Epigenetic Regulation of Nuclear Receptor Target Gene Expression

核受体靶基因表达的表观遗传调控

• 批准号: 8741472
• 负责人: Kai Ge
• 金额: $ 11.71万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8741472
• 关键词: Acetylation; Affinity Chromatography; Agonist; Biological Assay; Biological Models; Burn injury; Cell Line; Cells; Complex; DNA Damage; DNA Methylation; Development; Diabetes Mellitus; EP300 gene; Embryo; Epigenetic Process; Family; Fatty acid glycerol esters; Fibroblasts; Gene Activation; Gene Expression; Gene Targeting; Genetic Transcription; Histone Acetylation; Histone H3; Histones; Ligand Binding Domain; Ligands; Lysine; Mediating; Methylation; Methyltransferase; Molecular; Mus; Non-Insulin-Dependent Diabetes Mellitus; Nuclear Extract; Nuclear Protein; Nuclear Receptors; Obesity; PCAF gene; PPAR delta; PPAR gamma; Peroxisome Proliferator-Activated Receptors; Pharmaceutical Preparations; Phosphorylation; Play; Proteins; Proteomics; RNA Polymerase II; Recombinants; Recruitment Activity; Regulation; Reporter; Reporting; Role; Site; Specificity; Staging; Tissues; Transcription Coactivator; Transcription Repressor/Corepressor; Variant; activating transcription factor; base; chromatin remodeling; cofactor; drug candidate; gene repression; histone acetyltransferase; histone methyltransferase; histone modification; lipid metabolism; member; novel; response

My lab's initial effort was to isolate novel transcriptional cofactors for PPARgamma. Using GST-fused PPARgamma ligand binding domain (GST-PPARgLBD) as bait, we pulled down PTIP, a nuclear protein that has been implicated in DNA damage response, from cell nuclear extracts. PTIP functions as a transcription coactivator for PPARgamma in reporter assay. However, no direct interaction was observed between recombinant PTIP and PPARgamma proteins, suggesting that PPARgamma may interact indirectly with PTIP through PTIP-associated proteins.By using proteomic approaches to isolate PTIP-associated proteins, we found that in cells, endogenous PTIP and a novel protein PA1 are both subunits of a Set1-like histone H3K4 methyltransferase complex (i.e. MLL3/MLL4 complex) that contains H3K4 methyltransferases MLL3 and MLL4, and the JmjC domain-containing protein UTX (Cho, Y.-W., et al., J. Biol. Chem., 2007. 282: p. 20395-20406.) Further, we demonstrate that the JmjC domain-containing proteins UTX and JMJD3 are histone H3K27-specific demethylases (Hong, S., et al., PNAS, 2007. 104: p. 18439-18444). Methylation on H3K4 is an activating epigenetic mark while methylation on H3K27 is a repressive one. Based on our finding that H3K4 methyltransferases MLL3/MLL4 physically associate with H3K27 demethylase UTX, we propose that by adding an activating epigenetic mark and removing a repressive one, the MLL3/MLL4 complex may use two distinct histone modifying activities to synergistically activate target gene expression. We will use MEF cell lines derived from MLL3-/- and MLL4-flox/flox mice to investigate how MLL3/MLL4 complex regulates ligand-induced PPARgamma target gene expression. PPARdelta is a member of the PPAR family of nuclear receptors and is ubiquitously expressed. Activation of PPARdelta promotes fat burning. Highly specific synthetic PPARdelta ligands (agonists), such as GW501516 (GW), are promising drug candidates for obesity and diabetes. Endogenous PPARdelta is abundantly expressed in mouse embryonic fibroblasts (MEFs) but associates with histone deacetylases and behaves as a transcriptional repressor in the absence of ligand. Upon ligand treatment, endogenous PPARdelta switches from a repressor to an activator, which leads to a robust activation of target genes such as Angptl4. We are using the GW-induced Angptl4 expression in MEFs as a model system to investigate the roles of histone acetyltransferases GCN5/PCAF, CBP/p300, and associated histone acetylations in regulating expression of endogenous nuclear receptor target genes. We report that the two pairs of histone acetyltransferases (HATs), GCN5/PCAF and CBP/p300, are specifically required for H3K9 acetylation (H3K9ac) and H3K18/27 acetylation (H3K18/27ac), respectively, in cells. Further, we show that CBP/p300 and their HAT activities are essential, while GCN5/PCAF and associated H3K9ac are dispensable, for ligand-induced nuclear receptor target gene expression. These results highlight the substrate and site specificities of HATs in cells, demonstrate the distinct roles of GCN5/PCAF- and CBP/p300-mediated histone acetylations in gene activation, and suggest an important role of CBP/p300-mediated H3K18/27ac in nuclear receptor target gene expression (Jin Q. et al., EMBO J, 2011). We hypothesize that CBP/p300-mediated H3K18/27ac may be recognized by yet to be identified effector proteins, which recruit RNA polymerase II to initiate transcription. We will use affinity purification to isolate and determine the identities of the effector proteins.

我的实验室最初的努力是分离 PPARgamma 的新型转录辅助因子。使用 GST 融合的 PPARgamma 配体结合域 (GST-PPARgLBD) 作为诱饵，我们从细胞核提取物中提取了 PTIP，这是一种与 DNA 损伤反应有关的核蛋白。 PTIP 在报告基因检测中充当 PPARgamma 的转录共激活剂。然而，重组PTIP和PPARgamma蛋白之间没有观察到直接相互作用，这表明PPARgamma可能通过PTIP相关蛋白与PTIP间接相互作用。通过使用蛋白质组学方法分离PTIP相关蛋白，我们发现在细胞中，内源性PTIP和一种新蛋白PA1都是Set1样组蛋白H3K4的亚基 含有 H3K4 甲基转移酶 MLL3 和 MLL4 以及含有 JmjC 结构域的蛋白 UTX 的甲基转移酶复合物（即 MLL3/MLL4 复合物）（Cho, Y.-W., et al., J. Biol. Chem., 2007. 282: p. 20395-20406。）此外，我们证明含有 JmjC 结构域的蛋白 UTX 和 JMJD3 是组蛋白 H3K27 特异性去甲基酶 (Hong, S., et al., PNAS, 2007. 104: p. 18439-18444)。 H3K4 上的甲基化是一种激活的表观遗传标记，而 H3K27 上的甲基化是一种抑制性标记。基于我们发现 H3K4 甲基转移酶 MLL3/MLL4 与 H3K27 去甲基化酶 UTX 物理关联，我们提出，通过添加激活的表观遗传标记并去除抑制性标记，MLL3/MLL4 复合物可能使用两种不同的组蛋白修饰活性来协同激活靶基因表达。我们将使用源自 MLL3-/- 和 MLL4-flox/flox 小鼠的 MEF 细胞系来研究 MLL3/MLL4 复合物如何调节配体诱导的 PPARgamma 靶基因表达。 PPARδ 是 PPAR 核受体家族的成员，广泛表达。 PPARδ 的激活促进脂肪燃烧。高度特异性的合成 PPARδ 配体（激动剂），例如 GW501516 (GW)，是治疗肥胖和糖尿病的有前途的候选药物。内源性 PPARδ 在小鼠胚胎成纤维细胞 (MEF) 中大量表达，但与组蛋白脱乙酰酶相关，并且在没有配体的情况下充当转录抑制因子。配体处理后，内源性 PPARδ 从阻遏物转变为激活物，从而导致 Angptl4 等靶基因的强烈激活。我们使用 MEF 中 GW 诱导的 Angptl4 表达作为模型系统，研究组蛋白乙酰转移酶 GCN5/PCAF、CBP/p300 和相关组蛋白乙酰化在调节内源核受体靶基因表达中的作用。我们报告说，两对组蛋白乙酰转移酶 (HAT) GCN5/PCAF 和 CBP/p300 分别是细胞中 H3K9 乙酰化 (H3K9ac) 和 H3K18/27 乙酰化 (H3K18/27ac) 所特别需要的。此外，我们表明，对于配体诱导的核受体靶基因表达，CBP/p300 及其 HAT 活性是必需的，而 GCN5/PCAF 和相关的 H3K9ac 是可有可无的。这些结果突出了细胞中 HAT 的底物和位点特异性，证明了 GCN5/PCAF 和 CBP/p300 介导的组蛋白乙酰化在基因激活中的独特作用，并表明 CBP/p300 介导的 H3K18/27ac 在核受体靶基因表达中的重要作用 (Jin Q. et al., EMBO J, 2011)。我们假设 CBP/p300 介导的 H3K18/27ac 可能被尚未确定的效应蛋白识别，这些效应蛋白招募 RNA 聚合酶 II 来启动转录。 我们将使用亲和纯化来分离和确定效应蛋白的身份。