Role of PRC1 in RUNX1-ETO-mediated transcriptional control

这个应用程序的目标是了解t（8; 21），最常见的染色体 与急性髓细胞白血病（AML）相关的易位，为继发性白血病奠定了基础。 突变积累并发展成AML。了解编码的AML1-ETO 融合蛋白改变了表观遗传连接，对于寻找减少衰弱的治疗方法至关重要， 更好的结果。AML1（RUNX1）和ETO/MTG家族成员也存在点突变 在实体瘤中，ETO家族成员Mtgr 1（CBFA 2T2）和Mtg 16（CBFA 2T3）是肿瘤 在小鼠肠肿瘤模型中的抑制因子，因此了解ETO如何接触组蛋白 修饰酶在t（8; 21）之外具有很大的影响。在我们的初步数据中， CRISPR/Cas9技术用FKBP12F36V修饰内源性AML1-ETO的3 '末端- HA或3XFLAG标签选择性和快速降解AML1-ETO。我们把这个系统 利用最先进的基因组学，如精确核连续转录测序（PROseq） 和Cut & Run建立一个化学遗传系统，以明确定义 AML1-ETO的转录控制。重要的是，这使我们能够定义最早的， 在融合蛋白失活后直接改变转录。我们的初步PROseq数据 证明了关键造血调控基因（如CEBPA）中的增强子 在向Kasumi-1细胞培养物中加入dTAG 47后2小时内重新活化。这些新试剂让我们 定义组蛋白修饰和RNA聚合酶动力学的变化，以定义 AML1-ETO在确定的基因座和整个基因组中。此外，我们的初步数据已经 提供了一个范式转变：尽管AML1-ETO结合增强子自 这些细胞系的建立，它们被重新激活的时间过程， 融合蛋白的降解。因此，需要AML1-ETO的持续表达来维持 抑制，在许多位点，而其他位点更永久沉默。最后，我们用CRISPR 允许快速纯化与MUDPIT偶联的AML1-ETO，并鉴定了一种新的染色质 修饰复合物可能介导AML 1-ETO依赖性抑制。我们假设 AML1-ETO招募组蛋白修饰酶重新连接表观遗传景观，以抑制 CEBPA、PU.1和GFI1B损害骨髓分化。这就为中学的发展奠定了基础。 表观遗传突变加强了这些变化，如ASXL1/2的失活。我们会直接 通过定义控制AML1-ETO募集的分子接触来验证这一假设。 阻遏复合物，并使用化学遗传学来测试这些接触是否需要AML1- ETO调节的转录。

The goal of this application is to understand how the t(8;21), the most frequent chromosomal translocation associated with acute myeloid leukemia (AML), sets the stage for secondary mutations to accumulate and develop into AML. Understanding how the encoded AML1-ETO fusion protein alters epigenetic wiring, is critical to finding less debilitating therapies that yield better outcomes. Both AML1 (RUNX1) and ETO/MTG family members also suffer point mutations in solid tumors, and the ETO family members Mtgr1 (CBFA2T2) and Mtg16 (CBFA2T3) are tumor suppressors in mouse models of intestinal neoplasia, so understanding how ETO contacts histone modifying enzymes has great impact outside of the t(8;21). In our preliminary data, we have used CRISPR/Cas9 technology to modify the 3’ end of the endogenous AML1-ETO with FKBP12F36V- HA or 3XFLAG tags to selectively and rapidly degrade AML1-ETO. We have coupled this system with state-of-the-art genomics such as precision nuclear run-on transcription sequencing (PROseq) and Cut&Run to establish a chemical genetic system to unambiguously define the mechanism of transcriptional control by AML1-ETO. Critically, this allows us to define the earliest, and presumably direct, changes in transcription upon inactivation of the fusion protein. Our preliminary PROseq data demonstrate that enhancers within key hematopoietic regulatory genes such as CEBPA are reactivated within 2 hr of adding dTAG47 to Kasumi-1 cell cultures. These novel reagents allow us to define changes in histone modifications and RNA polymerase dynamics to define the action of AML1-ETO at defined loci and throughout the genome. Moreover, our preliminary data already provide a paradigm shift: even though AML1-ETO bound enhancers have been repressed since the establishment of these cell lines, they were reactivated with a time course that matched the degradation of the fusion protein. Thus, continued expression of AML1-ETO is needed to maintain repression, at many loci, while other loci are more permanently silenced. Finally, we used CRISPR to allow rapid purification of AML1-ETO coupled with MUDPIT and identified a new chromatin modifying complex as potentially mediating AML1-ETO-dependent repression. We hypothesize that AML1-ETO recruits histone modifying enzymes to rewire the epigenetic landscape to suppress CEBPA, PU.1 and GFI1B to impair myeloid differentiation. This sets the stage for secondary epigenetic mutations that reinforce these changes such as inactivation of ASXL1/2. We will directly test this hypothesis by defining the molecular contacts that control AML1-ETO recruitment of repression complexes and use chemical genetics to test if these contacts are required for AML1- ETO-regulated transcription.