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: 10378336
• 负责人: JOHN Hackett BUSHWELLER
• 金额: $ 6.78万
• 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/10378336
• 关键词: Acetylation; Automobile Driving; Binding; Biochemical; Blood; Blood Cells; Bromodomain; C-terminal; ChIP-seq; Chimeric Proteins; Chromatin; Chromosomal translocation; Complex; Development; Disease; Epigenetic Process; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Hematopoiesis; Histones; Homologous Gene; In Vitro; K-18 conjugate; Laboratories; Link; MLL gene; MLLT1 gene; MLLT2 gene; MLLT3 gene; Mixed-Lineage Leukemia; Peptides; Point Mutation; Prognosis; Proteins; RNA; RNA Binding; RNA Recognition Motif; Reader; Regulator Genes; Role; SWP29; Site; Specificity; Structure; Work; base; drug development; gene repression; in vivo; leukemia; mutant; novel; recruit; stem cell function; 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（ENL）最初被鉴定为染色体易位 在混合谱系白血病（MLL）中观察到的MLL（KMT2A）基因的伴侣。 mllt3的氨基终端， MLLT1蛋白包含几乎相同的染色质结合Yeats结构域，优先结合 双链型组蛋白部位（KCR）。这将Yeats域区分为互联物读取器模块 到其他乙酰化读取器模块，例如溴结构域。 Mllt3 Yeats域直接连接组蛋白 KCR读数为活性基因转录，但具有特定募集的机制直接靶基因 不了解。来自包括我们的不同实验室的工作已经揭示了Mllt3和Mllt1在 至少四个具有关键基因调节函数的不同复合物，基于与C末端的直接结合 ANC1同源域（AHD）。这些复合物中的两个的规范功能（含有AF4的超级 伸长复合物； dot1l）是激活基因转录，而其他两个（cbx8，bcor）最常 基因抑制的功能。决定招募这四个不同复合物中哪种的因素，以及 尚不清楚招募一种复杂的促进还是抑制招募另一个复杂的招募。 AIM 1：CBX8和BCOR募集对MLLT3/1（MLL-AF9/-ENL）功能的功能效应。 我们已经确定了MLLT3 AHD-CBX8和AHD-BCOR复合物的3D结构，并使用了结构 信息以开发点突变以选择性破坏CBX8和BCOR的募集。这些将被使用 专门描述了CBX8和BCOR直接募集到mll-mllt3和mll-mllt1中的作用 像我们先前针对AF4和DOT1L相互作用所做的那样，改变了基因表达和驱动白血病。 AIM 2：MLLT3（AF9）Yeats域是H3K9（和K18，K27）的双重读取器和RNA。 我们已经使用了一种生化方法来证明Mllt3 Yeats域也与RNA结合，此外 特异性结合与双旋式H3，表明该结构域是表观遗传标记和RNA的双重读取器。 我们建议完全表征该域在MLLT3函数中的RNA结合的作用。这包括 RNA结合特异性的描述，Yeats域-H3K9CRO-RNA三元复合物的结构研究， 以及可以选择性破坏RNA结合并与探针结合的H3肽结合的点突变的发展 这些相互作用的功能作用。类似的研究将使用MLLT1进行。 AIM 3：MLLT3（AF9）和MLLT1（ENL）在造血茎和祖细胞中具有非冗余的作用 细胞（HSPC）基因调节，需要其Yeats域和C端AHD功能。使用野生型 MLLT3和MLLT1的点突变形式，可以选择性地破坏组蛋白或RNA结合，我们 将探测H3KCR和RNA相互作用的功能作用，并通过芯片序列，RNA-Seq以及对体外的影响 和体内HSPC函数。野生型和突变体mllt3和mllt1，特别破坏与af4的结合， DOT1L，BCOR和CBX8将探测这些相互作用在基因表达和HSPC函数上的作用。