Escape from X inactivation in mice and humans

A subset of X-linked genes escapes silencing by X inactivation and is expressed from both X chromosomes in mammalian females. Species-specific differences in the identity of these genes have recently been discovered, suggesting a role in the evolution of sex differences. Chromatin analyses have aimed to discover how genes remain expressed within a repressive environment

Sex-biased and parental allele-specific gene regulation by KDM6A.

BackgroundKDM6A is a demethylase encoded by a gene with female-biased expression due to escape from X inactivation. Its main role is to facilitate gene expression through removal of the repressive H3K27me3 mark, with evidence of some additional histone demethylase-independent functions. KDM6A mutations have been implicated in congenital disorders such as Kabuki Syndrome, as well as in sex differences in cancer.MethodsKdm6a was knocked out using CRISPR/Cas9 gene editing in F1 male and female mouse embryonic stem cells (ES) derived from reciprocal crosses between C57BL6 x Mus castaneus. Diploid and allelic RNA-seq analyses were done to compare gene expression between wild-type and Kdm6a knockout (KO) clones. The effects of Kdm6a KO on sex-biased gene expression were investigated by comparing gene expression between male and female ES cells. Changes in H3K27me3 enrichment and chromatin accessibility at promoter regions of genes with expression changes were characterized by ChIP-seq and ATAC-seq followed by diploid and allelic analyses.ResultsWe report that Kdm6a KO in male and female embryonic stem (ES) cells derived from F1 hybrid mice cause extensive gene dysregulation, disruption of sex biases, and specific parental allele effects. Among the dysregulated genes are candidate genes that may explain abnormal developmental features of Kabuki syndrome caused by KDM6A mutations in human. Strikingly, Kdm6a knockouts result in a decrease in sex-biased expression and in preferential downregulation of the maternal alleles of a number of genes. Most promoters of dysregulated genes show concordant epigenetic changes including gain of H3K27me3 and loss of chromatin accessibility, but there was less concordance when considering allelic changes.ConclusionsOur study reveals new sex-related roles of KDM6A in the regulation of developmental genes, the maintenance of sex-biased gene expression, and the differential expression of parental alleles

Female Bias in <i>Rhox6</i> and <i>9</i> Regulation by the Histone Demethylase KDM6A

<div><p>The <i>Rho</i>x cluster on the mouse X chromosome contains reproduction-related homeobox genes expressed in a sexually dimorphic manner. We report that two members of the <i>Rhox</i> cluster, <i>Rhox6</i> and <i>9</i>, are regulated by de-methylation of histone H3 at lysine 27 by KDM6A, a histone demethylase with female-biased expression. Consistent with other homeobox genes, <i>Rhox6</i> and <i>9</i> are in bivalent domains prior to embryonic stem cell differentiation and thus poised for activation. In female mouse ES cells, KDM6A is specifically recruited to <i>Rhox6</i> and <i>9</i> for gene activation, a process inhibited by <i>Kdm6a</i> knockdown in a dose-dependent manner. In contrast, KDM6A occupancy at <i>Rhox6</i> and <i>9</i> is low in male ES cells and knockdown has no effect on expression. In mouse ovary where <i>Rhox6</i> and <i>9</i> remain highly expressed, KDM6A occupancy strongly correlates with expression. Our study implicates <i>Kdm6a</i>, a gene that escapes X inactivation, in the regulation of genes important in reproduction, suggesting that KDM6A may play a role in the etiology of developmental and reproduction-related effects of X chromosome anomalies.</p></div

KDM6A is preferentially recruited to <i>Rhox6</i> and <i>9</i> in female ES cells.

<p>(A) ChIP-qPCR analysis of KDM6A occupancy at the 5′ end of <i>Rhox6</i> and <i>9</i> is higher in female (PGK12.1 and E8) than male (WD44 and E14) undifferentiated ES cells (*p<0.05). (B) H3K4me3 enrichment during differentiation of female PGK12.1 and male WD44 ES cells shows lower levels in male ES cells and a decrease of between day 0 and 15 in agreement with gene silencing after differentiation of these ES cells (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003489#pgen-1003489-g001" target="_blank">Figure 1</a>). (C) KDM6A occupancy at the 5′ end of <i>Rhox6</i> and <i>9</i> during differentiation of female PGK12.1 ES cells and male WD44 ES cells. (D) H3K27me3 levels at the 5′ end of <i>Rhox6</i> and <i>9</i> mirror KDM6A occupancy changes. The increase at day 15 is due to X inactivation in female PGK12.1 ES cells (see also Figures S2B, S4, and S6). Average enrichment/occupancy for two separate ChIP experiments is shown as ChIP/input (A, B, C).</p

<i>Rhox6</i> and <i>9</i> are bivalent and preferentially occupied by KDM6A in female ES cells.

<p>H3K27me3, H3K4me3 and KDM6A enrichment profiles in undifferentiated female PGK12.1 (pink) and male WD44 ES (blue) cells at representative genes from each <i>Rhox</i> subcluster (α, β, and γ) demonstrate that only <i>Rhox6</i> and <i>9</i> are highly enriched with both histone modifications and are bound by KDM6A (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003489#pgen.1003489.s006" target="_blank">Figure S6</a>). <i>Rhox3e</i> (α cluster) is enriched in H3K27me3 but not H3K4me3 or KDM6A, and <i>Rhox12</i> (γ cluster) shows little enrichment for the proteins analyzed. Significant enrichment peaks based on Nimblescan analysis (FDR score <.05) are shown. Data uploaded to UCSC genome browser (NCBI36/mm8).</p

<i>Rhox6</i> and <i>9</i> expression and KDM6A occupancy are high in ovary where the genes are imprinted.

<p>(A) <i>Rhox6</i> and <i>9</i> have significantly higher expression in mouse ovary than in testis, based on re-analyses of published expression array data for 14 testis and 12 ovary specimens (*p<0.05, **p<0.001). Expression normalized to array mean (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003489#pgen-1003489-g001" target="_blank">Figure 1C</a>). (B) KDM6A occupancy measured by ChIP-qPCR at the 5′end of <i>Rhox6</i> and <i>9</i> is higher in ovary than in testis, and is very low to undetectable in brain where these genes are not expressed <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003489#pgen.1003489-Maclean1" target="_blank">[19]</a>. Occupancy levels were normalized to input fractions. (C) <i>Kdm6a</i> has high expression in female tissues especially ovary based on analyses of published expression array data (***p<0.0001) (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003489#pgen-1003489-g001" target="_blank">Figure 1F</a>). (D) <i>Rhox6</i> and <i>9</i> are expressed from the maternal allele only in ovary because of imprinting. DNA sequence chromatograms of gDNA and RT-PCR (cDNA) products derived from ovary from female F1 mice obtained by mating <i>M. spretus</i> males with C57BL/6J females with or without an <i>Xist</i> mutation (<i>Xist^Δ</i> and <i>Xist^Δ−</i>). SNPs to distinguish <i>Rhox6</i> and <i>9</i> alleles on the active X (Xa) and on inactive X (Xi) are indicated below. In ovary from both <i>Xist^Δ</i> and <i>Xist^Δ−</i> mice the gDNA shows heterozygosity at the SNPs while the cDNA shows only the maternal allele, consistent with paternal imprinting. (E) By qRT-PCR <i>Rhox6</i> and <i>9</i> are more highly expressed in ovary from <i>Xist^Δ</i> mice in which the maternal X chromosome is expressed in all cells, compared to <i>Xist^Δ−</i> mice in which there is random X inactivation (1.7-fold and 3-fold, respectively), suggesting that <i>Rhox6</i> and <i>9</i> are silenced by X inactivation. Values represent the expression ratio between <i>Xist^Δ</i> and <i>Xist^Δ−</i> ovaries.</p

Identification of genes escaping X inactivation by allelic expression analysis in a novel hybrid mouse model

X chromosome inactivation (XCI) is a female-specific mechanism that serves to balance gene dosage between the sexes whereby one X chromosome in females is inactivated during early development. Despite this silencing, a small portion of genes escape inactivation and remain expressed from the inactive X (Xi). Little is known about the distribution of escape from XCI in different tissues in vivo and about the mechanisms that control tissue-specific differences. Using a new binomial model in conjunction with a mouse model with identifiable alleles and skewed X inactivation we are able to survey genes that escape XCI in vivo. We show that escape from X inactivation can be a common feature of some genes, whereas others escape in a tissue specific manner. Furthermore, we characterize the chromatin environment of escape genes and show that expression from the Xi correlates with factors associated with open chromatin and that CTCF co-localizes with escape genes. Here, we provide a detailed description of the experimental design and data analysis pipeline we used to assay allele-specific expression and epigenetic characteristics of genes escaping X inactivation. The data is publicly available through the GEO database under ascension numbers GSM1014171, GSE44255, and GSE59779. Interpretation and discussion of these data are included in a previously published study (Berletch et al., 2015) [1]

Allele-specific non-CG DNA methylation marks domains of active chromatin in female mouse brain.

DNA methylation at gene promoters in a CG context is associated with transcriptional repression, including at genes silenced on the inactive X chromosome in females. Non-CG methylation (mCH) is a distinct feature of the neuronal epigenome that is differentially distributed between males and females on the X chromosome. However, little is known about differences in mCH on the active (Xa) and inactive (Xi) X chromosomes because stochastic X-chromosome inactivation (XCI) confounds allele-specific epigenomic profiling. We used whole-genome bisulfite sequencing in a mouse model with nonrandom XCI to examine allele-specific DNA methylation in frontal cortex. Xi was largely devoid of mCH, whereas Xa contained abundant mCH similar to the male X chromosome and the autosomes. In contrast to the repressive association of DNA methylation at CG dinucleotides (mCG), mCH accumulates on Xi in domains with transcriptional activity, including the bodies of most genes that escape XCI and at the X-inactivation center, validating this epigenetic mark as a signature of transcriptional activity. Escape genes showing CH hypermethylation were the only genes with CG-hypomethylated promoters on Xi, a well-known mark of active transcription. Finally, we found extensive allele-specific mCH and mCG at autosomal imprinted regions, some with a negative correlation between methylation in the two contexts, further supporting their distinct functions. Our findings show that neuronal mCH functions independently of mCG and is a highly dynamic epigenomic correlate of allele-specific gene regulation

Genome-Wide Distribution of MacroH2A1 Histone Variants in Mouse Liver Chromatin ▿ #

Studies of macroH2A histone variants indicate that they have a role in regulating gene expression. To identify direct targets of the macroH2A1 variants, we produced a genome-wide map of the distribution of macroH2A1 nucleosomes in mouse liver chromatin using high-throughput DNA sequencing. Although macroH2A1 nucleosomes are widely distributed across the genome, their local concentration varies over a range of 100-fold or more. The transcribed regions of most active genes are depleted of macroH2A1, often in sharply localized domains that show depletion of 4-fold or more relative to bulk mouse liver chromatin. We used macroH2A1 enrichment to help identify genes that appear to be directly regulated by macroH2A1 in mouse liver. These genes functionally cluster in the area of lipid metabolism. All but one of these genes has increased expression in macroH2A1 knockout mice, indicating that macroH2A1 functions primarily as a repressor in adult liver. This repressor activity is further supported by the substantial and relatively uniform macroH2A1 enrichment along the inactive X chromosome, which averages 4-fold. Genes that escape X inactivation stand out as domains of macroH2A1 depletion. The rarity of such genes indicates that few genes escape X inactivation in mouse liver, in contrast to what has been observed in human cells