Female-specific CTCF binding on the inactive X chromosome in mouse

In mammals, genes located on the X chromosome are present in one copy in XY males and two in XX females. To balance the dosage of X-linked gene expression between the sexes one of the two X chromosomes in females is silenced by X inactivation initiated by up-regulation of the lncRNA (long non-coding RNA) Xist and recruitment of specific chromatin modifiers for silencing. The inactivated X chromosome becomes heterochromatic and visits a specific nuclear compartment adjacent to the nucleolus. We report a novel role for the X-linked lncRNA Firre in anchoring the inactive mouse X chromosome and preserving one of its main epigenetic features, trimethylation of histone H3 at lysine 27 (H3K27me3). Similar to Dxz4, Firre is expressed from a macrosatellite repeat locus associated with a cluster of CTCF and cohesin binding specifically on the inactive X. CTCF binding initially present in both male and female mouse embryonic stem cells was found to be lost from the active X during development. The Firre and Dxz4 loci on the inactive X were preferentially located adjacent to the nucleolus. Knockdown of Firre RNA disrupted perinucleolar targeting and H3K27me3 levels in mouse fibroblasts, demonstrating an important role for this lncRNA in maintenance of one of the main epigenetic features of the X chromosome. There was no X-linked gene reactivation after Firre knockdown; however, a compensatory increase in the expression of chromatin modifier genes implicated in X silencing was observed. In female ES cells Firre RNA knockdown did not disrupt Xist expression/coating nor silencing of G6pdx during differentiation, suggesting that Firre does not play a role in the onset of X inactivation. We conclude that the X-linked lncRNA Firre helps position the inactive X chromosome near the nucleolus and preserve one of its main epigenetic features. Examination of allelic protein-binding or histone modification profiles in Patski cells.

在哺乳动物中，X染色体上的基因在雄性XY个体中仅含1份拷贝，而在雌性XX个体中则含有2份拷贝。为平衡两性间X连锁基因的表达剂量，雌性个体的两条X染色体中会有一条通过X染色体失活（X inactivation）途径被沉默：该过程由长链非编码RNA（long non-coding RNA，lncRNA）Xist的上调所启动，并招募特异性染色质修饰因子以介导基因沉默。发生失活的X染色体将转变为异染色质，并定位于核仁附近的特定核区室内。本研究报道了X连锁lncRNA Firre的一项全新功能：其可锚定失活的小鼠X染色体，并维持其主要表观遗传特征之一——组蛋白H3第27位赖氨酸三甲基化（H3K27me3）。与Dxz4类似，Firre在与CCCTC结合因子（CTCF）和黏连蛋白（cohesin）结合簇相关的大卫星重复序列位点处表达，且该结合特异性仅存在于失活的X染色体上。研究发现，最初在雌雄小鼠胚胎干细胞中均存在的CTCF结合位点，会在发育过程中从活跃的X染色体上丢失。失活X染色体上的Firre与Dxz4位点优先定位于核仁附近。在小鼠成纤维细胞中敲低Firre RNA会破坏核仁周靶向定位以及H3K27me3的水平，这证明该lncRNA在维持X染色体主要表观遗传特征之一的过程中发挥着关键作用。敲低Firre后并未出现X连锁基因的重新激活，但研究观察到参与X染色体失活的染色质修饰基因的表达出现了代偿性升高。在雌性胚胎干细胞中，敲低Firre RNA并不会破坏Xist的表达与包被，也不会影响分化过程中G6pdx基因的沉默，这表明Firre并不参与X染色体失活的起始过程。综上，X连锁lncRNA Firre可协助将失活的X染色体定位至核仁附近，并维持其主要表观遗传特征之一。本研究还对Patski细胞中的等位基因蛋白结合谱与组蛋白修饰谱展开了分析。