Thyroid hormone receptors - regulation and function

描述（由申请人提供）：甲状腺激素受体（TRs）是配体依赖性的代谢转录调节因子。TRs在缺乏激素的情况下抑制基因表达，这是其他核受体（nr）在无配体状态下作为抑制物的范例。抑制是通过与N- CoR（核受体辅抑制因子）和SMRT（类视黄醇和甲状腺受体沉默介质）的相互作用介导的，它们与染色质修饰酶组蛋白去乙酰化酶3 （HDAC3）存在化学计量关联。而HDAC3则通过其独特的去乙酰化酶激活结构域（DAD）与N-CoR/SMRT相互作用，从而获得其催化活性。dad依赖的N-CoR/SMRT7HDAC3复合物在体外对TR和其他nr的抑制至关重要，但这种相互作用在体内的作用尚不清楚。在这里，我们建议使用最先进的基因靶向和小鼠表型分析方法，首次测试N-CoR7HDAC3和SMRT7HDAC3辅抑制因子复合物的生理相关性。我们假设这些依赖于dad的相互作用是非常重要的，并且影响涉及NRs的不同生理途径。具体目的1是确定N-CoR DAD结构域的生理功能。敲除N-CoR是胚胎致死；我们假设与HDAC3的相互作用支持N-CoR的一个子集的发育和生理功能。为了发现这些功能是什么，我们产生了在N-CoR DAD结构域中具有阻止HDAC3相互作用的点突变的小鼠。初步数据表明，这种突变的纯合小鼠是可行的，具有有趣的异常，指出N-CoR7HDAC3的生物学重要性。具体目的2是确定SMRT DAD结构域的生理功能。与N-CoR类似，SMRT7HDAC3的生理作用尚不清楚。我们假设SMRT支持由HDAC3介导的独特功能，并且也可能具有与N-CoR冗余的HDAC3依赖功能。我们将通过在SMRT DAD结构域产生点突变的敲入小鼠来验证这些假设。将仔细分析N-CoR和SMRT纯合突变小鼠，以及双纯合突变小鼠，以确定N-CoR/SMRT7HDAC3相互作用的生理功能。特异性目的3是确定HDAC3的生理、组织特异性功能。我们将在小鼠中删除HDAC3，以验证HDAC3功能缺失会导致双纯合N-CoR/SMRT DAD突变小鼠表型的假设。HDAC3基因敲除是有条件的，这使我们能够研究HDAC3的组织特异性功能。总之，这些创新和独特的研究将阐明TR和其他nr在生理背景下调节转录抑制的机制。从这项工作中获得的见解将为包括代谢和炎症在内的关键生物途径的转录和表观遗传控制提供新的视角。这有可能导致对代谢紊乱，如肥胖、糖尿病、心血管疾病以及癌症的新的和更深入的认识。

DESCRIPTION (provided by applicant): Thyroid hormone receptors (TRs) are ligand-dependent, transcriptional regulators of metabolism. TRs repress gene expression in the absence of hormone, which is paradigmatic for other nuclear receptors (NRs) that function as repressors in the unliganded state. Repression is mediated by interaction with corepressors N- CoR (Nuclear Receptor Corepressor) and SMRT (Silencing Mediator of Retinoid and Thyroid receptors), which exist in stoichiometric association with the chromatin-modifiying enzyme, histone deacetylase 3 (HDAC3). HDAC3, in turn, derives its catalytic activity from interacting with N-CoR/SMRT via their unique Deacetylase Activation Domain (DAD). The DAD-dependent N-CoR/SMRT7HDAC3 complex is critical for repression by TR and other NRs in vitro, but the role of this interaction in vivo is unknown. Here we propose to use state of the art methods of gene targeting and mouse phenotyping to test, for the first time, the physiological relevance of the N-CoR7HDAC3 and SMRT7HDAC3 corepressor complexes. We hypothesize that these DAD-dependent interactions are very important, and affect distinct physiological pathways involving NRs. Specific Aim 1 is to determine the physiological function of the N-CoR DAD domain. Knockout of N-CoR is embryonic lethal; we hypothesize that interaction with HDAC3 subserves a subset of N-CoR's developmental and physiological functions. To discover what those functions are, we have generated mice with a point mutation in the N-CoR DAD domain that prevents HDAC3 interaction. Preliminary data demonstrate that mice homozygous for this mutation are viable, with intriguing abnormalities that point to the biological importance of N-CoR7HDAC3. Specific Aim 2 is to determine the physiological function of the SMRT DAD domain. Similar to N-CoR, the physiological role of SMRT7HDAC3 is unknown. We hypothesize that SMRT subserves unique functions that are mediated by HDAC3, and may also have HDAC3-dependent functions that are redundant with those of N-CoR. We will test these hypotheses by generating knockin mice with a point mutation in the SMRT DAD domain. The N-CoR and SMRT homozygous mutant mice, and doubly homozygous mutants, will be carefully analyzed to determine the physiological function of the N-CoR/SMRT7HDAC3 interaction. Specific Aim 3 is to determine the physiological, tissue-specific functions of HDAC3. HDAC3 will be deleted in mice to test the hypothesis that losses of HDAC3 function will phenocopy the doubly homozygous N-CoR/SMRT DAD mutant mice. The HDAC3 knockout will be conditional, enabling us to investigate tissue-specific functions of HDAC3. Together, these innovative and unique studies will elucidate mechanisms regulating transcription repression by TR and other NRs in a physiological context. The insights gained from this work will shed new light on the transcriptional and epigenetic control of key biological pathways, including metabolism and inflammation. This has the potential to lead to new and deeper insights into metabolic disorders, such as obesity, diabetes, and cardiovascular disease, as well as cancer. Relevance: In the past decade, corepressors have emerged as critical regulators of hormone receptors. The proposed studies will innovatively and uniquely elucidate mechanisms regulating the action of hormones and other metabolic regulators. The insights gained from this work will shed new light on key biological pathways, with the potential to lead to new and deeper insights into metabolic disorders, including obesity, diabetes, and cardiovascular disease, as well as cancer.