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

• 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
• 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

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.

项目摘要 白血病是一种罕见但通常是致命的癌症形式，是由血液中的祖细胞引起的 细胞分化有缺陷。一种特别侵略性和难以处理的形式，称为混合乳清白血病 （MLL）来自MLL基因的异常染色体易位，该基因具有编码伸长的各种基因 因素。这些mll互及不正确地驱动像Hox家族中的同型基因的转录，从而 保持高度增殖的干细胞样人群。这些“白血病干细胞”成熟到白血病 可以通过血流在整个体内形成肿瘤并在整个体内转移的爆炸。但是，具体 MLL融合驱动的白血病的机制仍然不太了解，高频的高频 在治疗时复发，要求进一步表征该癌症的主要驱动因素。一个这样一个因素 最近已证明对调节MLL融合驱动性白血病生成至关重要是组蛋白ASH1L H3K36二甲基转移酶，带有假定的BRD-PHD-BAH HISSTONE READER域模块。脚手架蛋白 称为LEDGF被证明与H3K36Me2结合，并在ASH1L依赖性中募集MLL与同源基因 方式，但尚不清楚H3K36Me2和/或ASH1L是否直接招募LEDGF-MLL综合体 这些基因。此外，完全未知ASH1L如何专门定位于同源基因 比其他基因组的大部分。为了解决ASH1L在非白血病和白血病细胞中的功能，I 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AIM 1，我将确定假定ASH1L读取器域模块的组蛋白结合特异性和模式 使用现代化的组蛋白肽和核小体结合测定与纯化ASH1L的冷冻EM并行 与修饰的核小体结合。对于AIM 2，我将询问ASH1L如何阅读染色质景观 经过良好的研究，易于使用基因敲除/补充系统来易于操纵HEK293T细胞系。我 将评估ASH1L是否与我们在AIM 1和蛋白质中识别的组蛋白修饰共定位 被认为使用剪切和运行将与ledgf这样的ASH1L关联。此外，我将研究如何 这些细胞中ASH1L的丧失/互补会影响已知的基因靶标表达。对于AIM 3，我将采用一个 MLL融合白血病模型中的敲低/互补系统至1）评估是否需要ASH1L 对于生存能力，2）确定ASH1L基因在MLL中的位置，3）表征ASH1L丢失和 互补会影响共定位基因调节因子影响转录的募集。这项工作旨在 进一步破译“组蛋白代码”，并提供有关ASH1L作为MLL融合驱动器的功能的见解 白血病。我还将为测试ASH1L作为治疗这些侵略性的药物目标奠定基础 疾病。最后，这项工作是结构和细胞生物学方法的强大培训平台 我要学习并申请将来的研究职位。