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：
10652591
负责人：
SCOTT W HIEBERT
金额：
$ 42.26万
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/10652591
关键词：
AML1-ETO fusion protein Abbreviations Acute Myelocytic Leukemia Affect Affinity Chromatography Alleles Binding Biological Assay CBFA2T1 gene CD34 gene CEBPA gene CRISPR/Cas technology Cancer Etiology Cell Culture Techniques Cell Nucleus Cell fusion Cells ChIP-seq Chimeric Proteins Chromatin Chromosomal translocation Cloning Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Compensation Complex Coupled Coupling Cytoplasm 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 Complementation Test 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 Preleukemia 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 cell transformation chemical genetics chemotherapy differential expression established cell line histone modification inhibitor insight leukemogenesis mouse genetics mouse model novel permissiveness 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.

该应用的目的是了解最常见的染色体T（8; 21）如何与急性髓样白血病（AML）相关的易位，为次级设定了舞台突变积累并发展成AML。了解编码的AML1-Eto 融合蛋白改变表观遗传接线，对于找到较少的衰弱疗法至关重要更好的结果。 AML1（RUNX1）和ETO/MTG家族成员都遭受点突变在实体瘤中，ETO家族成员MTGR1（CBFA2T2）和MTG16（CBFA2T3）是肿瘤肠道肿瘤的小鼠模型中的补充，因此了解ETO如何接触组蛋白修饰酶在t之外的影响（8; 21）之外具有很大的影响。在我们的初步数据中，我们使用了 CRISPR/CAS9技术通过FKBP12F36V-修改内源性AML1-Eto的3'端 HA或3XFLAG标签有选择地和快速降级AML1-Eto。我们耦合了这个系统具有最先进的基因组学，例如精确的核跑步转录测序（Proseq）并剪切并建立一个化学遗传系统，以明确定义 AML1-Eto的转录控制。至关重要的是，这使我们能够最早定义直接，融合蛋白失活后转录变化。我们的初步Proseq数据证明关键造血调节基因（例如Cebpa）中的增强子是将DTAG47添加到Kasumi-1细胞培养物的2小时内重新激活。这些新颖的试剂允许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在定义的基因座和整个基因组中。而且，我们的初步数据已经提供范式转移：即使自从AML1-Eto绑定增强器从那以后复制了建立这些细胞系，它们通过与该细胞线相匹配的时间课程重新激活融合蛋白的降解。这是需要持续表达AML1-Eto来维持在许多地方的镇压，而其他地方则更永久地沉默。最后，我们使用了CRISPR 为了允许快速纯化AML1-Eto与泥锅结合并鉴定出新的染色质修改复合物作为潜在介导的AML1-Eto依赖性表示。我们假设这一点 AML1-Eto招募了Hisstone修改酶以重新布线表观遗传景观以抑制 CEBPA，PU.1和GFI1B损害髓样分化。这为次级设定了舞台增强这些变化的表观遗传突变，例如ASXL1/2的失活。我们将直接通过定义控制AML1-Eto募集的分子接触来检验该假设抑制复合物并使用化学遗传学来测试是否需要这些接触的AML1-- ETO调节的转录。