Prox1 directly interacts with LSD1 and recruits the LSD1/NuRD complex to epigenetically co-repress CYP7A1 transcription.
Prox1 directly interacts with LSD1 and recruits the LSD1/NuRD complex to epigenetically co-repress CYP7A1 transcription.
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Prox1 直接与 LSD1 相互作用并招募 LSD1/NuRD 复合物以表观遗传方式共同抑制 CYP7A1 转录
DOI：
10.1371/journal.pone.0062192
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Liu J
中科院分区：
文献类型：
--
作者：
Ouyang H;Qin Y;Liu Y;Xie Y;Liu J
胆固醇7α-羟化酶（CYP 7A 1）催化肝脏胆汁酸合成的第一步和限速步骤，对维持脂质稳态至关重要。肝细胞核因子4α（HNF 4 α）和甲胎蛋白转录因子（FTF）是驱动肝细胞CYP 7A 1启动子活性的两个主要转录因子。Prospero-related homeobox（Prox 1）与HNF 4 α和FTF直接相互作用，通过未知的机制共同抑制CYP 7A 1的转录和胆汁酸的合成。在这项工作中，参与Prox 1介导的共阻遏的机制进行了探索，通过识别Prox 1相关蛋白，使用免疫沉淀，然后质谱（IP-MS）方法。表观遗传抑制性赖氨酸特异性脱甲基酶1（LSD 1）/核小体重塑和组蛋白脱乙酰酶（NuRD）复合物的多个组分，最值得注意的是LSD 1和组蛋白脱乙酰酶2（HDAC 2），被发现与Prox 1和GST下拉试验证明，Prox 1直接与LSD 1相互作用。连续染色质免疫沉淀（ChIP）分析显示Prox 1与HepG 2细胞中的HNF 4 α、LSD 1和HDAC 2共定位于CYP 7A 1启动子上。此外，通过在HepG 2细胞上使用ChIP试验，通过RNA干扰敲低内源性Prox 1，Prox 1显示将LSD 1和HDAC 2募集到CYP 7A 1启动子上，并导致H3 K4去甲基化增加。最后，胆汁酸处理HepG 2细胞，显著抑制CYP 7A 1转录，导致CYP 7A 1启动子上Prox 1和LSD 1/NuRD复合物占用增加，同时增加H3 K4去甲基化和H3/H4去乙酰化。这些结果表明，Prox 1与LSD 1相互作用，将抑制性LSD 1/NuRD复合物募集到CYP 7A 1启动子，并通过表观遗传机制共同抑制转录。此外，这种Prox 1介导的表观遗传抑制参与胆汁酸对CYP 7A 1转录的生理学上必要的负反馈抑制。
Cholesterol 7α-hydroxylase (CYP7A1) catalyzes the first and rate-limiting step in the classical pathway of bile acids synthesis in liver and is crucial for maintaining lipid homeostasis. Hepatocyte nuclear factor 4α (HNF4α) and α1-fetoprotein transcription factor (FTF) are two major transcription factors driving CYP7A1 promoter activity in hepatocytes. Previous researches have shown that Prospero-related homeobox (Prox1) directly interacts with both HNF4α and FTF and potently co-represses CYP7A1 transcription and bile acid synthesis through unidentified mechanisms. In this work, mechanisms involved in Prox1-mediated co-repression were explored by identifying Prox1-associated proteins using immunoprecipitation followed by mass spectrometry (IP-MS) methodology. Multiple components of the epigenetically repressive lysine-specific demethylase 1 (LSD1)/nucleosome remodeling and histone deacetylase (NuRD) complex, most notably LSD1 and histone deacetylase 2 (HDAC2), were found to be associated with Prox1 and GST pulldown assay demonstrated that Prox1 directly interacts with LSD1. Sequential chromatin immunoprecipitation (ChIP) assays showed that Prox1 co-localizes with HNF4α, LSD1 and HDAC2 on CYP7A1 promoter in HepG2 cells. Furthermore, by using ChIP assay on HepG2 cells with endogenous Prox1 knocked down by RNA interference, Prox1 was shown to recruit LSD1 and HDAC2 onto CYP7A1 promoter and cause increased H3K4 demethylation. Finally, bile acids treatment of HepG2 cells, which significantly repressed CYP7A1 transcription, resulted in increased Prox1 and LSD1/NuRD complex occupancy on CYP7A1 promoter with a concurrent increase in H3K4 demethylation and H3/H4 deacetylation. These results showed that Prox1 interacts with LSD1 to recruit the repressive LSD1/NuRD complex to CYP7A1 promoter and co-represses transcription through epigenetic mechanisms. In addition, such Prox1-mediated epigenetic repression is involved in the physiologically essential negative feedback inhibition of CYP7A1 transcription by bile acids.
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