CTCF/cohesin organize the ground state of chromatin-nuclear speckle association [RNA-Seq]

The interchromatin space in the cell nucleus contains various membrane-less nuclear bodies. Recent findings indicate that nuclear speckles, comprising a distinct nuclear body, exhibit interactions with certain chromatin regions in a ground state. Key questions are how this ground state of chromatin-nuclear speckle association is established and what are the gene regulatory roles of this layer of nuclear organization. We report here that chromatin structural factors CTCF and cohesin are required for full ground state association between DNA and nuclear speckles. Disruption of ground state DNA-speckle contacts via either CTCF depletion or cohesin depletion had minor effects on basal level expression of speckle-associated genes, however we show strong negative effects on stimulus-dependent induction of speckle-associated genes. We identified a putative speckle targeting motif (STM) within cohesin subunit RAD21 and demonstrated that the STM is required for chromatin-nuclear speckle association. In contrast to reduction of CTCF or RAD21, depletion of the cohesin releasing factor WAPL stabilized cohesin on chromatin and DNA-speckle contacts, resulting in enhanced inducibility of speckle-associated genes. In addition, we observed disruption of chromatin-nuclear speckle association in patient derived cells with Cornelia de Lange syndrome (CdLS), a congenital neurodevelopmental diagnosis involving defective cohesin pathways, thus revealing nuclear speckles as an avenue for therapeutic inquiry. In summary, our findings reveal a mechanism to establish the ground organizational state of chromatin-speckle association, to promote gene inducibility, and with relevance to human disease. Comparative gene expression profiling analysis of full-length RNA-seq data for HeLa, IMR90, and patient derived LCL cells with deficiency in CTCF, RAD21 or WAPL, with or without designated external stimuli.

细胞核内染色质间区室含有多种无膜核体。最新研究表明，作为一类独特核体的核斑（nuclear speckles）在基础状态下可与特定染色质区域发生相互作用。当前亟待解答的关键科学问题包括：这种染色质-核斑关联的基础状态是如何建立的？这一层核组织架构承担着怎样的基因调控功能？

本研究证实，染色质结构因子CTCF与黏连蛋白（cohesin）是DNA与核斑之间完整基础状态关联所必需的调控因子。通过敲除CTCF或黏连蛋白破坏基础状态下DNA-核斑接触，仅对核斑关联基因的基础表达水平产生微弱影响；但本研究发现，该操作会显著抑制依赖刺激诱导的核斑关联基因的表达。

我们在黏连蛋白亚基RAD21内鉴定出一段潜在的核斑靶向基序（speckle targeting motif, STM），并证实该基序是染色质-核斑关联所必需的结构元件。与敲除CTCF或RAD21的效果相反，敲除黏连蛋白释放因子WAPL可增强黏连蛋白在染色质上的滞留，稳定DNA-核斑接触，最终提升核斑关联基因的诱导表达能力。

此外，我们在来自Cornelia de Lange综合征（Cornelia de Lange syndrome, CdLS）患者的细胞中观察到染色质-核斑关联被破坏——该疾病是一类以黏连蛋白通路异常为特征的先天性神经发育病症，这一发现将核斑确立为治疗探索的潜在方向。

综上，本研究揭示了建立染色质-核斑关联基础组织状态的分子机制，该机制可促进基因的诱导表达，且与人类疾病密切相关。本研究针对HeLa细胞、IMR90细胞以及CTCF、RAD21或WAPL缺陷的患者来源LCL细胞的全长RNA测序（RNA-seq）数据开展了比较基因表达谱分析，实验设置了施加指定外源刺激与未施加刺激两组对照。