-level approaches. For expression-based analysis, genes with FPKM > 2 were retained, and a bootstrapping strategy 74 was applied to account for the smaller number of X-linked genes: in each iteration, a random autosomal subset equal in size to the X-linked gene set was sampled, and median expression values were compared over 10,000 iterations to derive X:A ratios and confidence intervals. For a gene-annotation–independent estimate, chromosome-wise read counts and lengths were extracted using samtools 75 idxstats, and a length-normalized X:A read-density ratio was computed. Statistical analyses and visualization were performed in R using ggplot2 76 , with group comparisons conducted using the Wilcoxon rank-sum test and results displayed as boxplots with jittered points. Transposable element (TE) expression analysis Genomic annotations for repetitive elements were obtained from the UCSC Genome Browser for the respective mm39 and rn7 assemblies. TE expression was quantified using the SQuIRE 77 software (v0.9.9) under default parameters. Differentially expressed TEs were identified using the same thresholds as for genes: |log 2 FoldChange| > 1 and p-value < 0.05. Evolutionary analysis of mouse and rat TEs The evolutionary ages of TEs were estimated based on RepeatMasker output files acquired from the UCSC Genome Browser for the mouse (mm39) and rat (rn7) genomes. For each repeat element locus, the "milliDiv" value (representing the percentage divergence from the consensus sequence) was extracted directly from the output. The Jukes-Cantor method was applied using species-specific substitution rates: 5.2 × 10 − 9 per base per year for mouse and 5.5 × 10 − 9 per base per year for rat 4 , 78 . The evolutionary age (in years) was then calculated using the formula: age = divergence rate / substitution rate, yielding an estimated age for each repetitive element locus 79 . Phylogenetic analysis of mouse and rat full length LINE1 . Full-length LINE1 sequences (> 6.5 kb) were extracted from the telomere-to-telomere (T2T) mouse genome and the rat genome (RN8) using RepeatMasker (v4.1.1) 80 based on the Repbase repeat database. Sequences were subsequently clustered using CD-HIT (v4.8.1) 81 with the parameters “-d 0 -c 0.8 -aL 0.80” to reduce redundancy and define representative sequences. Multiple sequence alignment of the clustered sequences was performed with MAFFT (v7.475) 82 under default settings. A phylogenetic tree was constructed from the aligned sequences using FastTree (v2.1.11) 83 with default parameters. Finally, the resulting tree was visualized and annotated using the iTOL platform 84 . Statistics and reproducibility Statistical analyses were performed in R using ggplot2 76 or Graphpad Prism. Levels of significance were calculated using the two-tailed Student’s t test. In all figures: *, p value < 0.05; **, p value < 0.01, ***, p value < 0.001; ****, p value < 0.0001. The number of independent biological replicates and statistical approach for each experiment were described in the figure legends or the Methods section. Data availability The sequencing data are available at Genome Sequence Archive (GSA) of China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences ( https://ngdc.cncb.ac.cn/gsa/ ) under accession CRA035509. Previously published raw data used in this paper is available at Gene Expression Omnibus under accessions GSE44150, GSE97966 85 , GSE178701 86 , GSE220805 87 , and GSE90516 88 . Declarations Acknowledgments This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDC0200000 to W.L. and XDA0510400); the National Key Research and Development Program (2022YFA1103600 to L.W. and 2019YFA0903800); the National Natural Science Foundation of China (32225030 and 82488301 to W.L.); the CAS Project for Young Scientists in Basic Research (YSBR-012 to W.L.); the Beijing Natural Science Foundation (Z230011 to W.L.); Initiative Scientific Research Program, Institute of Zoology, Chinese Academy of Sciences (2023IOZ0101). We acknowledge Runze Ma, Xiaohua Shen and Bing Zhu for their helpful suggestions. We thank Lingyun Wei, Youjia Shu, Qing Meng, Peipei Long, Ming Ge, Weiyu Jin, Shiwen Li, Xili Zhu, Hua Qin and Xia Yang for their technical assistance. Author contributions W.L., Q.Z., Y.M. and L.W. conceived and designed the study. 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12:45:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8995396/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8995396/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107066531,"identity":"03a60b04-7f9d-4b2b-b8df-eb68c92364cf","added_by":"auto","created_at":"2026-04-16 11:13:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":357304,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of the CROSS platform for intact chromosome transfer and substitution in mESCs\u003c/strong\u003e. \u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eevolutionary distance\u003cem\u003e \u003c/em\u003e(\u003cem\u003eLeft\u003c/em\u003e) and conserved synteny\u003cem\u003e \u003c/em\u003e(\u003cem\u003eRight\u003c/em\u003e) between rat and mouse genomes. Orthologous chromosomal segments are shown based on mouse chromosomes. Xm, mouse X chromosome (Chr.X); Xr, rat Chr.X. \u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003e c\u003c/strong\u003e, Overlap of protein-coding genes (\u003cstrong\u003eb\u003c/strong\u003e) and transposable elements (\u003cstrong\u003ec\u003c/strong\u003e) between Xm and Xr. \u003cstrong\u003ed\u003c/strong\u003e, Schematic of rat Chr.X replacement in mouse ESCs. Engineered rat Chr.X (with PuroR-EGFP) was transferred via microcells into mESCs bearing edited mouse Chr.X (with TK-DsRed-NeoR). Puro and HAT selection first isolated X-transferred mESCs (X\u003csup\u003etra\u003c/sup\u003e mESCs), then GCV selection isolated X-substituted mESCs (X\u003csup\u003esub\u003c/sup\u003e mESCs) losing mouse Chr.X. \u003cstrong\u003ee\u003c/strong\u003e, Design of CRISPR/Cas9-mediated engineering on rat (\u003cem\u003eleft\u003c/em\u003e) and mouse (\u003cem\u003eright\u003c/em\u003e) Chr.X. \u003cstrong\u003ef\u003c/strong\u003e, Morphology of X\u003csup\u003esub\u003c/sup\u003e mESCs in bright-field, GFP, and RFP channels. Scale bar, 50 μm.\u003cstrong\u003e g\u003c/strong\u003e, Genotyping validation of X\u003csup\u003esub\u003c/sup\u003e mESCs using primers (listed in Supplementary Table 1 and 2) specific across rat and mouse Chr.X. \u003cstrong\u003eh\u003c/strong\u003e, Representative karyotype of X\u003csup\u003esub\u003c/sup\u003e mESCs showing retention of the rat Chr.X and the absence of mouse Chr.X. \u003cstrong\u003ei\u003c/strong\u003e, Whole-genome sequencing analysis of X\u003csup\u003esub\u003c/sup\u003e mESCs (n = 2).\u003cem\u003e \u003c/em\u003e\u003cstrong\u003ej\u003c/strong\u003e, Immunofluorescence of ESC marker proteins in X\u003csup\u003esub\u003c/sup\u003e mESCs. Scale bar, 70 μm. \u003cstrong\u003ek\u003c/strong\u003e, Scatter plot comparing autosomal gene expression between mESCs (n = 2) and X\u003csup\u003esub\u003c/sup\u003e mESCs (n=4). ESC marker genes were highlighted in red. Linear regression line was shown. \u003cem\u003eP\u003c/em\u003e values were calculated by two-sided one-sample \u003cem\u003et\u003c/em\u003e-test. \u003cstrong\u003el\u003c/strong\u003e, Teratomas sections (day 30) derived from X\u003csup\u003esub\u003c/sup\u003e mESCs. Scale bar, 100 μm.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8995396/v1/43d9559739399b6c8c46f11d.png"},{"id":107704983,"identity":"4ad84429-c031-4327-97c2-5e20e6230231","added_by":"auto","created_at":"2026-04-24 09:05:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2057766,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e3D architecture of rat X chromosome in the X\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003esub\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mESCs\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003e\u0026nbsp;\u003cstrong\u003ea\u003c/strong\u003e, Hi-C contact matrices of the Chr.X in rESCs (\u003cem\u003eleft, \u003c/em\u003e2 biological replicates), X\u003csup\u003esub\u003c/sup\u003e mESCs (\u003cem\u003emiddle\u003c/em\u003e,\u003cem\u003e \u003c/em\u003e2 biological replicates), and log\u003csub\u003e2\u003c/sub\u003e(X\u003csup\u003esub\u003c/sup\u003e mESC/rESC) (\u003cem\u003eright\u003c/em\u003e) at 100-kb resolution. The intensity of each pixel reflects the normalized contact frequency between pairs of loci. \u003cstrong\u003eb\u003c/strong\u003e, Saddle plots of Chr.X compartmentalization strength, calculated as the ratio of observed to expected contact frequencies between A and B compartments (i.e., (AA+BB)/(AB+BA)). Regions are grouped by their eigenvector value (active, high; inactive, low). Red indicates enrichment (A-A, B-B interactions), and blue indicates depletion (A-B interactions) of contacts. The bar charts on the left and top indicate the distribution of signal intensity. \u003cstrong\u003ec\u003c/strong\u003e, compartment saddle strength curve of Chr.X, based on (\u003cstrong\u003eb\u003c/strong\u003e). \u003cstrong\u003ed\u003c/strong\u003e, Compartment changes in the Chr.X of X\u003csup\u003esub\u003c/sup\u003e mESCs relative to rESCs. The dashed line marks regions unchanged; the pink curve represented the fitted trend line. \u003cstrong\u003ee\u003c/strong\u003e, Representative genome browser tracks showing compartment switch of Chr.X in X\u003csup\u003esub\u003c/sup\u003e mESCs V.S. rESCs. \u003cstrong\u003ef\u003c/strong\u003e, Compartment changes in rat Chr.X (compared to Chr.X of rESCs) and Chr.1 (compared to Chr.1 of mESCs) in X\u003csup\u003esub\u003c/sup\u003e mESCs. \u003cstrong\u003eg\u003c/strong\u003e, 3D structural models of the Chr.X. Blue and pink denote compartments B and A, respectively.\u003cstrong\u003e h\u003c/strong\u003e, Surface Area: Volume (S: V) ratio of Chr.X in rESCs and X\u003csup\u003esub\u003c/sup\u003e mESCs. \u003cstrong\u003ei\u003c/strong\u003e, Alterations in topologically associating domains (TADs) of the X chromosome in X\u003csup\u003esub\u003c/sup\u003e mESCs. Insulation score of TAD boundaries, including gained, lost, unchanged in X\u003csup\u003esub\u003c/sup\u003e mESCs compared to rESCs. \u003cstrong\u003ej\u003c/strong\u003e, A genome browser screenshot shows altered TADs across 65.5-67 Mb on Chr.X. \u003cstrong\u003ek\u003c/strong\u003e, Changes in TAD boundaries of the rat Chr.X (compared to Chr.X of rESCs) and Chr.1 (compared to Chr.1 of mESCs) in X\u003csup\u003esub\u003c/sup\u003e mESCs. rES, male rat ESCs; mES, male mouse ESCs\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8995396/v1/c640bb43620a0350ec1d167c.png"},{"id":107066534,"identity":"9ba7f34c-75c3-4faa-badb-5974fcacaee8","added_by":"auto","created_at":"2026-04-16 11:13:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1170848,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMulti-omic characterization of localized epigenetic and structural divergence on the substituted chromosome\u003c/strong\u003e. \u003cstrong\u003ea\u003c/strong\u003e, Genome browser tracks showing enrichment profiles of histone marks, accessibility (ATAC-seq), and transcription (RNA-seq) on Chr.X in rat ESCs and X\u003csup\u003esub\u003c/sup\u003e mESCs. \u003cstrong\u003eb\u003c/strong\u003e, Dynamic of related signals on the rat Chr.X (compared to Chr.X of rESCs) and autosomes (compared to autosomes of mESCs) in X\u003csup\u003esub\u003c/sup\u003e mESCs. \u003cstrong\u003ec\u003c/strong\u003e, Violin plots comparing the H3K9me3 signal intensities across the entire Chr.X in rat ESCs and X\u003csup\u003esub\u003c/sup\u003e mESCs (n = 2 biologically independent samples per group). Dashed line indicates the median value. \u003cstrong\u003ed\u003c/strong\u003e, Correlation between H3K9me3 fold change and chromatin compartment changes of the Chr.X in X\u003csup\u003esub\u003c/sup\u003e mESCs. \u003cstrong\u003ee\u003c/strong\u003e, H3K9me3 fold change in regions near gained, lost, unchanged TAD boundaries in Chr.X of X\u003csup\u003esub\u003c/sup\u003e mESCs, respectively. \u003cstrong\u003ef\u003c/strong\u003e, Correlation between H3K9me3 fold change and TAD boundary changes of the Chr.X of. X\u003csup\u003esub\u003c/sup\u003e mESCs. \u003cstrong\u003eg\u003c/strong\u003e, Pairwise correlation of Chr.X-linked gene expression across X\u003csup\u003esub\u003c/sup\u003e mESCs (n=4) vs. rat ESCs (n=2).\u003cstrong\u003e h\u003c/strong\u003e, Statistical analysis of gene expression changes on the Chr.X (compared to rat ESCs) and autosomes (compared to mESCs) in X\u003csup\u003esub\u003c/sup\u003e mESCs, respectively. Pink, blue, gray indicated up-regulated, down-regulated and unchanged genes, respectively. \u003cstrong\u003ei\u003c/strong\u003e, Box plot showing the X-to-autosome (X: A) expression ratio of coding genes in X\u003csup\u003esub\u003c/sup\u003e mESCs (n=4), male mouse ESCs (n=13), and male rat ESCs (n=15). Data for X\u003csup\u003esub\u003c/sup\u003e mESCs were from this study, while mouse and rat ESCs were combined from this study and public datasets (GSE44150, GSE97966, GSE178701, GSE220805, and GSE90516). \u003cem\u003eP\u003c/em\u003e values were calculated using Wilcoxon rank-sum test. \u003cstrong\u003ej\u003c/strong\u003e, Correlation between TAD boundary changes and nearby gene expression on Chr.X of X\u003csup\u003esub\u003c/sup\u003e mESCs. 2 biological replicates of each group for \u003cstrong\u003ea-j\u003c/strong\u003e, except \u003cstrong\u003eg\u003c/strong\u003e and \u003cstrong\u003ei\u003c/strong\u003e. Statistical significance was assessed using an unpaired two-sided \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8995396/v1/9aecad9fce3def88a4b316d9.png"},{"id":107480649,"identity":"5e67fdbd-46c0-445a-a1a3-0c235830472d","added_by":"auto","created_at":"2026-04-22 02:12:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1174191,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular basis of heterochromatin erosion linked to defective SETDB1 recruitment at species-specific repeats\u003c/strong\u003e. \u003cstrong\u003ea\u003c/strong\u003e, Pie chart showing the genomic distribution of H3K9me3-lost regions on the Chr.X in X\u003csup\u003esub\u003c/sup\u003e mESCs. \u003cstrong\u003eb\u003c/strong\u003e, Bar chart showing the proportion of repeat types within H3K9me3-lost regions on the Chr.X in X\u003csup\u003esub\u003c/sup\u003e mESCs and within all repeats across Chr.X. LTR, long terminal repeat; SINE, short interspersed nuclear element; LINE, long interspersed nuclear element; DNA, DNA transposon; Others, other types of repeats. \u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eThe comparative analysis of transposable elements (TEs) composition in the whole genome and the Chr.X of mouse and rat. The proportion of rat-specific TE was highlighted. \u003cstrong\u003ed\u003c/strong\u003e, Conservation analysis of top repeat types suffering from H3K9me3 loss on the Chr.X. \u003cstrong\u003ee\u003c/strong\u003e, Fold change of H3K9me3 peak intensity (X\u003csup\u003esub\u003c/sup\u003e mESCs vs rat ESCs) between conserved and rat-specific LINE1s (L1s)\u003cem\u003e,\u003c/em\u003e and rat-specific RatSatRep2 on the Chr.X.\u003cstrong\u003e f\u003c/strong\u003e, Phylogenetic analysis of full-length L1s in rat and mouse. Colors denoted genomic origin: grey, mouse or rat autosomes; pink, mouse Chr.X; green, rat Chr.X. Branch labels indicated L1 subfamily and copy number (n). Scale bar, 0.1 substitutions per site. Summary counts of full-length L1s per genomic compartment were shown. \u003cstrong\u003eg\u003c/strong\u003e, Fold change of H3K9me3 on X-linked L1 subfamilies, plotted against their evolutionary age (million year ago, MYA). Dashed line marked rat-mouse divergence. Point sizes indicated basal H3K9me3 level in rat ESCs. \u003cstrong\u003eh\u003c/strong\u003e, SETDB1 binding at sites of gained, lost, and unchanged H3K9me3 on Chr.X of X\u003csup\u003esub\u003c/sup\u003e mESCs (n = 2) and rESCs (n = 2), respectively. \u003cstrong\u003ei\u003c/strong\u003e, Fold change of SETDB1 (X\u003csup\u003esub\u003c/sup\u003e mESCs vs rat ESCs) between conserved and rat-specific L1s on the H3K9me3-lost region of Chr.X. Statistical significance was assessed by unpaired two-sided \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8995396/v1/ff200ef356ba7127bdeafe9d.png"},{"id":107480710,"identity":"281a3dbd-a465-4777-bc20-0d3364f2f491","added_by":"auto","created_at":"2026-04-22 02:13:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5284863,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCROSS-mediated discovery of functionally active TAD boundaries at the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eRhox5\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e locus linked to cellular differentiation\u003c/strong\u003e. \u003cstrong\u003ea\u003c/strong\u003e, SCNT embryos of X\u003csup\u003esub\u003c/sup\u003e mESCs across several developmental stages. Scale bar, 100 μm. \u003cstrong\u003eb\u003c/strong\u003e, SCNT embryos derived from X\u003csup\u003esub\u003c/sup\u003e mESCs at E4.5 (embryonic day 4.5). Scale bar, 70 μm. \u003cstrong\u003ec\u003c/strong\u003e, X\u003csup\u003esub\u003c/sup\u003e-mESC (GFP\u003csup\u003e+ \u003c/sup\u003ecells)-containing chimeric embryos at E4.5 after diploid blastocyst injection. Scale bar, 100 μm. \u003cstrong\u003ed\u003c/strong\u003e, Expression of ESC maker genes in mESC- and X\u003csup\u003esub\u003c/sup\u003e mESC-derived EBs. \u003cstrong\u003ee\u003c/strong\u003e, Heatmap showing expression of the top 10 most highly expressed genes near those de novo TAD boundaries on the Chr.X in X\u003csup\u003esub\u003c/sup\u003e mESCs. \u003cstrong\u003ef\u003c/strong\u003e, Volcano plots comparing X-linked gene expression. The \u003cem\u003eleft\u003c/em\u003e and \u003cem\u003eright\u003c/em\u003e panels show differential expression in EpiLCs and teratomas, respectively, derived from mESCs versus X\u003csup\u003esub\u003c/sup\u003e mESCs. n = 2 for each group and P value calculated by \u003cem\u003eWald \u003c/em\u003etest. The \u003cem\u003eRhox5\u003c/em\u003e was labeled. \u003cstrong\u003eg\u003c/strong\u003e, Changes in TAD, epigenetic marks and transcription around the \u003cem\u003eRhox5\u003c/em\u003e locus of X\u003csup\u003esub\u003c/sup\u003e mESCs, alongside gene and rat-specific TE annotations.\u003cstrong\u003e \u003c/strong\u003eTwo biological replicates used for each group, except in the RNA-seq where X\u003csup\u003esub\u003c/sup\u003e mESCs had four replicates\u003cstrong\u003e. h\u003c/strong\u003e, Comparing \u003cem\u003eRhox5\u003c/em\u003e mRNA expression between X\u003csup\u003esub\u003c/sup\u003e mESCs and \u003cem\u003eRhox5\u003c/em\u003e-knockout X\u003csup\u003esub\u003c/sup\u003e mESCs in RT-qPCR assay.\u003cstrong\u003e i\u003c/strong\u003e, Expression of related genes in X\u003csup\u003esub\u003c/sup\u003e-mESC-derived EBs and \u003cem\u003eRhox5\u003c/em\u003e-knockout-X\u003csup\u003esub\u003c/sup\u003e-mESC-derived EBs. \u003cstrong\u003ej\u003c/strong\u003e, Temporal expression dynamics of naïve pluripotency genes in EpiLCs derived from X\u003csup\u003esub\u003c/sup\u003e mESCs and \u003cem\u003eRhox5\u003c/em\u003e-knockout X\u003csup\u003esub\u003c/sup\u003e mESCs, respectively. \u003cstrong\u003ek\u003c/strong\u003e, Developmental analysis of SCNT embryos derived from \u003cem\u003eRhox5\u003c/em\u003e-knockout X\u003csup\u003esub\u003c/sup\u003e mESCs at E6.5. Scale bar, 100 μm. EB, embryoid body; SCNT, somatic cell nuclear transfer; EpiLC, epiblast-like cells.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8995396/v1/50c88e7e73079bd876379ae3.png"},{"id":107708708,"identity":"2227b66c-e24f-46a6-aa60-5084c4ba1ae2","added_by":"auto","created_at":"2026-04-24 09:31:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10424459,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8995396/v1/f09b8a6d-3b5b-46f9-8e47-35ded9825acb.pdf"},{"id":107480865,"identity":"b400cee8-28a5-4d4c-9156-38475955e2e3","added_by":"auto","created_at":"2026-04-22 02:14:01","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":192944,"visible":true,"origin":"","legend":"supplementary tables","description":"","filename":"Supplementarytables.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8995396/v1/3f08e270ba8e0f0d9fd63786.pdf"},{"id":107066533,"identity":"e5a3494b-e186-46db-a4a5-12e7e8ddf719","added_by":"auto","created_at":"2026-04-16 11:13:28","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":929380,"visible":true,"origin":"","legend":"","description":"","filename":"Extendeddatafigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8995396/v1/0cab0b13a939326be3d53027.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"CROSS: A cross-species chromosome substitution platform for dissecting chromosome architecture and activity","fulltext":[{"header":"Main","content":"\u003cp\u003eThe 3D organization and function of mammalian genome are shaped by its long-term co-evolution with the nuclear environment. How this co-evolution dictates chromosome folding and transcriptional regulation, and ultimately governs cellular function is a central question. In simpler eukaryotes, large-scale genomic substitution has recently emerged as a transformative strategy for deciphering these sequence-dependent principles; notably, the integration of billion-year-diverged bacterial genomes into yeast demonstrated that foreign DNA can spontaneously adopt distinct chromatin archetypes even in the absence of host-sequence co-evolution\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. However, while these \"top-down\" evolutionary perturbations have redefined our understanding of nuclear compartmentalization in yeast, achieving the stable, scarless replacement of an entire endogenous chromosome remains a formidable challenge in mammalian systems. Current \"bottom-up\" synthetic approaches, such as the SynNICE system\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, provide powerful means to study de novo assembled DNA in vivo; yet, recapitulating the full structural complexity of a native mammalian chromosome\u0026mdash;characterized by its dense landscape of repetitive elements\u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e and multi-layered 3D architecture\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e\u0026mdash;requires engineering methods capable of handling hundred-megabase-scale templates.\u003c/p\u003e \u003cp\u003eTo bridge this gap, we developed a robust cross-species chromosome substitution (CROSS) platform. This method employs an \"add-then-delete\" strategy, integrating microcell-mediated chromosome transfer (MMCT)\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e with CRISPR/Cas9\u003csup\u003e16,17\u003c/sup\u003e to achieve the high-fidelity replacement of an endogenous chromosome with a heterologous ortholog. We demonstrate the utility of this platform by substituting the mouse X chromosome with its 158-Mb rat ortholog in mouse embryonic stem cells (mESCs). The X chromosome serves as a high-stringency benchmark due to its requirement for chromosome-wide regulatory programs\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e and its high degree of synteny (gene-order) and gene-content conservation\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) contrasted by rapidly evolving non-coding sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur CROSS platform provided a \"living stress test\" to evaluate the structural and functional fidelity of large-scale exogenous DNA within a non-native nuclear environment. We showed that the substitution was stable and that the engineered mESCs maintained self-renewal and pluripotency. Beyond replacement, this system functioned as a discovery tool for identifying functional genomic elements sensitive to host regulatory machinery. By using the rat X chromosome as an evolutionary perturbation, we identified specific failures in host-dependent epigenetic recruitment. Species-specific LINE1 and RatSatRep2 repeats failed to adequately recruit the host methyltransferase SETDB1, leading to localized H3K9me3 erosion. This epigenetic incompatibility subsequently triggered local 3D structural remodeling, such as the de novo formation of topologically associating domain (TAD) boundaries, which were linked to the regulation of developmental loci including \u003cem\u003eRhox5\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eBy providing a streamlined workflow for whole-chromosome substitution, this study offers a powerful methodology for the functional annotation of non-coding sequences and establishes a critical framework for assessing the performance of large-scale heterologous or synthetic DNA in mammalian cells.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eA robust CROSS platform for scarless, intact, and cross-species chromosome substitution in mammalian cells\u003c/h2\u003e \u003cp\u003eTo achieve X chromosome substitution, a two-step process is required: First, transfer of the rat Chr.X from donor cells (rat ESC-A9 hybrid cells) to recipient cells (male mouse ESCs); Second, loss of endogenous mouse Chr.X (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). To enable the isolation of chromosome-transferred and subsequent chromosome-substituted clones, distinct selectable markers were introduced into male rat ESCs (hereafter referred to as rESCs) and male mouse ESCs (hereafter referred to as mESCs) by their X chromosomes through CRISPR/Cas9 system \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). The Chr.X of rESCs was labeled by puromycin resistant gene (Puro\u003csup\u003eR\u003c/sup\u003e) and enhanced green fluorescent protein (EGFP) at the downstream of the X-linked housekeeping gene \u003cem\u003ePgk1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, e and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). To generate donor cells, the above rESCs were fused with A9 cells, yielding rESC-A9 hybrids. For recipient cells, while X-linked Hypoxanthine Phosphoribosyltransferase (\u003cem\u003eHprt)\u003c/em\u003e gene was simultaneously disrupted via targeted insertion of pEF1α-Thymidine kinase (TK)-DsRed-NeoR cassette into its coding sequence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, e and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). By using MMCT\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, rat Chr.X can be randomly transfer from donor cells to recipient cells. mESC clones with rat Chr.X (X\u003csup\u003etra\u003c/sup\u003e mESCs) were first isolated based on EGFP fluorescence and subsequently selected using HAT and puromycin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Previous research has demonstrated that exogenous chromosomes exhibit instability within host cells and are susceptible to loss\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Under puromycin treatment, clones were capable of maintaining rat Chr.X (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). However, upon drug withdrawal, rat Chr.X underwent rapidly lost. After ten passages, proportion of GFP\u003csup\u003e+\u003c/sup\u003e cells dropped below 20% (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Clones were further validated by polymerase chain reaction (PCR) amplification (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, e), combined with karyotypic analysis (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eTo derive mESC lines with X chromosome substitution, we next subjected the X\u003csup\u003etra\u003c/sup\u003e mESCs to ganciclovir (GCV) selection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). This strategy exploited the spontaneous loss of one X chromosome in XX ESCs, thereby enriching for clones that had lost the endogenous mouse Chr.X (which carries the TK negative selection marker) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, e). The resulting clones, designated X\u003csup\u003esub\u003c/sup\u003e mESCs, were isolated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). PCR and karyotyping confirmed that these lines retained the rat Chr.X but lacked the mouse Chr.X (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, h). The transferred rat Chr.X remained stable during extended culture of Xsub mESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Clones with possibly intact Chr.X were selected for whole-genome sequencing. Results showed that the mouse Chr.X and rat autosomes were absent, whereas the rat Chr.X was present and intact, indicating successful replacement of the Chr.X (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). X\u003csup\u003esub\u003c/sup\u003e mESCs maintained morphology similar to ESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef), expressed ESC marker genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej, k), and formed all three germ layers after teratoma differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003el). Collectively, these findings demonstrated the successful construction of a male mouse ESC line harboring only rat Chr.X.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHigh-fidelity conservation of 3D chromatin architecture on the substituted rat X chromosome\u003c/h3\u003e\n\u003cp\u003eWe next explored the 3D organization of the rat Chr.X in X\u003csup\u003esub\u003c/sup\u003e mESCs through high-throughput chromatin conformation capture (Hi-C). Hi-C contact maps indicated that the distal interactions of the rat Chr.X in X\u003csup\u003esub\u003c/sup\u003e mESCs were slightly downregulated when compared to rESCs, whereas the proximal interactions were slightly enhanced (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Chromatin DNA folds at multiple scales, including compartment, TAD, chromatin loops, to build chromosomes\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Chromatin segregates into two compartments: compartment A (open, gene-active, and broadly euchromatic) and compartment B (closed, gene-inactive, and broadly heterochromatic)\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. We first assessed Chr.X compartmentalization. The level of compartmentalization in both compartment A and compartment B decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c). Next, compartment changes were analyzed by plotting the E1 values, which revealed a bidirectional shift involving both compaction and decompaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-f). This analysis identified a pattern of global weakening, where the most definitive A and B compartments showed significant weaken strength (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Furthermore, compartment switching events were observed in specific genomic regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Correspondingly, 3D remodeling revealed that the chromatin underwent a moderate decompaction, resulting in a disorganized and more open architecture (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). This was further confirmed by the increased ratio of the surface area (S) to the volume (V) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTAD organization of rat Chr.X was subsequently analyzed. Profiling insulation scores across TAD boundaries (\u0026plusmn;\u0026thinsp;50 kb) confirmed that boundaries were characterized by local minima (characteristic troughs), forming distinct troughs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). Analysis of boundary dynamics revealed that gained boundaries exhibited a decrease in insulation score, whereas lost boundaries showed an increase; unchanged boundaries remained stable (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). Representative genomic regions exhibiting both gained and lost TADs were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej). The TAD landscape was largely stable, with 86% of boundaries remaining unchanged, while a small but detectable fraction of boundaries were gained (7.9%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek). In summary, the TAD architecture remained predominantly conserved, with a subset of novel TADs emerging. As a control, the 3D structure of Chr.1 in X\u003csup\u003esub\u003c/sup\u003e mESCs showed slighter changes when compared to Chr.1 in mouse ESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, k; and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-f), reinforcing that the observed structural reorganization was more specific to the exogenous Chr.X.\u003c/p\u003e \u003cp\u003eTaken together, no drastic alterations were observed in the overall 3D chromatin architecture of rat X chromosome in X\u003csup\u003esub\u003c/sup\u003e mESCs. However, reduction in compartment strength, a weakened segregation between compartments A and B, and moderate decompaction were evident on Chr.X. At a finer scale, while the majority of TAD boundaries in Chr.X were unchanged, the emergence of de novo TADs at specific loci was noted. This TAD stability aligns with the established role of CTCF binding sites in defining TAD boundaries and loops\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. As mentioned above, previous studies have demonstrated that heterochromatin is the primary factor driving the formation of chromosomal compartments\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Besides, it has been reported that \u003cem\u003eSetdb1\u003c/em\u003e knockdown/knockout resulted in H3K9me3 loss and gained ectopic binding of CTCF in mouse ESCs\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, suggested a potential role of H3K9me3 in prevent genomic CTCF binding, a core protein for TAD boundary formation. Thus, we extended our analysis to investigate the related epigenetic landscape of Chr.X, including heterochromatic histone modifications, to figure out the mechanisms of alterations in 3D genome organization.\u003c/p\u003e\n\u003ch3\u003eMulti-omic characterization of H3K9me3 depletion and its association with local chromatin organization and transcriptional activity\u003c/h3\u003e\n\u003cp\u003eWe characterized the epigenetic landscapes in X\u003csup\u003esub\u003c/sup\u003e mESCs by analyzing multi-omics data from chromatin immunoprecipitation followed by sequencing (ChIP-Seq), Cleavage Under Targets and Tagmentation (CUT\u0026amp;Tag) assay, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) and RNA-Seq (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). Global erosion of the constitutive heterochromatin mark H3K9me3\u0026mdash;the most pronounced change among all these epigenetic marks\u0026mdash;characterized the substituted chromosome, as revealed by ChIP-seq and confirmed by CUT\u0026amp;Tag assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Notably, the longest of these H3K9me3 loss regions was a 3.2-Mb region (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), indicating large-scale epigenetic reorganization. Meanwhile, we also observed focal increase in the facultative repressive mark H3K27me3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b)\u0026mdash;a potential compensatory response to the loss of global heterochromatin\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Consistent with the observed changes of A-B compartments mentioned above, the active marks H3K4me3 and H3K27ac, along with chromatin accessibility, exhibited concordant and overlapping patterns of focal upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). In contrast, all analyses showed less pronounced alterations on mouse autosomes (ranging from Chr.1 to Chr.19) in X\u003csup\u003esub\u003c/sup\u003e mESCs when compared to autosomes in mouse ESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Notably, decompacted regions across Chr.X were closely associated with regions exhibiting pronounced loss of H3K9me3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Similarly, de novo TAD boundaries formed on Chr.X in X\u003csup\u003esub\u003c/sup\u003e mESCs were also associated with substantial H3K9me3 loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, f). Multiple megabase-scale regions on Chr.X exhibited substantial H3K9me3 loss (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d), and displayed changes in chromatin 3D architecture (compartments and TADs) (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). To summarize, our data demonstrated a profound epigenetic remodeling of Chr.X in X\u003csup\u003esub\u003c/sup\u003e mESCs when compared to Chr.X in rat ESCs, marked specifically by H3K9me3-dependent heterochromatin loss, which possibly contributed to changes in 3D organization of rat Chr.X.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, to assess the transcriptional impact of the chromosomal engineering, we performed pairwise correlation analysis of gene expression across samples. Importantly, X-linked gene expression in the X\u003csup\u003esub\u003c/sup\u003e mESCs most closely resembled that of the male rat ESCs (\u003cem\u003eR\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.911), exhibiting a higher correlation than X\u003csup\u003esub\u003c/sup\u003e mESCs vs. male mouse ESCs (\u003cem\u003eR\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.759) and mouse ESCs vs. rat ESCs (\u003cem\u003eR\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.835) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The expression profile of key ESC marker genes was largely maintained (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek). However, our data demonstrated a partial increase in transcriptional activity of rat Chr.X in X\u003csup\u003esub\u003c/sup\u003e mESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, h). About 23% of the Chr.X-linked genes in X\u003csup\u003esub\u003c/sup\u003e mESCs were dysregulated (13% upregulated and 10% downregulated) compared to rat ESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). This high degree of transcriptional conservation indicated that the rat Chr.X largely retained its native gene expression program with moderate disruption following its introduction into the mouse cellular environment. Meanwhile, 95% of the mouse autosome genes maintained their expression in the X\u003csup\u003esub\u003c/sup\u003e mESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). And the correlation patterns for autosomal genes were consistent across all sample pairs (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Then we assessed the X-chromosome-to-autosome (X: A) expression ratio to evaluate X chromosome dosage compensation. The X\u003csup\u003esub\u003c/sup\u003e mESCs exhibited an X: A (X\u003csub\u003erat\u003c/sub\u003e: A\u003csub\u003emouse\u003c/sub\u003e) ratio of coding genes comparable to male mouse ESCs, which was slightly higher than that of male rat ESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). This establishment of a mouse-like expression balance was mediated by a genome-wide upregulation of the rat Chr.X but not downregulation of autosomal genes, evidenced by an elevated X-derived RNA: total RNA ratio of X\u003csup\u003esub\u003c/sup\u003e mESCs compared to rat ESCs (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed) and the conservation of autosomal gene transcription mentioned above (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Besides, analysis of the Chr.X of X\u003csup\u003esub\u003c/sup\u003e mESCs identified specific pathway enrichments in differentially expressed genes (DEGs) (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). GO analysis of autosomal DEGs indicated different pathways (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Overall, while autosomes maintained a high degree of expression conservation, the Chr.X globally conserved its expression pattern but underwent specific upregulation, resetting the X:A ratio to a level comparable to that in male mESCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo elucidate the mechanism underlying the transcriptional upregulation of rat X-linked genes in the mouse nuclear environment, we analyzed the chromatin landscape across gene bodies (\u0026plusmn;\u0026thinsp;3 kb from TSS to TES). We observed a pronounced global reduction of the heterochromatin mark H3K9me3, along with a focal increase in chromatin accessibility and H3K4me3 at transcription start sites (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Previous study suggested that novel TAD boundaries were related to proximal gene upregulation\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Building on the above finding that the loss of H3K9me3 was associated with the formation of new TAD boundaries (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, f), we asked whether this structural remodeling contributed to gene upregulation. Importantly, genes located near these de novo TAD boundaries exhibited a significant tendency for transcriptional activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej). Thus, our data supported a model in which H3K9me3 erosion facilitates the formation of new chromatin TAD boundaries, which promotes the upregulation of boundary-proximal genes.\u003c/p\u003e \u003cp\u003eTaken together, our results delineated the epigenetic fate of the rat Chr.X within a mouse nuclear environment. the chromosome underwent a profound epigenetic remodeling, characterized by a global loss of H3K9me3 and a consequent collapse of its native 3D architecture. While overall gene expression was broadly conserved, it underwent specific upregulation on the Chr.X-linked genes, resetting the X:A ratio. Critically, we established that the formation of new TAD boundaries at sites of H3K9me3 depletion provided a mechanistic link, explaining the upregulation of adjacent genes. The central role of H3K9me3 erosion prompted a question: what leads to the dissolution of this repressive mark in a foreign nuclear environment?\u003c/p\u003e\n\u003ch3\u003eDefective SETDB1 recruitment at species-specific repeats as the molecular basis of heterochromatin erosion\u003c/h3\u003e\n\u003cp\u003eWe first mapped the genomic regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) and their top enriched motifs (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) with reduced H3K9me3 and revealed that H3K9me3 loss were predominantly localized to distal intergenic sequences. This finding, together with the established role of H3K9me3 in silencing repetitive DNA, TE composition within these depleted regions was further examined. Our findings revealed a significant enrichment of specific TE subfamilies, including LINE: LINE1 (L1), Unknown: RatSatRep2, LTR: ERVK, and LTR: ERV1, among the H3K9me3-lost regions on Chr.X (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Comparative genomic analysis of repetitive elements revealed that LINEs are highly abundant in both the rat and mouse genomes and are further enriched on their Chr.X (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, c). Notably, the rat Chr.X exhibits a substantial proportion of rat-specific LINEs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Previous studies have reported the evolutionary arms race between TEs and host defense mechanisms\u003csup\u003e\u003cspan additionalcitationids=\"CR34 CR35 CR36 CR37\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Thus, we hypothesized that the rat-specific TE sequences may no longer be efficiently recognized by the mouse heterochromatin machinery. To test this, the evolutionary conservation of those top TE types within H3K9me3-lost regions were analyzed. Importantly, rat-specific TEs constituted the majority of these analyzed TE types, with RatSatRep2 being exclusively rat-specific (100%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Consistent with this, species-specific LINEs displayed significantly sharper decreases in H3K9me3 levels than conserved LINEs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). And the completely species-specific RatSatRep2 elements also exhibited substantial H3K9me3 reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Phylogenetic analysis of full-length L1 retrotransposons from mouse and rat further demonstrated species-specific clustering, with distinct clades corresponding to mouse and rat L1 sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Within each species, L1s derived from the Chr.X did not form separate clusters but were intermixed with autosomal L1s (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Both the total number and the Chr.X-linked count of full-length L1s were higher in the rat genome compared to the mouse genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Further analysis revealed that those young L1s emerging after rat-mouse divergence showed high H3K9me3 in their native state and suffered from greater H3K9me3 loss in X\u003csup\u003esub\u003c/sup\u003e mESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). This heterochromatin breakdown was accompanied by the aberrant transcriptional activation of the Chr.X-linked TEs (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). This indicated preferential epigenetic erosion of recently evolved repetitive elements.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the mechanism of this recognition failure, the key H3K9 methyltransferase SET Domain Bifurcated Histone Lysine Methyltransferase 1 (SETDB1) occupancy was examined by ChIP-seq.\u0026nbsp;SETDB1 showed markedly reduced binding at H3K9me3-lost regions in Chr.X of X\u003csup\u003esub\u003c/sup\u003e mESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh), despite unchanged expression level of \u003cem\u003eSetdb1\u003c/em\u003e, \u003cem\u003eSuv39h2\u003c/em\u003e, \u003cem\u003eKdm4a\u003c/em\u003e, and \u003cem\u003eKdm4c\u003c/em\u003e (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Within the H3K9me3-lost region, rat-specific L1s showed sharper decreases in SETDB1 levels than conserved L1s (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). These results indicated impaired recruitment of the H3K9me3 silencing machinery at these regions.\u003c/p\u003e \u003cp\u003eIn summary, our data traced the origin of H3K9me3 erosion to an evolutionary incompatibility: the sequence divergence of rat-specific transposable elements compromises their targeting by the mouse heterochromatin machinery. This defect manifests through impaired SETDB1 recruitment, ultimately leading to large-scale heterochromatin loss of the rat X chromosome in a mouse cellular environment.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCROSS-mediated discovery of functional TAD boundaries at the\u003c/b\u003e \u003cb\u003eRhox5\u003c/b\u003e \u003cb\u003elocus regulating cellular differentiation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNext, the developmental potential of X\u003csup\u003esub\u003c/sup\u003e mESCs was assessed using both in vivo and in vitro assays. Although X\u003csup\u003esub\u003c/sup\u003e mESCs expressed core ESC marker genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej, k) and formed teratomas containing all three germ layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003el), they exhibited profound defects in differentiation capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-d and Extended Data Fig.\u0026nbsp;6a-d). Diploid blastocyst injection and somatic cell nuclear transfer (SCNT) assays showed that X\u003csup\u003esub\u003c/sup\u003e mESCs contributed only to pre-implantation embryos but not to post-implantation stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-c and Extended Data Fig.\u0026nbsp;6a, b). Consistently, X\u003csup\u003esub\u003c/sup\u003e-mESC-derived teratomas retained abundant KI67\u003csup\u003e+\u003c/sup\u003e undifferentiated cells (Extended Data Fig.\u0026nbsp;6c), indicating impaired terminal differentiation. In vitro, X\u003csup\u003esub\u003c/sup\u003e mESCs formed embryoid bodies (EBs) with delayed silencing of pluripotency genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Given the in vivo developmental arrest around implantation, we further examined the naive-to-primed transition using an ESCs to epiblast-like cells (EpiLCs) differentiation model in vitro, which revealed a significant delay in the downregulation of naive pluripotency genes (Extended Data Fig.\u0026nbsp;6d).\u003c/p\u003e \u003cp\u003eTo further investigate the potential contribution of the aforementioned epigenetic remodeling to the differentiation repair, we focused on genes near de novo TAD boundaries associated with H3K9me3 loss on Chr.X. We ranked these genes by expression level and selected the top 10 candidates (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee) for further functional validation. The majority of them were upregulated in the X\u003csup\u003esub\u003c/sup\u003e mESCs compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Among them, we focused on \u003cem\u003eRhox5\u003c/em\u003e (reproductive homeobox 5), a gene thought to be related to survival of male germ cell and tumor progress\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. This gene was highly expressed in our mouse ESCs but silenced upon differentiation (data not shown). Concomitantly, \u003cem\u003eRhox5\u003c/em\u003e failed to silence properly during differentiation, maintaining aberrantly high expression in X\u003csup\u003esub\u003c/sup\u003e mESC-derived- EpiLCs, teratomas and EBs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef and Extended Data Fig.\u0026nbsp;6e). In X\u003csup\u003esub\u003c/sup\u003e mESCs, the locus surrounding \u003cem\u003eRhox5\u003c/em\u003e exhibited substantial epigenetic alterations: a prominent H3K9me3 domain immediately downstream was eroded, coinciding with the formation of a new TAD boundary, a local decrease in CTCF binding, and a gain of H3K4me3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). We found that the newly formed TAD boundaries were enriched with rat-specific TEs, specifically L1VL4a, RatSatRep2, and RNIAP1bLTR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eTo test whether \u003cem\u003eRhox5\u003c/em\u003e dysregulation contribute to differentiation delay, we performed knockout (KO) of \u003cem\u003eRhox5\u003c/em\u003e in X\u003csup\u003esub\u003c/sup\u003e mESCs using CRISPR/Cas9 system (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh and Extended Data Fig.\u0026nbsp;6f). \u003cem\u003eRhox5\u003c/em\u003e KO of X\u003csup\u003esub\u003c/sup\u003e mESCs leaded to larger EBs with improved lineage gene expression and decreased ESC marker gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei and Extended Data Fig.\u0026nbsp;6g); improved na\u0026iuml;ve-to-primed differentiation in vitro (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej); and reduced KI67\u003csup\u003e+\u003c/sup\u003e cells after teratoma differentiation (Extended Data Fig.\u0026nbsp;6h). Importantly, \u003cem\u003eRhox5\u003c/em\u003e KO enabled development to E6.5 in SCNT assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek). However, embryos did not survive to later stages in both SCNT and diploid blastocyst injection assays (Extended Data Fig.\u0026nbsp;6i and data not shown), indicating that \u003cem\u003eRhox5\u003c/em\u003e dysregulation was an important, but not the sole, contributor to the developmental failure.\u003c/p\u003e \u003cp\u003eThe case of \u003cem\u003eRhox5\u003c/em\u003e demonstrated how the epigenetic instability of the rat Chr.X\u0026mdash;initiated by TE-related H3K9me3 loss and 3D remodeling\u0026mdash;propagates to disrupt key developmental genes. This connects a chromosome-scale epigenetic incompatibility to an observed developmental arrest, revealing of the important role of a single, X-linked gene during development. Our model thus provides a platform to identify X-linked dosage-sensitive genes involved in developmental regulation and investigate X chromosome dosage compensation evolution.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we established CROSS as a high-fidelity platform for the intact and scarless substitution of hundred-megabase-scale chromosomes in mammalian cells. By replacing the mouse X chromosome with its rat ortholog, we demonstrated that the exogenous rat Chr.X is stably maintained in mouse ESCs in the absence of mouse Chr.X. The resulting mouse ESCs harboring only rat Chr.X (X\u003csup\u003esub\u003c/sup\u003e mESCs) could be stably maintained in culture, expressed key pluripotency markers, and retained the capacity to differentiate into all three germ layers. This system directly tests how a chromosome’s native sequence governs its architecture and function within a heterologous nuclear environment.\u003c/p\u003e \u003cp\u003eA key technical challenge in large-scale mammalian genome engineering is maintaining the integrity of the epigenetic and structural landscape. Our findings directly linked the evolutionary divergence of species-specific transposable elements (L1/RatSatRep2) to a failure in recruiting the histone methyltransferase SETDB1, resulting in pervasive H3K9me3 loss. This provides direct experimental support for the model that TEs act as genomic platforms for heterochromatin machinery, whose recognition is shaped by sequence-host co-evolution\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Notably, a recent study reported that synthetic megabase human DNA delivered into mouse embryos also lacked H3K9me3 enrichment\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. This shared lack of heterochromatin in both cross-species systems suggests that H3K9me3 deposition on foreign DNA is not an automatic outcome of chromatinization and likely depends on co‑evolved, sequence‑specific recognition mechanisms. At the level of 3D organization, the rat X chromosome maintained broadly conserved architecture, consistent with previous reports suggesting the deterministic role of DNA sequence features like LINE/SINE distribution in establishing compartment identity\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e and CTCF-binding cite distribution in TAD boundary formation by CTCF/cohesin-mediated loop extrusion mechanism\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Nevertheless, However, heterochromatin erosion mediated localized structural changes, including reduced compartmentalization and the emergence of de novo TAD boundaries. These alterations were consistent with studies suggesting that \u003cem\u003eSetdb1\u003c/em\u003e knockdown/knockout results in H3K9me3 loss and ectopic binding of CTCF binding, and thereby influence TAD and chromatin loop landscapes\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Thus, we suggested that beyond the deterministic role of DNA sequence, the co-evolution of DNA and trans acting heterochromatin machinery dynamically shapes chromatin organization.\u003c/p\u003e \u003cp\u003eBesides, our system offers a discovery tool to systematically identify functional non-coding elements that regulate epigenetic patterning and 3D genome folding. The characterization of the \u003cem\u003eRhox5\u003c/em\u003e locus—where H3K9me3 loss and de novo TAD boundary formation correlate with differentiation defects—exemplifies the platform’s capacity to reveal functional dependencies between 3D organization and developmental phenotypes. Unlike traditional knockout screens, the \"evolutionary perturbation\" approach offered by CROSS allows for the identification of regulatory elements within their native chromosomal context, providing insights into the functional requirements of the non-coding genome.\u003c/p\u003e \u003cp\u003eIn parallel with very recent progress in high-fidelity chromosome transfer and engineering pipelines\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, our work addressed a distinct and important question: the functional outcome of cross-species chromosomal replacement. While existing methods enable the physical transfer of intact chromosomes, our model demonstrated that epigenetic compatibility—especially at species-specific repeats—constitutes a newly identified, requirement for stable chromosomal function.\u003c/p\u003e \u003cp\u003eIn conclusion, the CROSS platform bridges the gap between small-scale gene editing and whole-genome synthesis. It provides a standardized workflow for probing genomic compatibility and offers a critical framework for the functional validation of large-scale synthetic DNA. As the field moves toward the de novo design of mammalian chromosomes, the principles of repeat-mediated epigenetic stability identified here will likely inform the engineering of functional and robust synthetic genomes.\u003c/p\u003e "},{"header":"Methods","content":"\u003ch2\u003eAnimal care and use\u003c/h2\u003e\u003cp\u003e All animal experiments were approved by the Institutional Animal Care and Use Committee of the Institute of Zoology, Chinese Academy of Sciences (IOZ, CAS). The C57BL/6, B6D2F1 and CD-1 mice and Dark Agouti (DA) rats were obtained from Beijing Vital River Laboratories. All the mice used were raised in the SPF environment of the Laboratory Animal Center of the IOZ, CAS.\u003c/p\u003e\u003ch3\u003eESC derivation and culture\u003c/h3\u003e\u003cp\u003eMouse and rat ESCs were derived from embryos at E3.5 and cultured on feeder cells in 2i/LIF medium\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e plus 5% knockout serum replacement. The 2i/LIF medium consists of N2B27 medium (DMEM/F12, Neurobasal, N2, B27, GlutaMAX, β-Mercaptoethanol, 2% Bovine Serum Albumin, 10 mg/mL Insulin, and Penicillin-Streptomycin) supplemented with 1 µM MEK inhibitor PD0325901 (Stemgent), 3 µM GSK3b inhibitor CHIR99021 (Stemgent), and 10\u003csup\u003e3\u003c/sup\u003e units/mL mLIF (Millipore) for mouse ESCs and X\u003csup\u003esub\u003c/sup\u003e mESCs, or rLIF (Millipore) for rat ESCs.\u003c/p\u003e\u003ch2\u003eCulture of other cell types\u003c/h2\u003e\u003cp\u003eThe A9 cells were purchased from the Cell Bank of the Chinese Academy of Sciences, and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. rES-A9 hybrid cells were also cultured in the same medium. Feeder cells, mouse embryonic fibroblasts inactivated by treatment with 10 µg/ml mitomycin C, were cultured in the above medium.\u003c/p\u003e\u003ch2\u003eGenetic engineering of chromosome donor cells and recipient cells\u003c/h2\u003e\u003cp\u003eTo enable site-specific DNA cleavage and integration, we used the CRISPR-Cas9 system to generate double-strand breaks at designated genomic loci. Single-guide RNAs (sgRNAs) were designed using Cas-Designer (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.rgenome.net/cas-designer/\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Cas-OFFinder (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.rgenome.net/cas-offinder/\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). SgRNAs with a score higher than 60 and minimal predicted off-target activity in the genome were selected. Each sgRNA spacer sequences was cloned into a BsaI-digested pUC19-U6-sgRNA vector by ligation of annealed oligonucleotides. SgRNA spacer sequences were listed in Supplementary Table\u0026nbsp;4.\u003c/p\u003e\u003cp\u003e \u003cem\u003eDonor cell lines\u003c/em\u003e. To engineer the donor cell line, we inserted an pEF1α-EGFP-PuroR cassette downstream of the \u003cem\u003ePgk1\u003c/em\u003e gene on the X chromosome of DA rat ESCs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed, e). The engineered rat ESCs were further fused with A9 cell lines to produce rES-A9 hybrid cells (see \u003cem\u003eCell fusion\u003c/em\u003e section), which served as Chr.X donor cells in MMCT assay.\u003c/p\u003e\u003cp\u003e \u003cem\u003eRecipient cell lines\u003c/em\u003e. We used a male C57BL/6 mESC line that is competent for diploid aggregation and tetraploid complementation and carries a random ERT2-Cre-ERT2-DsRed integration. On this parental line, we knocked out the \u003cem\u003eHprt\u003c/em\u003e gene in X chromosome and inserted an pEF1α-TK-DsRed-NeoR cassette at the same locus in one step of this ESC line (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed, e). For each targeting experiment, we co-delivered a pCAG-SpCas9-EGFP plasmid, the relevant U6-sgRNA plasmid, and the appropriate homology-directed repair (HDR) donor plasmid. The transfection was carried out using the electroporation transfection instrument Neon transfection system (Invitrogen). 48–72 hours after electroporation, GFP- or DsRed-positive cells were sorted by flow cytometry, respectively. Following drug selection, single-cell clones were isolated, expanded, and genotyped to confirm correct modifications.\u003c/p\u003e\u003ch2\u003eCell fusion\u003c/h2\u003e\u003cp\u003eCell fusion was performed using Polyethylene Glycol 1500 (PEG 1500, ROCHE). The A cells and B cells to be fused were digested, terminated, and washed with PBS. Then, the cells were resuspended in PBS and counted. In brief, taking 10\u003csup\u003e8\u003c/sup\u003e rat ESCs and 2 × 10\u003csup\u003e7\u003c/sup\u003e A9 cells as an example. The mixed cell suspension was centrifuged: at a speed of 1000–1500 rpm for 2–3 minutes. Remove the supernatant completely. Place the centrifuge tube in a 37 °C water bath to maintain the fusion process at 37 °C. Slowly and continuously add 1 mL of 50% PEG 1500 solution preheated to 37 °C to the centrifuge tube within 1 minute, and continue to stir. Continue stirring the cells for 1–2 minutes. Slowly and continuously add 1 mL of preheated KnockOut™ DMEM (Gibco) medium at 37 °C to the centrifuge tube and continue to stir. Add 3 mL of preheated KnockOut™ DMEM medium at 37 °C slowly and continuously, and continue to stir. Slowly add 10 mL of preheated KnockOut™ DMEM medium at 37 °C, and the cell suspension will be incubated at 37 °C for 5 minutes. Centrifuge, discard the supernatant. Resuspend the cell clumps in fresh culture medium, and after 24 hours of culture, add the drug sieve culture medium.\u003c/p\u003e\u003ch2\u003eMicrocell-mediated chromosome transfer (MMCT)\u003c/h2\u003e\u003cp\u003eThe MMCT procedures were primarily adapted from previously established protocols\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e with modifications. In brief, we first screened several candidate cell lines (rat ESC-A9 hybrids harboring rat Chr.X) for micronucleation efficiency and selected efficient donors for subsequent MMCT experiments. Donor cell lines were treated with 0.04–0.05 µg/mL colcemid for 48 h to form micronuclei. After trypsinization and washing, cells were resuspended in KnockOut™ DMEM containing 10 µg/mL cytochalasin B and subjected to density gradient centrifugation in Percoll: KnockOut™ DMEM (1: 1) at 16,000 × g for 60–75 min. The microcell enriched fraction was collected, washed, and sequentially filtered through 8 µm and 5 µm membranes to isolate microcells containing one to several chromosomes. After additional washing and counting, purified microcells were fused with recipient cells using PEG 1500 as described in the \u003cem\u003eCell Fusion\u003c/em\u003e section.\u003c/p\u003e\u003ch2\u003eScreening and identification of chromosome-transfer cell lines (X\u003csup\u003etra\u003c/sup\u003e mESC lines)\u003c/h2\u003e\u003cp\u003e48 hours after MMCT, cells dual positive for GFP and RFP were enriched by fluorescence activated cell sorting (FACS). Puromycin and HAT were then added to the culture medium for selection. After 1–2 weeks, the clones were picked and expanded. Genomic DNA was extracted from each single clone cell line, and successful transfer of the rat X chromosome and the absence of contaminating other chromosomes (including rat autosomes) were verified by genotyping, karyotyping, and whole-genome sequencing. The primer sequences used for genotyping were listed in Supplementary Tables\u0026nbsp;1 and 3.\u003c/p\u003e\u003ch2\u003eScreening and identification of chromosome-substituted cell lines (X\u003csup\u003esub\u003c/sup\u003e mESC lines)\u003c/h2\u003e\u003cp\u003eTo obtain subclones that had lost the endogenous mouse X chromosome (and its linked HSV-TK negative selection marker) from the established X\u003csup\u003etra\u003c/sup\u003e mESC lines, cells were treated with ganciclovir (GCV). GCV-resistant clones were picked and expanded. Initial genotypic screening for loss of the mouse X chromosome was conducted using locus-species PCR (primers listed in Supplementary Table\u0026nbsp;2). Finally, putative positive clones were then subjected to karyotype analysis, and the complete replacement of the mouse X chromosome by the rat X chromosome was confirmed by whole-genome sequencing.\u003c/p\u003e\u003ch2\u003eKaryotyping analysis\u003c/h2\u003e\u003cp\u003eTreat the cells with 0.05 µg/mL colcemid for 2 to 6 hours. After trypsin digestion, wash the cells with phosphate buffered saline (PBS). Resuspend the cells in 0.075 M KCl hypotonic solution and incubate at 37°C for 30 minutes. Then, fix the cells on a 3:1 (volume ratio) methanol-acetic acid fixative at 4°C for 30 minutes. The cell suspension is dropped onto a pre-cooled glass slide under the influence of gravity. The cells were stained using Giemsa Stain.\u003c/p\u003e\u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e\u003cp\u003eImmunofluorescence staining was performed as previously described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.Fix the cells with 4% paraformaldehyde for 10 minutes, then permeabilize and block the cells with 0.5% Triton X-100 containing 2% BSA for 1 hour. Add the primary antibodies at 4°C for overnight incubation. Incubate with the secondary antibody at room temperature for 1 hour. DNA was stained with Hoechst 33342 for 10 minutes. Imaging was performed using a two-photon confocal microscope (Leica, TCS Sp8). Antibodies: anti-OCT4 (ab19857, Abcam); anti-NANOG (ab80892, Abcam); anti-SSEA1 (ab16285, Abcam); anti-KI67 (ab16667, Abcam).\u003c/p\u003e\u003ch2\u003eFormation of teratomas\u003c/h2\u003e\u003cp\u003eTeratoma assay was performed as previously published\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Briefly, 0.05% pancreatic trypsin was used to digest and collect ESCs. Then 10\u003csup\u003e7\u003c/sup\u003e cells were then subcutaneously injected into both sides of the abdomen of SCID mice. After 21 days or one month, the tumors were excised and subjected to histological analysis through fixation and H\u0026amp;E staining or immunofluorescence staining.\u003c/p\u003e\u003ch2\u003eFormation of Embryoid bodies (EBs)\u003c/h2\u003e\u003cp\u003eFirst, the ESCs are digested into single cells using 0.05% pancreatic trypsin. Subsequently, the ESCs were suspended and cultured in the N2B27 medium. After approximately 8 days, the differentiated EB spheres were picked out.\u003c/p\u003e\u003ch2\u003eOocyte collection\u003c/h2\u003e\u003cp\u003eOocyte collection was performed as previously described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Eight-week-old female mice were super-ovulated by consecutive injection of 7.5 IU human chorionic gonadotropin (hCG) and 7.5 IU pregnant mare's serum gonadotropin (PMSG). MII oocytes were harvested from ovarian oviduct 13–15 hours after the injections. Based on the instructions, derived oocytes were cultured in M16 medium after being washed with M2 medium. Hyaluronidase was used to remove the cumulus cells. Oocytes were then cultured in M2 medium at 37°C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003ch2\u003eSomatic cell nuclear transfer (SCNT)\u003c/h2\u003e\u003cp\u003eSCNT was conducted follow protocols of the published article\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. In brief, inject ESCs into enucleated oocytes to form embryos. The embryos are incubated in M16 medium for 1 hour, then activated in the CZB medium containing 5 mg/mL Cytochalasin B (Abcam) and 10 mM SrCl\u003csub\u003e2\u003c/sub\u003e (Sigma) for 5 to 6 hours. Subsequently, the embryos are transferred to M16 medium and cultured for 18 to 22 hours at 5% CO\u003csub\u003e2\u003c/sub\u003e and 37°C. Then they are transferred to the KSOM solution and cultured for 3.5 days at 5% CO\u003csub\u003e2\u003c/sub\u003e and 37°C.\u003c/p\u003e\u003ch2\u003eDiploid blastocyst injection\u003c/h2\u003e\u003cp\u003eThis assay was carried out following the published article\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e.3.5 days after the mating of superovulated female CD-1 mice with male CD-1 mice, blastocysts were taken from their uteri. The ESCs were digested with trypsin and 12 to 15 cells were microinjected into each blastocyst. After culturing for 1 to 4 hours, the treated embryos were transferred to the oviducts of pseudo-pregnant CD-1 mice at 0.5 days post-conception. Identification of the chimeras were based on GFP and coat color.\u003c/p\u003e\u003ch2\u003eQuantitative real-time PCR\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted using TRIzol® Reagent (Life Technologies) and further purified with the PureLink® RNA Mini Kit (Invitrogen). Genomic DNA was removed by treatment with DNase I (TIANGEN). Subsequently, the RNA was reverse-transcribed into cDNA using the ReverTra Ace® qPCR RT Master Mix with gDNA Remover Kit. The real-time fluorescent quantitative PCR system contained SYBR qPCR Mix and 50 ⋅ ROX reference dye. The fluorescence quantitative PCR experiment was performed using the QuantStudio 6 Pro Real-Time PCR System (Thermo Fisher Scientific). The reference gene was \u003cem\u003eGapdh\u003c/em\u003e. The primer sequences used were listed in Supplementary Table\u0026nbsp;5.\u003c/p\u003e\u003cp\u003e \u003cb\u003eCRISPR/Cas9-mediated deletion of\u003c/b\u003e \u003cb\u003eRhox5\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe design and construction of CRISPR/Cas9 system was described in the above \u003cem\u003eGenetic engineering of chromosome donor cells and recipient cells\u003c/em\u003e section. Briefly, we co-delivered a pCAG-SpCas9-EGFP plasmid and the relevant U6-sgRNA plasmid into X\u003csup\u003esub\u003c/sup\u003e mESCs to knockout \u003cem\u003eRhox5\u003c/em\u003e gene. SgRNA spacer sequences were listed in Supplementary Table\u0026nbsp;4.\u003c/p\u003e\u003cp\u003e \u003cb\u003eReference Genome Construction\u003c/b\u003e \u003c/p\u003e\u003cp\u003eReference genomes for \u003cem\u003eMus musculus\u003c/em\u003e (mm39) and \u003cem\u003eRattus norvegicus\u003c/em\u003e (rn7) were downloaded from the Ensembl database (Release 106). A combined mm39-rn7 reference genome was generated by merging the sequences and annotations from both assemblies to facilitate integrated cross-species analysis.\u003c/p\u003e\u003ch2\u003eWhole genome sequencing\u003c/h2\u003e\u003cp\u003eGenomic DNA was extracted using the E.Z.N.A.® MicroElute Genomic DNA Kit (Omega Bio-tek) according to the manufacturer’s instructions. DNA quality was assessed by agarose gel electrophoresis and quantified using a Qubit® 3.0 Fluorometer. Library construction and high-throughput sequencing were performed on Illumina NovaSeq 6000 platform by Annoroad Gene Technology (Beijing) Co., Ltd.\u003c/p\u003e\u003cp\u003eRaw reads underwent quality assessment using FastQC (v0.11.5) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Adapter sequences and low-quality bases were subsequently trimmed using trim_galore (v0.6.6) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/FelixKrueger/\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003eTrimGalore). The cleaned reads were aligned to the above reference genome (mm39-rn7) using BWA-MEM (v0.7.17)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e with default parameters, followed by duplicate marking and removal with GATK (v4.1.8)\u003csup\u003e56\u003c/sup\u003e. The resulting BAM files were used for copy number variation (CNV) analysis with SCCNV (v1.0.2)\u003csup\u003e57\u003c/sup\u003e, generating visualizations of genomic segments’ copy number states.\u003c/p\u003e\u003cp\u003e \u003cb\u003eHi-C library construction and data analysis.\u003c/b\u003e \u003c/p\u003e\u003cp\u003eHi-C libraries were constructed using standard protocol by Annoroad and were sequenced on Illumina platforms. Hi-C data were processed using HiC-Pro (v3.1.0)\u003csup\u003e58\u003c/sup\u003e to generate raw contact matrices at 10-kb and 100-kb genomic bin resolutions. Systematic biases in the contact matrices were corrected using the ‘hicCorrectMatrix’ function from HiCExplorer (v3.6)\u003csup\u003e59\u003c/sup\u003e. The corrected 100 kb matrix was utilized for global structural assessment. Contact matrix visualization and saddle plots were generated with cooltools (v0.7.1)\u003csup\u003e60\u003c/sup\u003e, and three-dimensional (3D) genome structures were inferred using miniMDS\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eChromatin compartment (A/B) analysis was performed using cscoreTool (v1.1)\u003csup\u003e62\u003c/sup\u003e. A compartment switch was defined as a genomic bin where the absolute difference in compartment score between samples exceeded 0.3.\u003c/p\u003e\u003cp\u003eTopologically Associating Domains (TADs) were identified at 10-kb resolution by calculating the insulation score with cooltools\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Dynamic TAD boundaries were classified as: \u003cem\u003eGain boundary\u003c/em\u003e, a genomic locus identified as a TAD boundary exclusively in the X\u003csup\u003esub\u003c/sup\u003e mESC group; \u003cem\u003eLost boundary\u003c/em\u003e, a genomic locus identified as a TAD boundary exclusively in the control (ctrl) group; \u003cem\u003eMaintained boundary\u003c/em\u003e, a TAD boundary called in both groups. In all analyses, the ctrl was rat ESCs when assessing the rat X chromosome, and mouse ESCs when assessing autosomes. TADs were visualized using pyGenomeTracks\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e, and insulation scores distribution plot were generated with deepTools\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003ch2\u003eChIP-seq library preparation\u003c/h2\u003e\u003cp\u003eChromatin immunoprecipitation was performed using the SimpleChIP® Plus Sonication Chromatin IP Kit (Cell Signaling Technology, 56383) following the manufacturer’s protocol. Briefly, cells were cross-linked with formaldehyde, lysed, and chromatin was fragmented by sonication. Immunoprecipitation was carried out with target-specific antibodies. Antibodies: anti-H3K9me3 (ab8898, Abcam), anti-H3K27me3 (9733s, Cell Signaling technology), anti-H3K27ac (ab4729, Abcam), anti-H3K4me3 (ab8580, Abcam), anti-CTCF (3418S, Cell Signaling technology), anti-SETDB1 (11231-1-AP, Proteintech). After reversal of cross-links, DNA was purified using the kit’s spin columns. Libraries were constructed and sequenced by Annoroad on Illumina NovaSeq platform.\u003c/p\u003e\u003ch2\u003eATAC-seq library preparation\u003c/h2\u003e\u003cp\u003eATAC‑seq libraries were prepared by Annoroad using their standard protocol, based on hyperactive Tn5 transposase\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. In brief, 50,000 viable cells were used for transposase-mediated tagmentation, followed by PCR amplification with indexed primers.\u003c/p\u003e\u003ch2\u003eChIP-seq and ATAC-seq data analysis\u003c/h2\u003e\u003cp\u003eChIP-seq and ATAC-seq libraries were sequenced on the Illumina NovaSeq platform by Annoroad. ChIP-seq and ATAC-seq datasets were processed through an identical pipeline. Following quality control with FastQC (v0.11.5), adapter sequences were removed using trim_galore (v0.6.6). The processed reads were aligned to the reference genome with Bowtie2 (v2.3.5)\u003csup\u003e66\u003c/sup\u003e under the parameters “--sensitive --end-to-end”, and PCR duplicates were eliminated using a custom script. Read coverage was normalized to Reads Per Kilobase per Million mapped reads (RPKM) using the ‘bamCoverage’ utility from deepTools (v3.5.1)\u003csup\u003e64\u003c/sup\u003e. The bigwig files were visualized using pyGenomeTracks (v3.6)\u003csup\u003e63\u003c/sup\u003e. The mean read count distribution plot was generated using deeptools. Peak calling was executed with MACS2 (v2.1.2)\u003csup\u003e67\u003c/sup\u003e with the parameters “-p 0.01 --nomodel --nolambda”. All peaks were segmented into 1-kb windows using the sliding window method. These regions were categorized as follows: \u003cem\u003egain regions\u003c/em\u003e, peaks present in the X\u003csup\u003esub\u003c/sup\u003e mESC group but absent in the control group (ctrl), exhibiting an RPM fold-change (X\u003csup\u003esub\u003c/sup\u003e mESC/ctrl) ≥ 4; \u003cem\u003elost regions\u003c/em\u003e, peaks present in the ctrl group but absent in the X\u003csup\u003esub\u003c/sup\u003e mESC group, exhibiting an RPM fold-change (ctrl/ X\u003csup\u003esub\u003c/sup\u003e mESC) ≥ 4; \u003cem\u003emaintained regions\u003c/em\u003e, peaks called in both X\u003csup\u003esub\u003c/sup\u003e mESC and ctrl group. In all analyses, the ctrl was rat ESCs when assessing the rat X chromosome, and mouse ESCs when assessing autosomes.\u003c/p\u003e\u003cp\u003e \u003cstrong\u003eMotif enrichment\u003c/strong\u003e \u003c/p\u003e\u003cp\u003ede novo motif discovery within specific peaks was performed using the MEME suite (v5.5.2)\u003csup\u003e68\u003c/sup\u003e.\u003c/p\u003e\u003ch2\u003eCUT\u0026amp;Tag library preparation and data processing\u003c/h2\u003e\u003cp\u003eCUT\u0026amp;Tag libraries were prepared using the Hyperactive Universal CUT\u0026amp;Tag Assay Kit for Illumina Pro (Vazyme, TD904) following the manufacturer’s instructions. Cells were bound to ConA beads, permeabilized with digitonin, and incubated with primary antibodies (anti-H3K9me3, ab8898, Abcam) and secondary antibodies. Protein A/G-Tn5 transposase was then targeted to antibody-bound regions for tagmentation. DNA was extracted, amplified with indexed primers, and purified using DNA Clean Beads. Libraries were quantified and sequenced on the Illumina NovaSeq platform.\u003c/p\u003e\u003cp\u003eAfter removing the adapter sequences with trim_galore, CUT\u0026amp;Tag reads were mapped to the reference genome (mm39-rn7) using bowtie2\u003csup\u003e66\u003c/sup\u003e with the parameters “--local --very-sensitive --no-mixed --no-discordant”. The redundant reads were removed using our own script. The mean read count distribution plot was generated using deeptools\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003ch3\u003eRNA-Seq library construction and data analysis\u003c/h3\u003e\u003cp\u003eTotal RNA was extracted as described in the \u003cem\u003eQuantitative real-time PCR\u003c/em\u003e section. Libraries were quantified and sequenced on Illumina platforms. Raw RNA-seq reads underwent quality assessment using FastQC. Adapter sequences and low-quality bases were trimmed with trim_galore. The resulting high-quality reads were aligned to the composite mm39-rn7 genome using STAR (v2.7.1a)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Uniquely mapped reads were isolated using a custom script for subsequent quantification. Transcript abundance was estimated with StringTie (v2.0)\u003csup\u003e70\u003c/sup\u003e and normalized to Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Differential gene expression analysis was conducted using the DESeq2 package (v1.38.0)\u003csup\u003e71\u003c/sup\u003e in R. Genes demonstrating an absolute log2 fold change \u0026gt; 1 with p-value \u0026lt; 0.05 were designated as differentially expressed. Functional enrichment analysis for Gene Ontology (GO) terms was performed on these gene sets using the DAVID\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e bioinformatics database.\u003c/p\u003e\u003cp\u003eX:A ratio: StringTie\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e-derived gene expression tables were merged into a unified matrix, retaining annotated protein-coding genes on autosomes and the X chromosome; genes in the top 5% of maximal expression were excluded. Two gene sets were used: all expressed protein-coding genes and one-to-one mouse–rat homologous protein-coding genes obtained from Ensembl BioMart\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. X:A ratio was assessed using both expression-based and read-level approaches. For expression-based analysis, genes with FPKM \u0026gt; 2 were retained, and a bootstrapping strategy\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e was applied to account for the smaller number of X-linked genes: in each iteration, a random autosomal subset equal in size to the X-linked gene set was sampled, and median expression values were compared over 10,000 iterations to derive X:A ratios and confidence intervals. For a gene-annotation–independent estimate, chromosome-wise read counts and lengths were extracted using samtools\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e idxstats, and a length-normalized X:A read-density ratio was computed. Statistical analyses and visualization were performed in R using ggplot2\u003csup\u003e76\u003c/sup\u003e, with group comparisons conducted using the Wilcoxon rank-sum test and results displayed as boxplots with jittered points.\u003c/p\u003e\u003ch2\u003eTransposable element (TE) expression analysis\u003c/h2\u003e\u003cp\u003eGenomic annotations for repetitive elements were obtained from the UCSC Genome Browser for the respective mm39 and rn7 assemblies. TE expression was quantified using the SQuIRE\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e software (v0.9.9) under default parameters. Differentially expressed TEs were identified using the same thresholds as for genes: |log\u003csub\u003e2\u003c/sub\u003eFoldChange| \u0026gt; 1 and p-value \u0026lt; 0.05.\u003c/p\u003e\u003ch2\u003eEvolutionary analysis of mouse and rat TEs\u003c/h2\u003e\u003cp\u003eThe evolutionary ages of TEs were estimated based on RepeatMasker output files acquired from the UCSC Genome Browser for the mouse (mm39) and rat (rn7) genomes. For each repeat element locus, the \"milliDiv\" value (representing the percentage divergence from the consensus sequence) was extracted directly from the output. The Jukes-Cantor method was applied using species-specific substitution rates: 5.2 × 10\u003csup\u003e− 9\u003c/sup\u003e per base per year for mouse and 5.5 × 10\u003csup\u003e− 9\u003c/sup\u003e per base per year for rat\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. The evolutionary age (in years) was then calculated using the formula: age = divergence rate / substitution rate, yielding an estimated age for each repetitive element locus\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e \u003cem\u003ePhylogenetic analysis of mouse and rat full length LINE1\u003c/em\u003e. Full-length LINE1 sequences (\u0026gt; 6.5 kb) were extracted from the telomere-to-telomere (T2T) mouse genome and the rat genome (RN8) using RepeatMasker (v4.1.1)\u003csup\u003e80\u003c/sup\u003e based on the Repbase repeat database. Sequences were subsequently clustered using CD-HIT (v4.8.1)\u003csup\u003e81\u003c/sup\u003e with the parameters “-d 0 -c 0.8 -aL 0.80” to reduce redundancy and define representative sequences. Multiple sequence alignment of the clustered sequences was performed with MAFFT (v7.475)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e under default settings. A phylogenetic tree was constructed from the aligned sequences using FastTree (v2.1.11)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e with default parameters. Finally, the resulting tree was visualized and annotated using the iTOL platform\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003ch2\u003eStatistics and reproducibility\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed in R using ggplot2\u003csup\u003e76\u003c/sup\u003e or Graphpad Prism. Levels of significance were calculated using the two-tailed Student’s t test. In all figures: *, p value \u0026lt; 0.05; **, p value \u0026lt; 0.01, ***, p value \u0026lt; 0.001; ****, p value \u0026lt; 0.0001. The number of independent biological replicates and statistical approach for each experiment were described in the figure legends or the \u003cem\u003eMethods\u003c/em\u003e section.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eThe sequencing data are available at Genome Sequence Archive (GSA) of China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ngdc.cncb.ac.cn/gsa/\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) under accession CRA035509. Previously published raw data used in this paper is available at Gene Expression Omnibus under accessions GSE44150, GSE97966\u003csup\u003e85\u003c/sup\u003e, GSE178701\u003csup\u003e86\u003c/sup\u003e, GSE220805\u003csup\u003e87\u003c/sup\u003e, and GSE90516\u003csup\u003e88\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDC0200000 to W.L. and XDA0510400); the National Key Research and Development Program (2022YFA1103600 to L.W. and 2019YFA0903800); the National Natural Science Foundation of China (32225030 and 82488301 to W.L.); the CAS Project for Young Scientists in Basic Research (YSBR-012 to W.L.); the Beijing Natural Science Foundation (Z230011 to W.L.); \u0026nbsp;Initiative Scientific Research Program, Institute of Zoology, Chinese Academy of Sciences (2023IOZ0101). We acknowledge Runze Ma, Xiaohua Shen and Bing Zhu for their\u0026nbsp;helpful suggestions. We thank Lingyun Wei, Youjia Shu, Qing Meng, Peipei Long, Ming Ge, Weiyu Jin, Shiwen Li, Xili Zhu, Hua Qin and Xia Yang for their technical assistance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eW.L., Q.Z., Y.M. and L.W. conceived and designed the study. Y.M., L.W., Y.Z., K.X., B.Z., and S.M. performed the experiments; N.Y., Y.M., W.L., L.W., G.F., Q.Z., Y.D., J.L., Y.Z. and Q.N. analyzed the data; W.L. and Q.Z. supervised the research; W.L., Y.M., Y.Z., N.Y., Q.Z., Y.C., and Q.Z. wrote the manuscript, assisted by the other authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence and requests for materials\u003c/strong\u003e should be addressed to Wei Li.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMeneu L et al (2025) Sequence-dependent activity and compartmentalization of foreign DNA in a eukaryotic nucleus. 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Biol Sex Differ 8:28. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13293-017-0150-x\u003c/span\u003e\u003cspan address=\"10.1186/s13293-017-0150-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8995396/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8995396/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe functional integrity of a mammalian chromosome is shaped by its long-term, co-evolution with species-specific nuclear environment. How chromosomes co-adapt with their native environment to define 3D architecture and transcriptional activity remain poorly understood, largely due to a lack of experimental models capable of systematically dissecting this co-evolution relationship. Here, we report a cross-species chromosome substitution (CROSS) method, a robust genomic engineering method that enables the stable, scarless replacement of host chromosomes with evolutionarily divergent orthologs. By integrating microcell-mediated chromosome transfer with CRISPR/Cas9, we imported the intact 158-Mb rat X chromosome into mouse embryonic stem cells, and subsequently achieved targeted substitution of its endogenous mouse counterpart, maintaining stability and integrity. Using this model, we found that rat-specific LINE1 and RatSatRep2 repeats failed to adequately recruit host SETDB1 in mouse cells, leading to localized erosion of H3K9me3 heterochromatin. This further triggered 3D structural remodeling, characterized by the de novo formation of topologically associating domain (TAD) boundaries that aberrantly activated adjacent genes\u0026mdash;including \u003cem\u003eRhox5\u003c/em\u003e, the master regulator of the \u003cem\u003eRhox\u003c/em\u003e cluster\u0026mdash;impairing cellular differentiation. Our method provides a powerful chromosome engineering platform for dissecting how genomic sequences and epigenetic mechanisms cooperate in regulating chromosome architecture and function, and for evaluating the structural and functional fidelity of large-scale synthetic or heterologous DNA across species.\u003c/p\u003e","manuscriptTitle":"CROSS: A cross-species chromosome substitution platform for dissecting chromosome architecture and activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-16 11:13:17","doi":"10.21203/rs.3.rs-8995396/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b2a7af64-90f5-4f0b-9635-274b9a339916","owner":[],"postedDate":"April 16th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-05-06T04:46:53+00:00","index":2,"fulltext":"This content is not available."}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":66393545,"name":"Biological sciences/Biotechnology"},{"id":66393546,"name":"Biological sciences/Evolution/Coevolution"}],"tags":[],"updatedAt":"2026-04-16T11:13:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-16 11:13:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8995396","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8995396","identity":"rs-8995396","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}