The role of the PAFc subunit Cdc73 in normal hematopoiesis and transformation

染色质调控元件的表观遗传修饰深刻影响基因表达并发挥作用 在细胞分化和转化等过程中发挥作用。这些翻译后修饰 发生在DNA和组蛋白上，由表观遗传修饰蛋白介导。与之相关的聚合酶 因子复合体(PAFC)是一种表观遗传修饰复合体，是几种表观遗传学沉积所必需的 与转录激活相关的修饰(包括H2 Bub、H3K4me和H3K79me)。我们有 最近发现，PAFC对于人类白血病来说是必不可少的，因为它含有混合的重排 谱系白血病(MLL)基因以及其他几种急性髓系白血病(AML)亚型。我们的 初步数据显示，PAFC对胎儿造血也是必要的。此外，我们发现PAFC是 受抑制白血病转化的H3K9甲基转移酶SETDB1相互作用调节。 目的：PAFC在造血中的作用和细胞所需的不同的表观遗传功能 转型仍不明朗。我们的初步研究揭示了PAFC之间的一种新的相互作用 以及H3K9甲基转移酶SETDB1和PAFC在胎儿造血中的作用。我们假设 PAFC-SETDB1相互作用促进造血分化及其干扰 艾滋病在转化中通过促进转录激活一种阻止分化的基因程序。 具体目标：我们的目标是(1)表征PAFC亚单位CDC73在造血中的作用，(2)验证 PAFC相互作用伙伴SETDB1作为造血肿瘤抑制因子的作用和(3)定义 SETDB1调节PAFC介导的转录激活的机制。 研究设计：我们开发了一种允许条件删除和后续删除的小鼠模型 成人造血组织中PAFC亚单位cdc73的特征。我们将使用CDC73的突变体 这改变了与SETDB1的相互作用，以及过度表达和敲除以评估SETDB1的作用 分化过程中PAFC-SETDB1的相互作用。我们还将结合使用生化、分子 以及高通量方法，以查询SETDB1与PAFC相互作用的转录结果。 健康影响：表观遗传修饰物被认为是细胞分化的关键因素。他们也会玩 在血液病中发挥重要作用，并已被证实为可行的治疗靶点。要理解 像造血分化和转化这样的过程，我们必须定义调控机制 表观遗传修饰物。由于PAFC在造血和几种疾病中发挥作用，拟议的研究 将揭示以前未知的表观遗传调节复合体在造血中的作用，同时也 确定蛋白质相互作用，如SETDB1，如何在造血分化过程中模块PAFC的功能。

Epigenetic modifications of regulatory elements on chromatin profoundly impact gene expression and play a role in processes such as cellular differentiation and transformation. These post-translational modifications occur on DNA and histones and are mediated by epigenetic modifying proteins. The Polymerase Associated Factor complex (PAFc) is an epigenetic modifying complex necessary for the deposition of several epigenetic modifications associated with transcriptional activation (including H2Bub, H3K4me and H3K79me). We have recently shown that the PAFc is essential for human leukemias harboring rearrangements of the Mixed Lineage Leukemia (MLL) gene as well as several other subtypes of acute myeloid leukemia (AML). Our preliminary data shows the PAFc is also necessary for fetal hematopoiesis. Further, we found the PAFc is regulated by interaction with the H3K9 methyltransferase SETDB1, which suppresses leukemic transformation. Objective: The role of the PAFc in hematopoiesis and the distinct epigenetic functions required for cellular transformation remains unclear. Our preliminary studies have revealed a novel interaction between the PAFc and an H3K9 methyltransferase, SETDB1 and a role for the PAFc in fetal hematopoiesis. We hypothesize that the PAFc-SETDB1 interaction promotes hematopoietic differentiation and that interference of this interaction aids in transformation by promoting transcriptional activation of a gene program blocking differentiation. Specific Aims: We aim to (1) Characterize the role of the PAFc subunit, Cdc73, in hematopoiesis, (2) Validate the role of the PAFc interaction partner, SETDB1, as a hematopoietic tumor suppressor and (3) Define the mechanism by which SETDB1 modulates PAFc mediated transcriptional activation. Study Design: We have developed a mouse model that allows for the conditional deletion and subsequent characterization of the PAFc subunit, Cdc73, in adult hematopoietic tissues. We will use mutants of CDC73 that alter interaction with SETDB1, as well as overexpression and knock down to evaluate the role of SETDB1 and the PAFc-SETDB1 interaction in differentiation. We will also use a combination of biochemical, molecular and high throughput methods to query the transcriptional consequences of SETDB1 interaction with the PAFc. Health Impact: Epigenetic modifiers are recognized as critical players in cellular differentiation. They also play important roles in hematologic disease and have been validated as viable therapeutic targets. To understand processes like hematopoietic differentiation and transformation, we must define the mechanisms regulating epigenetic modifiers. As the PAFc plays a role in hematopoiesis and several diseases, the proposed research will reveal the role of a previously uncharacterized epigenetic regulator complex in hematopoiesis while also defining how protein interactions, like SETDB1, module the PAFc function during hematopoietic differentiation.