Human-specific epigenomic states in spermatogenesis.

Infertility is becoming increasingly common, affecting one in six people globally. Half of these cases can be attributed to male factors, many driven by abnormalities in the process of sperm development. Emerging evidence from genome-wide association studies, genetic screening of patient cohorts, and animal models highlights an important genetic contribution to spermatogenic defects, but comprehensive identification and characterization of the genes critical for male fertility remain lacking. High divergence of gene regulation in spermatogenic cells across species poses challenges for delineating the genetic pathways required for human spermatogenesis using common model organisms. In this study, we leveraged post-translational histone modification and gene transcription data for 15,491 genes in four mammalian species (human, rhesus macaque, mouse, and opossum), to identify human-specific patterns of gene regulation during spermatogenesis. We combined H3K27me3 ChIP-seq, H3K4me3 ChIP-seq, and RNA-seq data to define epigenetic states for each gene at two stages of spermatogenesis, pachytene spermatocytes and round spermatids, in each species. We identified 239 genes that are uniquely active, poised, or dynamically regulated in human spermatogenic cells distinct from the other three species. While some of these genes have been implicated in reproductive functions, many more have not yet been associated with human infertility and may be candidates for further molecular and epidemiologic studies. Our analysis offers an example of the opportunities provided by evolutionary and epigenomic data for broadly screening candidate genes implicated in reproduction, which might lead to discoveries of novel genetic targets for diagnosis and management of male infertility and male contraception.

KDM6A/UTX promotes spermatogenic gene expression across generations and is not required for male fertility†.

Paternal chromatin undergoes extensive structural and epigenetic changes during mammalian spermatogenesis, producing sperm with an epigenome optimized for the transition to embryogenesis. Lysine demethylase 6a (KDM6A, also called UTX) promotes gene activation in part via demethylation of H3K27me3, a developmentally important repressive modification abundant throughout the epigenome of spermatogenic cells and sperm. We previously demonstrated increased cancer risk in genetically wild-type mice derived from a paternal germ line lacking Kdm6a (Kdm6a cKO), indicating a role for KDM6A in regulating heritable epigenetic states. However, the regulatory function of KDM6A during spermatogenesis is not known. Here, we show that Kdm6a is transiently expressed in spermatogenesis, with RNA and protein expression largely limited to late spermatogonia and early meiotic prophase. Kdm6a cKO males do not have defects in fertility or the overall progression of spermatogenesis. However, hundreds of genes are deregulated upon loss of Kdm6a in spermatogenic cells, with a strong bias toward downregulation coinciding with the time when Kdm6a is expressed. Misregulated genes encode factors involved in chromatin organization and regulation of repetitive elements, and a subset of these genes was persistently deregulated in the male germ line across two generations of offspring of Kdm6a cKO males. Genome-wide epigenetic profiling revealed broadening of H3K27me3 peaks in differentiating spermatogonia of Kdm6a cKO mice, suggesting that KDM6A demarcates H3K27me3 domains in the male germ line. Our findings highlight KDM6A as a transcriptional activator in the mammalian male germ line that is dispensable for spermatogenesis but important for safeguarding gene regulatory state intergenerationally.

DOT1L bridges transcription and heterochromatin formation at mammalian pericentromeres.

Repetitive DNA elements are packaged in heterochromatin, but many require bursts of transcription to initiate and maintain long-term silencing. The mechanisms by which these heterochromatic genome features are transcribed remain largely unknown. Here, we show that DOT1L, a conserved histone methyltransferase that modifies lysine 79 of histone H3 (H3K79), has a specialized role in transcription of major satellite repeats to maintain pericentromeric heterochromatin and genome stability. We find that H3K79me3 is selectively enriched relative to H3K79me2 at repetitive elements in mouse embryonic stem cells (mESCs), that DOT1L loss compromises pericentromeric satellite transcription, and that this activity involves possible coordination between DOT1L and the chromatin remodeler SMARCA5. Stimulation of transcript production from pericentromeric repeats by DOT1L participates in stabilization of heterochromatin structures in mESCs and cleavage-stage embryos and is required for preimplantation viability. Our findings uncover an important role for DOT1L as a bridge between transcriptional activation of repeat elements and heterochromatin stability, advancing our understanding of how genome integrity is maintained and how chromatin state is set up during early development.

DOT1L promotes spermatid differentiation by regulating expression of genes required for histone-to-protamine replacement.

Unique chromatin remodeling factors orchestrate dramatic changes in nuclear morphology during differentiation of the mature sperm head. A crucial step in this process is histone-to-protamine exchange, which must be executed correctly to avoid sperm DNA damage, embryonic lethality and male sterility. Here, we define an essential role for the histone methyltransferase DOT1L in the histone-to-protamine transition. We show that DOT1L is abundantly expressed in mouse meiotic and postmeiotic germ cells, and that methylation of histone H3 lysine 79 (H3K79), the modification catalyzed by DOT1L, is enriched in developing spermatids in the initial stages of histone replacement. Elongating spermatids lacking DOT1L fail to fully replace histones and exhibit aberrant protamine recruitment, resulting in deformed sperm heads and male sterility. Loss of DOT1L results in transcriptional dysregulation coinciding with the onset of histone replacement and affecting genes required for histone-to-protamine exchange. DOT1L also deposits H3K79me2 and promotes accumulation of elongating RNA Polymerase II at the testis-specific bromodomain gene Brdt. Together, our results indicate that DOT1L is an important mediator of transcription during spermatid differentiation and an indispensable regulator of male fertility.

Widespread association of the Argonaute protein AGO2 with meiotic chromatin suggests a distinct nuclear function in mammalian male reproduction.

Argonaute 2 (AGO2) is a ubiquitously expressed protein critical for regulation of mRNA translation and vital to animal development. AGO2 protein is found in both cytoplasmic and nuclear compartments, and although its cytoplasmic role is well studied, the biological relevance of nuclear AGO2 is unclear. Here, we address this problem in vivo using spermatogenic cells as a model. We find that AGO2 transiently binds both chromatin and nucleus-specific mRNA transcripts of hundreds of genes required for sperm production during male meiosis in mice, and that germline conditional knockout (cKO) of Ago2 causes depletion of the encoded proteins. Correspondingly, Ago2 cKO males show abnormal sperm head morphology and reduced sperm count, along with reduced postnatal viability of offspring. Together, our data reveal an unexpected nuclear role for AGO2 in enhancing expression of developmentally important genes during mammalian male reproduction.

Deconvolution of in vivo protein-RNA contacts using fractionated eCLIP-seq.

Thousands of RNA-binding proteins orchestrate RNA processing and altered protein-RNA interactions frequently lead to disease. Here, we present experimental and computational analysis pipelines of fractionated eCLIP-seq (freCLIP-seq), a modification of enhanced UV-crosslinking and RNA immunoprecipitation followed by sequencing. FreCLIP-seq allows transcriptome-wide analysis of protein-RNA interactions at single-nucleotide level and provides an additional level of resolution by isolating binding signals of individual RNA-binding proteins within a multicomponent complex. Binding occupancy can be inferred from read counts and crosslinking events. For complete details on the use and execution of this protocol, please refer to Biancon et al. (2022).

Neuron-specific chromosomal megadomain organization is adaptive to recent retrotransposon expansions.

Regulatory mechanisms associated with repeat-rich sequences and chromosomal conformations in mature neurons remain unexplored. Here, we map cell-type specific chromatin domain organization in adult mouse cerebral cortex and report strong enrichment of Endogenous Retrovirus 2 (ERV2) repeat sequences in the neuron-specific heterochromatic B2NeuN+ megabase-scaling subcompartment. Single molecule long-read sequencing and comparative Hi-C chromosomal contact mapping in wild-derived SPRET/EiJ (Mus spretus) and laboratory inbred C57BL/6J (Mus musculus) reveal neuronal reconfigurations tracking recent ERV2 expansions in the murine germline, with significantly higher B2NeuN+ contact frequencies at sites with ongoing insertions in Mus musculus. Neuronal ablation of the retrotransposon silencer Kmt1e/Setdb1 triggers B2NeuN+ disintegration and rewiring with open chromatin domains enriched for cellular stress response genes, along with severe neuroinflammation and proviral assembly with infiltration of dendrites . We conclude that neuronal megabase-scale chromosomal architectures include an evolutionarily adaptive heterochromatic organization which, upon perturbation, results in transcriptional dysregulation and unleashes ERV2 proviruses with strong neuronal tropism.

Epigenetic states in the human placenta: A singular epigenome for an exceptional tissue.

The placenta is a dynamic, short-lived organ, and many features of its epigenome are unique among human tissues. In this issue of Developmental Cell, Zhang et al. define new aspects of epigenetic regulation in normal and disease-associated placental cells and relate them to the specialized biological requirements of this organ.

H3K4me1 Distribution Predicts Transcription State and Poising at Promoters.

Monomethylation on lysine 4 of histone H3 (H3K4me1) is commonly associated with distal enhancers, but H3K4me1 is also present at promoter regions proximal to transcription start sites. To assess a possible role for H3K4me1 in dictating gene regulatory states at promoters, we examined H3K4me1 peak density around promoters in human and mouse germ cells using an analytic strategy that allowed us to assess relationships between different epigenetic marks on a promoter-by-promoter basis. We found that H3K4me1 exhibits either a bimodal pattern at active promoters, where it flanks H3K4me3, or a unimodal pattern at poised promoters, where it coincides with both H3K4me3 and H3K27me3. This pattern is correlated with gene expression level, but is more strongly linked to a poised chromatin state, defined by the simultaneous presence of H3K4me3 and H3K27me3, than to transcriptional activity. The pattern is especially prominent in germ cells, but is also present in other cell types, including embryonic stem cells and differentiated somatic cells. We propose that H3K4me1 is a key feature of the poised epigenetic state, and suggest possible roles for this mark in epigenetic memory.

Sperm Go to (Transcription) Extremes.

Xia and colleagues show that heritable mutations are kept in check in the male germline partly by "transcriptional scanning," wherein the majority of genes are transcribed and therefore subject to transcription-coupled repair. They provide a new model for understanding the mechanisms of genome surveillance and evolution.

A universal transportin protein drives stochastic choice of olfactory neurons via specific nuclear import of a sox-2-activating factor.

Stochastic neuronal cell fate choice involving notch-independent mechanisms is a poorly understood biological process. The Caenorhabditis elegans AWC olfactory neuron pair asymmetrically differentiates into the default AWCOFF and induced AWCON subtypes in a stochastic manner. Stochastic choice of the AWCON subtype is established using gap junctions and SLO BK potassium channels to repress a calcium-activated protein kinase pathway. However, it is unknown how the potassium channel-repressed calcium signaling is translated into the induction of the AWCON subtype. Here, we identify a detailed working mechanism of how the homeodomain-like transcription factor NSY-7, previously described as a repressor in the maintenance of AWC asymmetry, couples SLO BK potassium channels to transactivation of sox-2 expression for the induction of the AWCON subtype through the identification of a unique imb-2 (transportin 1) allele. imb-2 loss-of-function mutants are not viable; however, we identify a viable imb-2 allele from an unbiased forward genetic screen that reveals a specific role of imb-2 in AWC olfactory neuron asymmetry. IMB-2 specifically drives nuclear import of NSY-7 within AWC neurons to transactivate the expression of the high mobility group (HMG)-box transcription factor SOX-2 for the specification of the AWCON subtype. This study provides mechanistic insight into how NSY-7 couples SLO BK potassium channels to transactivation of sox-2 expression for the induction of the AWCON subtype. Our findings also provide structure-function insight into a conserved amino acid residue of transportins in brain development and suggest its dysfunction may lead to human neurological disorders.

Intergenerational epigenetic inheritance of cancer susceptibility in mammals.

Susceptibility to cancer is heritable, but much of this heritability remains unexplained. Some 'missing' heritability may be mediated by epigenetic changes in the parental germ line that do not involve transmission of genetic variants from parent to offspring. We report that deletion of the chromatin regulator Kdm6a (Utx) in the paternal germ line results in elevated tumor incidence in genetically wild type mice. This effect increases following passage through two successive generations of Kdm6a male germline deletion, but is lost following passage through a wild type germ line. The H3K27me3 mark is redistributed in sperm of Kdm6a mutants, and we define approximately 200 H3K27me3-marked regions that exhibit increased DNA methylation, both in sperm of Kdm6a mutants and in somatic tissue of progeny. Hypermethylated regions in enhancers may alter regulation of genes involved in cancer initiation or progression. Epigenetic changes in male gametes may therefore impact cancer susceptibility in adult offspring.

Parallel evolution of male germline epigenetic poising and somatic development in animals.

Changes in gene regulation frequently underlie changes in morphology during evolution, and differences in chromatin state have been linked with changes in anatomical structure and gene expression across evolutionary time. Here we assess the relationship between evolution of chromatin state in germ cells and evolution of gene regulatory programs governing somatic development. We examined the poised (H3K4me3/H3K27me3 bivalent) epigenetic state in male germ cells from five mammalian and one avian species. We find that core genes poised in germ cells from multiple amniote species are ancient regulators of morphogenesis that sit at the top of transcriptional hierarchies controlling somatic tissue development, whereas genes that gain poising in germ cells from individual species act downstream of core poised genes during development in a species-specific fashion. We propose that critical regulators of animal development gained an epigenetically privileged state in germ cells, manifested in amniotes by H3K4me3/H3K27me3 poising, early in metazoan evolution.

Poised chromatin in the mammalian germ line.

Poised (bivalent) chromatin is defined by the simultaneous presence of histone modifications associated with both gene activation and repression. This epigenetic feature was first observed at promoters of lineage-specific regulatory genes in embryonic stem cells in culture. More recent work has shown that, in vivo, mammalian germ cells maintain poised chromatin at promoters of many genes that regulate somatic development, and that they retain this state from fetal stages through meiosis and gametogenesis. We hypothesize that the poised chromatin state is essential for germ cell identity and function. We propose three roles for poised chromatin in the mammalian germ line: prevention of DNA methylation, maintenance of germ cell identity and preparation for totipotency. We discuss these roles in the context of recently proposed models for germline potency and epigenetic inheritance.

A chromatin-dependent role of the fragile X mental retardation protein FMRP in the DNA damage response.

Fragile X syndrome, a common form of inherited intellectual disability, is caused by loss of the fragile X mental retardation protein FMRP. FMRP is present predominantly in the cytoplasm, where it regulates translation of proteins that are important for synaptic function. We identify FMRP as a chromatin-binding protein that functions in the DNA damage response (DDR). Specifically, we show that FMRP binds chromatin through its tandem Tudor (Agenet) domain in vitro and associates with chromatin in vivo. We also demonstrate that FMRP participates in the DDR in a chromatin-binding-dependent manner. The DDR machinery is known to play important roles in developmental processes such as gametogenesis. We show that FMRP occupies meiotic chromosomes and regulates the dynamics of the DDR machinery during mouse spermatogenesis. These findings suggest that nuclear FMRP regulates genomic stability at the chromatin interface and may impact gametogenesis and some developmental aspects of fragile X syndrome.

A set of genes critical to development is epigenetically poised in mouse germ cells from fetal stages through completion of meiosis.

In multicellular organisms, germ cells carry the hereditary material from one generation to the next. Developing germ cells are unipotent gamete precursors, and mature gametes are highly differentiated, specialized cells. However, upon gamete union at fertilization, their genomes drive a totipotent program, giving rise to a complete embryo as well as extraembryonic tissues. The biochemical basis for the ability to transition from differentiated cell to totipotent zygote is unknown. Here we report that a set of developmentally critical genes is maintained in an epigenetically poised (bivalent) state from embryonic stages through the end of meiosis. We performed ChIP-seq and RNA-seq analysis on flow-sorted male and female germ cells during embryogenesis at three time points surrounding sexual differentiation and female meiotic initiation, and then extended our analysis to meiotic and postmeiotic male germ cells. We identified a set of genes that is highly enriched for regulators of differentiation and retains a poised state (high H3K4me3, high H3K27me3, and lack of expression) across sexes and across developmental stages, including in haploid postmeiotic cells. The existence of such a state in embryonic stem cells has been well described. We now demonstrate that a subset of genes is maintained in a poised state in the germ line from the initiation of sexual differentiation during fetal development and into postmeiotic stages. We propose that the epigenetically poised condition of these developmental genes is a fundamental property of the mammalian germ-line nucleus, allowing differentiated gametes to unleash a totipotent program following fertilization.

The ligand binding domain of GCNF is not required for repression of pluripotency genes in mouse fetal ovarian germ cells.

In mice, successful development and reproduction require that all cells, including germ cells, transition from a pluripotent to a differentiated state. This transition is associated with silencing of the pluripotency genes Oct4 and Nanog. Interestingly, these genes are repressed at different developmental timepoints in germ and somatic cells. Ovarian germ cells maintain their expression until about embryonic day (E) 14.5, whereas somatic cells silence them much earlier, at about E8.0. In both somatic cells and embryonic stem cells, silencing of Oct4 and Nanog requires the nuclear receptor GCNF. However, expression of the Gcnf gene has not been investigated in fetal ovarian germ cells, and whether it is required for silencing Oct4 and Nanog in that context is not known. Here we demonstrate that Gcnf is expressed in fetal ovarian germ cells, peaking at E14.5, when Oct4 and Nanog are silenced. However, conditional ablation of the ligand-binding domain of Gcnf using a ubiquitous, tamoxifen-inducible Cre indicates that Gcnf is not required for the down-regulation of pluripotency genes in fetal ovarian germ cells, nor is it required for initiation of meiosis and oogenesis. These results suggest that the silencing of Oct4 and Nanog in germ cells occurs via a different mechanism from that operating in somatic cells during gastrulation.

Genetics of germ cell development.

The germ line represents a continuous cellular link between generations and between species, but the germ cells themselves develop in a specialized, organism-specific context. The model organisms Caenorhabditis elegans, Drosophila melanogaster and the mouse display striking similarities, as well as major differences, in the means by which they control germ cell development. Recent developments in genetic technologies allow a more detailed comparison of the germ cells of these three organisms than has previously been possible, shedding light not only on universal aspects of germline regulation, but also on the control of the pluripotent state in vivo and on the earliest steps of embryogenesis. Here, we highlight themes from the comparison of these three alternative strategies for navigating the fundamental cycle of sexual reproduction.

Microtubule-based localization of a synaptic calcium-signaling complex is required for left-right neuronal asymmetry in C. elegans.

The axons of C. elegans left and right AWC olfactory neurons communicate at synapses through a calcium-signaling complex to regulate stochastic asymmetric cell identities called AWC(ON) and AWC(OFF). However, it is not known how the calcium-signaling complex, which consists of UNC-43/CaMKII, TIR-1/SARM adaptor protein and NSY-1/ASK1 MAPKKK, is localized to postsynaptic sites in the AWC axons for this lateral interaction. Here, we show that microtubule-based localization of the TIR-1 signaling complex to the synapses regulates AWC asymmetry. Similar to unc-43, tir-1 and nsy-1 loss-of-function mutants, specific disruption of microtubules in AWC by nocodazole generates two AWC(ON) neurons. Reduced localization of UNC-43, TIR-1 and NSY-1 proteins in the AWC axons strongly correlates with the 2AWC(ON) phenotype in nocodazole-treated animals. We identified kinesin motor unc-104/kif1a mutants for enhancement of the 2AWC(ON) phenotype of a hypomorphic tir-1 mutant. Mutations in unc-104, like microtubule depolymerization, lead to a reduced level of UNC-43, TIR-1 and NSY-1 proteins in the AWC axons. In addition, dynamic transport of TIR-1 in the AWC axons is dependent on unc-104, the primary motor required for the transport of presynaptic vesicles. Furthermore, unc-104 acts non-cell autonomously in the AWC(ON) neuron to regulate the AWC(OFF) identity. Together, these results suggest a model in which UNC-104 may transport some unknown presynaptic factor(s) in the future AWC(ON) cell that non-cell autonomously control the trafficking of the TIR-1 signaling complex to postsynaptic regions of the AWC axons to regulate the AWC(OFF) identity.