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

项目摘要 白血病是一种罕见但往往是致命的癌症，源于血液中的祖细胞 细胞分化有缺陷的一种特别侵袭性和难以治疗的形式，称为混合血统白血病 (MLL)是由MLL基因与各种编码延长的基因的染色体异常易位引起的 各种因素。这些MLL融合不适当地驱动同源异型基因的转录，如HOX家族中的基因，从而 维持高度增殖的干细胞样种群。这些“白血病干细胞”成熟为白血病。 可以形成肿瘤并通过血液在全身转移的母细胞。然而，具体的 MLL融合驱动的白血病发生的机制仍不清楚，而高频 治疗后复发需要进一步描述这种癌症的主要驱动因素。其中一个因素就是 最近被证明对调节MLL融合驱动的白血病发生起关键作用的是组蛋白Ash1 H3K36二甲基转移酶，可能含有BRD-PHD-BAH组蛋白阅读器结构域模块。支架蛋白 一种名为LEDGF的基因被证明与H3K36me2结合，并将MLL招募到Ash11依赖的同源异型基因上 方式，但尚不清楚H3K36me2和/或Ash1是否直接招募LEDGF-MLL复合体 这些基因。此外，我们完全不知道Ash1是如何定位到同源异型基因的。 比基因组的其余大部分都要多。为了解决Ash1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分析、结构方法和基于细胞的分析。 对于目标1，我将确定假定的Ash1阅读器结构域模块的组蛋白结合特异性和模式 用现代化的组蛋白多肽和核小体结合分析与冷冻-EM法平行检测纯化的Ash1 结合到修饰的核小体上。对于目标2，我将询问Ash1如何阅读 利用基因敲除/互补系统，对HEK293T细胞系进行了深入研究，并易于进行基因操作。我 将评估Ash1是否与我们在Aim 1中确定的组蛋白修饰以及蛋白质共定位 被认为像LEDGF一样使用Cut&Run与Ash1联系在一起。此外，我还将研究如何 在这些细胞中，Ash1l的缺失/互补会影响已知的基因靶标表达。对于目标3，我将聘请一名 MLL融合白血病模型中的敲除/互补系统1)评估是否需要Ash1 对于生存能力，2)确定Ash1在MLL中的基因定位，以及3)表征Ash1的损失和 互补作用影响共定位基因调控因子的招募，从而影响转录。这项工作旨在 进一步破译“组蛋白密码”，并提供对Ash1如何作为MLL-融合的驱动因素的见解 白血病的发生。我还将为测试Ash1作为治疗这些侵略性疾病的药物靶点奠定基础。 疾病。最后，这项工作作为结构和细胞生物学方法论的强大培训平台 希望我能在未来的研究岗位上学习和应用。

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.