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

• 批准号: 10322679
• 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
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10322679
• 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 Mouse; Knowledge; Lead; Ligands; Light; Malignant Neoplasms; Mammary Gland Parenchyma; Mammary Neoplasms; Mediating; Mediator of activation protein; 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; base; 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

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.

抽象的 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 与许多共激活因子的相互作用。这些共激活因子首先起作用局部染色质位点 可访问，随后与调解人一起建立功能性pol II预原始复合物 （图片）核心促进者。我们已经对Med1（含NR盒的介体）表示了关键要求 亚基，在正常的乳腺发育和乳腺肿瘤形成中。异常相互作用 他莫昔芬与MED1的配合ER也与对他莫昔芬治疗的抵抗力有关。 其他与调解人显示交互和/或合作功能的共激活因子包括SRC，PGC1， CCAR1和可可。我们还有证据表明与MLL3/4复合物直接相互作用 具有组蛋白H3K4单甲基转移酶和H3K27脱甲基酶活性，以及​​先驱 因子FOXA1，在增强子激活的最早阶段起作用。部分鉴于 在增强子促销通信中的调解人，我们假设（i）这些辅助因子促进了ER- Med1/介体相互作用和功能通过充当增强剂过渡中的中间因素 激活和染色质重塑/修改PIC形成和功能以及（ii）这些相互作用 在他莫昔芬的抵抗力中出现问题，以促进原本压抑条件下的转录。在这里，我们 提议通过增强子激活的阶段阐明关键ER-CACTOR相互作用的机理细节， 增强器促销相互作用和PIC组装/功能。在AIM 1中，我们将使用集成的生化（体外 转录），基于细胞的（全基因组分析与CRISPR/CAS9介导的突变体Med1结合 敲击蛋白）和小鼠肿瘤异种移植方法阐明（i）Med1 NR盒的作用和机制 ER的依赖招聘/功能； （ii）替代方法的作用和机制，med1 nr盒 - ER进行调解员招募的独立途径； （iii）在 酪氨酸激酶HER2和（磷酸化的）Med1介体亚基有助于对他莫昔芬的抗性 治疗。在AIM 2中，我们将采用类似的方法来调查（i）建立主动增强器 带有H3K4ME1和H3K27AC标记的风景，重点是这些修饰的机制 通过p300/cbp，mll3/4C以及SRC和PGC-1B之间的基于ER和FOXA1的协作来实现 共激活因子； （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 粘素）。在AIM 3中，我们将采用Cryo-EM，XL-MS和计算集成建模来阐明细节 ER和关键辅因子之间的物理相互作用（介体和MLL3/4C）。基于此，我们将设计 并测试稳定的肽型和相关的治疗剂，靶向关键的蛋白质蛋白质相互作用。 因此，我们的研究将从核受体和乳腺癌的角度受到广泛影响。