Thyroid hormone receptors - regulation and function

描述(由申请人提供)：甲状腺激素受体(TRs)是一种依赖配体的、转录的新陈代谢调节剂。TRs在没有激素的情况下抑制基因的表达，这对其他核受体(NRs)来说是一个范例，这些NRs在去连接状态下发挥抑制作用。抑制是通过与辅阻遏子N-COR(核受体辅阻遏子)和SMRT(维甲酸和甲状腺受体的沉默介体)相互作用而介导的，它们与染色质修饰酶组蛋白脱乙酰基酶3(HDAC3)存在化学计量关系。反过来，HDAC3通过其独特的脱乙酰酶激活结构域(DAD)与N-COR/SMRT相互作用，从而获得其催化活性。依赖于DAD的N-COR/SMRT7HDAC3复合体在体外对TR和其他NRs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打靶和小鼠表型鉴定方法来首次测试N-CoR7HDAC3和SMRT7HDAC3辅阻遏子复合体的生理相关性。我们假设，这些依赖于DAD的相互作用是非常重要的，并影响涉及NRS的不同生理通路。具体目的1是确定N-COR-DAD结构域的生理功能。N-COR的敲除是胚胎致死的；我们假设与HDAC3的相互作用亚于N-COR的发育和生理功能的子集。为了发现这些功能是什么，我们培育了N-COR DAD结构域点突变的小鼠，该突变阻止了HDAC3的相互作用。初步数据表明，这种突变的纯合子小鼠是可行的，具有有趣的异常，表明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从属于HDAC3介导的独特功能，也可能具有HDAC3依赖的功能，这些功能与N-COR的功能是多余的。我们将通过在SMRT DAD结构域中产生点突变的敲门小鼠来检验这些假设。将仔细分析N-COR和SMRT纯合子突变小鼠以及双纯合子突变小鼠，以确定N-COR/SMRT7HDAC3相互作用的生理功能。具体目标3是确定HDAC3的生理和组织特异性功能。将在小鼠中删除HDAC3，以检验HDAC3功能丧失将复制双纯合子N-COR/SMRT DAD突变小鼠的假设。HDAC3基因敲除将是有条件的，使我们能够研究HDAC3的组织特异性功能。综上所述，这些创新和独特的研究将阐明在生理环境中调节转录抑制的机制。从这项工作中获得的见解将为关键生物途径的转录和表观遗传控制提供新的线索，包括新陈代谢和炎症。这有可能导致对代谢紊乱的新的和更深入的见解，如肥胖症、糖尿病、心血管疾病以及癌症。 相关性：在过去的十年中，辅抑制子已经成为激素受体的关键调节因子。拟议的研究将创新性地和独特地阐明调节激素和其他代谢调节剂的作用的机制。从这项工作中获得的见解将为关键的生物学途径提供新的线索，有可能导致对代谢紊乱的新的、更深入的见解，包括肥胖、糖尿病、心血管疾病以及癌症。

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.