Antoine H. F. M. Peters

Oncogene-induced senescence as an initial barrier in lymphoma development

Acute induction of oncogenic Ras provokes cellular senescence involving the retinoblastoma (Rb) pathway, but the tumour suppressive potential of senescence in vivo remains elusive. Recently, Rb-mediated silencing of growth-promoting genes by heterochromatin formation associated with methylation of histone H3 lysine 9 (H3K9me) was identified as a critical feature of cellular senescence, which may depend on the histone methyltransferase Suv39h1. Here we show that Eµ-N-Ras transgenic mice harbouring targeted heterozygous lesions at the Suv39h1, or the p53 locus for comparison, succumb to invasive T-cell lymphomas that lack expression of Suv39h1 or p53, respectively. By contrast, most N-Ras-transgenic wild-type (‘control’) animals develop a non-lymphoid neoplasia significantly later. Proliferation of primary lymphocytes is directly stalled by a Suv39h1-dependent, H3K9me-related senescent growth arrest in response to oncogenic Ras, thereby cancelling lymphomagenesis at an initial step. Suv39h1-deficient lymphoma cells grow rapidly but, unlike p53-deficient cells, remain highly susceptible to adriamycin-induced apoptosis. In contrast, only control, but not Suv39h1-deficient or p53-deficient, lymphomas senesce after drug therapy when apoptosis is blocked. These results identify H3K9me-mediated senescence as a novel Suv39h1-dependent tumour suppressor mechanism whose inactivation permits the formation of aggressive but apoptosis-competent lymphomas in response to oncogenic Ras.

Repressive and active histone methylation mark distinct promoters in human and mouse spermatozoa

In higher eukaryotes, histone methylation is involved in maintaining cellular identity during somatic development. As most nucleosomes are replaced by protamines during spermatogenesis, it is unclear whether histone modifications function in paternal transmission of epigenetic information. Here we show that two modifications important for Trithorax- and Polycomb-mediated gene regulation have methylation-specific distributions at regulatory regions in human spermatozoa. Histone H3 Lys4 dimethylation (H3K4me2) marks genes that are relevant in spermatogenesis and cellular homeostasis. In contrast, histone H3 Lys27 trimethylation (H3K27me3) marks developmental regulators in sperm, as in somatic cells. However, nucleosomes are only moderately retained at regulatory regions in human sperm. Nonetheless, genes with extensive H3K27me3 coverage around transcriptional start sites in particular tend not to be expressed during male and female gametogenesis or in preimplantation embryos. Promoters of orthologous genes are similarly modified in mouse spermatozoa. These data are compatible with a role for Polycomb in repressing somatic determinants across generations, potentially in a variegating manner.

PRC1 and Suv39h specify parental asymmetry at constitutive heterochromatin in early mouse embryos

In eukaryotes, Suv39h H3K9 trimethyltransferases are required for pericentric heterochromatin formation and function. In early mouse preimplantation embryos, however, paternal pericentric heterochromatin lacks Suv39h-mediated H3K9me3 and downstream marks. Here we demonstrate Ezh2-independent targeting of maternally provided polycomb repressive complex 1 (PRC1) components to paternal heterochromatin. In Suv39h2 maternally deficient zygotes, PRC1 also associates with maternal heterochromatin lacking H3K9me3, thereby revealing hierarchy between repressive pathways. In Rnf2 maternally deficient zygotes, the PRC1 complex is disrupted, and levels of pericentric major satellite transcripts are increased at the paternal but not the maternal genome. We conclude that in early embryos, Suv39h-mediated H3K9me3 constitutes the dominant maternal transgenerational signal for pericentric heterochromatin formation. In absence of this signal, PRC1 functions as the default repressive back-up mechanism. Parental epigenetic asymmetry, also observed along cleavage chromosomes, is resolved by the end of the 8-cell stage—concurrent with blastomere polarization—marking the end of the maternal-to-embryonic transition.

Polycomb Group Proteins Ezh2 and Rnf2 Direct Genomic Contraction and Imprinted Repression in Early Mouse Embryos

Genomic imprinting regulates parental-specific expression of particular genes and is required for normal mammalian development. How imprinting is established during development is, however, largely unknown. To address this question, we studied the mouse Kcnq1 imprinted cluster at which paternal-specific silencing depends on expression of the noncoding RNA Kcnq1ot1. We show that Kcnq1ot1 is expressed from the zygote stage onward and rapidly associates with chromatin marked by Polycomb group (PcG) proteins and repressive histone modifications, forming a discrete repressive nuclear compartment devoid of RNA polymerase II, a configuration also observed at the Igf2r imprinted cluster. In this compartment, the paternal Kcnq1 cluster exists in a three-dimensionally contracted state. In vivo the PcG proteins Ezh2 and Rnf2 are independently required for genomic contraction and imprinted silencing. We propose that the formation of a parental-specific higher-order chromatin organization renders imprint clusters competent for monoallelic silencing and assign a central role to PcG proteins in this process.

In utero undernourishment perturbs the adult sperm methylome and intergenerational metabolism

Introduction The rapid global rise in metabolic disease suggests that nongenetic environmental factors contribute to disease risk. Early life represents a window of phenotypic plasticity important for adult metabolic health and that of future generations. Epigenetic inheritance has been implicated in the paternal transmission of environmentally induced phenotypes, but the mechanisms responsible remain unknown. In utero undernourishment alters the adult germ cell methylome. Undernourishment during PGC reprogramming results in hypomethylation of discrete loci in adult sperm. These regions are enriched in nucleosomes and are low-methylated regions. Although partially resistant to blastocyst reprogramming, differential methylation does not persist in the next generation. However, dysregulated expression of genes neighboring DMRs is observed in F2 offspring. Rationale We investigated the role of DNA methylation in epigenetic inheritance in an established murine model of intergenerational developmental programming. F1 offspring of undernourished dams (UN) have low birth weight and multiple metabolic defects. Metabolic phenotypic inheritance to the F2 generation is observed through the paternal line, even though the F1 mice did not experience postnatal environmental perturbation. The timing of nutritional restriction coincides with methylation reacquisition in F1 male primordial germ cells (PGCs). Therefore, we assessed F1 sperm whole-genome methylation using immunoprecipitation of methylated DNA, combined with high-throughput sequencing, followed by independent validation. We characterized the regions susceptible to methylation change and investigated the legacy of such methylation change in the phenotypic development of the next generation. Results In UN mice, 111 regions are hypomethylated relative to control sperm, and these changes are validated by bisulfite pyrosequencing. Methylation differences span multiple CpGs, with robust absolute changes of 10 to 30% (relative reduction ~50%). The absolute methylation level is consistent with differentially methylated regions (DMRs) being “low-methylated regions,” known to be enriched in regulatory elements. Indeed, luciferase assays suggest a role for these DMRs in transcriptional regulation. Hypomethylated DMRs are significantly depleted from coding and repetitive regions and enriched in intergenic regions and CpG islands. They are also enriched in nucleosome-retaining regions, which suggests that, at some loci, paternal germline hypomethylation induced by in utero undernutrition is transmitted in a chromatin context. DMRs are late to regain methylation in normal male PGCs. This may render them particularly susceptible to environmental perturbations that delay or impair remethylation in late gestation. Except for imprinted loci, gene-associated male germline methylation has generally been thought to be largely erased in the zygote,although recent studies suggest that resistance to reprogramming is more widespread. Indeed, 43% of hypomethylated DMRs persist and thus have the potential to affect development of the next generation. We show that differential methylation is lost in late-gestation F2 tissues, but considerable tissue-specific differences in expression of metabolic genes neighboring DMRs are present. Thus, it is unlikely that these expression changes are directly mediated by altered methylation; rather, the cumulative effects of dysregulated epigenetic patterns earlier in development may yield sustained alterations in chromatin architecture, transcriptional regulatory networks, differentiation, or tissue structure. Conclusion Prenatal undernutrition can compromise male germline epigenetic reprogramming and thus permanently alter DNA methylation in the sperm of adult offspring at regions resistant to zygotic reprogramming. However, persistence of altered DNA methylation into late-gestation somatic tissues of the subsequent generation is not observed. Nonetheless, alterations in gamete methylation may serve as a legacy of earlier developmental exposures and may contribute to the intergenerational transmission of environmentally induced disease. The nutritional sins of the mother… Prenatal exposures of a mother can affect the health of her offspring, but how? Radford et al. found that the male progeny of undernourished pregnant mice had altered DNA chemistry in their sperm. In addition, the offspring displayed compromised metabolic health. The specific affected genes not only lost DNA methylation but also lacked the normal sperm DNA packaging factors (protamines) and instead were enriched in nucleosomes. Thus, when subjected to a suboptimal prenatal environment, offspring feel the effects of the maternal assault. Science, this issue p. 10.1126/science.1255903 Prenatal assaults change DNA methylation and chromatin structure in sperm and affect offspring. [Also see Perspective by Susiarjo and Bartolomei] Adverse prenatal environments can promote metabolic disease in offspring and subsequent generations. Animal models and epidemiological data implicate epigenetic inheritance, but the mechanisms remain unknown. In an intergenerational developmental programming model affecting F2 mouse metabolism, we demonstrate that the in utero nutritional environment of F1 embryos alters the germline DNA methylome of F1 adult males in a locus-specific manner. Differentially methylated regions are hypomethylated and enriched in nucleosome-retaining regions. A substantial fraction is resistant to early embryo methylation reprogramming, which may have an impact on F2 development. Differential methylation is not maintained in F2 tissues, yet locus-specific expression is perturbed. Thus, in utero nutritional exposures during critical windows of germ cell development can impact the male germline methylome, associated with metabolic disease in offspring.

Chromosome-wide nucleosome replacement and H3.3 incorporation during mammalian meiotic sex chromosome inactivation

In mammalian males, the first meiotic prophase is characterized by formation of a separate chromatin domain called the sex body. In this domain, the X and Y chromosomes are partially synapsed and transcriptionally silenced, a process termed meiotic sex-chromosome inactivation (MSCI). Likewise, unsynapsed autosomal chromatin present during pachytene is also silenced (meiotic silencing of unsynapsed chromatin, MSUC). Although it is known that MSCI and MSUC are both dependent on histone H2A.X phosphorylation mediated by the kinase ATR, and cause repressive H3 Lys9 dimethylation, the mechanisms underlying silencing are largely unidentified. Here, we demonstrate an extensive replacement of nucleosomes within unsynapsed chromatin, depending on and initiated shortly after induction of MSCI and MSUC. Nucleosomal eviction results in the exclusive incorporation of the H3.3 variant, which to date has primarily been associated with transcriptional activity. Nucleosomal exchange causes loss and subsequent selective reacquisition of specific histone modifications. This process therefore provides a means for epigenetic reprogramming of sex chromatin presumably required for gene silencing in the male mammalian germ line.

Molecular determinants of nucleosome retention at CpG-rich sequences in mouse spermatozoa

In mammalian spermatozoa, most but not all of the genome is densely packaged by protamines. Here we reveal the molecular logic underlying the retention of nucleosomes in mouse spermatozoa, which contain only 1% residual histones. We observe high enrichment throughout the genome of nucleosomes at CpG-rich sequences that lack DNA methylation. Residual nucleosomes are largely composed of the histone H3.3 variant and are trimethylated at Lys4 of histone H3 (H3K4me3). Canonical H3.1 and H3.2 histones are also enriched at CpG-rich promoters marked by Polycomb-mediated H3K27me3, a modification predictive of gene repression in preimplantation embryos. Histone variant–specific nucleosome retention in sperm is strongly associated with nucleosome turnover in round spermatids. Our data show evolutionary conservation of the basic principles of nucleosome retention in mouse and human sperm, supporting a model of epigenetic inheritance by nucleosomes between generations.

Mechanisms of transcriptional repression by histone lysine methylation

During development, covalent modification of both, histones and DNA contribute to the specification and maintenance of cell identity. Repressive modifications are thought to stabilize cell type specific gene expression patterns, reducing the likelihood of reactivation of lineage-unrelated genes. In this report, we review the recent literature to deduce mechanisms underlying Polycomb and H3K9 methylation mediated repression, and describe the functional interplay with activating H3K4 methylation. We summarize recent data that indicate a close relationship between GC density of promoter sequences, transcription factor binding and the antagonizing activities of distinct epigenetic regulators such as histone methyltransferases (HMTs) and histone demethylases (HDMs). Subsequently, we compare chromatin signatures associated with different types of transcriptional outcomes from stable repression to highly dynamic regulated genes, strongly suggesting that the interplay of different epigenetic pathways is essential in defining specific types of heritable chromatin and associated transcriptional states.

A double-stranded RNA binding protein required for activation of repressed messages in mammalian germ cells.

Chromatin packaging in mammalian spermatozoa requires an ordered replacement of the somatic histones by two classes of spermatid-specific basic proteins, the transition proteins and the protamines. Temporal expression of transition proteins and protamines during spermatid differentiation is under translational control, and premature translation of protamine 1 leads to precocious nuclear condensation and sterility. We have previously suggested that the double-stranded (ds) RNA binding protein Prbp (encoded by the gene Tarbp2) functions as a translational regulator during mouse spermatogenesis. Here we show that Prbp is required for proper translational activation of the mRNAs encoding the protamines. We generated mice that carry a targeted disruption of Tarbp2 and determined that they were sterile and severely oligospermic. Using immunohistological analysis, we determined that the endogenous Prm2 mRNA and a reporter mRNA carrying protamine 1 translational-control elements were translated in a mosaic pattern. We showed that failure to synthesize the protamines resulted in delayed replacement of the transition proteins and subsequent failure of spermiation. The timing of Prbp expression suggests that it may function as a chaperone in the assembly of specific translationally regulated ribonucleoprotein particles.

Disruption of histone methylation in developing sperm impairs offspring health transgenerationally

Generations affected by histone changes Parent and even grandparent environmental exposure can transmit adverse health effects to offspring. The mechanism of transmission is unclear, but some studies have implicated variations in DNA methylation. In a mouse model, Siklenka et al. found that alterations in histone methylation during sperm formation in one generation leads to reduced survival and developmental abnormalities in three subsequent generations (see the Perspective by McCarrey). Although changes in DNA methylation were not observed, altered sperm RNA content and abnormal gene expression in offspring were measured. Thus, chromatin may act as a mediator of molecular memory in transgenerational inheritance. Science, this issue p. 10.1126/science.aab2006; see also p. 634 Overexpression of a histone demethylase in the mouse germ line reveals a mode of transgenerational epigenetic inheritance. [Also see Perspective by McCarrey] INTRODUCTION Despite the father transmitting half of the heritable information to the embryo, the focus of preconception health has been the mother. Paternal effects have been linked to complex diseases such as cancer, diabetes, and obesity. These diseases are increasing in prevalence at rates that cannot be explained by genetics alone and highlight the potential for disease transmission via nongenetic inheritance, through epigenetic mechanisms. Epigenetic mechanisms include DNA methylation, posttranslational modifications of histones, and noncoding RNA. Studies in humans and animals suggest that epigenetic mechanisms may serve in the transmission of environmentally induced phenotypic traits from the father to the offspring. Such traits have been associated with altered gene expression and tissue function in first and second offspring generations, a phenomenon known as intergenerational or transgenerational inheritance, respectively. The mechanisms underlying such paternal epigenetic transmission are unclear. RATIONALE Sperm formation involves rapid cell division and distinctive transcription programs, resulting in a motile cell with highly condensed chromatin. Within the highly compacted sperm nucleus, few histones are retained in a manner that suggests an influential role in development. Despite being the major focus of studies in epigenetic inheritance, the role of DNA methylation in paternal epigenetic inheritance is unresolved, as only minor changes in DNA methylation in sperm at CpG-enriched regions have been associated with transmission of environmentally induced traits. Instead, there may be a combination of molecular mechanisms underlying paternal transgenerational epigenetic inheritance involving changes in histone states and/or RNA in sperm. The function of sperm histones and their modifications in embryonic development, offspring health, and epigenetic inheritance is unknown. By overexpressing the human KDM1A histone lysine 4 demethylase during mouse spermatogenesis, we generated a mouse model producing spermatozoa with reduced H3K4me2 within the CpG islands of genes implicated in development, and we studied the development and fitness of the offspring. RESULTS Male transgenic offspring were bred with C57BL/6 females, generating the experimental heterozygous transgenic (TG) and nontransgenic (nonTG) brothers. Each generation from TG and nonTG animals (F1 to F3 in our transgenerational studies) was bred with C57BL/6 females, and the offspring (pups from generations F1 to F4) were analyzed for intergenerational and transgenerational effects. We found that KDM1A overexpression in one generation severely impaired development and survivability of offspring. These defects lasted for two subsequent generations in the absence of KDM1A germline expression. We characterized histone and DNA methylation states in the sperm of TG and nonTG sires. Overexpression of KDM1A was associated with a specific loss of H3K4me2 at more than 2300 genes, including many developmental regulatory genes. Unlike in other examples of paternal transgenerational inheritance, we observed no changes in sperm DNA methylation associated with primarily CpG-enriched regions. Instead, we measured robust and analogous changes in sperm RNA content of TG and nonTG descendants, as well as in their offspring, at the two-cell stage. These changes in expression and the phenotypic abnormalities observed in offspring correlated with altered histone methylation levels at genes in sperm. This study demonstrates that KDM1A activity during sperm development has major developmental consequences for offspring and implicates histone methylation and sperm RNA as potential mediators of transgenerational inheritance. Our data emphasize the complexity of transgenerational epigenetic inheritance likely involving multiple molecular factors, including the establishment of chromatin states in spermatogenesis and sperm-borne RNA. CONCLUSION Correct histone methylation during spermatogenesis is critical for offspring development and survival over multiple generations. These findings demonstrate the potential of histone methylation as a molecular mechanism underlying paternal epigenetic inheritance. Its modification by environmental influences may alter embryo development and complex disease transmission across generations. An essential next step is to establish functional links between environmental exposures, the composition of the sperm epigenome, and consequent altered gene expression and metabolic processes in offspring. Considering the mounting evidence, it may soon be reasonable to suggest that future fathers protect their sperm epigenome. Disruption of histone methylation in developing sperm by exposure to the KDM1A transgene in one generation severely impaired development and survivability of offspring. These defects were transgenerational and occurred in nonTG descendants in the absence of KDM1A germline expression. Developmental defects in offspring and embryos were associated with altered RNA expression in sperm and embryos. A father’s lifetime experiences can be transmitted to his offspring to affect health and development. However, the mechanisms underlying paternal epigenetic transmission are unclear. Unlike in somatic cells, there are few nucleosomes in sperm, and their function in epigenetic inheritance is unknown. We generated transgenic mice in which overexpression of the histone H3 lysine 4 (H3K4) demethylase KDM1A (also known as LSD1) during spermatogenesis reduced H3K4 dimethylation in sperm. KDM1A overexpression in one generation severely impaired development and survivability of offspring. These defects persisted transgenerationally in the absence of KDM1A germline expression and were associated with altered RNA profiles in sperm and offspring. We show that epigenetic inheritance of aberrant development can be initiated by histone demethylase activity in developing sperm, without changes to DNA methylation at CpG-rich regions.