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Molecular mechanisms of estrogen receptor-dependent transcription regulation

雌激素受体依赖性转录调控的分子机制

基本信息

批准号：
10545758
负责人：
ROBERT G ROEDER
金额：
$ 65.08万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10545758
关键词：
Affect Biochemical Biological Assay Breast Cancer Model Breast Cancer therapy CRISPR/Cas technology Cells ChIP-seq Chromatin Chromatin Loop Chromatin Structure Cocoa Powder Communication Complex Cryoelectron Microscopy DNA Polymerase II Disease Distal EP300 gene ERBB2 gene Enhancers Ensure Epigenetic Process Estrogen Receptors Estrogens Exhibits Gene Activation Genes Genetic Transcription Histones Hormone Responsive Human In Vitro Investigation Knock-in Knowledge acquisition Ligands Light Malignant Neoplasms Mammary Gland Parenchyma Mammary Neoplasms Mediating Mediator Methyltransferase Mitogen-Activated Protein Kinases Modeling Modification Molecular Mus Nuclear Receptors Pathway interactions Phosphorylation Physiological Processes Protein Tyrosine Kinase Reporting Resistance Resolution Role Signal Transduction Site System Tamoxifen Testing Transcription Coactivator Transcriptional Regulation Xenograft Model Xenograft procedure chemical synthesis chromatin modification chromatin remodeling cofactor cohesin design genome-wide analysis in vivo knock-down malignant breast neoplasm mammary gland development mutant next generation novel therapeutics p300/CBP-Associated Factor peptidomimetics promoter protein protein interaction rational design receptor binding receptor function reconstitution recruit small molecule inhibitor targeted treatment transcription factor transcriptome sequencing tumor xenograft

项目摘要

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.

摘要与靶基因增强子结合的配体雌激素受体(ER)通过序列激活转录与多个共激活剂的相互作用。这些共活化子首先作用于使局部染色质部位更多可利用，并随后与调解人一起建立功能性POL II预引发复合体 (图)在核心推动者。我们已经展示了对包含NR盒的调解器MED1的关键要求亚单位，在正常乳腺发育和乳腺肿瘤形成中。反常的相互作用他莫昔芬与MED1连接的ER也与获得对他莫昔芬治疗的耐药性有关。显示与Mediator的交互和/或协作功能的其他共激活器包括SRCs、PGC1、 CCAR1和可可。我们也有证据表明内质网与MLL3/4复合体直接相互作用，这具有组蛋白H3K4单甲基转移酶和H3K27去甲基酶活性，并与先锋 FOXA1因子在增强子激活的最早阶段发挥作用。这在一定程度上是因为在增强子-启动子通信中，我们假设(I)这些辅助因子中的各种促进ER- MED1/Mediator相互作用及其在增强子转变过程中的中介作用激活和染色质重塑/修饰对PIC的形成和功能以及(Ii)这些相互作用在他莫昔芬抗性方面出现错误，以促进在其他抑制条件下的转录。在这里，我们建议通过增强子激活的阶段来阐明关键的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，我们将使用集成生化(体外转录)，基于细胞的(结合CRISPR/Cas9介导的突变体MED1的全基因组分析 (I)MED1NRbox的作用和机制。 ER对调解人的依赖招募/职能；(Ii)替代方案MED1NR box的作用和机制-- ER招募调解人的独立途径；以及(Iii) 酪氨酸激酶HER2和(磷酸化)MED1亚单位参与对他莫昔芬的耐药性心理治疗。在目标2中，我们将使用类似的方法来研究(I)活性增强子的建立带有H3K4me1和H3K27ac标志的景观，重点是这些修改的机制是通过p300/CBP、MLL3/4C、SRC和PGC-1b之间基于ER和FOXA1的协同作用而影响的共激活子；和(Ii)通过启动子相互作用发挥远端增强子的功能，重点是在体外通过附加因素(包括粘附素)。在目标3中，我们将使用冷冻-EM、XL-MS和计算集成建模来阐明细节 ER和关键辅因子(Mediator和MLI3/4C)之间的物理相互作用。在此基础上，我们将设计并测试以关键蛋白质-蛋白质相互作用为目标的稳定的肽模拟剂和相关治疗剂。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。