Epigenetic Regulation of Nuclear Receptor Target Gene Expression

我的实验室最初的努力是分离PPAR伽马的新的转录辅助因子。以GST融合的PPARGamma配体结合域(GST-PPARgLBD)为诱饵，我们从细胞核提取液中提取了参与DNA损伤反应的核蛋白PTIP。在报告实验中，PTIP作为PPARGamma的转录共激活因子发挥作用。通过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和一个新的蛋白PA1都是含有H3K4甲基转移酶ML3和MLL4的Set1样组蛋白H3K4甲基转移酶复合体(即MLL3/MLL4复合体)和含有JmjC结构域的蛋白UTX(Cho，Y.-W.等，J.Biol)的亚基。化学，2007年。第282页：20395-20406页。)此外，我们证明了含有JmjC结构域的蛋白UTX和JMJD3是组蛋白H3K27特异的去甲基酶(Hong，S.，等人，PNAS，2007。第104页：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标记。根据我们的发现，H3K4甲基转移酶MLL3/MLL4物理上与H3K27去甲基酶UTX结合，我们认为，通过增加一个激活的表观遗传标记和去除一个抑制标记，MLL3/MLL4复合体可能使用两种不同的组蛋白修饰活性来协同激活靶基因的表达。我们将使用来自MLL3-/-和MLL4-FLOX/FLOX小鼠的MEF细胞系来研究MLL3/MLL4复合体如何调节配体诱导的PPAR伽马靶基因的表达。 PPAR Delta是核受体PPAR家族的一员，广泛表达。PPARDelta的激活促进脂肪燃烧。高度特异的合成PPAR Delta配体(激动剂)，如GW501516(GW)，是治疗肥胖症和糖尿病的有前途的候选药物。内源性PPARDelta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，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从抑制子转换为激活子，从而导致ANGPTL4等靶基因的强劲激活。我们正在使用GW诱导的ANGPTL4在MEF中的表达作为一个模型系统来研究组蛋白乙酰转移酶GCN5/PCAF、CBP/p300及其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。我们报道了两对组蛋白乙酰转移酶(HATS)，GCN5/PCAF和CBP/p300，分别是细胞中H3K9乙酰化(H3K9ac)和H3K18/27乙酰化(H3K18/27ac)所必需的。进一步，我们证明了CBP/p300及其HAT活性对于配体诱导的核受体靶基因表达是必需的，而GCN5/PCAF和相关的H3K9ac是必不可少的。这些结果突出了HATS在细胞中底物和位点的特异性，证明了GCN5/PCAF-和CBP/p300介导组蛋白乙酰化在基因激活中的不同作用，并表明CBP/p300介导的H3K18/27ac在核受体靶基因表达中发挥重要作用(金强等，EMBO J，2011)。我们推测CBP/p300介导的H3K18/27ac可能被尚未鉴定的效应蛋白识别，这些效应蛋白招募RNA聚合酶II启动转录。我们将使用亲和纯化来分离和确定效应蛋白的特性。

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.