Zygotic genome activation by the totipotency pioneer factor Nr5a2.

Life begins with a switch in genetic control from the maternal to the embryonic genome during zygotic genome activation (ZGA). Despite its importance, the essential regulators of ZGA remain largely unknown in mammals. On the basis of de novo motif searches, we identified the orphan nuclear receptor Nr5a2 as a key activator of major ZGA in mouse two-cell embryos. Nr5a2 is required for progression beyond the two-cell stage. It binds to its motif within SINE B1/Alu retrotransposable elements found in cis-regulatory regions of ZGA genes. Chemical inhibition suggests that 72% of ZGA genes are regulated by Nr5a2 and potentially other orphan nuclear receptors. Nr5a2 promotes chromatin accessibility during ZGA and binds nucleosomal DNA in vitro. We conclude that Nr5a2 is an essential pioneer factor that regulates ZGA.

MCM complexes are barriers that restrict cohesin-mediated loop extrusion.

Eukaryotic genomes are compacted into loops and topologically associating domains (TADs)1-3, which contribute to transcription, recombination and genomic stability4,5. Cohesin extrudes DNA into loops that are thought to lengthen until CTCF boundaries are encountered6-12. Little is known about whether loop extrusion is impeded by DNA-bound machines. Here we show that the minichromosome maintenance (MCM) complex is a barrier that restricts loop extrusion in G1 phase. Single-nucleus Hi-C (high-resolution chromosome conformation capture) of mouse zygotes reveals that MCM loading reduces CTCF-anchored loops and decreases TAD boundary insulation, which suggests that loop extrusion is impeded before reaching CTCF. This effect extends to HCT116 cells, in which MCMs affect the number of CTCF-anchored loops and gene expression. Simulations suggest that MCMs are abundant, randomly positioned and partially permeable barriers. Single-molecule imaging shows that MCMs are physical barriers that frequently constrain cohesin translocation in vitro. Notably, chimeric yeast MCMs that contain a cohesin-interaction motif from human MCM3 induce cohesin pausing, indicating that MCMs are 'active' barriers with binding sites. These findings raise the possibility that cohesin can arrive by loop extrusion at MCMs, which determine the genomic sites at which sister chromatid cohesion is established. On the basis of in vivo, in silico and in vitro data, we conclude that distinct loop extrusion barriers shape the three-dimensional genome.

Ovulation suppression protects against chromosomal abnormalities in mouse eggs at advanced maternal age.

The frequency of egg aneuploidy and trisomic pregnancies increases with maternal age. To what extent individual approaches can delay the "maternal age effect" is unclear because multiple causes contribute to chromosomal abnormalities in mammalian eggs. We propose that ovulation frequency determines the physiological aging of oocytes, a key aspect of which is the ability to accurately segregate chromosomes and produce euploid eggs. To test this hypothesis, ovulations were reduced using successive pregnancies, hormonal contraception, and a pre-pubertal knockout mouse model, and the effects on chromosome segregation and egg ploidy were examined. We show that each intervention reduces chromosomal abnormalities in eggs of aged mice, suggesting that ovulation reduction delays oocyte aging. The protective effect can be partly explained by retention of chromosomal Rec8-cohesin that maintains sister chromatid cohesion in meiosis. In addition, single-nucleus Hi-C (snHi-C) revealed deterioration in the 3D chromatin structure including an increase in extruded loop sizes in long-lived oocytes. Artificial cleavage of Rec8 is sufficient to increase extruded loop sizes, suggesting that cohesin complexes maintaining cohesion restrict loop extrusion. These findings suggest that ovulation suppression protects against Rec8 loss, thereby maintaining both sister chromatid cohesion and 3D chromatin structure and promoting production of euploid eggs. We conclude that the maternal age effect can be delayed in mice. An implication of this work is that long-term ovulation-suppressing conditions can potentially reduce the risk of aneuploid pregnancies at advanced maternal age.

Wapl releases Scc1-cohesin and regulates chromosome structure and segregation in mouse oocytes.

Cohesin is essential for genome folding and inheritance. In somatic cells, these functions are both mediated by Scc1-cohesin, which in mitosis is released from chromosomes by Wapl and separase. In mammalian oocytes, cohesion is mediated by Rec8-cohesin. Scc1 is expressed but neither required nor sufficient for cohesion, and its function remains unknown. Likewise, it is unknown whether Wapl regulates one or both cohesin complexes and chromosome segregation in mature oocytes. Here, we show that Wapl is required for accurate meiosis I chromosome segregation, predominantly releases Scc1-cohesin from chromosomes, and promotes production of euploid eggs. Using single-nucleus Hi-C, we found that Scc1 is essential for chromosome organization in oocytes. Increasing Scc1 residence time on chromosomes by Wapl depletion leads to vermicelli formation and intra-loop structures but, unlike in somatic cells, does not increase loop size. We conclude that distinct cohesin complexes generate loops and cohesion in oocytes and propose that the same principle applies to all cell types and species.

Polycomb Group Proteins Regulate Chromatin Architecture in Mouse Oocytes and Early Embryos.

In mammals, chromatin organization undergoes drastic reorganization during oocyte development. However, the dynamics of three-dimensional chromatin structure in this process is poorly characterized. Using low-input Hi-C (genome-wide chromatin conformation capture), we found that a unique chromatin organization gradually appears during mouse oocyte growth. Oocytes at late stages show self-interacting, cohesin-independent compartmental domains marked by H3K27me3, therefore termed Polycomb-associating domains (PADs). PADs and inter-PAD (iPAD) regions form compartment-like structures with strong inter-domain interactions among nearby PADs. PADs disassemble upon meiotic resumption from diplotene arrest but briefly reappear on the maternal genome after fertilization. Upon maternal depletion of Eed, PADs are largely intact in oocytes, but their reestablishment after fertilization is compromised. By contrast, depletion of Polycomb repressive complex 1 (PRC1) proteins attenuates PADs in oocytes, which is associated with substantial gene de-repression in PADs. These data reveal a critical role of Polycomb in regulating chromatin architecture during mammalian oocyte growth and early development.

Single-nucleus Hi-C of mammalian oocytes and zygotes.

The 3D folding of the genome is linked to essential nuclear processes including gene expression, DNA repair, and replication. Chromatin conformation capture assays such as Hi-C are providing unprecedented insights into higher-order chromatin structure. Bulk Hi-C of millions of cells enables detection of average chromatin features at high resolution but is challenging to apply to rare cell types. This chapter describes our recently developed single-nucleus Hi-C (snHi-C) approach for detection of chromatin contacts in single nuclei of murine oocytes and one-cell embryos (zygotes). The step-by-step protocol includes isolation of these cells, extraction of nuclei, fixation, restriction digestion, ligation, and whole genome amplification. Contacts obtained by snHi-C allow detection of chromatin features including loops, topologically associating domains, and compartments when averaged over the genome. The combination of snHi-C with other single-cell techniques in these and other rare cell types will likely provide a comprehensive picture of how chromatin architecture shapes cell identity.

A mechanism of cohesin-dependent loop extrusion organizes zygotic genome architecture.

Fertilization triggers assembly of higher-order chromatin structure from a condensed maternal and a naïve paternal genome to generate a totipotent embryo. Chromatin loops and domains have been detected in mouse zygotes by single-nucleus Hi-C (snHi-C), but not bulk Hi-C. It is therefore unclear when and how embryonic chromatin conformations are assembled. Here, we investigated whether a mechanism of cohesin-dependent loop extrusion generates higher-order chromatin structures within the one-cell embryo. Using snHi-C of mouse knockout embryos, we demonstrate that the zygotic genome folds into loops and domains that critically depend on Scc1-cohesin and that are regulated in size and linear density by Wapl. Remarkably, we discovered distinct effects on maternal and paternal chromatin loop sizes, likely reflecting differences in loop extrusion dynamics and epigenetic reprogramming. Dynamic polymer models of chromosomes reproduce changes in snHi-C, suggesting a mechanism where cohesin locally compacts chromatin by active loop extrusion, whose processivity is controlled by Wapl. Our simulations and experimental data provide evidence that cohesin-dependent loop extrusion organizes mammalian genomes over multiple scales from the one-cell embryo onward.

Single-nucleus Hi-C reveals unique chromatin reorganization at oocyte-to-zygote transition.

Chromatin is reprogrammed after fertilization to produce a totipotent zygote with the potential to generate a new organism. The maternal genome inherited from the oocyte and the paternal genome provided by sperm coexist as separate haploid nuclei in the zygote. How these two epigenetically distinct genomes are spatially organized is poorly understood. Existing chromosome conformation capture-based methods are not applicable to oocytes and zygotes owing to a paucity of material. To study three-dimensional chromatin organization in rare cell types, we developed a single-nucleus Hi-C (high-resolution chromosome conformation capture) protocol that provides greater than tenfold more contacts per cell than the previous method. Here we show that chromatin architecture is uniquely reorganized during the oocyte-to-zygote transition in mice and is distinct in paternal and maternal nuclei within single-cell zygotes. Features of genomic organization including compartments, topologically associating domains (TADs) and loops are present in individual oocytes when averaged over the genome, but the presence of each feature at a locus varies between cells. At the sub-megabase level, we observed stochastic clusters of contacts that can occur across TAD boundaries but average into TADs. Notably, we found that TADs and loops, but not compartments, are present in zygotic maternal chromatin, suggesting that these are generated by different mechanisms. Our results demonstrate that the global chromatin organization of zygote nuclei is fundamentally different from that of other interphase cells. An understanding of this zygotic chromatin 'ground state' could potentially provide insights into reprogramming cells to a state of totipotency.