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

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

<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

Validation of <i>Rlim</i>, <i>Shroom4</i>, <i>Car5b</i>, <i>Hdac6</i>, <i>5530601H04Rik</i> expression profiles and <i>Firre</i> mRNA profiles.

<p>(A) Sanger sequencing tracings of <i>Rlim</i> cDNA confirm bi-allelic expression in Patski cells but not brain, while gDNA sequence tracings show SNP heterozygosity (C in BL6 and T in <i>spretus</i>). Arrows indicate SNP positions. (B, C) Sanger sequencing tracings of <i>Shroom4</i> (B) and <i>Carb5</i> (C) cDNA confirm that these genes are subject to XCI in F1 kidney while they were shown to escape XCI in Patski cells [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005079#pgen.1005079.ref018" target="_blank">18</a>]. gDNA sequence tracings show SNP heterozygosity (<i>Shroom4</i>—G in BL6 and A in <i>spretus</i>; <i>Car5b</i>—G and C in BL6; A and T in <i>spretus</i>). Arrows indicate SNP positions. (D, E) Validation of escape from XCI for <i>Hdac6</i>. (D) Gel electrophoresis of RT-PCR products using non-species-specific primers, <i>spretus</i>-specific primers, and BL6-specific primers (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005079#pgen.1005079.s014" target="_blank">S10 Table</a>) in BL6, <i>spretus</i>, Patski cells and F1 kidney. <i>ActinB</i> was used as a control. Control reactions include "No RT" (no reverse transcriptase) and H₂O (instead of primers). Sanger sequencing tracing confirms heterozygosity (A in BL6 and G in <i>spretus</i>) in the left primer (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005079#pgen.1005079.s014" target="_blank">S10 Table</a>). (E) Xi expression of <i>Hdac6</i> was determined to be 9% of total expression in Patski cells by gel band quantification measured by ImageJ. (F) Sanger sequencing tracings of <i>5530601H04Rik</i> cDNA confirms that the lncRNA escapes XCI in kidney and Patski cells, while gDNA sequence tracings show heterozygosity (T and A in BL6; A and G in <i>spretus</i>). Arrows indicate SNP positions. (G) mRNA SNP read distribution profiles on the Xi and Xa for <i>Firre</i>, a lncRNA that escapes XCI in mouse tissues and Patski cells. Note that <i>Firre</i> is classified as a variable escape gene in brain (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005079#pgen.1005079.s015" target="_blank">S1 Dataset</a>). Xa SNP reads are in blue and Xi SNP reads in green.</p

Evaluation of escape from XCI in mouse tissues.

<p>(A) Example of mRNA SNP read distribution profiles on the Xi and Xa for <i>Kdm5c</i>, an escape gene common to all mouse tissues tested (brain, spleen and ovary). SNP reads specific to the Xa (blue) and Xi (green) are visualized in the UCSC genome browser. RNA-seq read quantification was done by normalizing reads from the Xi to total reads (Xi + Xa) in two biological replicates. (B) Example of mRNA SNP read distribution profiles on the Xi and Xa for <i>Cfp</i>, a gene that escapes XCI only in spleen. SNP reads specific to the Xa (blue) and Xi (green) are visualized in the UCSC genome browser. (C, D) Validation of escape from XCI for <i>Cfp</i>. (C) Gel electrophoresis of RT-PCR products using non-species-specific primers and <i>spretus</i>-specific primers (sp) (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005079#pgen.1005079.s014" target="_blank">S10 Table</a>) in BL6, <i>spretus</i>, and F1 brain in which the Xi is from <i>spretus</i>. <i>ActinB</i> was used as a control. Control reactions include "No RT" (no reverse transcriptase) and H₂O (instead of primers). (D) Graph comparing RT-PCR <i>Cfp</i> gel band quantification measured by ImageJ with SNP read quantification measured by RNA-seq. Xi product abundance measured by RT-PCR using <i>spretus</i>-specific primers in F1 spleen was normalized to total RT-PCR product abundance measured by non-species specific primers.</p

Escape from X Inactivation Varies in Mouse Tissues

<div><p>X chromosome inactivation (XCI) silences most genes on one X chromosome in female mammals, but some genes escape XCI. To identify escape genes in vivo and to explore molecular mechanisms that regulate this process we analyzed the allele-specific expression and chromatin structure of X-linked genes in mouse tissues and cells with skewed XCI and distinguishable alleles based on single nucleotide polymorphisms. Using a binomial model to assess allelic expression, we demonstrate a continuum between complete silencing and expression from the inactive X (Xi). The validity of the RNA-seq approach was verified using RT-PCR with species-specific primers or Sanger sequencing. Both common escape genes and genes with significant differences in XCI status between tissues were identified. Such genes may be candidates for tissue-specific sex differences. Overall, few genes (3–7%) escape XCI in any of the mouse tissues examined, suggesting stringent silencing and escape controls. In contrast, an in vitro system represented by the embryonic-kidney-derived Patski cell line showed a higher density of escape genes (21%), representing both kidney-specific escape genes and cell-line specific escape genes. Allele-specific RNA polymerase II occupancy and DNase I hypersensitivity at the promoter of genes on the Xi correlated well with levels of escape, consistent with an open chromatin structure at escape genes. Allele-specific CTCF binding on the Xi clustered at escape genes and was denser in brain compared to the Patski cell line, possibly contributing to a more compartmentalized structure of the Xi and fewer escape genes in brain compared to the cell line where larger domains of escape were observed.</p></div

Xi-associated but not Xa-associated CTCF peak clusters co-localize with escape regions.

<p>(A) Significant CTCF Xi-binding clusters were mapped along the Xi in brain and Patski cells. Xi- and both-preferred peaks were determined by a binomial model and used for density analysis. Red bars represent merger of clusters of CTCF Xi-binding peaks, while purple dots represent escape genes. Significant Xi-binding CTCF binding clusters tend to co-localize with chromatin containing escape genes. Little change was seen after removal of promoter-associated CTCF binding (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005079#pgen.1005079.s003" target="_blank">S3B Fig</a>). Horizontal axis represents the Xi in Mb. The vertical axis is the negative log of the calculated binomial p-value (-log (p-value)). The thin red dashed line represents a 0.01 p-value cutoff. (B) Similar analysis for CTCF Xa- and both-preferred peaks. There was no significant CTCF co-localization with escape genes on the Xa in either brain or Patski cells. (C) Average CTCF Xi-SNP read counts in ten 100bp windows at promoters (0.5kb upstream and downstream of the TSS) is plotted against mRNA-seq Xi-SNP read counts escape genes (purple) and for genes subject to XCI (gray) in brain and Patski cells. In brain, a higher proportion of escape genes (6/14; Fisher’s exact test, p = 5e^-9) had an average ≥10 reads (black line) at their promoter compared to genes subject to XCI (0/403). Similarly, in Patski cells a higher proportion of escape genes (9/65; Fisher’s exact test, p = 0.0004) had an average of ≥1 read (black line) at their promoter compared to genes subject to XCI (3/204).</p

Enrichment in PolII-S5p and DNase I hypersensitivity on the Xi allele at escape genes.

<p>(A, B) Examples of allele-specific PolII-S5p occupancy profiles and expression (mRNA) profiles at <i>Ddx3x</i>, <i>Rlim</i> and <i>Igbp1</i> in two systems: brain (A) and Patski cells (B). <i>Ddx3x</i> escapes XCI in both systems, <i>Rlim</i> escapes XCI in Patski cells only, and <i>Igbp1</i> is subject to XCI in both systems. PolII-S5p is enriched at the promoter regions (highlighted by a red box) of escape genes on both the Xa and the Xi, whereas enrichment is limited to the Xa for genes subject to XCI. DNase I hypersensitivity tested in Patski cells only is also increased at the promoter regions (highlighted by a red box) of escape genes on both the Xa and Xi, but is limited to the Xa for genes subject to XCI. Genes that escape XCI are labeled orange and genes subject to XCI blue. Color-coded profiles are shown for the Xa (blue) and Xi (green) SNP reads, and for the total reads (Xt, black). See additional examples in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005079#pgen.1005079.s002" target="_blank">S2 Fig</a>.</p

DNase I hypersensitivity at the promoters of X-linked genes correlates with expression and escape from XCI.

<p>(A) Metagene analyses of DNase I hypersensitivity (DHS) in Patski cells show average Xa- (left) and Xi- (right) SNP read counts in 100bp windows 3kb upstream and downstream of the TSS for escape genes (purple; 43 genes in both replicates for Patski cells) and for genes subject to XCI (gray; 203 genes with <2 Xi-SRPM in both replicates for Patski cells). (B) Scatter plot shows a positive correlation between DHS at the promoter (log₂ of all reads within a region ±500bp from the TSS) and expression (log₂ RPKM) for all X-linked genes in Patski cells. (C) Scatter plot of Xi-specific DHS at the promoter (reads within a region ±500bp from the TSS) against expression (Xi SNP-reads) for escape genes (purple) and genes subject to XCI (gray) shows a correlation between DHS and level of escape from XCI in Patski cells. (D) Same analysis as in C but for Xa-specific DHS at the promoter region. Escape genes generally overlap with genes subject to XCI although escape genes tend to have high expression and high DHS. (E). Scatter plot shows a good correlation between DHS and enrichment in PolII-S5p at the promoter of genes on the Xi. DHS and PolII-S5p are shown as reads within a region ±500bp from the TSS for escape genes (purple) and genes subject to XCI (gray) in Patski cells.</p