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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.

这个应用程序的目标是了解t(8;21)，最常见的染色体