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

染色质上调控元件的表观遗传修饰深刻地影响基因表达， 在细胞分化和转化等过程中的作用。这些翻译后修饰 发生在DNA和组蛋白上，并由表观遗传修饰蛋白介导。聚合酶相关 因子复合物（PAFc）是一种表观遗传修饰复合物，是多种表观遗传修饰因子沉积所必需的。 与转录激活相关的修饰（包括H2 Bub、H3 K4 me和H3 K79 me）。我们有 最近表明，PAFc是人类白血病所必需的， 谱系白血病（MLL）基因以及其他几种急性髓细胞白血病（AML）亚型。我们 初步数据显示PAFc也是胎儿造血所必需的。此外，我们发现PAFc是 通过与H3 K9甲基转移酶SETDB 1相互作用调节，后者抑制白血病转化。 目的：研究PAFc在造血中的作用以及细胞增殖所需的不同表观遗传功能。 转型仍不明朗。我们的初步研究揭示了PAFc之间的新的相互作用， 和H3 K9甲基转移酶、SETDB 1以及PAFc在胎儿造血中的作用。我们假设 PAFc-SETDB 1相互作用促进造血分化，这种相互作用干扰 通过促进阻断分化的基因程序的转录激活来帮助转化。 具体目的：我们的目标是（1）表征PAFc亚基Cdc 73在造血中的作用，（2）研究PAFc亚基Cdc 73在造血中的作用。 PAFc相互作用配偶体SETDB 1作为造血肿瘤抑制因子的作用，以及（3）定义PAFc相互作用配偶体SETDB 1作为造血肿瘤抑制因子的作用。 SETDB 1调节PAFc介导的转录激活的机制。 研究设计：我们开发了一种小鼠模型，允许条件性缺失和随后的 在成人造血组织中的PAFc亚基Cdc 73的表征。我们将使用CDC 73的突变体 改变与SETDB 1的相互作用，以及过表达和敲低，以评估SETDB 1的作用 以及PAFc-SETDB 1在分化中的相互作用。我们还将结合生物化学，分子， 和高通量方法来查询SETDB 1与PAFc相互作用的转录结果。 健康影响：表观遗传修饰剂被认为是细胞分化的关键参与者。他们也发挥 在血液病中起重要作用，并已被证实为可行的治疗靶点。了解 在造血分化和转化等过程中，我们必须确定调控机制， 表观遗传修饰由于PAFc在造血和几种疾病中起作用，因此建议的研究 将揭示一个以前没有特征的表观遗传调节复合物在造血中的作用， 定义蛋白质相互作用，如SETDB 1，如何在造血分化过程中模块PAFc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.