Relaxed Polymerase Pausing as a Driver of Epigenetic Plasticity and Cancer Cell Invasion

项目摘要 远处转移的发展占乳腺癌死亡率的很大比例; 因为30%在早期阶段最初诊断的患者最终将发展为转移性疾病。一把钥匙早 转移进展中的一个步骤是细胞可塑性的增加，这使得一部分肿瘤细胞失去了 残留的上皮特征和获得的迁移和侵袭行为，在分子水平上反映在上皮细胞上， 间充质转化（EMT）。现在有相当多的证据表明，癌症相关的EMT是一种高度 扩散性癌细胞表现出许多混合中间状态的动态和可逆过程 （部分EMT）被认为具有最大的攻击性，干样，行为的潜力。但 控制这种表型可塑性的表观遗传机制以及这一过程在早期入侵中的作用 仍然不完全理解。最近我们发现RNA聚合酶（Pol II）的局部调节 组蛋白甲基转移酶SUV 420 H2的暂停释放在稳定上皮细胞中起重要作用。 乳腺癌细胞的“身份”，并在这样做时抑制乳腺癌细胞的侵袭。我们特别 发现SUV 420 H2将H4 K20 me 1局部转化为me 3，通过阻断RNA聚合酶， M0 F/MSL复合物的募集，这又是H4 K16的乙酰化所必需的， pTEFb和Pol II暂停释放。我们进一步发现，SUV 420 H2介导的抑制抑制了细胞的增殖。 在管腔乳腺上皮细胞中的间充质程序，但针对TGF-β诱导的EMT的新位点。 SUV 420 H2在乳腺癌的三阴性/基底亚型中下调，其强制下调 或抑制促进在3D中生长的乳腺细胞球状体中的集体侵袭。这些和其他发现使我们 提出SUV 420 H2介导的Pol II暂停控制的放松是表观遗传的一个来源， 乳腺癌细胞适应和出现的可塑性和转录异质性 具有侵袭性的肿瘤细胞。使用精确运行测序（Pro-seq）和天然测序的组合， 通过CUT&Tag技术进行染色质分析，我们将确定SUV 420 H2如何介导暂停 限制强制表型稳定性以及这些限制的丧失如何允许转录混杂。 我们将探讨HEXIM 1/ 7SK snRNP复合物作为组蛋白H4修饰的新型“阅读器”的作用。 及其在SUV 420 H2介导的停顿控制中的作用。最后，我们将确定Pol II失调的影响， 暂停转录多样性的动态和具有侵入性“领导者”潜力的乳腺细胞的出现 在一个集体入侵的三维球体模型中。从长远来看，我们的研究结果将提供重要的 深入了解表观遗传可塑性的机制及其在肿瘤细胞适应中的作用，以及一个框架 用于开发新的重编程策略以阻断乳腺癌转移进展。

Project Summary The development of distant metastases accounts for a significant proportion of breast cancer mortality; as many as 30% of patients initially diagnosed at an early stage will eventually progress to metastatic disease. A key early step in metastatic progression is an increase in cellular plasticity that enables a subset of tumor cells to lose residual epithelial features and gain migratory and invasive behavior, molecularly reflected in the epithelial to mesenchymal transition (EMT). Considerable evidence now points to cancer-associated EMT as a highly dynamic and reversible process with disseminated cancer cells exhibiting many hybrid intermediate states (partial-EMT) proposed to possess the greatest potential for aggressive, stem-like, behavior. However, the epigenetic mechanisms that control such phenotypic plasticity and the role of this process in early invasion remain incompletely understood. Recently we discovered that the local regulation of RNA polymerase (Pol II) pause release by the histone methyltransferase SUV420H2 plays an important role in stabilizing the epithelial ‘identity’ of luminal breast cancer cells and in so doing, suppresses breast cancer cell invasion. Specifically, we find that the local conversion of H4K20me1 to me3 by SUV420H2 enforces RNA polymerase pausing by blocking recruitment of the MOF/MSL complex, which is in turn necessary for the acetylation of H4K16, recruitment of pTEFb and Pol II pause release. We further find that SUV420H2-mediated repression constrains the mesenchymal program in luminal breast epithelial cells, yet is directed to new sites upon TGF-β induced EMT. SUV420H2 is downregulated in triple negative/basal subtype of breast cancers, and its forced downregulation or inhibition promotes collective invasion in breast cell spheroids grown in 3D. These and other findings lead us to propose that the relaxation of SUV420H2-mediated Pol II pausing control is one source of the epigenetic plasticity and transcriptional heterogeneity that underlies breast cancer cell adaptation and the emergence of tumor cells with invasive properties. Using a combination of precision run-on sequencing (Pro-seq) and native chromatin analyses via CUT&Tag technology, we will determine the how the SUV420H2 mediated pause constraints enforces phenotypic stability and how loss of these constraints allows for transcriptional promiscuity. We will explore the role of the HEXIM1 / 7SK snRNP complex as a novel ‘reader’ of histone H4 modifications and its role in SUV420H2-mediated pause control. Lastly, we will determine the impact of dysregulated Pol II pausing dynamics on transcriptional diversity and the emergence of breast cells with invasive “leader” potential in a 3D spheroid model of collective invasion. Over the long term, the results of our studies will provide important insight into the mechanisms underlying epigenetic plasticity and its role in tumor cell adaptation, and a framework for the development of novel reprogramming strategies to block breast cancer metastatic progression.