Coordinated Changes in DNA Methylation and Histone Modifications Regulate Silencing/Derepression of Luteinizing Hormone Receptor Gene Transcription
Coordinated Changes in DNA Methylation and Histone Modifications Regulate Silencing/Derepression of Luteinizing Hormone Receptor Gene Transcription
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DOI：
10.1128/mcb.25.18.7929-7939.2005
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发表时间：
2005-09
影响因子：
5.3
通讯作者：
Ying Zhang;N. Fatima;M. Dufau
中科院分区：
文献类型：
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作者：
Ying Zhang;N. Fatima;M. Dufau
摘要我们以前已经证明，黄体生成素受体(LHR)基因的转录受到其启动子区域组蛋白去乙酰化的抑制，组蛋白去乙酰化酶(HDAC)/mSin3A复合体锚定在Sp1的近端。目前的研究表明，LHR基因的表观遗传沉默和激活是通过在组蛋白和DNA水平上的协调调节实现的。HDAC抑制剂曲古抑素A(TSA)在JAR细胞中引起LHR基因的强烈激活，但显著低于MCF-7细胞。这种效应被定位在176个碱基的启动子区域，该区域在JAR中高度甲基化，在MCF-7细胞中轻度甲基化。因此，TSA和DNA去甲基化试剂5-氮杂胞苷(5-azaC)可显著协同激活JAR细胞中的LHR基因，但对MCF-7细胞中的LHR基因激活作用不明显。H3/H4的多个位点特异性赖氨酸乙酰化与LHR基因的激活有关。H3在K9的甲基化或乙酰化分别存在于沉默的LHR启动子和去抑制的LHR启动子上。虽然DNA甲基化水平不影响LHR基因启动子的组蛋白编码，但为了最大限度地刺激该基因，启动子CpG位点的去甲基化是必要的。在机制上，TSA和5-AzaC的联合作用，但不是5-AzaC或TSA单独作用，导致JAR细胞中LHR基因启动子完全去甲基化。在这两种细胞中，从LHR基因启动子释放抑制性HDAC/mSin3A复合体需要TSA诱导组蛋白修饰的变化，同时需要去甲基化的启动子。此外，在TSA或TSA+5-AzaC存在下，Dnmt1基本上与LHR基因启动子解离，并且当启动子转换到去甲基化状态时，Mbd2在JAR细胞中的结合减少。这些变化导致染色质更加宽容，其中聚合酶II和TFIIB对启动子的募集显著增加。在表达LHR的PLC细胞中，基本观察到TSA和5-azaC诱导的JAR和MCF-7细胞中LHR基因的激活状态，在这种状态下，启动子未甲基化，并与高乙酰化组蛋白相关。因此，PLC细胞对药物治疗没有反应。这些发现阐明了一种调控机制，通过组蛋白超乙酰化和DNA去甲基化，抑制物的同时解离以及激活物和基础转录成分的结合导致LHR基因表达的去抑制。
ABSTRACT We have previously demonstrated that transcription of the luteinizing hormone receptor (LHR) gene is subject to repression by histone deacetylation at its promoter region, where a histone deacetylase (HDAC)/mSin3A complex is anchored at a proximal Sp1 site. The present studies have shown that epigenetic silencing and activation of the LHR gene is achieved through coordinated regulation at both the histone and DNA levels. The HDAC inhibitor trichostatin A (TSA) evoked robust but significantly lower activation of the LHR gene in JAR than in MCF-7 cells. This effect was localized to the 176-bp promoter region, which is highly methylated in JAR and lightly methylated in MCF-7 cells. Consequently, TSA and the DNA demethylating reagent 5-azacytidine (5-AzaC) caused marked synergistic activation of the LHR gene in JAR but not in MCF-7 cells. Multiple site-specific lysine acetylation of H3/H4 is associated with such LHR gene activation. Methylation or acetylation of H3 at K9 is present at the silenced and derepressed LHR promoter, respectively. While DNA methylation levels did not affect the histone code of the LHR gene promoter, demethylation of the promoter CpG sites was necessary for maximal stimulation of this gene. Mechanistically, the combined actions of TSA and 5-AzaC, but not either 5-AzaC or TSA alone, resulted in complete demethylation of the LHR gene promoter in JAR cells. Release of the repressive HDAC/mSin3A complex from the LHR gene promoter in both cell types required both TSA-induced changes of histone modifications and, concurrently, a demethylated promoter. Also, Dnmt1 was largely dissociated from the LHR gene promoter in the presence of TSA or TSA plus 5-AzaC, and binding of MBD2 in JAR cells was diminished upon conversion of the promoter to a demethylated state. Such changes induced a more permissive chromatin where recruitment of polymerase II and TFIIB to the promoter was significantly increased. The activated state of the LHR gene induced by TSA and 5-AzaC in JAR and MCF-7 cells was observed basally in LHR-expressing PLC cells, in which the promoter is unmethylated and associated with hyperacetylated histones. Consequently, PLC cells are unresponsive to drug treatment. These findings have elucidated a regulatory mechanism whereby concurrent dissociation of repressors and association of activators and basal transcriptional components, resulting from coordinated histone hyperacetylation and DNA demethylation, lead to derepression of the LHR gene expression.