Rise and SINE: roles of transcription factors and retrotransposons in zygotic genome activation.

In sexually reproducing organisms, life begins with the fusion of transcriptionally silent gametes, the oocyte and sperm. Although initiation of transcription in the embryo, known as zygotic genome activation (ZGA), is universally required for development, the transcription factors regulating this process are poorly conserved. In this Perspective, we discuss recent insights into the mechanisms of ZGA in totipotent mammalian embryos, namely ZGA regulation by several transcription factors, including by orphan nuclear receptors (OrphNRs) such as the pioneer transcription factor NR5A2, and by factors of the DUX, TPRX and OBOX families. We performed a meta-analysis and compiled a list of pan-ZGA genes, and found that most of these genes are indeed targets of the above transcription factors. Remarkably, more than a third of these ZGA genes appear to be regulated both by OrphNRs such as NR5A2 and by OBOX proteins, whose motifs co-occur in SINE B1 retrotransposable elements, which are enriched near ZGA genes. We propose that ZGA in mice is activated by recruitment of multiple transcription factors to SINE B1 elements that function as enhancers, and discuss a potential relevance of this mechanism to Alu retrotransposable elements in human ZGA.

Control of inducible gene expression links cohesin to hematopoietic progenitor self-renewal and differentiation.

Cohesin is important for 3D genome organization. Nevertheless, even the complete removal of cohesin has surprisingly little impact on steady-state gene transcription and enhancer activity. Here we show that cohesin is required for the core transcriptional response of primary macrophages to microbial signals, and for inducible enhancer activity that underpins inflammatory gene expression. Consistent with a role for inflammatory signals in promoting myeloid differentiation of hematopoietic stem and progenitor cells (HPSCs), cohesin mutations in HSPCs led to reduced inflammatory gene expression and increased resistance to differentiation-inducing inflammatory stimuli. These findings uncover an unexpected dependence of inducible gene expression on cohesin, link cohesin with myeloid differentiation, and may help explain the prevalence of cohesin mutations in human acute myeloid leukemia.

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.

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.

Nucleome programming is required for the foundation of totipotency in mammalian germline development.

Germ cells are unique in engendering totipotency, yet the mechanisms underlying this capacity remain elusive. Here, we perform comprehensive and in-depth nucleome analysis of mouse germ-cell development in vitro, encompassing pluripotent precursors, primordial germ cells (PGCs) before and after epigenetic reprogramming, and spermatogonia/spermatogonial stem cells (SSCs). Although epigenetic reprogramming, including genome-wide DNA de-methylation, creates broadly open chromatin with abundant enhancer-like signatures, the augmented chromatin insulation safeguards transcriptional fidelity. These insulatory constraints are then erased en masse for spermatogonial development. Notably, despite distinguishing epigenetic programming, including global DNA re-methylation, the PGCs-to-spermatogonia/SSCs development entails further euchromatization. This accompanies substantial erasure of lamina-associated domains, generating spermatogonia/SSCs with a minimal peripheral attachment of chromatin except for pericentromeres-an architecture conserved in primates. Accordingly, faulty nucleome maturation, including persistent insulation and improper euchromatization, leads to impaired spermatogenic potential. Given that PGCs after epigenetic reprogramming serve as oogenic progenitors as well, our findings elucidate a principle for the nucleome programming that creates gametogenic progenitors in both sexes, defining a basis for nuclear totipotency.

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.

Control of DNA replication and its potential clinical exploitation.

Multiple conserved mechanisms limit DNA replication to once per cell cycle. One vital level of control focuses on the loading of the heterohexameric ring of minichromosome maintenance proteins (MCMs) onto chromatin in the hierarchical assembly of the pre-replication complex at origins of replication. An essential role in proliferation for MCMs and their regulators makes them potentially important biomarkers for routine clinical use in cancer detection and prognosis.

Genome folding and zygotic genome activation in mammalian preimplantation embryos.

The totipotent one-cell embryo, or zygote, gives rise to all germ layers and extraembryonic tissues that culminate in the development of a new organism. A zygote is produced at fertilisation by the fusion of differentiated germ cells, egg and sperm. The chromatin of parental genomes is reprogrammed and spatially reorganised in the early embryo. The 3D chromatin organisation is established de novo after fertilisation by a cohesin-dependent mechanism of loop extrusion that forms chromatin loops and topologically associating domains (TADs). Strengthening of TAD insulation is concomitant with the transcriptional 'awakening' of the embryo known as zygotic genome activation (ZGA). Whether and how these processes are causally linked remains poorly understood. In this review, we discuss recent findings of 3D chromatin organisation in mammalian gametes and embryos and how these are potentially related to ZGA.

Changes in DNA repair compartments and cohesin loss promote DNA damage accumulation in aged oocytes.

Oocyte loss, a natural process that accelerates as women approach their mid-30s, poses a significant challenge to female reproduction. Recent studies have identified DNA damage as a primary contributor to oocyte loss, but the mechanisms underlying DNA damage accumulation remain unclear. Here, we show that aged oocytes have a lower DNA repair capacity and reduced mobility of DNA damage sites compared to young oocytes. Incomplete DNA repair in aged oocytes results in defective chromosome integrity and partitioning, thereby compromising oocyte quality. We found that DNA repair proteins are arranged in spatially distinct DNA repair compartments that form during the late stages of oocyte growth, accompanied by changes in the activity of DNA repair pathways. We demonstrate alterations in these compartments with age, including substantial changes in the levels of key DNA repair proteins and a shift toward error-prone DNA repair pathways. In addition, we show that reduced cohesin levels make aged oocytes more vulnerable to persistent DNA damage and cause changes in DNA repair compartments. Our study links DNA damage accumulation in aged oocytes, a leading cause of oocyte loss, to cohesin deterioration and changes in the organization, abundance, and response of DNA repair machinery.

Nucleosome-bound NR5A2 structure reveals pioneer factor mechanism by DNA minor groove anchor competition.

Gene expression during natural and induced reprogramming is controlled by pioneer transcription factors that initiate transcription from closed chromatin. Nr5a2 is a key pioneer factor that regulates zygotic genome activation in totipotent embryos, pluripotency in embryonic stem cells and metabolism in adult tissues, but the mechanism of its pioneer activity remains poorly understood. Here, we present a cryo-electron microscopy structure of human NR5A2 bound to a nucleosome. The structure shows that the conserved carboxy-terminal extension (CTE) loop of the NR5A2 DNA-binding domain competes with a DNA minor groove anchor of the nucleosome and releases entry-exit site DNA. Mutational analysis showed that NR5A2 D159 of the CTE is dispensable for DNA binding but required for stable nucleosome association and persistent DNA 'unwrapping'. These findings suggest that NR5A2 belongs to an emerging class of pioneer factors that can use DNA minor groove anchor competition to destabilize nucleosomes and facilitate gene expression during reprogramming.

Centromere-specifying nucleosomes persist in aging mouse oocytes in the absence of nascent assembly.

Centromeres direct genetic inheritance but are not themselves genetically encoded. Instead, centromeres are defined epigenetically by the presence of a histone H3 variant, CENP-A.1 In cultured somatic cells, an established paradigm of cell-cycle-coupled propagation maintains centromere identity: CENP-A is partitioned between sisters during replication and replenished by new assembly, which is restricted to G1. The mammalian female germ line challenges this model because of the cell-cycle arrest between pre-meiotic S phase and the subsequent G1, which can last for the entire reproductive lifespan (months to decades). New CENP-A chromatin assembly maintains centromeres during prophase I in worm and starfish oocytes,2,3 suggesting that a similar process may be required for centromere inheritance in mammals. To test this hypothesis, we developed an oocyte-specific conditional knockout (cKO) mouse for Mis18α, an essential component of the assembly machinery. We find that embryos derived from Mis18α knockout oocytes fail to assemble CENP-A nucleosomes prior to zygotic genome activation (ZGA), validating the knockout model. We show that deletion of Mis18α in the female germ line at the time of birth has no impact on centromeric CENP-A nucleosome abundance, even after 6-8 months of aging. In addition, there is no detectable detriment to fertility. Thus, centromere chromatin is maintained long-term, independent of new assembly during the extended prophase I arrest in mouse oocytes.

Centromere-specifying nucleosomes persist in aging mouse oocytes in the absence of nascent assembly.

Centromeres direct genetic inheritance but are not themselves genetically encoded. Instead, centromeres are defined epigenetically by the presence of a histone H3 variant, CENP-A 1 . In cultured somatic cells, an established paradigm of cell cycle-coupled propagation maintains centromere identity: CENP-A is partitioned between sisters during replication and replenished by new assembly, which is restricted to G1. The mammalian female germline challenges this model because of the cell cycle arrest between pre-meiotic S-phase and the subsequent G1, which can last for the entire reproductive lifespan (months to decades). New CENP-A chromatin assembly maintains centromeres during prophase I in worm and starfish oocyte 2,3 , suggesting that a similar process may be required for centromere inheritance in mammals. However, we show that centromere chromatin is maintained long-term independent of new assembly during the extended prophase I arrest in mouse oocytes. Conditional knockout of Mis18α, an essential component of the assembly machinery, in the female germline at the time of birth has almost no impact on centromeric CENP-A nucleosome abundance nor any detectable detriment to fertility.

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.

Biallelic PAX5 mutations cause hypogammaglobulinemia, sensorimotor deficits, and autism spectrum disorder.

The genetic causes of primary antibody deficiencies and autism spectrum disorder (ASD) are largely unknown. Here, we report a patient with hypogammaglobulinemia and ASD who carries biallelic mutations in the transcription factor PAX5. A patient-specific Pax5 mutant mouse revealed an early B cell developmental block and impaired immune responses as the cause of hypogammaglobulinemia. Pax5 mutant mice displayed behavioral deficits in all ASD domains. The patient and the mouse model showed aberrant cerebellar foliation and severely impaired sensorimotor learning. PAX5 deficiency also caused profound hypoplasia of the substantia nigra and ventral tegmental area due to loss of GABAergic neurons, thus affecting two midbrain hubs, controlling motor function and reward processing, respectively. Heterozygous Pax5 mutant mice exhibited similar anatomic and behavioral abnormalities. Lineage tracing identified Pax5 as a crucial regulator of cerebellar morphogenesis and midbrain GABAergic neurogenesis. These findings reveal new roles of Pax5 in brain development and unravel the underlying mechanism of a novel immunological and neurodevelopmental syndrome.

Awakening of the zygotic genome by pioneer transcription factors.

After fertilization, the genome of the totipotent embryo is transcriptionally inactive and then initiates bursts of transcription termed zygotic genome activation (ZGA). Despite the fundamental importance of initiating an embryonic transcription program for the start of life, the essential regulators and molecular mechanisms triggering ZGA in most organisms are poorly understood. One mechanism centers on pioneer factors that function in cellular reprogramming and differentiation. Recent studies revealed that not only a single but multiple pioneer factors bind cooperatively to the genome to open chromatin, resulting in changes in epigenetic modifications and triggering ZGA. Here, we review recent insights into the functions of pioneer factors during ZGA and discuss the potential relevance to three-dimensional chromatin organization during embryonic development.

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.

The emergence of genome architecture and zygotic genome activation.

The fusion of two transcriptionally silent gametes, egg and sperm, generates a totipotent zygote that activates zygotic transcription to support further development. Although the molecular details of zygotic genome activation (ZGA) are not well understood in most species, an emerging concept is that one or more pioneer transcription factors trigger zygotic transcription. Concomitantly, extensive changes in 3D chromatin organization occur during development. In this review, we discuss recent advances in understanding when and how genome architecture emerges in early metazoan embryos, how the zygotic genome is activated, and how these events might be coordinated. We also highlight some of the unknowns that may be critical to address in the future.

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.

Genomic insights into chromatin reprogramming to totipotency in embryos.

The early embryo is the natural prototype for the acquisition of totipotency, which is the potential of a cell to produce a whole organism. Generation of a totipotent embryo involves chromatin reorganization and epigenetic reprogramming that alter DNA and histone modifications. Understanding embryonic chromatin architecture and how this is related to the epigenome and transcriptome will provide invaluable insights into cell fate decisions. Recently emerging low-input genomic assays allow the exploration of regulatory networks in the sparsely available mammalian embryo. Thus, the field of developmental biology is transitioning from microscopy to genome-wide chromatin descriptions. Ultimately, the prototype becomes a unique model for studying fundamental principles of development, epigenetic reprogramming, and cellular plasticity. In this review, we discuss chromatin reprogramming in the early mouse embryo, focusing on DNA methylation, chromatin accessibility, and higher-order chromatin structure.