人角质形成细胞分化中染色质高级结构图谱及其调控机制研究

Dramatic changes of higher-order chromatin architecture occur in epidermal keratinocytes (KCs) differentiation, accompanied by transcriptional silencing and nuclear condensation until its complete disappearance. The nucleus and chromatin remained in KCs lead to parakeratosis and impaired skin barrier function, and cause various skin diseases such as psoriasis. Although the chromatin structure in KCs is dynamically regulated and plays an important role in the terminal differentiation and barrier function of the skin, but the molecular mechanism is not yet clear. Recent studies have reported the three-dimensional structure of chromatin at an individual genomic locus in KCs, however, the dynamic changes in genome-wide structure and spatial organization of chromatin during KC differentiation are still poorly understood, due to limitations of experimental procedure and sample collection. We speculate that spatial chromatin folding, organization of the enhancer-promoter networks and their relevance to Topologically Associating Domains (TADs) appear unique changes in KC differentiation. We aim to use the sensitive sisHi-C to comprehensively and systematically dissect the dynamic landscape of chromatin structure and the molecular mechanisms during human KC differentiation in vivo. This will greatly deepen our understanding of the roles for 3D chromatin architecture in genomic transcriptional regulation and seek novel ideas and targets for the prevention and treatment of skin diseases such as psoriasis.

染色质高级结构在表皮角质形成细胞（KC）分化中发生了剧烈变化，并伴随转录沉默和细胞核凝缩直至完全消失。细胞核和染色质的残留导致了角化不全和皮肤屏障受损，并引起银屑病等多种皮肤病。尽管染色质高级结构的动态调控对于KC分化和皮肤屏障功能极其重要，但其分子机制尚不清楚。虽然最近报道了KC中个别基因座上的染色质三维结构，由于实验材料和研究方法的限制，KC分化中基因组染色质高级结构的动态变化仍鲜为人知。我们猜测，染色质的空间折叠、增强子启动子互作网络和拓扑相关结构域在KC分化中出现了独特的变化。我们拟利用灵敏的sisHi-C技术来全面解析染色质高级结构在人KC分化中的动态图谱及其分子调控机理。这将大大加深人们对细胞分化中染色质高级结构特征与功能相互关联的认识，为银屑病等皮肤病的防治提供新思路和新靶点。

真核生物的染色质在细胞核中呈现出紧密盘绕高度折叠的三维结构。特定的染色质构象能够选择性地激活或沉默基因，从而调控基因转录、细胞分化、组织稳态等生命过程。免疫荧光染色结果显示，染色质高级结构在表皮角质形成细胞KC分化中发生了剧烈变化。但在全基因组范围的动态调控机制尚不清楚。利用灵敏的sisHi-C技术，我们发现基底层及棘层KC的染色质呈现相对松散、高度活跃转录的状态。这些经典的染色质三维结构具有清晰的拓扑结构域和区室结构。然而在分化的颗粒层KC中，染色质经典的区室结构整个出现了明显减弱或消失，并逐渐转化成转录沉默、染色体高度压缩的状态。我们发现伴随这一过程的是，在基底层KC向棘层、颗粒层等上层KC分化过程中，基因表达模式呈现连续变化状态，即常规报道的KRT14和KRT10等标志基因存在共表达窗口，一个可能的原因是这些角蛋白编码基因的转录表达水平很高，以致其在KC分化过程中的代谢周期较长。而KRT14与IVL等颗粒层标志基因则未出现如此明显的共表达现象。另外，KC在分化前后的染色质开放状态出现明显差异，整体上分化前的染色质更加开放，而分化后大量的染色质开放信号在启动子区消失了。我们发现病变皮肤KC中表皮分化复合物基因座缺失了大部分的染色质开放信号。这些数据提示，皮肤KC中染色质结构异常可能与角化不全相关。我们还发现其所富集的关键转录因子发生了巨大改变。例如，皮肤KC分化前的ATAC-seq信号主要富集AP-1和P63等转录因子，而分化后则主要富集SCL和BMYB等转录因子。在敲除关键因子如P63后，我们发现KC不能完成正常的谱系分化，Krt5等关键标志基因的表达呈现缺失，纯合敲除小鼠出生因皮肤缺陷致死，而在抑制或RNA干扰后，KC增殖生长受阻且分化异常。我们的研究加深了人们对细胞分化中染色质高级结构特征与功能相互关联的认识，为银屑病等皮肤病的防治提供新思路和新靶点。