Deciphering the histone interactions and reader functions of ASH1L in biology and leukemia

破译 ASH1L 在生物学和白血病中的组蛋白相互作用和阅读器功能

基本信息

批准号：
10389050
负责人：
Nathaniel T Burkholder
金额：
$ 4.9万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-03-01 至 2022-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10389050
关键词：
ASH1L gene Address Affect Anemia Animals Apoptosis Automobile Driving Binding Biological Assay Biology Blood Blood Circulation Bone Pain Cell Line Cell Transplantation Cell fusion Cell model Cells Cellular biology Chromatin Chromosomal translocation Chromosome Mapping Clinical Trials Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Complement Complex Cryoelectron Microscopy Development Disease Docking Doctor of Philosophy Drug Targeting Elongation Factor Epigenetic Process Equilibrium Family Foundations Frequencies Gene Expression Gene Targeting Genes Genetic Transcription Genome Hematopoietic Hematopoietic Neoplasms Histone Acetylation Histone Code Histones Homeobox Genes In Vitro Knock-out Learning Leukemic Cell Leukocytes MLL gene MLLT2 gene MLLT3 gene Malignant Bone Neoplasm Malignant Neoplasms Mediating Methodology Methods Mixed-Lineage Leukemia Modeling Modernization Mutagenesis Mutation Nature Neoplasm Metastasis Neurodevelopmental Disorder Nucleosomes Pathway interactions Peptides Phenotype Play Pliability Polycomb Population Positioning Attribute Post-Translational Protein Processing Process Proliferating Proteins Radiation therapy Reader Regulator Genes Relapse Resistance Rest Role Scaffolding Protein Site Specificity Stream Survival Rate System Testing Training Work X-Ray Crystallography base complement system genetic manipulation histone methylation histone methyltransferase histone modification in vivo inhibitor innovation insight knock-down leukemia leukemic stem cell leukemogenesis mouse model mutant new therapeutic target overexpression preference prevent recruit screening stem stem cells stem-like cell structural biology therapeutic target tool transcriptome sequencing tumor tumorigenesis

项目摘要

Project Summary Leukemia is a rare, but often fatal, form of cancer stemming from progenitor cells in the blood that are defective in cellular differentiation. A particularly aggressive and hard to treat form called mixed-lineage leukemia (MLL) arises from aberrant chromosomal translocations of the MLL gene with various genes encoding elongation factors. These MLL-fusions improperly drive transcription of homeotic genes like those in the Hox family, thereby maintaining a highly proliferative, stem cell-like population. These “leukemic stem cells” mature into leukemic blasts that can form tumors and metastasize throughout the body via the blood stream. However, the specific mechanisms of MLL-fusion driven leukemogenesis are still not well understood and the high frequency of relapses upon treatment call for further characterization of the main drivers of this cancer. One such factor that has recently been shown to be critical for regulating MLL-fusion driven leukemogenesis is ASH1L, a histone H3K36 dimethyltransferase with a putative BRD-PHD-BAH histone reader domain module. A scaffold protein called LEDGF was shown to bind to H3K36me2 and recruit MLL to homeotic genes in an ASH1L-dependent manner, but it is unclear whether H3K36me2 and/or ASH1L are directly recruiting LEDGF-MLL complexes to these genes. Additionally, it is completely unknown how ASH1L specifically localizes to homeotic genes rather than much of the rest of the genome. To address how ASH1L functions in non-leukemic and leukemic cells, I will employ a suite of innovative histone-binding assays, structural methodologies, and cellular-based analyses. For Aim 1, I will determine the histone binding specificity and mode of the putative ASH1L reader domain module using modernized histone peptide and nucleosome binding assays in parallel with Cryo-EM of purified ASH1L bound to modified nucleosomes. For Aim 2, I will interrogate how ASH1L reads the chromatin landscape in the well-studied and easy to genetically manipulate HEK293T cell line using a knockout/complementation system. I will assess whether ASH1L colocalizes with histone modifications that we identify in Aim 1 as well as proteins thought to associate with ASH1L like LEDGF using CUT&RUN. Additionally, I will examine how loss/complementation of ASH1L in these cells affects known gene target expression. For Aim 3, I will employ a knockdown/complementation system in MLL-fusion leukemia models to 1) assess whether ASH1L is required for viability, 2) determine where ASH1L genomically localizes in MLL, and 3) characterize how ASH1L loss and complementation affects recruitment of co-localizing gene regulators to impact transcription. This work aims to further decipher the “Histone Code” and provide insights into how ASH1L functions as a driver of MLL-fusion leukemogenesis. I will also lay out a foundation for testing ASH1L as a drug target for treating these aggressive diseases. Finally, this work serves as a strong training platform in structural and cell biology methodologies for me to learn and apply in future research positions.

项目概要白血病是一种罕见但通常致命的癌症，源自血液中的祖细胞一种特别具有侵袭性且难以治疗的细胞分化缺陷，称为混合谱系白血病。 (MLL) 由 MLL 基因与编码伸长的各种基因的异常染色体易位引起这些 MLL 融合不正确地驱动同源基因（如 Hox 家族中的基因）的转录，从而维持高度增殖的干细胞样群，这些“白血病干细胞”成熟为白血病。原始细胞可以形成肿瘤并通过血流转移到全身。 MLL 融合驱动的白血病发生机制仍不清楚，并且发生频率很高治疗后复发需要进一步确定这种癌症的主要驱动因素。最近被证明对调节 MLL 融合驱动的白血病发生至关重要的是 ASH1L，一种组蛋白 H3K36 二甲基转移酶与假定的 BRD-PHD-BAH 组蛋白阅读器结构域模块 A 支架蛋白。 LEDGF 被证明可以与 H3K36me2 结合，并在 ASH1L 依赖的同源异型基因中招募 MLL 方式，但尚不清楚 H3K36me2 和/或 ASH1L 是否直接招募 LEDGF-MLL 复合物此外，完全不知道 ASH1L 如何特异性定位于同源异型基因。为了解决 ASH1L 在非白血病和白血病细胞中的功能，我将采用一套创新的组蛋白结合测定、结构方法和基于细胞的分析。对于目标 1，我将确定假定的 ASH1L 阅读器域模块的组蛋白结合特异性和模式使用现代化组蛋白肽和核小体结合测定与纯化 ASH1L 的 Cryo-EM 并行对于目标 2，我将询问 ASH1L 如何读取染色质景观。经过充分研究且易于使用敲除/互补系统 I 进行基因操作的 HEK293T 细胞系。将评估 ASH1L 是否与我们在 Aim 1 中识别的组蛋白修饰以及蛋白质共定位另外，考虑使用 CUT&RUN 与 ASH1L（如 LEDGF）相关联，我将研究如何进行。这些细胞中 ASH1L 的缺失/互补会影响已知的基因靶标表达。对于目标 3，我将采用 MLL 融合白血病模型中的敲低/互补系统 1) 评估是否需要 ASH1L 为了确定活力，2) 确定 ASH1L 在 MLL 中的基因组定位位置，以及 3) 描述 ASH1L 丢失和丢失的方式这项工作的目的是进一步破译“组蛋白密码”并深入了解 ASH1L 如何作为 MLL 融合的驱动程序我还将为测试 ASH1L 作为治疗这些侵袭性白血病的药物靶点奠定基础。最后，这项工作为结构和细胞生物学方法学提供了强大的培训平台。我学习并应用于未来的研究职位。