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 白血病是一种罕见的，但往往是致命的癌症，起源于血液中的祖细胞， 在细胞分化上有缺陷。一种特别具有侵略性和难以治疗的形式称为混合谱系白血病 (MLL)由MLL基因的异常染色体易位与各种基因编码延长 因素这些MLL融合体不适当地驱动同源异型基因的转录，如Hox家族中的同源异型基因， 维持高度增殖的干细胞样群体。这些“白血病干细胞”成熟为白血病细胞。 可以形成肿瘤并通过血流转移到全身的胚细胞。但具体 MLL融合驱动白血病发生的机制仍然没有很好地理解， 治疗后的复发要求进一步表征这种癌症的主要驱动因素。其中一个因素是， 最近显示，调控MLL融合驱动的白血病发生的关键是ASH 1 L， 具有推定的BRD-PHD-BAH组蛋白阅读器结构域模块的H3 K36二甲基转移酶。支架蛋白 一种名为LEDGF的基因被证明可以与H3 K36 me 2结合，并在ASH 1 L依赖的同源异型基因中招募MLL。 H3 K36 me 2和/或ASH 1 L是否直接募集LEDGF-MLL复合物， 这些基因。此外，完全不知道ASH 1 L如何特异性定位于同源异型基因，而不是 比基因组的其他部分都要多。为了解决ASH 1 L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试验，结构方法，和基于细胞的分析。 对于目标1，我将确定假定的ASH 1 L阅读器结构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的ASH 1 L的Cryo-EM平行 与修饰的核小体结合。对于目标2，我将询问ASH 1 L如何读取染色体中的染色质景观。 使用敲除/互补系统充分研究并且易于遗传操作HEK 293 T细胞系。我 将评估ASH 1 L是否与我们在Aim 1中鉴定的组蛋白修饰以及蛋白质共定位 被认为与ASH 1 L相关联，如使用CUT&RUN的LEDGF。此外，我将研究如何 这些细胞中ASH 1 L的缺失/互补影响已知的基因靶表达。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，以1）评估是否需要ASH 1 L 对于生存力，2）确定ASH 1 L基因组定位在MLL中的位置，以及3）表征ASH 1 L损失和 互补作用影响共定位基因调节子的募集以影响转录。这项工作旨在 进一步破译“组蛋白密码”，并提供关于ASH 1 L如何作为MLL-fusion驱动程序的见解 白血病发生我还将为测试ASH 1 L作为治疗这些侵袭性疾病的药物靶点奠定基础。 疾病最后，这项工作作为一个强大的培训平台，在结构和细胞生物学方法， 我学习和应用在未来的研究职位。