Role of PRC1 in RUNX1-ETO-mediated transcriptional control

PRC1 在 RUNX1-ETO 介导的转录控制中的作用

基本信息

批准号：
10298288
负责人：
SCOTT W HIEBERT
金额：
$ 45.72万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-05 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10298288
关键词：
AML1-ETO fusion protein Acute Myelocytic Leukemia Affect Affinity Chromatography Alleles Biological Assay CBFA2T1 gene CD34 gene CEBPA gene CRISPR/Cas technology Cancer Etiology Cell Culture Techniques Cell Nucleus Cells Chimeric Proteins Chromatin Chromosomal translocation Cloning Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Complement Complex Coupled Coupling DNA Binding Domain DNA-Directed RNA Polymerase DRPLA protein Data Development Drug Combinations Drug Design Enhancers Environment Enzymes Epigenetic Process Family member Future GFI1B gene Gene Expression Genes Genetic Genetic Transcription Genome Genomics Goals HDAC3 gene Hematopoietic Hematopoietic stem cells Histones Human Impairment Intestinal Neoplasms KAI1 gene Leukemic Cell Link Mass Spectrum Analysis Mediating Methods Methylation Modeling Molecular Mutate Mutation Myelogenous Normal Cell Nuclear Nucleosomes Outcome PRC1 Protein Patients Phenotype Point Mutation Prognostic Marker Protac Proteins RNA Interference RUNX1 gene Reagent Regulator Genes Relapse Repression Role Running Sodium Chloride Solid Neoplasm System Testing Thalidomide Time Transcript Transcriptional Regulation Tumor Suppressor Proteins Work Yeasts analog base cell transformation chemical genetics chemotherapy differential expression established cell line histone modification inhibitor/antagonist insight leukemogenesis mouse genetics mouse model novel programs protein degradation recruit side effect small hairpin RNA success t(8 21) acute myeloid leukemia t(8 21)(q22 q22)targeted treatment tool transcription factor transcriptome sequencing tumorigenesis ubiquitin isopeptidase ubiquitin-protein ligase yeast two hybrid system

项目摘要

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.

此应用程序的目标是了解最常见的染色体 t(8;21) 如何与急性髓系白血病（AML）相关的易位，为继发性白血病奠定了基础了解如何编码 AML1-ETO。融合蛋白改变表观遗传线路，对于寻找更少衰弱的疗法至关重要 AML1 (RUNX1) 和 ETO/MTG 家族成员也遭受点突变。在实体瘤中，ETO家族成员Mtgr1（CBFA2T2）和Mtg16（CBFA2T3）是肿瘤肠道肿瘤小鼠模型中的抑制因子，因此了解 ETO 如何接触组蛋白在我们的初步数据中，修饰酶对 t(8;21) 之外有很大影响。 CRISPR/Cas9技术用FKBP12F36V修饰内源性AML1-ETO的3'端 HA 或 3XFLAG 标签可选择性地快速降解 AML1-ETO。采用最先进的基因组学，例如精密核连续转录测序 (PROseq) 和 Cut&Run 建立化学遗传系统，以明确定义其机制至关重要的是，这使我们能够定义最早的、大概的。我们的初步 PROseq 数据表明，融合蛋白失活后转录发生了直接变化。证明关键造血调节基因（如 CEBPA）内的增强子将 dTAG47 添加到 Kasumi-1 细胞培养物中后 2 小时内重新激活，这些新型试剂使我们能够使用。定义组蛋白修饰和 RNA 聚合酶动力学的变化，定义作用此外，我们的初步数据已经在确定的位点和整个基因组中进行了。提供了范式转变：尽管 AML1-ETO 结合增强子自此以来一直受到抑制这些细胞系的建立后，它们被重新激活，时间过程与因此，需要 AML1-ETO 的持续表达来维持。在许多位点上受到抑制，而其他位点则更加永久地沉默。最后，我们使用了 CRISPR。允许快速纯化 AML1-ETO 与 MUDPIT 结合并鉴定出新的染色质修改复合体作为潜在介导 AML1-ETO 依赖性抑制。 AML1-ETO 招募组蛋白修饰酶来重新连接表观遗传景观以抑制 CEBPA、PU.1 和 GFI1B 会损害骨髓分化，这为继发性奠定了基础。加强这些变化的表观遗传突变，例如 ASXL1/2 的失活，我们将直接进行。通过定义控制 AML1-ETO 募集的分子接触来检验这一假设抑制复合物并使用化学遗传学来测试这些接触是否是 AML1-所必需的 ETO 调节的转录。