Molecular mechanisms of estrogen receptor-dependent transcription regulation

摘要 与靶基因增强子结合的配体雌激素受体（ER）通过顺序激活转录 与许多辅活化剂的相互作用。这些辅激活因子首先作用于局部染色质位点， 随后，与介体结合，建立功能性Pol II预起始复合物 （PICs）在核心推动者。我们已经证明了MED1的关键要求，MED1是一种含有NR框的中介体， 亚基，在正常乳腺发育和乳腺肿瘤形成中。异常相互作用 具有MED1他莫昔芬配体的ER也与获得对他莫昔芬治疗的抗性有关。 显示与介体相互作用和/或协同功能的其它共激活因子包括SRC，PGC1， CCAR 1和可可酸。我们也有证据表明ER与MLL3/4复合物直接相互作用， 具有组蛋白H3 K4单甲基转移酶和H3 K27脱甲基酶活性，并且沿着先锋 因子FOXA 1在增强子激活的最早阶段起作用。部分原因是， 作为增强子-启动子通讯的介导者，我们假设（i）这些辅因子中的各种促进ER-1表达， MED1/Mediator相互作用和功能，作为从增强子转换的中介因子 激活和染色质重塑/修饰PIC的形成和功能，以及（ii）这些相互作用 在他莫昔芬抗性中出错，以促进在其他抑制条件下的转录。这里我们 提出通过增强子激活的各个阶段阐明关键ER-辅因子相互作用的机制细节， 增强子-启动子相互作用和PIC组装/功能。在目标1中，我们将使用整合的生物化学（体外 转录）、基于细胞的（全基因组分析与CRISPR/Cas9介导的突变MED1 敲入）和小鼠肿瘤异种移植方法来阐明（i）MED1 NR盒的作用和机制- ER依赖性募集/调节剂功能;（ii）替代方案MED1 NR盒的作用和机制- ER募集介体的独立途径;和（iii） 酪氨酸激酶HER2和Mediator的（磷酸化）MED1亚基有助于对他莫昔芬的耐药性 疗法在目标2中，我们将采用类似的方法来研究（i）活性增强子的建立 带有H3K4me1和H3K27ac标记的景观，重点是这些修改的机制， 通过p300/CBP、MLL3/4C、SRC和PGC-1b之间基于ER和FOXA 1的协同作用实现 辅激活子;和（ii）通过启动子相互作用的远端增强子功能，重点是体外 通过其他因素（包括 粘着蛋白）。在目标3中，我们将采用冷冻EM，XL-MS和计算综合建模来阐明细节 ER和关键辅因子（Mediator和MLL3/4C）之间的物理相互作用。基于此，我们将设计 并测试稳定的肽模拟物和靶向关键蛋白质-蛋白质相互作用的相关治疗剂。 因此，我们的研究将从核受体和乳腺癌的角度产生广泛的影响。

ABSTRACT Liganded estrogen receptor (ER) bound to target gene enhancers activates transcription through sequential interactions with a number of coactivators. These coactivators act first to make local chromatin sites more accessible and, subsequently, in conjunction with the Mediator, establish functional Pol II preinitiation complexes (PICs) at core promoters. We have shown both a critical requirement for MED1, an NR box-containing Mediator subunit, in normal mammary gland development and in mammary tumor formation. Aberrant interactions of tamoxifen-liganded ER with MED1 have also been implicated in acquisition of resistance to tamoxifen therapy. Other coactivators that display interactions and/or cooperative functions with Mediator include SRCs, PGC1, CCAR1, and CoCoA. We also have evidence for direct ER interactions with the MLL3/4 complex, which possesses histone H3K4 monomethyltransferase and H3K27 demethylase activities and, along with pioneer factor FOXA1, functions at the earliest stages of enhancer activation. Partly in light of an emerging role of Mediator in enhancer-promoter communication, we hypothesize (i) that various of these cofactors facilitate ER- MED1/Mediator interactions and functions by acting as intermediary factors in the transitions from enhancer activation and chromatin remodeling/modification to PIC formation and function and (ii) that these interactions go awry in tamoxifen resistance to facilitate transcription under otherwise repressive conditions. Here, we propose to elucidate mechanistic details of key ER-cofactor interactions through stages of enhancer activation, enhancer-promoter interactions and PIC assembly/function. In Aim 1, we will use integrated biochemical (in vitro transcription), cell-based (genome-wide analyses in combination with CRISPR/Cas9-mediated mutant MED1 knockins) and mouse tumor xenograft approaches to elucidate (i) the role and mechanism of MED1 NR box- dependent recruitment/function of Mediator by ER; (ii) the role and mechanism of an alternative, MED1 NR box- independent pathway for Mediator recruitment by ER; and (iii) the mechanism by which crosstalk between the tyrosine kinase HER2 and the (phosphorylated) MED1 subunit of Mediator contributes to resistance to tamoxifen therapy. In Aim 2, we will employ similar approaches to investigate (i) establishment of active enhancer landscapes bearing H3K4me1 and H3K27ac marks, with emphasis on mechanisms by which these modifications are effected through ER- and FOXA1-based cooperativity between p300/CBP, MLL3/4C, and SRC and PGC-1b coactivators; and (ii) distal enhancer function through promoter interactions, with emphasis on the in vitro recapitulation and mechanistic analysis of Mediator-dependent ER function through additional factors (including cohesin). In Aim 3, we will employ cryo-EM, XL-MS and computational integrative modeling to elucidate details of physical interactions between ER and critical cofactors (Mediator and MLL3/4C). Based on this, we will design and test stabilized peptidomimetic and related therapeutic agents that target key protein-protein interactions. Thus, our studies will have widespread impact from both nuclear receptor and breast cancer perspectives.