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； 因为早期诊断的患者中有30%最终会发展为转移性疾病。一把钥匙早到了 转移进展的一步是细胞可塑性的增加，使一部分肿瘤细胞失去 残留的上皮特征和获得的迁移和侵袭行为，分子反映在上皮到 间充质转化(EMT)。现在有大量证据表明，与癌症相关的EMT是一种高度 播散性癌细胞呈现多种杂交中间状态的动态和可逆过程 (Partial-EMT)被认为具有最大的攻击性、干状行为的潜力。然而， 控制这种表型可塑性的表观遗传机制及其在早期入侵中的作用 仍然没有完全被理解。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SUV420H2的暂停释放在稳定上皮细胞中起重要作用 这样做可以抑制乳腺癌细胞的侵袭。具体来说，我们 发现SUV420H2将H4K20me1局部转化为ME3通过阻断RNA聚合酶来强制暂停 招募MOF/MSL复合体，这反过来是H4K16乙酰化所必需的，招募 PTEFb和POL II暂停释放。我们进一步发现，SUV420H2介导的抑制作用抑制了 在转化生长因子-β诱导的子宫内膜上皮细胞中，间质细胞程序被定向到新的部位。 SUV420H2在三重阴性/基础亚型乳腺癌中表达下调及其强制下调 或抑制促进在3D中生长的乳腺细胞球体的集体侵袭。这些和其他发现引导我们 提示SUV420H2介导的POL II暂停控制的松弛是表观遗传学的来源之一 可塑性和转录异质性是乳腺癌细胞适应和出现的基础 具有侵袭性的肿瘤细胞。使用精确连续测序(Pro-SEQ)和原生测序相结合 通过Cut&Tag技术进行染色质分析，我们将确定SUV420H2如何介导停顿 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。 我们将探索HEXIM1/7SK SnRNP复合体作为组蛋白H4修饰的新的读取器的作用 以及它在SUV420H2介导的停顿控制中的作用。最后，我们将确定监管失调的POL II的影响 转录多样性和具有侵袭性“先导”潜能的乳腺细胞出现的停顿动态 在集体入侵的三维椭球体模型中。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，并提出一个框架 用于开发新的重新编程策略来阻止乳腺癌的转移进展。

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.