Trans- and cis-acting effects of the lncRNA Firre on epigenetic and structural features of the inactive X chromosome [ChIP-seq]

Firre encodes a lncRNA involved in nuclear organization in mammals. Here we find that Firre RNA is transcribed from the active X chromosome (Xa) and exerts trans-acting effects on the inactive X chromosome (Xi). Allelic deletion of Firre on the Xa in a mouse hybrid fibroblast cell line results in a dramatic loss of the histone modification H3K27me3 and of components of the PRC2 complex on the Xi as well as the disruption of the perinucleolar location of the Xi. These features are measurably rescued by ectopic expression of a mouse or human Firre/FIRRE cDNA transgene, strongly supporting a conserved trans-acting role of the Firre transcript in maintaining the Xi heterochromatin environment. Surprisingly, CTCF occupancy is decreased on the Xi upon loss of Firre RNA, but is partially recovered by ectopic transgene expression, suggesting a functional link between Firre RNA and CTCF in maintenance of epigenetic features and/or location of the Xi. Loss of Firre RNA results in dysregulation of genes implicated in cell division and development, but not in reactivation of genes on the Xi, which retains its bipartite structure despite some changes in chromatin contact distribution. Allelic deletion or inversion of Firre on the Xi causes localized redistribution of chromatin contacts, apparently dependent on the orientation of CTCF binding sites clustered at the locus. Thus, the Firre locus and its RNA have roles in the maintenance of epigenetic features and structure of the Xi. H3K27me3 ChIP-seq on F1 hybrid wild-type and CRISPR/cas9-modified Patski cells

Firre基因编码一种参与哺乳动物细胞核组织结构构建的长链非编码RNA（lncRNA）。本研究发现，Firre RNA由活性X染色体（active X chromosome，Xa）转录而来，并对失活X染色体（inactive X chromosome，Xi）发挥反式调控作用。在小鼠杂交成纤维细胞系中，对X染色体上的Firre进行等位基因敲除，会导致Xi上的组蛋白修饰H3K27me3以及多梳抑制复合体2（Polycomb Repressive Complex 2，PRC2）组分显著丢失，同时破坏Xi的核仁周定位。通过异位表达小鼠或人类Firre/FIRRE互补DNA（complementary DNA，cDNA）转基因，可显著挽救上述表型，有力证实了Firre转录本在维持Xi异染色质环境中具有保守的反式调控功能。令人意外的是，Firre RNA缺失会导致Xi上的CCCTC结合因子（CCCTC-binding factor，CTCF）结合占有率降低，但可通过异位转基因表达部分恢复，这表明Firre RNA与CTCF在维持Xi的表观遗传特征及/或定位方面存在功能关联。Firre RNA的缺失会导致参与细胞分裂与发育的基因表达失调，但不会引发Xi上基因的重新激活；尽管染色质互作分布发生了一定改变，Xi仍保留其二分结构。对Xi上的Firre进行等位基因敲除或倒位，会导致染色质互作发生局部重分布，这一过程明显依赖于该位点聚集的CTCF结合位点的方向。综上，Firre基因座及其RNA在维持Xi的表观遗传特征与结构方面发挥着重要作用。针对F1杂交野生型及CRISPR/Cas9修饰的Patski细胞开展H3K27me3染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing，ChIP-seq）实验。