MLL3/4-complexes in nuclear receptor-mediated metabolism

描述（由申请人提供）：胆汁酸（BA）信号传导的稳态调节需要复杂的核受体（NR）网络，但人们对它们的共激活剂（重塑染色质并响应 BA 信号传导调节基因转录）知之甚少。我们对 NR 共激活剂 ASC-2 的发现导致我们随后纯化了“ASC-2-复合物”(ASCOM)，这是第一个包含 H3 赖氨酸 4 甲基转移酶 (H3K4MT) MLL3 或其旁系同源物 MLL4 的哺乳动物复合物。后来，ASCOM还被发现含有H3-赖氨酸27-去甲基酶（H3K27DM）UTX。三甲基化 H3K4 和三甲基化 H3K27 分别标记转录活性和非活性染色质。因此，ASCOM 包含两种与转录激活相关的酶。令人兴奋的是，我们发现 ASCOM 的主要生理功能是在各种不同条件下调节代谢，这主要归因于 ASC-2 能够将 ASCOM 募集到多种代谢 NR 中，包括 PPAR？、LXR 和 FXR（BA 的 NR）。特别是，我们发现 ASCOM 在调节 BA 合成中充当 FXR 的关键共激活剂。一致地，我们的 MLL3 突变小鼠最显着的表型之一是 BA 水平显着增加，表明 MLL3 介导的 ASCOM H3K4MT 活性对于维持 BA 稳态至关重要。有趣的是，我们的 MLL3 突变小鼠也表现出良好的代谢特征，我们认为这是由于 ASCOM 拮抗 Tgr5（BA 的质膜受体）信号传导能力的缺陷所致。 Tgr5 触发一条信号通路，导致“环腺苷酸依赖性甲状腺激素激活酶 2 型碘甲状腺氨酸脱碘酶”(D2) 上调，并增强胰高血糖素样肽-1 (GLP-1)（一种胰岛素促分泌剂）的分泌，从而增加能量消耗并改善葡萄糖稳态。我们的初步结果表明，ASCOM 不仅通过降低 BA 水平来抑制 Tgr5 信号传导，而且还直接通过调节编码 Tgr5 信号传导关键修饰因子“二肽基肽酶-4”(Dpp4) 的基因来抑制 Tgr5 信号传导，该基因可使 GLP-1 失活。总之，这些结果支持了本研究的中心假设：ASCOM 通过控制调节 BA 代谢和信号传导的多个途径中的基因表达，充当“BA 信号传导的稳态调节”的主辅激活剂。此次更新有两个目标：1) 我们将利用上一资助期间的发现来测试 ASCOM 在 NR 反式激活（主要针对 FXR 和 RAR）中的作用机制。 2）通过特别关注ASCOM在BA稳态中的作用，我们将建立一个新的范式来理解ASCOM的多种代谢作用。这是一项综合性良好的研究，因为该研究的第一部分对于理解 ASCOM 在 BA 稳态和信号传导中的功能的分子基础至关重要（该研究的第二部分）。我们将结合生物化学、细胞和遗传学方法，通过三个具体目标来解决这两个问题。这项研究将帮助我们了解 NR 如何调节转录和代谢的分子基础。

DESCRIPTION (provided by applicant): The homeostatic regulation of bile acid (BA) signaling requires a complex network of nuclear receptors (NRs), but their coactivators that remodel chromatin and regulate gene transcription in response to BA signaling are poorly understood. Our discovery of the NR coactivator ASC-2 led to our subsequent purification of 'ASC-2- complex' (ASCOM), the first mammalian complex that contains the H3 lysine 4 methyltransferase (H3K4MT) MLL3 or its paralogue MLL4. Later, ASCOM has also been found to contain the H3-lysine 27-demethylase (H3K27DM) UTX. Trimethylated H3K4 and trimethylated H3K27 mark transcriptionally active and inactive chromatin, respectively. Thus, ASCOM contains two types of enzymes that are linked to transcriptional activation. Excitingly, we found that the major physiological function of ASCOM is to regulate metabolism under a variety of different conditions primarily attributed by the ability of ASC-2 to recruit ASCOM to multiple metabolic NRs, including PPAR?, LXRs, and FXR, the NR for BAs. In particular, we discovered that ASCOM functions as a critical coactivator for FXR in regulating BA synthesis. Consistently, one of the most salient phenotypes of our MLL3 mutant mice was a significant increase in BA levels, suggesting that MLL3-mediated H3K4MT activity of ASCOM is essential for maintaining BA homeostasis. Intriguingly, our MLL3 mutant mice also displayed favorable metabolic profiles, which we propose is via defects in the ability of ASCOM to antagonize signaling by Tgr5, the plasma membrane receptor for BAs. Tgr5 triggers a signaling pathway that leads to upregulation of 'the cyclic-AMP-dependent thyroid hormone activating enzyme type 2 iodothyronine deiodinase' (D2) and to enhance secretion of glucagon-like peptide-1 (GLP-1), an insulin secretagogue, thereby resulting in enhanced energy expenditure and improved glucose homeostasis. Our preliminary results suggest that ASCOM inhibits Tgr5 signaling not only through decreasing BA levels but also directly through the regulation of a gene encoding a key modifier of Tgr5 signaling, 'dipeptidyl peptidase-4' (Dpp4), which inactivates GLP-1. Together, these results support the central hypothesis of this study: ASCOM functions as a master coactivator of 'the homeostatic regulation of BA signaling' by controlling the expression of genes in multiple pathways that regulate BA metabolism and signaling. This renewal has two objectives: 1) We will test the mechanisms of action for ASCOM in NR transactivation (primarily for FXR and RAR) by using the discoveries made during the previous funding period. 2) By focusing specifically on the role of ASCOM in BA homeostasis, we will establish a new paradigm for understanding the diverse metabolic roles of ASCOM. This is a well-integrated study, as the first part of the study is critical to understand the molecular basis for the functio of ASCOM in BA homeostasis and signaling (the second part of the study). We will tackle these two issues in three specific aims, utilizing a combination of biochemical, cellular and genetic approaches. This study will help us to understand the molecular basis for how NRs regulate transcription and metabolism.