RORA proximal enhancer regulates its expression during early human Th17 cell differentiation [ATAC]

Distal regulatory elements, such as enhancers, play a pivotal role in dictating cell identity by controlling the transcriptional status of specific cells. The comprehensive understanding of the epigenetic landscape, encompassing enhancer establishment and chromatin accessibility, during the differentiation of human Th17 cells remains incomplete. Leveraging ATAC-seq based chromatin accessibility profile along with the profiling of key histone marks we identified potential enhancers crucial for fate specification of Th17 cells. We found that 24 single nucleotide polymorphisms (SNPs) associated with autoimmune diseases were located near Th17-enhancers. Interestingly, these SNPs overlapped the binding sites of transcription factor active in Th17 cells. Among the Th17 specific enhancers, we identified an enhancer in the intron of RAR-related orphan receptor alpha (RORA) that was earlier predicted to regulate RORA expression. Functional validation through luciferase reporter assays confirmed that this enhancer positively regulate the transcription. Moreover, employing CRISPR-Cas9-mediated deletion of a transcription factor binding site-rich region within the identified RORA enhancer, we demonstrated its role in regulating RORA transcription. These findings provide insights into the potential mechanism by which the RORA enhancer modulates Th17 differentiation and into the role of regulatory SNPs within noncoding regions in conferring resistance or susceptibility to Th17 cell-mediated autoimmune pathologies. Human CD4+ T cels are isolated from cord blood. Cells were activated under Th0 and Th17 condition for 2 h, 4 h, 24 h, 48 h, and 72 h and ATAC-seq was performed on harvested nucleii.

远端调控元件（distal regulatory elements），例如增强子（enhancer），通过调控特定细胞的转录状态，在细胞身份决定过程中发挥核心作用。目前学界对人类Th17细胞分化过程中的表观遗传景观（涵盖增强子建立与染色质可及性）仍缺乏全面认知。本研究基于ATAC-seq（Assay for Transposase-Accessible Chromatin using sequencing）染色质可及性谱与关键组蛋白修饰谱，鉴定出对Th17细胞命运特化至关重要的潜在增强子。我们发现，24个与自身免疫疾病相关的单核苷酸多态性（Single Nucleotide Polymorphisms, SNPs）位点位于Th17细胞增强子附近。有趣的是，这些SNPs位点与Th17细胞中活化的转录因子结合位点存在重叠。在Th17细胞特异性增强子中，我们于视黄酸相关孤儿受体α（RAR-related orphan receptor alpha, RORA）的内含子区域鉴定出一个此前被预测可调控RORA表达的增强子。通过荧光素酶报告基因实验开展的功能验证证实，该增强子可正向调控基因转录。此外，我们利用CRISPR-Cas9介导的基因编辑技术，对鉴定出的RORA增强子内富含转录因子结合位点的区域进行敲除，证实了该区域在调控RORA转录中的功能。本研究结果为RORA增强子调控Th17细胞分化的潜在机制，以及非编码区域内调控性SNPs如何影响Th17细胞介导的自身免疫疾病易感性与抗性提供了全新见解。本研究从脐带血中分离人类CD4+ T细胞，将其分别在Th0与Th17极化条件下激活2小时、4小时、24小时、48小时及72小时，随后对收集的细胞核进行ATAC-seq测序。