Background & Aims

Biliary disease is associated with a proliferative/fibrogenic ductular reaction (DR). p300 is an epigenetic regulator that acetylates lysine 27 on histone 3 (H3K27ac) and is activated during fibrosis. Long non-coding RNAs (lncRNAs) are aberrantly expressed in cholangiopathies, but little is known about how they recruit epigenetic complexes and regulate DR. We investigated epigenetic complexes, including transcription factors (TFs) and lncRNAs, contributing to p300-mediated transcription during fibrosis.

Methods

We evaluated p300 in vivo using tamoxifen-inducible, cholangiocyte-selective, p300 knockout (KO) coupled with bile duct ligation (BDL) and Mdr KO mice treated with SGC-CBP30. Primary cholangiocytes and liver tissue were analyzed for expression of Acta2-as1 lncRNA by qPCR and RNA in situ hybridization. In vitro, we performed RNA-sequencing in human cholangiocytes with a p300 inhibitor. Cholangiocytes were exposed to lipopolysaccharide (LPS) as an injury model. We confirmed formation of a p300/ELK1 complex by immunoprecipitation (IP). RNA IP was used to examine interactions between ACTA2-AS1 and p300. Chromatin IP assays were used to evaluate p300/ELK1 occupancy and p300-mediated H3K27ac. Organoids were generated from ACTA2-AS1-depleted cholangiocytes.

Results

BDL-induced DR and fibrosis were reduced in Krt19-Cre^ERT/p300^fl/fl mice. Similarly, Mdr KO mice were protected from DR and fibrosis after SGC-CBP30 treatment. In vitro, depletion of ACTA2-AS1 reduced expression of proliferative/fibrogenic markers, reduced LPS-induced cholangiocyte proliferation, and impaired organoid formation. ACTA2-AS1 regulated transcription by facilitating p300/ELK1 binding to the PDGFB promoter after LPS exposure. Correspondingly, LPS-induced H3K27ac was mediated by p300/ELK1 and was reduced in ACTA2-AS1-depleted cholangiocytes.

Conclusion

Cholangiocyte-selective p300 KO or p300 inhibition attenuate DR/fibrosis in mice. ACTA2-AS1 influences recruitment of p300/ELK1 to specific promoters to drive H3K27ac and epigenetic activation of proliferative/fibrogenic genes. This suggests that cooperation between epigenetic co-activators and lncRNAs facilitates DR/fibrosis in biliary diseases.

Lay summary

We identified a three-part complex containing an RNA molecule, a transcription factor, and an epigenetic enzyme. The complex is active in injured bile duct cells and contributes to activation of genes involved in proliferation and fibrosis.

中文翻译：

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长链非编码 RNA ACTA2-AS1 通过与 p300/ELK1 复合物相互作用促进导管反应

背景与目标

胆道疾病与增殖性/纤维化导管反应 (DR) 相关。p300 是一种表观遗传调节因子，可使组蛋白 3 (H3K27ac) 上的赖氨酸 27 乙酰化，并在纤维化过程中被激活。长链非编码 RNA (lncRNA) 在胆管病中异常表达，但关于它们如何募集表观遗传复合物和调节 DR 知之甚少。我们研究了表观遗传复合物，包括转录因子 (TF) 和 lncRNA，它们在纤维化过程中促进了 p300 介导的转录。

方法

我们使用他莫昔芬诱导的、胆管细胞选择性的、p300 敲除 (KO) 加上胆管结扎 (BDL) 和用 SGC-CBP30 处理的Mdr KO 小鼠在体内评估了 p300。通过 qPCR 和 RNA原位杂交分析原代胆管细胞和肝组织中 Acta2-as1 lncRNA的表达。在体外，我们用 p300 抑制剂对人胆管细胞进行了 RNA 测序。胆管细胞暴露于脂多糖 (LPS) 作为损伤模型。我们通过免疫沉淀 (IP) 确认了 p300/ELK1 复合物的形成。RNA IP 用于检查ACTA2-AS1之间的相互作用和 p300。染色质 IP 测定用于评估 p300/ELK1 占用和 p300 介导的 H3K27ac。类器官产生自ACTA2-AS1耗尽的胆管细胞。

结果

BDL 诱导的 DR 和纤维化在Krt19 -Cre ^ERT / p300 ^fl/fl小鼠中减少。类似地，Mdr KO 小鼠在 SGC-CBP30 治疗后免受 DR 和纤维化。在体外，ACTA2-AS1的消耗减少了增殖/纤维化标志物的表达，减少了 LPS 诱导的胆管细胞增殖，并损害了类器官的形成。ACTA2-AS1通过在 LPS 暴露后促进 p300/ELK1 与PDGFB启动子的结合来调节转录。相应地，LPS 诱导的 H3K27ac 由 p300/ELK1 介导，并在ACTA2-AS1耗尽的胆管细胞中减少。

结论

胆管细胞选择性 p300 KO 或 p300 抑制减弱小鼠的 DR/纤维化。ACTA2-AS1影响 p300/ELK1 向特定启动子的募集，以驱动 H3K27ac 和增殖/纤维化基因的表观遗传激活。这表明表观遗传共激活因子和 lncRNA 之间的合作促进了胆道疾病的 DR/纤维化。

外行总结

我们确定了一个包含 RNA 分子、转录因子和表观遗传酶的三部分复合物。该复合物在受损的胆管细胞中具有活性，并有助于激活参与增殖和纤维化的基因。