GRHL3 binding and the enhancer landscape are reorganized during transitions between different functional states of epidermal keratinocytes [siREST-proliferation]

The genomic mechanisms underlying progressive, irreversible cell lineage commitments and differentiation, which include large scale chromatin re-organization, transcription factor binding, and chromatin modifications, have been well defined. However, we know little about the chromatin changes during transitions between transient cell states such as cell migration. Here we demonstrate the formation of unique complements of typical enhancers and super-enhancers as human progenitor keratinocytes either differentiate or migrate. Unique super-enhancers for each cellular state are linked to gene expression that confer functions associated with each cell state, and sequence variants associated with skin diseases are enriched within super-enhancers. GRHL3, a factor that promotes both differentiation and migration, exhibits prominent super-enhancer interactions in differentiating keratinocytes, while during migration, it preferentially binds to promoters along with REST, repressing the expression of migration inhibitors. Key epidermal differentiation transcription factor genes, including GRHL3, are located within super-enhancers, and many of these transcription factors in turn bind to and regulate super-enhancers. Of note, GRHL3 also represses the formation of a number of progenitor and non-keratinocyte super-enhancers in differentiating keratinocytes. Thus, coordinated GRHL3 binding and enhancer rearrangements regulate the functional states of keratinocytes. pooled siRNA was used to knockdown REST in duplicate, with 2 scramble siRNA control replicates. Cells were collected 72 hours later. RNA was extracted and submitted for microarray.

渐进性、不可逆的细胞谱系定型（cell lineage commitments）与分化背后的基因组调控机制——包括大规模染色质重塑（chromatin re-organization）、转录因子（transcription factor）结合与染色质修饰（chromatin modifications）——已得到充分阐明。然而，我们对细胞迁移这类瞬时细胞状态（transient cell states）转换过程中的染色质变化仍知之甚少。
本研究证实，人表皮祖角质形成细胞（human progenitor keratinocytes）在分化或迁移过程中，会形成典型增强子（enhancer）与超级增强子（super-enhancer）的独特组合模式。对应每种细胞状态的独特超级增强子，与赋予该细胞状态相关功能的基因表达紧密关联；而与皮肤疾病相关的序列变异（sequence variants），在超级增强子区域中显著富集。
GRHL3作为同时促进分化与迁移的转录因子，在分化中的角质形成细胞内展现出显著的超级增强子互作；而在迁移过程中，GRHL3会与REST协同优先结合启动子（promoter）区域，抑制迁移抑制因子的表达。包括GRHL3在内的关键表皮分化（epidermal differentiation）转录因子基因，均位于超级增强子区域内；而这类转录因子中的多数，又会结合并调控超级增强子的活性。值得注意的是，在分化的角质形成细胞中，GRHL3还会抑制多种祖细胞型与非角质形成细胞（non-keratinocyte）型超级增强子的形成。
综上，GRHL3结合与增强子重排的协同调控，决定了角质形成细胞的功能状态。本研究采用混合小干扰RNA（siRNA）对REST进行敲低，设置2份生物学重复样本，并同步设置2份阴性对照小干扰RNA（scramble siRNA）重复样本；于转染72小时后收集细胞，提取RNA并提交进行基因芯片（microarray）检测。