Epigenetic modulation of the TAZ-CAMTA1 transcriptional program by the Ada2a-containing histone acetyltransferase complex

项目摘要/摘要 TAZ/YAP是河马信号转导途径的转录效应物和原癌蛋白 包括肉瘤在内的多种癌症中调节肿瘤生长和转移的途径。虽然已经发生了很多事情 关于TAZ和YAP的上游调控，人们对表观遗传调控知之甚少 他们的转录程序，代表着知识上的巨大差距。为了解决这一差距，我们利用 TAZ-CAMTA1(TC)和YAP-TFE3(YT)融合蛋白。TC和YT驱动上皮样细胞中的癌蛋白 血管内皮瘤(EHE)，一种发生在青少年的肉瘤(对PA-16-251有反应，基因融合在 儿童肉瘤)。TAZ和YAP的过度激活形式TC和YT是相关的，因为基因融合 是癌症中最常见的TAZ/YAP基因改变类型。我们在这项建议中的目标是 确定TC和YT是否通过改变表观基因组进一步刺激TAZ/YAP转录。我们的中央 假设TC和YT招募含有Ada2a(ATAC)组蛋白的YEATS2和ZZZ3 乙酰转移酶复合体，它增强了TAZ/YAP致癌转录程序。这一假设 得到了两种连续的无偏见方法的支持：BioID质谱学，随后是shRNA筛选， 确定YEATS2和ZZZ3是TC驱动的锚定非依赖性生长最关键的蛋白质。我们 计划利用主要的融合蛋白TC来测试我们的中心假设，具体目标如下 在EHE(占EHE的85%)： 目的1)确定YEATS2和ZZZ3是如何改变TAZ-CAMTA1转录程序的。 我们将进行CHIP-SEQ，并确定TC是否与YEATS2和ZZZ3在基因组上共定位。RNA序列 将在有和不有YEATS2或ZZZ3表达的TC表达细胞系中进行检测，以确定其 对TC转录组的影响。 目的2)鉴定细胞转化所需的TAZ-CAMTA1、YEATS2和ZZZ3结构域。 YEATS2和ZZZ3与TC的相互作用将通过免疫共沉淀法利用缺失进行剖析 变种人。这些突变体将在我们开发的锚定非依赖生长试验中进一步研究。 目的3)确定YEATS2和ZZZ3与TAZ-CAMTA1的相互作用在体内的贡献。 我们将使用遗传和药理学方法来抑制新型TC的YEATS2/ZZZ3/ATAC功能 转基因和异种移植肉瘤小鼠模型，并评价其对肿瘤发生和转移的影响。 该建议具有创新性，因为它利用了TC和YT以及两种连续的无偏见方法来 确定YEATS2/ZZZ3/ATAC是TAZ/YAP转录组的新的表观遗传调控因子，扩展了 这一领域的重点是解决知识方面的重大差距。该项目意义重大，因为它确定了 YEATS2和ZZZ3是肉瘤中新的癌蛋白和驱动因素，并为抑制组蛋白提供了理论基础 修饰复合体是靶向河马途径的一种新方法，目前河马途径缺乏治疗靶点。

Project Summary/Abstract TAZ/YAP are transcriptional effectors and proto-oncoproteins of the Hippo pathway, a signal transduction pathway regulating tumor growth and metastasis in multiple cancers including sarcomas. While much has been uncovered with regards to the upstream regulation of TAZ and YAP, little is known about epigenetic regulation of their transcriptional programs, representing a significant gap in knowledge. To address this gap, we utilized the TAZ-CAMTA1 (TC) and YAP-TFE3 (YT) fusion proteins. TC and YT are driving oncoproteins in epithelioid hemangioendothelioma (EHE), a sarcoma arising in adolescents (responsive to PA-16-251, Gene Fusions in Pediatric Sarcomas). TC and YT, hyperactivated forms of TAZ and YAP, are relevant because gene fusions are the most common type of genetic alterations of TAZ/YAP in cancer. Our objective in this proposal is to determine if TC and YT further stimulate TAZ/YAP transcription by altering the epigenome. Our central hypothesis is that TC and YT recruit YEATS2 and ZZZ3, part of the Ada2a-containing (ATAC) histone acetyltransferase complex, which potentiate the TAZ/YAP oncogenic transcriptional programs. This hypothesis is supported by two serial unbiased approaches: BioID mass spectrometry followed by an shRNA screen that identified YEATS2 and ZZZ3 as the proteins most critical for TC driven anchorage independent growth. We plan to test our central hypothesis with the following specific aims utilizing TC, the predominant fusion protein in EHE (85% of EHE): Aim 1) Determine how YEATS2 and ZZZ3 alter the TAZ-CAMTA1 transcriptional program. We will perform ChIP-seq and determine if TC co-localizes with YEATS2 and ZZZ3 on the genome. RNA-seq will be performed in TC expressing cell lines with and without expression of YEATS2 or ZZZ3 to identify their effects on the TC transcriptome. Aim 2) Identify domains of TAZ-CAMTA1, YEATS2, and ZZZ3 required for cellular transformation. The interaction of YEATS2 and ZZZ3 with TC will be dissected by co-immunoprecipitation utilizing deletion mutants. The mutants will be further studied in anchorage independent growth assays we have developed. Aim 3) Determine the contributions of the YEATS2 and ZZZ3 interactions with TAZ-CAMTA1 in vivo. We will use both genetic and pharmacological approaches to inhibit YEATS2/ZZZ3/ATAC function in novel TC transgenic and xenograft sarcoma mouse models and evaluate their effect on tumorigenesis and metastasis. The proposal is innovative because it utilizes TC and YT as well as two serial unbiased approaches to identify YEATS2/ZZZ3/ATAC as novel epigenetic regulators of the TAZ/YAP transcriptome, expanding the focus of the field and addressing a significant gap in knowledge. The project is significant because it identifies YEATS2 and ZZZ3 as novel oncoproteins and drivers in sarcoma and provides a rationale for inhibiting histone modifying complexes as a novel way of targeting the Hippo pathway, which currently lacks a therapeutic target.