RBBP4 dysfunction reshapes the genomic landscape of H3K27 methylation and acetylation and disrupts gene expression.

RBBP4 is a subunit of the chromatin remodeling complexes known as Polycomb repressive complex 2 and histone deacetylase 1/2-containing complexes. These complexes are responsible for histone H3 lysine 27 methylation and deacetylation, respectively. How RBBP4 modulates the functions of these complexes remains largely unknown. We generated viable Rbbp4 mutant alleles in mouse embryonic stem cell lines by CRISPR-Cas9. The mutations disrupted Polycomb repressive complex 2 assembly and H3K27me3 establishment on target chromatin and altered histone H3 lysine 27 acetylation genome wide. Moreover, Rbbp4 mutant cells underwent dramatic changes in transcriptional profiles closely tied to the deregulation of H3K27ac. The alteration of H3K27ac due to RBBP4 dysfunction occurred on numerous cis-regulatory elements, especially putative enhancers. These data suggest that RBBP4 plays a central role in regulating histone H3 lysine 27 methylation and acetylation to modulate gene expression.

EZH2 variants differentially regulate polycomb repressive complex 2 in histone methylation and cell differentiation.

BACKGROUND: Polycomb repressive complex 2 (PRC2) is responsible for establishing and maintaining histone H3K27 methylation during cell differentiation and proliferation. H3K27 can be mono-, di-, or trimethylated, resulting in differential gene regulation. However, it remains unknown how PRC2 specifies the degree and biological effects of H3K27 methylation within a given cellular context. One way to determine PRC2 specificity may be through alternative splicing of Ezh2, PRC2's catalytic subunit, during cell differentiation and tissue maturation. RESULTS: We fully characterized the alternative splicing of Ezh2 in somatic cells and male germ cells and found that Ezh's exon 14 was differentially regulated during mitosis and meiosis. The Ezh2 isoform containing exon 14 (ex14-Ezh2) is upregulated during cell cycle progression, consistent with a role in maintaining H3K27 methylation during chromatin replication. In contrast, the isoform lacking exon 14 (ex14D-Ezh2) was almost exclusively present in spermatocytes when new H3K27me2 is established during meiotic differentiation. Moreover, Ezh2's transcript is normally controlled by E2F transcription activators, but in spermatocytes, Ezh2's transcription is controlled by the meiotic regulator MYBL1. Compared to ex14-EZH2, ex14D-EZH2 has a diminished efficiency for catalyzing H3K27me3 and promotes embryonic stem cell differentiation. CONCLUSIONS: Ezh2's expression is regulated at transcriptional and post-transcriptional levels in a cellular context-dependent manner. EZH2 variants determine functional specificity of PRC2 in histone methylation during cell proliferation and differentiation.

EZH1 in germ cells safeguards the function of PRC2 during spermatogenesis.

We previously reported the requirement of Polycomb Repressive Complex 2 (PRC2) for spermatogenesis through transcriptional repression of somatic genes and meiosis-specific genes. To characterize how PRC2's two methyltransferase subunits, EZH1 and EZH2, regulate histone H3 lysine 27 (H3K27) methylation during germ cell development, we generated mouse models with a germline ablation of EZH1 and/or EHZ2. Only the combined loss of EZH1 and EZH2 caused a depletion of global H3K27me3 marks and meiotic arrest in spermatocytes. Genome-wide analysis of H3K27me3 in spermatogenic cells revealed that a noncanonical EZH1-PRC2 could establish and maintain this histone mark on somatic genes and certain meiotic genes. Consistent with it having active enhancers in testis, Ezh1 was not only abundant in highly differentiated spermatocytes but also in actively proliferating progenitor and stem germ cells. Taken together, our findings suggest that the expression level of Ezh1 determines the restoration of H3K27 methylation in the absence of the canonical EZH2-PRC2.

Histone H3.3 maintains genome integrity during mammalian development.

Histone H3.3 is a highly conserved histone H3 replacement variant in metazoans and has been implicated in many important biological processes, including cell differentiation and reprogramming. Germline and somatic mutations in H3.3 genomic incorporation pathway components or in H3.3 encoding genes have been associated with human congenital diseases and cancers, respectively. However, the role of H3.3 in mammalian development remains unclear. To address this question, we generated H3.3-null mouse models through classical genetic approaches. We found that H3.3 plays an essential role in mouse development. Complete depletion of H3.3 leads to developmental retardation and early embryonic lethality. At the cellular level, H3.3 loss triggers cell cycle suppression and cell death. Surprisingly, H3.3 depletion does not dramatically disrupt gene regulation in the developing embryo. Instead, H3.3 depletion causes dysfunction of heterochromatin structures at telomeres, centromeres, and pericentromeric regions of chromosomes, leading to mitotic defects. The resulting karyotypical abnormalities and DNA damage lead to p53 pathway activation. In summary, our results reveal that an important function of H3.3 is to support chromosomal heterochromatic structures, thus maintaining genome integrity during mammalian development.

A survey of imprinted gene expression in mouse trophoblast stem cells.

Several hundred mammalian genes are expressed preferentially from one parental allele as the result of a process called genomic imprinting. Genomic imprinting is prevalent in extra-embryonic tissue, where it plays an essential role during development. Here, we profiled imprinted gene expression via RNA-Seq in a panel of six mouse trophoblast stem lines, which are ex vivo derivatives of a progenitor population that gives rise to the placental tissue of the mouse. We found evidence of imprinted expression for 48 genes, 31 of which had been described previously as imprinted and 17 of which we suggest as candidate imprinted genes. An equal number of maternally and paternally biased genes were detected. On average, candidate imprinted genes were more lowly expressed and had weaker parent-of-origin biases than known imprinted genes. Several known and candidate imprinted genes showed variability in parent-of-origin expression bias between the six trophoblast stem cell lines. Sixteen of the 48 known and candidate imprinted genes were previously or newly annotated noncoding RNAs and six encoded for a total of 60 annotated microRNAs. Pyrosequencing across our panel of trophoblast stem cell lines returned levels of imprinted expression that were concordant with RNA-Seq measurements for all eight genes examined. Our results solidify trophoblast stem cells as a cell culture-based experimental model to study genomic imprinting, and provide a quantitative foundation upon which to delineate mechanisms by which the process is maintained in the mouse.

Coexistent ARID1A-PIK3CA mutations promote ovarian clear-cell tumorigenesis through pro-tumorigenic inflammatory cytokine signalling.

Ovarian clear-cell carcinoma (OCCC) is an aggressive form of ovarian cancer with high ARID1A mutation rates. Here we present a mutant mouse model of OCCC. We find that ARID1A inactivation is not sufficient for tumour formation, but requires concurrent activation of the phosphoinositide 3-kinase catalytic subunit, PIK3CA. Remarkably, the mice develop highly penetrant tumours with OCCC-like histopathology, culminating in haemorrhagic ascites and a median survival period of 7.5 weeks. Therapeutic treatment with the pan-PI3K inhibitor, BKM120, prolongs mouse survival by inhibiting the tumour cell growth. Cross-species gene expression comparisons support a role for IL-6 inflammatory cytokine signalling in OCCC pathogenesis. We further show that ARID1A and PIK3CA mutations cooperate to promote tumour growth through sustained IL-6 overproduction. Our findings establish an epistatic relationship between SWI/SNF chromatin remodelling and PI3K pathway mutations in OCCC and demonstrate that these pathways converge on pro-tumorigenic cytokine signalling. We propose that ARID1A protects against inflammation-driven tumorigenesis.

Repression of the soma-specific transcriptome by Polycomb-repressive complex 2 promotes male germ cell development.

Polycomb-repressive complex 2 (PRC2) catalyzes the methylation of histone H3 Lys27 (H3K27) and functions as a critical epigenetic regulator of both stem cell pluripotency and somatic differentiation, but its role in male germ cell development is unknown. Using conditional mutagenesis to remove the core PRC2 subunits EED and SUZ12 during male germ cell development, we identified a requirement for PRC2 in both mitotic and meiotic germ cells. We observed a paucity of mutant spermatogonial stem cells (SSCs), which appears independent of repression of the known cell cycle inhibitors Ink4a/Ink4b/Arf. Moreover, mutant spermatocytes exhibited ectopic expression of somatic lamins and an abnormal distribution of SUN1 proteins on the nuclear envelope. These defects were coincident with abnormal chromosome dynamics, affecting homologous chromosome pairing and synapsis. We observed acquisition of H3K27me3 on stage-specific genes during meiotic progression, indicating a requirement for PRC2 in regulating the meiotic transcriptional program. Together, these data demonstrate that transcriptional repression of soma-specific genes by PRC2 facilitates homeostasis and differentiation during mammalian spermatogenesis.

KDM6 demethylase independent loss of histone H3 lysine 27 trimethylation during early embryonic development.

The early mammalian embryo utilizes histone H3 lysine 27 trimethylation (H3K27me3) to maintain essential developmental genes in a repressive chromatin state. As differentiation progresses, H3K27me3 is removed in a distinct fashion to activate lineage specific patterns of developmental gene expression. These rapid changes in early embryonic chromatin environment are thought to be dependent on H3K27 demethylases. We have taken a mouse genetics approach to remove activity of both H3K27 demethylases of the Kdm6 gene family, Utx (Kdm6a, X-linked gene) and Jmjd3 (Kdm6b, autosomal gene). Male embryos null for active H3K27 demethylation by the Kdm6 gene family survive to term. At mid-gestation, embryos demonstrate proper patterning and activation of Hox genes. These male embryos retain the Y-chromosome UTX homolog, UTY, which cannot demethylate H3K27me3 due to mutations in catalytic site of the Jumonji-C domain. Embryonic stem (ES) cells lacking all enzymatic KDM6 demethylation exhibit a typical decrease in global H3K27me3 levels with differentiation. Retinoic acid differentiations of these ES cells demonstrate loss of H3K27me3 and gain of H3K4me3 to Hox promoters and other transcription factors, and induce expression similar to control cells. A small subset of genes exhibit decreased expression associated with reduction of promoter H3K4me3 and some low-level accumulation of H3K27me3. Finally, Utx and Jmjd3 mutant mouse embryonic fibroblasts (MEFs) demonstrate dramatic loss of H3K27me3 from promoters of several Hox genes and transcription factors. Our results indicate that early embryonic H3K27me3 repression can be alleviated in the absence of active demethylation by the Kdm6 gene family.

Evidence for local regulatory control of escape from imprinted X chromosome inactivation.

X chromosome inactivation (XCI) is an epigenetic process that almost completely inactivates one of two X chromosomes in somatic cells of mammalian females. A few genes are known to escape XCI and the mechanism for this escape remains unclear. Here, using mouse trophoblast stem (TS) cells, we address whether particular chromosomal interactions facilitate escape from imprinted XCI. We demonstrate that promoters of genes escaping XCI do not congregate to any particular region of the genome in TS cells. Further, the escape status of a gene was uncorrelated with the types of genomic features and gene activity located in contacted regions. Our results suggest that genes escaping imprinted XCI do so by using the same regulatory sequences as their expressed alleles on the active X chromosome. We suggest a model where regulatory control of escape from imprinted XCI is mediated by genomic elements located in close linear proximity to escaping genes.

fourSig: a method for determining chromosomal interactions in 4C-Seq data.

The ability to correlate chromosome conformation and gene expression gives a great deal of information regarding the strategies used by a cell to properly regulate gene activity. 4C-Seq is a relatively new and increasingly popular technology where the set of genomic interactions generated by a single point in the genome can be determined. 4C-Seq experiments generate large, complicated data sets and it is imperative that signal is properly distinguished from noise. Currently, there are a limited number of methods for analyzing 4C-Seq data. Here, we present a new method, fourSig, which in addition to being precise and simple to use also includes a new feature that prioritizes detected interactions. Our results demonstrate the efficacy of fourSig with previously published and novel 4C-Seq data sets and show that our significance prioritization correlates with the ability to reproducibly detect interactions among replicates.

Site-specific silencing of regulatory elements as a mechanism of X inactivation.

The inactive X chromosome's (Xi) physical territory is microscopically devoid of transcriptional hallmarks and enriched in silencing-associated modifications. How these microscopic signatures relate to specific Xi sequences is unknown. Therefore, we profiled Xi gene expression and chromatin states at high resolution via allele-specific sequencing in mouse trophoblast stem cells. Most notably, X-inactivated transcription start sites harbored distinct epigenetic signatures relative to surrounding Xi DNA. These sites displayed H3-lysine27-trimethylation enrichment and DNaseI hypersensitivity, similar to autosomal Polycomb targets, yet excluded Pol II and other transcriptional hallmarks, similar to nontranscribed genes. CTCF bound X-inactivated and escaping genes, irrespective of measured chromatin boundaries. Escape from X inactivation occurred within, and X inactivation was maintained exterior to, the area encompassed by Xist in cells subject to imprinted and random X inactivation. The data support a model whereby inactivation of specific regulatory elements, rather than a simple chromosome-wide separation from transcription machinery, governs gene silencing over the Xi.

Failure of extra-embryonic progenitor maintenance in the absence of dosage compensation.

Proper regulation of X-linked gene expression, termed dosage compensation, is required for the normal development of mammalian embryos. Through the process of X chromosome inactivation (XCI), somatic cells of mammalian females inactivate one of their two X chromosomes in order to balance X-linked gene dosage with their male counterparts. The process of XCI is dependent upon the long non-coding RNA Xist, which is expressed from and coats the inactivated X chromosome (Xi) in cis. During mouse embryogenesis, imprinted XCI inactivates the paternally inherited X chromosome (Xp) within the extra-embryonic lineages. Consequently, females harboring a paternally derived Xist mutation (X/X(Xist-)) die owing to failure of imprinted XCI and, presumably, poor trophoblast development. Here, we investigate the consequence of two active X chromosomes in the extra-embryonic ectoderm (ExE) of X/X(Xist-) female embryos. At embryonic day (E) 6.5, we find that the X/X(Xist-) ExE lacks the transcriptional regulator CDX2, a factor required to maintain the ExE in a progenitor state. In addition, spongiotrophoblast progenitors are not maintained. Surprisingly, we observe evidence of an Xi in a subpopulation of X/X(Xist-) ExE cells. We demonstrate further that trophectodermal stem cells derived from X/X(Xist-) embryos completely reverse normal imprinted XCI patterns. Taken together, our data suggest that, much like in the cells of the epiblast, the initial imprint that establishes imprinted XCI is probably erased in ExE cells. Conversely, unlike the epiblast, in which XCI is not required for progenitor cell maintenance, we demonstrate that dosage compensation is indispensable for the maintenance of trophoblast progenitors.

Nucleolar association and transcriptional inhibition through 5S rDNA in mammals.

Changes in the spatial positioning of genes within the mammalian nucleus have been associated with transcriptional differences and thus have been hypothesized as a mode of regulation. In particular, the localization of genes to the nuclear and nucleolar peripheries is associated with transcriptional repression. However, the mechanistic basis, including the pertinent cis- elements, for such associations remains largely unknown. Here, we provide evidence that demonstrates a 119 bp 5S rDNA can influence nucleolar association in mammals. We found that integration of transgenes with 5S rDNA significantly increases the association of the host region with the nucleolus, and their degree of association correlates strongly with repression of a linked reporter gene. We further show that this mechanism may be functional in endogenous contexts: pseudogenes derived from 5S rDNA show biased conservation of their internal transcription factor binding sites and, in some cases, are frequently associated with the nucleolus. These results demonstrate that 5S rDNA sequence can significantly contribute to the positioning of a locus and suggest a novel, endogenous mechanism for nuclear organization in mammals.

Differentiation-driven nucleolar association of the mouse imprinted Kcnq1 locus.

The organization of the genome within the mammalian nucleus is nonrandom, with physiologic processes often concentrated in specific three-dimensional domains. This organization may be functionally related to gene regulation and, as such, may play a role in normal development and human disease processes. However, the mechanisms that participate in nuclear organization are poorly understood. Here, we present data characterizing localization of the imprinted Kcnq1 alleles. We show that nucleolar association of the paternal allele (1) is stimulated during the differentiation of trophoblast stem cells, (ii) is dependent upon the Kcnq1ot1 noncoding RNA, (3) does not require polycomb repressive complex 2, and (4) is not sufficient to preclude transcription of imprinted genes. Although nucleolar positioning has been proposed as a mechanism to related to gene silencing, we find that silencing and perinucleolar localization through the Kcnq1ot1 noncoding RNA are separable events.

Nodal signaling regulates the bone morphogenic protein pluripotency pathway in mouse embryonic stem cells.

Members of the transforming growth factor-beta superfamily play essential roles in both the pluripotency and differentiation of embryonic stem (ES) cells. Although bone morphogenic proteins (BMPs) maintain pluripotency of undifferentiated mouse ES cells, the role of autocrine Nodal signaling is less clear. Pharmacological, molecular, and genetic methods were used to further understand the roles and potential interactions of these pathways. Treatment of undifferentiated ES cells with SB431542, a pharmacological inhibitor of Smad2 signaling, resulted in a rapid reduction of phosphorylated Smad2 and altered the expression of several putative downstream targets. Unexpectedly, inhibition of the Nodal signaling pathway resulted in enhanced BMP signaling, as assessed by Smad1/5 phosphorylation. SB431542-treated cells also demonstrated significant induction of the Id genes, which are known direct targets of BMP signaling and important factors in ES cell pluripotency. Inhibition of BMP signaling decreased the SB431542-mediated phosphorylation of Smad1/5 and induction of Id genes, suggesting that BMP signaling is necessary for some Smad2-mediated activity. Because Smad7, a known inhibitory factor to both Nodal and BMP signaling, was down-regulated following inhibition of Nodal-Smad2 signaling, the contribution of Smad7 to the cross-talk between the transforming growth factor-beta pathways in ES cells was examined. Biochemical manipulation of Smad7 expression, through shRNA knockdown or inducible gene expression, significantly reduced the SB431542-mediated phosphorylation of Smad1/5 and induction of the Id genes. We conclude that autocrine Nodal signaling in undifferentiated mouse ES cells modulates the vital pluripotency pathway of BMP signaling.

Polycomb repressive complex 2 is dispensable for maintenance of embryonic stem cell pluripotency.

Polycomb repressive complex 2 (PRC2) methylates histone H3 tails at lysine 27 and is essential for embryonic development. The three core components of PRC2, Eed, Ezh2, and Suz12, are also highly expressed in embryonic stem (ES) cells, where they are postulated to repress developmental regulators and thereby prevent differentiation to maintain the pluripotent state. We performed gene expression and chimera analyses on low- and high-passage Eed(null) ES cells to determine whether PRC2 is required for the maintenance of pluripotency. We report here that although developmental regulators are overexpressed in Eed(null) ES cells, both low- and high-passage cells are functionally pluripotent. We hypothesize that they are pluripotent because they maintain expression of critical pluripotency factors. Given that EED is required for stability of EZH2, the catalytic subunit of the complex, these data suggest that PRC2 is not necessary for the maintenance of the pluripotent state in ES cells. We propose a positive-only model of embryonic stem cell maintenance, where positive regulation of pluripotency factors is sufficient to mediate stem cell pluripotency. Disclosure of potential conflicts of interest is found at the end of this article.

Molecular and functional mapping of EED motifs required for PRC2-dependent histone methylation.

Polycomb group proteins represent a conserved family of developmental regulators that mediate heritable transcriptional silencing by modifying chromatin states. One Polycomb group complex, the PRC2 complex, is composed of several proteins, including the histone H3 lysine 27 (H3K27) methyltransferase enhancer of zeste homolog 2 and the WD-repeat protein embryonic ectoderm development (EED). Histone H3K27 can be monomethylated (H3K27me1), dimethylated (H3K27me2), or trimethylated (H3K27me3). However, it remains unclear what regulates the number of methyl groups added to H3K27 in a particular nucleosome. In mammalian cells, EED is present as four distinct isoforms, which are believed to be produced by utilizing four distinct, in-frame translation start sites in a common Eed mRNA. A mutation that disables all four EED isoforms produces defects in H3K27 methylation [Montgomery, N.D., Yee, D., Chen, A., Kalantry, S., Chamberlain, S.J., Otte, A.P. & Magnuson, T. (2005). The murine polycomb group protein Eed is required for global histone H3 lysine-27 methylation. Curr. Biol., 15, 942-947]. To assess the roles of individual EED isoforms in H3K27 methylation, we first characterized three of the four EED isoform start sites and then demonstrated that individual isoforms are not necessary for H3K27me1, H3K27me2, or H3K27me3. Instead, we show that the core WD-40 motifs and the histone-binding region of EED alone are sufficient for the generation of all three marks, demonstrating that EED isoforms do not control the number of methyl groups added to H3K27.

Reduced maternal expression of adrenomedullin disrupts fertility, placentation, and fetal growth in mice.

Adrenomedullin (AM) is a multifunctional peptide vasodilator that is essential for life. Plasma AM expression dramatically increases during pregnancy, and alterations in its levels are associated with complications of pregnancy including fetal growth restriction (FGR) and preeclampsia. Using AM+/- female mice with genetically reduced AM expression, we demonstrate that fetal growth and placental development are seriously compromised by this modest decrease in expression. AM+/- female mice had reduced fertility characterized by FGR. The incidence of FGR was also influenced by the genotype of the embryo, since AM-/- embryos were more often affected than either AM+/- or AM+/+ embryos. We demonstrate that fetal trophoblast cells and the maternal uterine wall have coordinated and localized increases in AM gene expression at the time of implantation. Placentas from growth-restricted embryos showed defects in trophoblast cell invasion, similar to defects that underlie human preeclampsia and placenta accreta. Our data provide a genetic in vivo model to implicate both maternal and, to a lesser extent, embryonic levels of AM in the processes of implantation, placentation, and subsequent fetal growth. This study provides the first genetic evidence to our knowledge to suggest that a modest reduction in human AM expression during pregnancy may have an unfavorable impact on reproduction.

A novel mouse Smad4 mutation reduces protein stability and wild-type protein levels.

Smad4 is a key signal transducer of the transforming growth factor-beta (TGF-beta) superfamily of growth factors that are critical regulators of embryonic patterning and adult tissue homeostasis. The biological activity of the TGF-beta signaling is tightly controlled at multiple levels, including the abundance of SMAD4 proteins. We previously recovered a novel allele of Smad4 in a gene-based screen in N-ethyl-N-nitrosourea (ENU)-mutagenized mouse embryonic stem cells. The mutation resulted in an unstable truncated protein that is degraded through proteasomal pathways. In the heterozygous state, this allele acts in a dominant negative fashion to reduce the wild-type protein level as well as signaling output. Biochemical characterization indicated that the truncated protein is able to form a complex with the wild-type protein, thus targeting it for proteasomal degradation as well. Phenotypic analyses of the heterozygous animals provided insight into the threshold requirement of Smad4-dependent signaling in vivo.

The Polycomb group protein Eed protects the inactive X-chromosome from differentiation-induced reactivation.

The Polycomb group (PcG) encodes an evolutionarily conserved set of chromatin-modifying proteins that are thought to maintain cellular transcriptional memory by stably silencing gene expression. In mouse embryos that are mutated for the PcG protein Eed, X-chromosome inactivation (XCI) is not stably maintained in extra-embryonic tissues. Eed is a component of a histone-methyltransferase complex that is thought to contribute to stable silencing in undifferentiated cells due to its enrichment on the inactive X-chromosome in cells of the early mouse embryo and in stem cells of the extra-embryonic trophectoderm lineage. Here, we demonstrate that the inactive X-chromosome in Eed(-/-) trophoblast stem cells and in cells of the trophectoderm-derived extra-embryonic ectoderm in Eed(-/-) embryos remain transcriptionally silent, despite lacking the PcG-mediated histone modifications that normally characterize the facultative heterochromatin of the inactive X-chromosome. Whereas undifferentiated Eed(-/-) trophoblast stem cells maintained XCI, reactivation of the inactive X-chromosome occurred when these cells were differentiated. These results indicate that PcG complexes are not necessary to maintain transcriptional silencing of the inactive X-chromosome in undifferentiated stem cells. Instead, PcG proteins seem to propagate cellular memory by preventing transcriptional activation of facultative heterochromatin during differentiation.