Mechanisms of enhancer regulation in leukemia

摘要 转录因子需要共激活子与通用转录机器进行通信， 因此，确保生物输入转化为特定的基因表达程序。《调解人》 复合体就是这样一种共激活因子，在转录因子增强子和辅助子之间起着分子桥梁的作用 启动子上的RNA聚合酶II(POL II)。它是一个大的大分子络合物，进一步排列成四个模块 这赋予了高度的灵活性：头部、中部、尾部和激酶模块。该激活剂的成员 模块介体12(MED12)在固体(子宫内膜、肺、宫颈、 结肠癌)和血液(DLBCL、CLL、ALL、AML)癌。然而，潜在的机制是 MED12突变及其在疾病发生和发展中的作用仍然难以捉摸。我们最近专注于 蛋白激酶模块和MED12在造血干细胞分化中的作用 和转型。我们发现MED12蛋白的表达受翻译后泛素的控制 连接酶FBXW7，一种频繁突变的肿瘤抑制因子。我们还发现MED12是一种必不可少的调节因子。 HSC的功能，因为体内MED12的缺失会影响HSC的存活，并导致小鼠死亡。同舟共济 与基本的造血转录因子，MED12共同占据HSC特异性增强子。MED12 耗竭破坏P300结合的稳定性，从而导致增强子的快速失活和KEY表达的丧失 HSC特异性基因。这些数据表明，MED12的表达和功能可能由于多种原因而改变 机制，包括针对基因本身或其调节器的体细胞突变(FBXW7)，并且这 功能异常可导致恶变。这项提议旨在阐明分子 在放松对增强子活性的关键调节因子，如MED12的管制后，机制发生了变化。虽然它已经 虽然有人认为MED12基因突变会带来“功能增益”，但还没有机制研究。 执行到最新。为了解决这个关键问题，我们正在研究慢性淋巴细胞白血病(CLL)， 西方世界最常见的成人白血病。剖析调解人功能的中断是如何促成 对于这种异质和复杂的疾病，我们使用了：a)转录/表观遗传的组合 携带MED12突变的人类患者样本的特征，b)CRISPR修饰和ES靶向 转基因小鼠模型，以研究MED12损伤引发和维持疾病的能力，以及，c)在 MED12基因修饰的CRISPR细胞系的体外转录组、表观遗传学和3D-染色体拓扑 突变。明确介体和增强子调控促进肿瘤发生的机制 转化将有利于针对血液恶性肿瘤和固体的新疗法的开发 肿瘤。最近发现的以介体为靶点的小分子药理学表明，这种 治疗方法触手可及。

ABSTRACT Transcription factors require coactivators to communicate with the general transcription machinery and, thereby, ensure that biological inputs are translated into specific gene-expression programs. The Mediator complex is such a coactivator and acts as a ‘molecular bridge’ between transcription factor at enhancers and RNA polymerase II (Pol II) at promoters. It is a large macromolecular complex further arranged in four modules that confer high flexibility: the head, the middle, the tail and the kinase module. The member of the kinase module Mediator 12 (MED12) has been found frequently mutated in both solid (endometrial, lung, cervical, colon carcinomas) and blood (DLBCL, CLL, ALL, AML) cancers. However, the underlying mechanisms of MED12 mutations and its role in disease initiation and progression remain elusive. We have recently focused on the function of the kinase module and specifically MED12 in hematopoietic stem cell (HSC) differentiation and transformation. We found that MED12 protein expression is controlled post-translationally by the ubiquitin ligase FBXW7, a frequently mutated tumor suppressor. We also found that MED12 is an essential regulator of HSC function, as in vivo deletion of MED12 compromises HSC survival and leads to mouse lethality. Together with essential hematopoietic transcription factors, MED12 co-occupies HSC-specific enhancers. MED12 depletion destabilizes P300 binding thus leading to rapid enhancer “inactivation”, and loss of expression of key HSC-specific genes. These data suggest that MED12 expression and function can be altered due to multiple mechanisms, including somatic mutations targeting the gene itself or its regulators (FBXW7), and that this aberrant function can lead to malignant transformation. This proposal aims to shed light on the molecular mechanisms altered upon deregulation of a crucial regulator of enhancer activity, such as MED12. While it has been suggested that MED12 mutations confer a “gain-of-function”, no mechanistic studies have been performed up to date. To address this key question, we are studying chronic lymphocytic leukemia (CLL), the most common adult leukemia in the western world. To dissect how disruption of Mediator function contributes to this heterogeneous and complex disease, we use a combination of: a) transcriptional/epigenetic characterization of human patient samples harboring MED12 mutations, b) CRISPR-modified and ES targeted transgenic mouse models to investigate the ability of MED12 lesions to initiate and maintain disease, and, c) in vitro transcriptome, epigenetic and 3D-chromosome topology in CRISPR-modified cell lines with MED12 mutations. Defining the mechanisms by which Mediator and enhancer regulation contribute to malignant transformation will be beneficial for the development of novel therapies targeting blood malignancies and solid tumors. The recent identification of small molecules targeting Mediator pharmacologically suggests that such therapies are within reach.