Renin Regulatory Pathway Inhibition Epigenetically Silences *Ren1* (ATAC-Seq)

Background: Renin-expressing cells are myoendocrine cells crucial for survival which detect changes in blood pressure and release renin to maintain homeostasis. Renin is regulated through several pathways including cAMP, p300/CBP, and BET proteins including Brd4. Binding to the cAMP-responsive element in the renin enhancer region amplifies renin transcription and relies on each of these factors for an appropriate response. The specific regulatory changes that occur following inhibition of these pathways remains understudied. Therefore, we aimed to evaluate chromatin changes and gene signatures that occur following inhibition of renin regulatory factors. Methods: We treated As4.1 cells (a tumoral cell line that constitutively expresses renin) with three inhibitors (H89: PKA inhibition; JQ1: Brd4 inhibition; A-485: p300/CBP inhibition) that target required factors for renin transcriptional regulation. We then performed ATAC-seq, scRNA-seq, and ChIP-seq for H3K27ac and P300 binding on biological replicates of treated and control As4.1 cells. Results: Ren1 expression is significantly, but transiently, reduced following each inhibitory treatment. Further, PKA inhibition leads to corresponding losses in H3K27ac and p300 binding at the locus. A restricted set of nine genes with overlapping dynamically accessible regions, differential gene expression, and H3K27ac and p300 binding were identified with roles across three primary renin regulatory paradigms. Conclusions: The data shows that renin expression is regulated through a switch from an active to poised state of epigenetic control, a shift toward a less differentiated cellular identity via the loss of enhancer elements, and the disruption of cAMP, baroreceptor, and Notch mediated renin regulatory pathways. Overall design: The regulation of renin was evaluated in As4.1 cells, a mouse cell line which expresses high levels of renin mRNA, by treating with either a control compound (DMSO) or separate inhibitors to either PKA, Brd4, or p300/CBP. The regulatory effects were evaluated using ATAC-seq, scRNA-seq, and ChIP-seq for H3K27ac and P300 binding.

背景：表达肾素（renin）的细胞为肌内分泌细胞，是机体生存必需的细胞类群，可感知血压变化并释放肾素以维持内环境稳态。肾素的调控涉及多条信号通路，包括cAMP、p300/CBP以及包含Brd4在内的BET蛋白家族。结合肾素增强子区域的cAMP应答元件可增强肾素的转录，且这一过程依赖上述各类因子以实现恰当的调控应答。目前针对上述通路受抑制后发生的特异性调控变化的研究仍较为匮乏。因此，本研究旨在探究肾素调控因子受抑制后发生的染色质变化与基因表达特征。
方法：本研究以组成型表达肾素的肿瘤细胞系As4.1细胞为研究对象，使用三种靶向肾素转录调控必需因子的抑制剂进行处理：H89（蛋白激酶A（PKA）抑制剂）、JQ1（Brd4抑制剂）以及A-485（p300/CBP抑制剂）。随后，我们对处理组与对照组的As4.1细胞的生物学重复样本开展了ATAC-seq、单细胞RNA测序（scRNA-seq），以及针对H3K27ac和P300结合的染色质免疫沉淀测序（ChIP-seq）。
结果：经每一种抑制剂处理后，Ren1的表达均出现显著但短暂的下调。进一步分析显示，PKA抑制会导致该基因座上H3K27ac修饰与p300结合水平出现相应下降。本研究筛选得到一组共9个基因，这些基因存在重叠的动态染色质可及区域、差异表达特征，且伴有H3K27ac修饰与p300结合变化，其功能涵盖肾素调控的三类主要范式。
结论：本研究数据表明，肾素的表达通过表观遗传调控从激活状态转向预激活状态，通过增强子元件的丢失实现细胞身份向更低分化程度的转变，并破坏了cAMP、压力感受器以及Notch介导的肾素调控通路。
整体实验设计：本研究以高表达肾素mRNA的小鼠细胞系As4.1为研究模型，分别使用二甲基亚砜（DMSO）作为对照溶剂，以及针对PKA、Brd4或p300/CBP的特异性抑制剂处理细胞，以探究肾素的调控机制。随后通过ATAC-seq、scRNA-seq以及针对H3K27ac和P300结合的ChIP-seq分析，评估其调控效应。