AF9(MLLT3) Function in Leukemia and Normal Hematopoiesis

AF9(MLLT3) 在白血病和正常造血中的功能

• 批准号: 10434785
• 负责人: JOHN Hackett BUSHWELLER
• 金额: $ 58.98万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10434785
• 关键词: Acetylation; Address; Automobile Driving; Binding; Binding Proteins; Biochemical; Blood; Blood Cells; Bromodomain; C-terminal; Cell Lineage; Cell Maintenance; Cells; ChIP-seq; Chimeric Proteins; Chromatin; Chromosomal translocation; Competitive Binding; Complex; Data; Development; Disease; Enhancers; Epigenetic Process; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Grant; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Histones; Homologous Gene; In Vitro; Individual; K-18 conjugate; Knockout Mice; Laboratories; Link; Lysine; MLL gene; MLLT1 gene; MLLT2 gene; MLLT3 gene; Mixed-Lineage Leukemia; Multiprotein Complexes; Peptides; Phenotype; Point Mutation; Prognosis; Proteins; RNA; RNA Binding; RNA Recognition Motif; Reader; Regulator Genes; Role; SWP29; Site; Specificity; Structure; Work; base; drug development; epigenetic regulation; experimental study; gene repression; in vivo; leukemia; leukemogenesis; mutant; novel; programs; promoter; recruit; stem cell function; stem cell homeostasis; stem cells; three dimensional structure; transcriptome sequencing

MLLT3 (AF9) and its homolog MLLT1 (ENL) were initially identified as chromosome translocation partners of the MLL (KMT2A) gene observed in Mixed Lineage Leukemia (MLL). The amino termini of MLLT3, and MLLT1 proteins contain a nearly identical chromatin-binding YEATS domain which preferentially binds crotonylated histone sites (Kcr). This distinguishes YEATS domains as crotonylation reader modules in contrast to other acetylation reader modules, such as bromodomains. The MLLT3 YEATS domain directly links histone Kcr readout to active gene transcription, but mechanisms underlying specific recruitment to direct target genes are not understood. Work from different laboratories, including ours, has revealed roles of MLLT3 and MLLT1 in at least four different complexes with critical gene regulatory functions based on direct binding to the C-terminal ANC1 homology domain (AHD). The canonical functions of two of these complexes (AF4-containing Super Elongation Complex; DOT1L) are to activate gene transcription whereas the other two (CBX8, BCOR) most often function in gene repression. The factors that decide which of these four different complexes are recruited, and whether recruitment of one complex facilitates or inhibits recruitment of another are not understood. Aim 1: Functional effects of CBX8 and BCOR recruitment on MLL-MLLT3/1 (MLL-AF9/-ENL) function. We have determined 3D structures of MLLT3 AHD-CBX8 and AHD-BCOR complexes and used the structural information to develop point mutations to selectively disrupt recruitment of CBX8 and BCOR. These will be used to specifically delineate the role of direct recruitment of CBX8 and BCOR to MLL-MLLT3 and MLL-MLLT1 in altering gene expression and driving leukemia, as we have done previously for the AF4 and DOT1L interactions. Aim 2: MLLT3 (AF9) YEATS domain is a dual reader of H3K9 (and K18, K27) crotonylation and RNA. We have used a biochemical approach to show that the MLLT3 YEATS domain also binds to RNA, in addition to specific binding to crotonylated H3, indicating this domain is a dual reader of both epigenetic marks and RNA. We are proposing to fully characterize the role of the RNA binding of this domain in MLLT3 function. This includes delineation of the RNA binding specificity, structural studies of a YEATS domain-H3K9cro-RNA ternary complex, and development of point mutations which can selectively disrupt RNA binding and H3 peptide binding to probe the functional role of these interactions. Similar studies will be carried out with MLLT1. Aim 3: MLLT3 (AF9) and MLLT1 (ENL) have non-redundant roles in hematooietic stem and progenitor cell (HSPC) gene regulation which require their YEATS domain and C-terminal AHD functions. Using wildtype and point mutant forms of MLLT3 and MLLT1 which can selectively disrupt either histone or RNA binding, we will probe the functional role of the H3Kcr and RNA interactions via ChIP-seq, RNA-seq, and effects on in vitro and in vivo HSPC functions. Wildtype and mutant MLLT3 and MLLT1 that specifically disrupt binding to AF4, DOT1L, BCOR, and CBX8 will probe the roles of these interactions on gene expression and HSPC functions.

MLLT 3（AF 9）及其同源物MLLT 1（ENL）初步鉴定为染色体易位 在混合谱系白血病（MLL）中观察到的MLL（KMT 2A）基因的配偶体。MLLT 3的氨基末端， 和MLLT 1蛋白含有几乎相同的染色质结合YEATS结构域， 巴豆酰化组蛋白位点（Kcr）。这将YEATS结构域区分为巴豆酰化阅读器模块， 到其他乙酰化读取器模块，例如溴结构域。MLLT 3 YEATS结构域直接连接组蛋白 Kcr读出到活跃的基因转录，但潜在的机制特异性募集直接靶基因 不被理解。包括我们在内的不同实验室的工作揭示了MLLT 3和MLLT 1在以下方面的作用： 至少四种不同的复合物，其基于与C-末端的直接结合而具有关键的基因调节功能， ANC 1同源结构域（AHD）。其中两个复合物（含AF_4的Super 延伸复合物; DOT 1 L）是激活基因转录，而其他两个（CBX 8，BCOR）最常 在基因阻遏中起作用。决定这四种不同复合体中哪一种被招募的因素，以及 一种复合物的募集是促进还是抑制另一种复合物的募集尚不清楚。 目的1：CBX 8和BCOR募集对MLL-MLLT 3/1（MLL-AF 9/-ENL）功能的功能影响。 我们已经确定了MLLT 3 AHD-CBX 8和AHD-BCOR复合物的3D结构，并使用了结构分析。 信息开发点突变以选择性地破坏CBX 8和BCOR的募集。这些将用于 明确描述CBX 8和BCOR直接募集至MLL-MLLT 3和MLL-MLLT 1的作用， 改变基因表达和驱动白血病，就像我们以前对AF 4和DOT 1 L相互作用所做的那样。 目的2：MLLT 3（AF 9）YEATS结构域是H3 K9（和K18、K27）巴豆酰化和RNA的双重阅读器。 我们已经使用生物化学方法显示MLLT 3 YEATS结构域也结合RNA，此外， 与巴豆酰化H3特异性结合，表明该结构域是表观遗传标记和RNA的双重阅读器。 我们建议充分表征该结构域在MLLT 3功能中的RNA结合作用。这包括 RNA结合特异性的描述，YEATS结构域-H3 K9 cro-RNA三元复合物的结构研究， 以及可以选择性地破坏RNA结合和H3肽与探针结合的点突变的开发 这些相互作用的功能。将对MLLT 1进行类似的研究。 目的3：MLLT 3（AF 9）和MLLT 1（ENL）在造血干细胞和祖细胞中具有非冗余作用 细胞（HSPC）基因调节需要其YEATS结构域和C末端AHD功能。使用野生型 以及MLLT 3和MLLT 1的点突变形式，它们可以选择性地破坏组蛋白或RNA结合，我们 将通过ChIP-seq，RNA-seq和体外对H3 Kcr和RNA相互作用的影响来探索H3 Kcr和RNA相互作用的功能作用。 和体内HSPC功能。特异性破坏与AF 4结合的野生型和突变MLLT 3和MLLT 1， DOT 1 L、BCOR和CBX 8将探测这些相互作用对基因表达和HSPC功能的作用。