Department of Pediatrics, Child Health Research Center (J.P.S., R.P., S.M., M.L.S.S.-L., R.A.G.), University of Virginia, Charlottesville, VA.
CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria (R.P.).
Hypertension. 2024 Sep;81(9):1869-1882. doi: 10.1161/HYPERTENSIONAHA.124.22886. Epub 2024 Jul 11.
Renin-expressing cells are myoendocrine cells crucial for the maintenance of homeostasis. Renin is regulated by cAMP, p300 (histone acetyltransferase p300)/CBP (CREB-binding protein), and Brd4 (bromodomain-containing protein 4) proteins and associated pathways. However, the specific regulatory changes that occur following inhibition of these pathways are not clear.
We treated As4.1 cells (tumoral cells derived from mouse juxtaglomerular cells that constitutively express renin) with 3 inhibitors that target different factors required for renin transcription: H-89-dihydrochloride, PKA (protein kinase A) inhibitor; JQ1, Brd4 bromodomain inhibitor; and A-485, p300/CBP inhibitor. We performed assay for transposase-accessible chromatin with sequencing (ATAC-seq), single-cell RNA sequencing, cleavage under targets and tagmentation (CUT&Tag), and chromatin immunoprecipitation sequencing for H3K27ac (acetylation of lysine 27 of the histone H3 protein) and p300 binding on biological replicates of treated and control As4.1 cells.
In response to each inhibitor, expression was significantly reduced and reversible upon washout. Chromatin accessibility at the locus did not markedly change but was globally reduced at distal elements. Inhibition of PKA led to significant reductions in H3K27ac and p300 binding specifically within the super-enhancer region. Further, we identified enriched TF (transcription factor) motifs shared across each inhibitory treatment. Finally, we identified a set of 9 genes with putative roles across each of the 3 renin regulatory pathways and observed that each displayed differentially accessible chromatin, gene expression, H3K27ac, and p300 binding at their respective loci.
Inhibition of renin expression in cells that constitutively synthesize and release renin is regulated by an epigenetic switch from an active to poised state associated with decreased cell-cell communication and an epithelial-mesenchymal transition. This work highlights and helps define the factors necessary for renin cells to alternate between myoendocrine and contractile phenotypes.
表达肾素的细胞是维持内环境稳定的肌内分泌细胞。肾素受 cAMP、p300（组蛋白乙酰转移酶 p300）/CBP（CREB 结合蛋白）和 Brd4（溴结构域蛋白 4）蛋白及相关途径调节。然而，这些途径被抑制后发生的特定调节变化尚不清楚。
我们用 3 种抑制剂处理 As4.1 细胞（源自小鼠肾小球旁器细胞的肿瘤细胞，这些细胞持续表达肾素），这 3 种抑制剂针对的是肾素转录所需的不同因素：H-89 二盐酸盐，蛋白激酶 A（PKA）抑制剂；JQ1，Brd4 溴结构域抑制剂；A-485，p300/CBP 抑制剂。我们对处理和对照的 As4.1 细胞的生物重复样本进行转座酶可及染色质测序（ATAC-seq）、单细胞 RNA 测序、靶向切割和标签化（CUT&Tag）以及 H3K27ac（组蛋白 H3 蛋白赖氨酸 27 的乙酰化）和 p300 结合的染色质免疫沉淀测序。
每种抑制剂都能显著降低 renin 的表达，且在洗涤后可恢复。 基因座的染色质可及性没有明显变化，但在远端元件处整体减少。PKA 的抑制导致 H3K27ac 和 p300 结合在 超级增强子区域内显著减少。此外，我们鉴定了跨每种抑制处理都富集的 TF（转录因子）基序。最后，我们鉴定了一组 9 个基因，这些基因在 3 种肾素调节途径中都有潜在作用，并观察到它们在各自的基因座上都表现出不同的染色质可及性、基因表达、H3K27ac 和 p300 结合。
在持续合成和释放肾素的细胞中抑制肾素的表达是由一种从活跃到静止的表观遗传开关调节的，这种开关与细胞间通讯减少和上皮-间充质转化有关。这项工作强调并有助于确定肾素细胞在肌内分泌和收缩表型之间交替所需的因素。
