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: 10524126
• 负责人: JOHN Hackett BUSHWELLER
• 金额: $ 6.58万
• 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/10524126
• 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.

MLLT3(AF9)及其同源物MLLT1(En1)最初被鉴定为染色体易位 在混合血统白血病(MLL)中观察到MLL(KMT2A)基因的配对。MLLT3的氨基端， 和MLLT1蛋白包含几乎相同的染色质结合的叶芝结构域，该结构域优先结合 巴豆化的组蛋白位点(KCR)。相比之下，这将叶芝结构域区分为巴豆化阅读器模块 到其他乙酰化阅读器模块，例如溴域。MLLT3的叶芝结构域直接与组蛋白相连 KCR读数激活基因转录，但潜在的特定招募机制直接靶基因 是不被理解的。包括我们在内的不同实验室的工作已经揭示了MLLT3和MLLT1在 至少四种不同的基于与C末端直接结合而具有关键基因调控功能的复合体 ANC1同源结构域(AHD)。其中两个含AF4的超分子络合物的正则函数 伸长复合体(DOT1L)是激活基因转录，而另外两个(CBX8，BCOR)最常 在基因抑制中的作用。决定这四个不同复合体中的哪一个被招募的因素，以及 一个综合体的招募是促进还是抑制另一个综合体的招募尚不清楚。 目的1：CBX8和BCOR募集对MLL-MLLT3/1(MLL-AF9/-ENL)功能的影响。 我们测定了MLLT3AHD-CBX8和AHD-BCOR络合物的三维结构，并用结构 开发点突变以选择性地干扰CBX8和BCOR的招募的信息。这些将被用到 具体描述CBX8和BCOR对MLL-MLLT3和MLL-MLLT1的直接招募在 改变基因表达和驱动白血病，就像我们之前对AF4和DOT1L相互作用所做的那样。 目的：MLLT3(AF9)Yeats结构域是H3K9(和K18，K27)巴豆化和RNA的双重阅读器。 我们已经使用生物化学的方法来证明MLLT3Yeats结构域也与RNA结合，此外 与巴豆化H3的特异性结合，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的RNA结合在MLLT3功能中的作用。这包括 RNA结合特异性的描绘，Yeats结构域-H3K9cro-RNA三元络合物的结构研究， 以及可选择性破坏RNA结合和H3肽与探针结合的点突变的发展 这些相互作用的功能作用。将对MLLT1进行类似的研究。 目的3：MLLT3(AF9)和MLLT1(ENL)在造血干和祖细胞中具有非多余的作用 细胞(HSPC)基因调控需要其Yeats结构域和C-末端的AHD功能。使用通配类型 以及MLLT3和MLLT1的点突变形式，它们可以选择性地扰断组蛋白或RNA结合，我们 将探讨H3Kcr和RNA通过ChIP-seq、RNA-seq相互作用的功能作用以及对体外的影响 和体内HSPC的功能。野生型和突变型MLLT3和MLLT1特异性地破坏与AF4的结合， DOT1L、BCOR和CBX8将探索这些相互作用对基因表达和HSPC功能的作用。