Repression of Cardiac Phospholamban Gene Expression Is Mediated by Thyroid Hormone Receptor-α1 and Involves Targeted Covalent Histone Modifications
Repression of Cardiac Phospholamban Gene Expression Is Mediated by Thyroid Hormone Receptor-α1 and Involves Targeted Covalent Histone Modifications
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DOI：
10.1210/en.2009-1241
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发表时间：
2010-06-01
期刊：
影响因子：
4.8
通讯作者：
Fondell, Joseph D.
中科院分区：
文献类型：
--
作者：
Belakavadi, Madesh;Saunders, Jason;Fondell, Joseph D.
受磷蛋白（PLB）是心肌细胞中钙循环的关键调节因子，其基因表达被T-3显著下调。尽管如此，对心肌中T-3依赖性基因沉默的分子机制知之甚少，并且仍不清楚甲状腺激素受体（TRs）是否直接在体内结合PLB基因并促进转录抑制。为了研究TRs在PLB转录中的调节作用，我们使用了一种生理性小鼠心肌细胞系（HL-1），该细胞系保留了心脏电生理特性，表达TR α 1和TR β 1亚型，并表现出T-3依赖性PLB表达沉默。通过对HL-1细胞进行RNA干扰试验，我们发现TR α 1，而不是TR β 1，是T3依赖性PLB基因抑制所必需的。有趣的是，仅包含核心启动子序列-156至+64的PLB报告基因在HL-1细胞中显示出强烈的T-3依赖性沉默，从而表明TR α 1通过PLB核心启动子促进转录抑制，PLB核心启动子是哺乳动物中高度保守的调节区。与此一致的是，染色质免疫沉淀和体外结合试验表明TR α 1直接结合在PLB核心启动子区。此外，T3的加入触发了与基因沉默相关的PLB启动子处的共价组蛋白修饰的改变，即组蛋白H3乙酰化和组蛋白H3赖氨酸4甲基化的显著降低。总之，我们的数据表明，T-3依赖性抑制心肌细胞中的PLB直接促进TR α 1，并涉及与转录沉默相关的组蛋白修饰酶的依赖性募集。（内分泌学151：2946-2956，2010）
Phospholamban (PLB) is a critical regulator of Ca2+ cycling in heart muscle cells, and its gene expression is markedly down-regulated by T-3. Nonetheless, little is known about the molecular mechanisms of T-3-dependent gene silencing in cardiac muscle, and it remains unclear whether thyroid hormone receptors (TRs) directly bind at the PLB gene in vivo and facilitate transcriptional repression. To investigate the regulatory role of TRs in PLB transcription, we used a physiological murine heart muscle cell line (HL-1) that retains cardiac electrophysiological properties, expresses both TR alpha 1 and TR beta 1 subtypes, and exhibits T-3-dependent silencing of PLB expression. By performing RNA interference assays with HL-1 cells, we found that TR alpha 1, but not TR beta 1, is essential for T3-dependent PLB gene repression. Interestingly, a PLB reporter gene containing only the core promoter sequences -156 to +64 displayed robust T-3-dependent silencing in HL-1 cells, thus suggesting that transcriptional repression is facilitated by TR alpha 1 via the PLB core promoter, a regulatory region highly conserved in mammals. Consistent with this notion, chromatin immunoprecipitationandin vitro binding assaysshowthat TR alpha 1 directly binds at the PLB core promoter region. Furthermore, addition of T3 triggered alterations in covalent histone modifications at the PLB promoter that are associated with gene silencing, namely a pronounced decrease in both histone H3 acetylation and histone H3 lysine 4 methylation. Taken together, our data reveal that T-3-dependent repression of PLB in cardiac myocytes is directly facilitated by TR alpha 1 and involves the hormone-dependent recruitment of histone-modifying enzymes associated with transcriptional silencing. (Endocrinology 151: 2946-2956, 2010)