Role of PRC1 in RUNX1-ETO-mediated transcriptional control

PRC1 在 RUNX1-ETO 介导的转录控制中的作用

• 批准号: 10445091
• 负责人: SCOTT W HIEBERT
• 金额: $ 42.29万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 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/10445091
• 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; 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

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最常见的染色体t(8；21)是如何 与急性髓细胞白血病(AML)相关的易位，为继发性 突变累积并发展为急性髓系白血病。了解编码的AML1-ETO如何 融合蛋白改变表观遗传连接，对于找到不那么虚弱的治疗方法至关重要 更好的结果。AML1(RUNX1)和ETO/MTG家族成员也都发生了点突变 在实体瘤中，ETO家族成员Mtgr1(CBFA2T2)和Mtg16(CBFA2T3)是肿瘤 小鼠肠道肿瘤模型中的抑制子，因此了解eto如何接触组蛋白 修饰酶在t(8；21)之外有很大的影响。在我们的初步数据中，我们使用了 CRISPR/Cas9技术用FKBP12F36V修饰内源AML1-ETO的3‘端 HA或3XFLAG标签选择性地快速降解AML1-ETO。我们已经将这个系统连接起来 拥有最先进的基因组学，如精确的核连续转录测序(PROseq) 和Cut&Run建立一个化学遗传系统，以明确定义 AML1-ETO的转录调控。关键的是，这使我们能够定义最早的，大概 当融合蛋白失活时，转录的直接变化。我们的初步PROSEQ数据 证明关键造血调控基因中的增强子，如CEBPA是 在Kasumi-1细胞培养中加入dTAG47后2小时内重新激活。这些新奇的试剂让我们 为了定义组蛋白修饰和RNA聚合酶动力学的变化，以定义 AML1-ETO位于已定义的基因座和整个基因组。此外，我们的初步数据已经 提供范式转变：尽管AML1-ETO结合增强子自 在这些细胞系的建立过程中，它们被重新激活，时间进程与 融合蛋白的降解。因此，AML1-ETO的持续表达需要维持 抑制，在许多基因座上，而其他基因座更永久地沉默。最后，我们使用CRISPR 利用泥坑快速纯化AML1-ETO并鉴定出一个新的染色质 将复合体修饰为潜在地介导AML1-ETO依赖的抑制。我们假设 AML1-ETO招募组蛋白修饰酶重新连接表观遗传格局以抑制 CEBPA、PU.1和GFI1B抑制髓系分化。这为第二阶段奠定了基础 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 通过定义控制AML1-ETO招募的分子接触来检验这一假设 并使用化学遗传学来测试AML1是否需要这些接触- ETO调控的转录。