CTCF/cohesin organize the ground state of chromatin-nuclear speckle association [CUT&RUN, CUT&Tag and RNA-seq]. CTCF/cohesin organize the ground state of chromatin-nuclear speckle association [CUT&RUN, CUT&Tag and 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. Overall design: Cut &Run against speckle protein SON and corresponding IGG control, under designated treatment.

细胞核内的染色质间区（interchromatin space）包含多种无膜核体（membrane-less nuclear body）。近期研究表明，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。当前亟待解答的核心问题包括：这种染色质-核斑关联的基础状态是如何建立的？这一层核组织结构又发挥着怎样的基因调控功能？本研究报道，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组分。通过敲除CTCF或黏连蛋白破坏基础状态下的DNA-核斑接触，对核斑关联基因的基础表达水平仅产生微弱影响；但我们的实验结果显示，该操作对刺激依赖型核斑关联基因的诱导过程存在显著的抑制作用。我们在黏连蛋白亚基RAD21中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。与敲除CTCF或RAD21的结果不同，敲除黏连蛋白释放因子WAPL会使黏连蛋白在染色质及DNA-核斑接触处的滞留增强，进而提升核斑关联基因的诱导活性。此外，我们在源自Cornelia de Lange综合征（Cornelia de Lange syndrome, CdLS）患者的细胞中观察到染色质-核斑关联被破坏的现象——该先天性神经发育疾病的致病机制与黏连蛋白通路缺陷相关，这一发现也将核斑列为治疗探索的潜在靶点。综上，本研究揭示了建立染色质-核斑关联基础组织结构状态的分子机制，该机制可促进基因的诱导表达，且与人类疾病密切相关。整体实验设计：在指定处理条件下，针对核斑蛋白SON开展Cut&Run实验，并设置相应的IgG（Immunoglobulin G）对照。