Epigenetic Modifications to H3K9 in Renal Tubulointerstitial Cells after Unilateral Ureteric Obstruction and TGF-β1 Stimulation.
Epigenetic Modifications to H3K9 in Renal Tubulointerstitial Cells after Unilateral Ureteric Obstruction and TGF-β1 Stimulation.
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DOI：
10.3389/fphar.2017.00307
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发表时间：
2017
影响因子：
5.6
通讯作者：
Smith ER
中科院分区：
文献类型：
--
作者：
Hewitson TD;Holt SG;Tan SJ;Wigg B;Samuel CS;Smith ER
通过翻译后组蛋白修饰（标记）进行的纤维发生的表观遗传调控可能是肾脏疾病进展的关键决定因素。在本研究中，我们检测了组蛋白3赖氨酸9 （H3K9）标记在损伤和促纤维化细胞因子TGF-β1刺激后的分布和获取。我们的重点是它们在活化的成纤维细胞（肌成纤维细胞）和上皮细胞（上皮-间充质转化）中的存在。方法与结果：采用免疫荧光显微镜观察小鼠单侧输尿管梗阻（UUO）后H3K9乙酰化（H3K9Ac）和三甲基化（H3K9Me3）的变化。采用共聚焦、超分辨显微镜和流式细胞术检测TGF-β1对这些标记结构排列的体外影响，以及它们与肌成纤维细胞和早期EMT标志物α-平滑肌肌动蛋白（αSMA）表达的关系。UUO后10天个体组蛋白标记的数量增加（p < 0.05），在包括近端小管和肌成纤维细胞在内的各种细胞类型中都可以清楚地看到这两种标记。在原代大鼠肾成纤维细胞和近端小管细胞系（NRK-52e）的亚核显微镜下显示，H3K9Ac与磷酸化ser2 RNA聚合酶II （pRNAPol II）共定位，而H3K9Me3则不与之共定位，分别对基因表达具有允许和抑制作用。在这两种细胞类型中，H3K9Ac弥漫性分布在整个细胞核中，而H3K9Me3在类似核仁的室室中被发现，在成纤维细胞中，也与核膜并列。TGF-β1对两种细胞中的H3K9Ac标记均无影响，但导致H3K9Me3在成纤维细胞细胞核内重新分布。这与有丝分裂发生的变化无关，但与α - sma表达增加有关。结论：这些发现强调了为什么单独考虑每个细胞的表观遗传学是重要的，因为尽管没有发生总体富集，但肾肌成纤维细胞分化伴随着组蛋白标记排列的明显变化。
Introduction: Epigenetic regulation of fibrogenesis through post-translational histone modifications (marks) may be a key determinant of progression in renal disease. In this study, we examined the distribution and acquisition of histone 3 Lysine 9 (H3K9) marks after injury and stimulation with the pro-fibrotic cytokine TGF-β1. Our focus was on their presence in activated fibroblasts (myofibroblasts) and epithelial cells (epithelial-mesenchymal transition). Methods and Results: Immunofluorescent microscopy was used to examine global H3K9 acetylation (H3K9Ac) and tri-methylation (H3K9Me3) after unilateral ureteric obstruction (UUO) in mice. Confocal, super resolution microscopy and flow cytometry were used to determine the in vitro effect of TGF-β1 on structural arrangement of these marks, and their relationship with α-smooth muscle actin (αSMA) expression, a marker of myofibroblasts and early EMT. The number of individual histone marks was increased 10 days after UUO (p < 0.05 vs. control), with both marks clearly seen in various cell types including proximal tubules and myofibroblasts. Sub-nuclear microscopy in primary rat renal fibroblasts and a proximal tubule cell line (NRK-52e) showed that H3K9Ac was co-localized with phosphorylated-Ser2 RNA polymerase II (pRNAPol II), while H3K9Me3 was not, consistent with permissive and repressive effects on gene expression respectively. In both cell types H3K9Ac was diffusely distributed throughout the nucleus, while H3K9Me3 was found in compartments resembling the nucleolus, and in the case of the fibroblast, also juxtapositioned with the nuclear membrane. TGF-β1 had no effect on H3K9Ac marks in either cell, but resulted in a redistribution of H3K9Me3 within the fibroblast nucleus. This was unrelated to any change in mitogenesis, but was associated with increased αSMA expression. Conclusion: These findings highlight why it is important to consider the epigenetics of each cell individually, because whilst no overall enrichment occurred, renal myofibroblast differentiation was accompanied by distinct changes in histone mark arrangements.