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

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