A new model for random X chromosome inactivation

X chromosome inactivation (XCI) reduces the number of actively transcribed X chromosomes to one per diploid set of autosomes, allowing for dosage equality between the sexes. In eutherians, the inactive X chromosome in XX females is randomly selected. The mechanisms for determining both how many X chromosomes are present and which to inactivate are unknown. To understand these mechanisms, researchers have created X chromosome mutations and transgenes. Here, we introduce a new model of X chromosome inactivation that aims to account for the findings in recent studies, to promote a re-interpretation of existing data and to direct future experiments

Detecting broad domains and narrow peaks in ChIP-seq data with hiddenDomains

Abstract Background Correctly identifying genomic regions enriched with histone modifications and transcription factors is key to understanding their regulatory and developmental roles. Conceptually, these regions are divided into two categories, narrow peaks and broad domains, and different algorithms are used to identify each one. Datasets that span these two categories are often analyzed with a single program for peak calling combined with an ad hoc method for domains. Results We developed hiddenDomains, which identifies both peaks and domains, and compare it to the leading algorithms using H3K27me3, H3K36me3, GABP, ESR1 and FOXA ChIP-seq datasets. The output from the programs was compared to qPCR-validated enriched and depleted sites, predicted transcription factor binding sites, and highly-transcribed gene bodies. With every method, hiddenDomains, performed as well as, if not better than algorithms dedicated to a specific type of analysis. Conclusions hiddenDomains performs as well as the best domain and peak calling algorithms, making it ideal for analyzing ChIP-seq datasets, especially those that contain a mixture of peaks and domains

Long Noncoding RNA Moderates MicroRNA Activity to Maintain Self-Renewal in Embryonic Stem Cells

Of the thousands of long noncoding RNAs expressed in embryonic stem cells (ESCs), few have known roles and fewer have been functionally implicated in the regulation of self-renewal and pluripotency, or the reprogramming of somatic cells to the pluripotent state. In ESCs, Cyrano is a stably expressed long intergenic noncoding RNA with no previously assigned role. We demonstrate that Cyrano contributes to ESC maintenance, as its depletion results in the loss of hallmarks of self-renewal. Delineation of Cyrano's network through transcriptomics revealed widespread effects on signaling pathways and gene expression networks that contribute to ESC maintenance. Cyrano shares unique sequence complementarity with the differentiation-associated microRNA, mir-7, and mir-7 overexpression reduces expression of a key self-renewal factor to a similar extent as Cyrano knockdown. This suggests that Cyrano functions to restrain the action of mir-7. Altogether, we provide a view into the multifaceted function of Cyrano in ESC maintenance

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

Abstract 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

Transcription precedes loss of Xist coating and depletion of H3K27me3 during X-chromosome reprogramming in the mouse inner cell mass

Repression of Xist RNA expression is considered a prerequisite to reversal of X-chromosome inactivation (XCI) in the mouse inner cell mass (ICM), and reactivation of X-linked genes is thought to follow loss of Xist RNA coating and heterochromatic markers of inactivation, such as methylation of histone H3. We analyzed X-chromosome activity in developing ICMs and show that reactivation of gene expression from the inactive-X initiates in the presence of Xist coating and H3K27me3. Furthermore, depletion of Xist RNA coating through forced upregulation of NANOG does not result in altered reactivation kinetics. Taken together, our observations suggest that in the ICM, X-linked gene transcription and Xist coating are uncoupled. These data fundamentally alter our perception of the reactivation process and support the existence of a mechanism to reactivate Xp-linked genes in the ICM that operates independently of loss of Xist RNA and H3K27me3 from the imprinted inactive-X

Evidence of Xist RNA-independent initiation of mouse imprinted X-chromosome inactivation

SummaryXX female mammals undergo transcriptional silencing of most genes on one of their two X-chromosomes to equalize X-linked gene dosage with XY males in a process referred to as X-chromosome inactivation (XCI). XCI is a paradigm of epigenetic regulation1. Once enacted in individual cells of the early female embryo, XCI is stably transmitted such that most descendant cells maintain silencing of that X-chromosome2. In eutherian mammals, XCI is thought to be triggered by the expression of the non-coding Xist RNA from the future inactive-X (Xi)3,4,5; Xist RNA in turn is proposed to recruit protein complexes that bring about heterochromatinization of the Xi6,7. Here we test whether imprinted XCI, which results in preferential inactivation of the paternal X-chromosome (Xp), occurs in mouse embryos inheriting an Xp lacking Xist. We find that silencing of Xp-linked genes can initiate in the absence of paternal Xist; Xist is, however, required to stabilize silencing of the Xp. Xp-linked gene silencing associated with mouse imprinted XCI, therefore, can initiate in the embryo independently of Xist RNA

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

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

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

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