Epigenetic Regulation of Nuclear Receptor 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，没有观察到重组PTIP和PPARgamma蛋白之间的直接相互作用，这表明PPARgamma可能通过PTIP相关蛋白与PTIP间接相互作用。内源性PTIP和一种新的蛋白PA 1都是Set 1样组蛋白H3 K4甲基转移酶复合物的亚基（即MLL 3/MLL 4复合物），其含有H3 K4甲基转移酶MLL 3和MLL 4，以及含有JmjC结构域的蛋白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。 PPARdelta是核受体的PPAR家族的成员，并且广泛表达。PPARdelta的激活促进脂肪燃烧。高度特异性的合成PPARdelta配体（激动剂），如GW 501516（GW），是用于肥胖症和糖尿病的有希望的候选药物。内源性PPARdelta在小鼠胚胎成纤维细胞（MEFs）中大量表达，但与组蛋白脱乙酰酶相关，并且在缺乏配体的情况下表现为转录抑制因子。在配体处理后，内源性PPARdelta从阻遏物转换为激活物，这导致靶基因如Angptl 4的稳健激活。我们使用GW诱导的Angptl 4在MEFs中的表达作为模型系统来研究组蛋白乙酰转移酶GCN 5/PCAF、CBP/p300以及相关的组蛋白乙酰化在调节内源性核受体靶基因表达中的作用。

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.