Epigenetic Regulation of Nuclear Receptor Target Gene Expression

我的实验室最初的努力是分离出PPARgamma的新型转录辅因子。以gst融合的PPARgamma配体结合域（GST-PPARgLBD）为诱饵，从细胞核提取物中提取了与DNA损伤反应有关的核蛋白PTIP。在报告基因分析中，PTIP作为PPARgamma的转录辅激活因子。然而，重组PTIP与PPARgamma蛋白之间没有直接相互作用，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和新蛋白PA1都是set1样组蛋白H3K4甲基转移酶复合物（即MLL3/MLL4复合物）的亚基，该复合物含有H3K4甲基转移酶MLL3和MLL4，以及含有JmjC结构域的蛋白UTX (Cho, y - w。，等。[j]。化学。, 2007年。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特异性去甲基化酶（Hong， S.等，PNAS, 2007）。104: 18439-18444页)。H3K4上的甲基化是一个激活的表观遗传标记，而H3K27上的甲基化是一个抑制的表观遗传标记。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表观遗传标记和去除抑制标记，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。我们将使用来自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。

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.