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: 7967496
• 负责人: Kai Ge
• 金额: $ 39.34万
• 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/7967496
• 关键词: Acetylation; Biological Assay; Cell Line; Cells; Complex; DNA Damage; DNA Methylation; Development; Epigenetic Process; Family; Gene Activation; Gene Expression; Histone Acetylation; Histone H3; Histones; Ligand Binding Domain; Ligands; Lysine; Methylation; Methyltransferase; Molecular; Mus; Non-Insulin-Dependent Diabetes Mellitus; Nuclear Extract; Nuclear Protein; Nuclear Proteins; Nuclear Receptors; Obesity; PPAR delta; PPAR gamma; Pharmaceutical Preparations; Phosphorylation; Play; Proteins; Proteomics; Recombinants; Regulation; Reporter; Repression; Role; Site; Staging; Tissues; Transcription Coactivator; Variant; activating transcription factor; base; chromatin remodeling; cofactor; drug candidate; gene repression; histone methyltransferase; histone modification; lipid metabolism; novel; response

My labs 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.

我实验室最初的努力是分离PPARgamma的新型转录辅因子。以GST融合的PPARgamma配体结合结构域（GST-PPARgLBD）为诱饵，从细胞核提取物中提取PTIP蛋白。PTIP在报告基因分析中作为PPARgamma的转录共激活因子发挥作用。然而，重组PTIP和PPARgamma蛋白之间没有观察到直接的相互作用，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和新蛋白质PA 1都是Set 1样组蛋白H3 K4甲基转移酶复合物（即MLL 3/MLL 4复合物）的亚基，所述复合物含有H3 K4甲基转移酶MLL 3和MLL 4，并且含有JmjC结构域的蛋白质UTX（Cho，Y. W.，例如，J. Biol. Chem.，2007. 282：第20395-20406页）。 此外，我们证明了含JmjC结构域的蛋白UTX和JMJD 3是组蛋白H3 K27特异性脱甲基酶（Hong，S.，例如，PNAS，2007年。104：第18439-18444页）。 H3 K4上的甲基化是活化的表观遗传标记，而H3 K27上的甲基化是抑制性的。基于我们发现H3 K4甲基转移酶MLL 3/MLL 4与H3 K27去甲基酶UTX物理相关，我们提出通过添加活化表观遗传标记并去除抑制性标记，MLL 3/MLL 4复合物可以使用两种不同的组蛋白修饰活性来协同活化靶基因表达。我们将使用来自MLL 3-/-和MLL 4-flox/flox小鼠的MEF细胞系来研究MLL 3/MLL 4复合物如何调节配体诱导的PPARgamma靶基因表达。