Chromatin accessibility landscape of mouse early embryos revealed by single-cell NanoATAC-seq2.

In mammals, fertilized eggs undergo genome-wide epigenetic reprogramming to generate the organism. However, our understanding of epigenetic dynamics during preimplantation development at single-cell resolution remains incomplete. Here, we developed scNanoATAC-seq2, a single-cell assay for transposase-accessible chromatin using long-read sequencing for scarce samples. We present a detailed chromatin accessibility landscape of mouse preimplantation development, revealing distinct chromatin signatures in the epiblast, primitive endoderm, and trophectoderm during lineage segregation. Differences between zygotes and two-cell embryos highlight reprogramming in chromatin accessibility during the maternal-to-zygotic transition. Single-cell long-read sequencing enables in-depth analysis of chromatin accessibility in noncanonical imprinting, imprinted X chromosome inactivation, and low-mappability genomic regions, such as repetitive elements and paralogs. Our data provide insights into chromatin dynamics during mammalian preimplantation development and lineage differentiation. Editor's summary: Mammalian development is a complex and intricately organized process that we are just beginning to understand, in part because it requires coordinated interactions between many distinct cell types that change over time. To gain insight into this process, Li et al. developed sequencing methods for assessing chromatin accessibility in individual cells. They then applied these methods to mouse embryos at 10 distinct stages, ranging from the zygote to the late blastocyst, mapping key changes in chromatin state in different cell lineages over the course of these key stages in early development. —Yevgeniya Nusinovich INTRODUCTION: Preimplantation development marks the beginning of mammalian life, generating the entire embryo proper and extraembryonic tissues from a totipotent zygote. However, owing to technical limitations, the epigenetic regulation of biological processes during this period, including zygotic genome activation (ZGA), lineage differentiation, and initiation of pluripotency, remains inadequately understood. RATIONALE: We hypothesized that the regulation of chromatin states, especially on repetitive elements, plays vital roles in preimplantation embryos. Furthermore, transcription factors (TFs) are fundamentally involved in their gene expression networks. Therefore, we developed scNanoATAC-seq2, a single-cell assay for transposase-accessible chromatin using long-read sequencing that can accurately depict the chromatin state of repetitive elements usually missed by the short-read ATAC-seq. RESULTS: We utilized scNanoATAC-seq2 to analyze the chromatin accessibility landscape across all key stages of mouse preimplantation development. We systematically identified TFs that are potentially important for each stage and lineage of preimplantation embryos based on their downstream target sites' chromatin accessibility features and elucidated their activity dynamics, including SOX2, OCT4, and KLF2 for epiblast (EPI); GATA6, SOX17, and HNF1B for primitive endoderm (PE); and TEAD4, GATA3, and CDX2 for trophectoderm (TE). We delineated the chromatin states of paternal and maternal X chromosomes in female embryos during their imprinted inactivation and reactivation. Examining two opposing regulators of X chromosome inactivation (XCI), the Xist and Tsix domains, we uncovered a notable shift of allele-specific chromatin accessibility between them. The paternal-specific chromatin accessibility of the Xist domain is primarily associated with the imprinted XCI (iXCI) from zygote to eight-cell stage. Subsequently, during lineage segregation into inner cell mass (ICM) and early TE, the paternal-specific feature of the Xist domain diminishes, whereas the Tsix domain exhibits a moderate increase in maternal-specific feature. In the extraembryonic lineages (TE and PE), iXCI is maintained and only associated with the strong maternal-specific chromatin accessibility of the Tsix domain. By contrast, in the EPI, both the Xist and Tsix domains transition to allele-balanced chromatin accessibility patterns, coinciding with the erasure of iXCI in this pluripotent lineage. Furthermore, we characterized chromatin states of repetitive elements at individual copy resolution. More than 100 copies of full-length long interspersed nuclear element–1 (LINE1) elements are activated at two-cell stage, potentially allowing their genomic transposition. During ZGA, activated copies from major repetitive element families, such as LINE1 and endogenous retrovirus–L (ERVL), exhibit clear positive correlations between their proximity to the nearest promoter and the degree of transcriptional activation of the corresponding target gene. This suggests their cis-regulatory function, particularly for murine ERVL, in regulating the ZGA-specific expression of protein-coding genes, such as Sp110, Zscan4c, and Tcstv3. CONCLUSION: Our results suggest that the synchronized activities of critical TFs are potentially responsible for the wavelike gene expression patterns during preimplantation development. These patterns likely underpin key events, such as ZGA, lineage segregation between embryonic and extraembryonic cells, the initiation of pluripotency in ICM, the establishment of multipotency in TE, and the maintenance of iXCI in extraembryonic lineages and its erasure in the pluripotent EPI in female embryos. Gene regulatory landscape of mouse preimplantation development.: Single-cell chromatin accessibility landscape of mouse embryos from zygote to blastula identified TFs potentially controlling key biological events, such as ZGA and lineage segregation between embryonic (EPI) and extraembryonic cells (TE and PE). Regulatory dynamics of imprinted X chromosome inactivation and reactivation were characterized, including a shift of allele-specific chromatin accessibility between the Xist and Tsix domains. [ABSTRACT FROM AUTHOR]