Gene expression, epigenetic modification, and chromatin landscape of skin dermal cells in different body positions [ChIP-Seq]

we report that regional expression of the Hoxc genes is regulated by antagonistic switch between repression and activation epigenetic modification. In freshly isolated dermal cells from P50 telogen Wt/Wt ear skin, no enrichment of H3K27ac was detected at Hoxc cluster, consistent with their lack of expression; in contrast, robust peaks of this marker are observed in 3’ region of the Hoxc cluster in dorsal skin dermal cells. Secondly we looked at H3K27 trimethylation (H3K27me3), a marker for polycomb-dependent gene repression, which antagonizes H3K27ac. Concomitantly, we found that H3K27me3 spread across the entire Hoxc cluster in adult ear skin dermal cells but was restricted to the 5’ region of the Hoxc cluster in adult dorsal skin dermal cells . In adult dorsal skin dermal cells, we found three occupied CTCF binding sites inside Hoxc cluster. The rostral CTCF binding site marks the discontinuity point of H3K27me3 modification at the intergenic region between Hoxc11 and Hoxc10 in dorsal skin dermal cells. This indicates the CTCF binding event within Hoxc cluster might forge a topological barrier that can insulate activation region from repression region and therefore lead to the region specific expression of the Hoxc cluster genes.To directly test whether there is ectopic interaction between Hoxc cluster and the active domain in proximal-TAD, we used circularized chromosome conformation capture (4C) with Hoxc4 as bait. In Wt/Koa ear skin dermal cells, there are clear ectopic interactions of Hoxc4 with the remaining active regulatory landscape in proximal-TAD; on the other hand, all of the interactions of Hoxc4 remained inside the distal-TAD with repressive domain in Wt/Wt ear skin. Examination of gene expression in wt/wt ear epidermal cells, wt/wt dorsal epidermal cells, and wt/koa ear epidermal cells; Examination of gene expression and 2 histone modification in wt/wt ear dermal cells, wt/wt dorsal dermal cells, and wt/koa ear dermal cells; examination of CTCF binding in wt/wt ear dermal cells and wt/wt dorsal dermal cells; examination of chromatin interaction using Hoxc as viewpoint in wt/wt ear dermal cells and wt/koa ear dermal cells.

本研究报道，Hoxc基因的区域表达受表观遗传修饰的拮抗开关——即沉默与激活之间的拮抗切换——调控。在取自P50期静止态野生型（Wt/Wt）小鼠耳皮肤的新鲜分离真皮细胞中，未检测到组蛋白H3第27位乙酰化修饰（H3K27ac）在Hoxc基因簇（Hoxc cluster）上的富集，这与其表达缺失状态相符；与之相反，在背部皮肤真皮细胞中，该修饰标记的显著富集峰出现在Hoxc基因簇的3'端区域。其次，我们检测了组蛋白H3第27位三甲基化（H3K27me3）——一种介导多梳蛋白依赖的基因沉默（polycomb-dependent gene repression）的标记，其可拮抗H3K27ac。与此同时，我们发现成年小鼠耳皮肤真皮细胞中，H3K27me3广泛分布于整个Hoxc基因簇；而在成年背部皮肤真皮细胞中，该修饰仅局限于Hoxc基因簇的5'端区域。在成年背部皮肤真皮细胞中，我们于Hoxc基因簇内部发现了3个CTCF结合位点（CTCF binding sites）。头侧CTCF结合位点标记了背部皮肤真皮细胞中Hoxc11与Hoxc10之间基因间区域的H3K27me3修饰的不连续点。这表明，Hoxc基因簇内部的CTCF结合事件可能形成了一种拓扑屏障，可将激活区域与沉默区域隔离开来，进而促成Hoxc基因簇基因的区域特异性表达。为直接验证Hoxc基因簇与近端TAD（proximal-TAD）内的激活结构域之间是否存在异位互作，我们以Hoxc4为诱饵位点，采用环状染色质构象捕获（4C，circularized chromosome conformation capture）技术进行检测。在Wt/Koa小鼠耳皮肤真皮细胞中，可观察到Hoxc4与近端TAD内其余活性调控区域存在明确的异位互作；与之相反，在Wt/Wt小鼠耳皮肤真皮细胞中，Hoxc4的所有互作均局限于远端TAD内的沉默结构域内部。本研究开展的检测内容包括：对野生型（Wt/Wt）耳表皮细胞、Wt/Wt背部表皮细胞及Wt/koa耳表皮细胞的基因表达检测；对Wt/Wt耳真皮细胞、Wt/Wt背部真皮细胞及Wt/koa耳真皮细胞的基因表达与两种组蛋白修饰检测；对Wt/Wt耳真皮细胞与Wt/Wt背部真皮细胞的CTCF结合情况检测；以及以Hoxc为检测视角，对Wt/Wt耳真皮细胞及Wt/koa耳真皮细胞开展的染色质互作分析。