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) 基因伴侣。 MLLT3 的氨基末端， MLLT1 蛋白含有几乎相同的染色质结合 YEATS 结构域，该结构域优先结合 巴豆酰化组蛋白位点 (Kcr)。相比之下，这将 YEATS 域区分为巴豆酰化阅读器模块 其他乙酰化阅读器模块，例如溴结构域。 MLLT3 YEATS 结构域直接连接组蛋白 Kcr 读出活跃基因转录，但具体招募直接靶基因的机制 不被理解。包括我们在内的不同实验室的工作揭示了 MLLT3 和 MLLT1 在 至少四种具有基于直接结合到 C 端的关键基因调控功能的不同复合物 ANC1 同源域 (AHD)。其中两个复合体（含 AF4 的超级复合体）的规范函数 伸长率复合物； DOT1L）用于激活基因转录，而其他两个（CBX8、BCOR）最常用于激活基因转录 在基因抑制中发挥作用。决定招募这四种不同复合体中哪一种的因素，以及 一种复合物的募集是否促进或抑制另一种复合物的募集尚不清楚。 目标 1：CBX8 和 BCOR 募集对 MLL-MLLT3/1 (MLL-AF9/-ENL) 功能的功能影响。 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 开发点突变以选择性破坏 CBX8 和 BCOR 招募的信息。这些都会用到 具体描述了 CBX8 和 BCOR 直接招募到 MLL-MLLT3 和 MLL-MLLT1 中的作用 改变基因表达并驱动白血病，正如我们之前对 AF4 和 DOT1L 相互作用所做的那样。 目标 2：MLLT3 (AF9) YEATS 结构域是 H3K9（和 K18、K27）巴豆酰化和 RNA 的双重读取器。 我们使用生化方法证明 MLLT3 YEATS 结构域还与 RNA 结合，此外 与巴豆酰化 H3 的特异性结合，表明该结构域是表观遗传标记和 RNA 的双重读取器。 我们建议全面表征该结构域的 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三元复合物的结构研究， 以及开发点突变，可以选择性地破坏 RNA 结合和 H3 肽与探针的结合 这些相互作用的功能作用。 MLLT1 也将进行类似的研究。 目标 3：MLLT3 (AF9) 和 MLLT1 (ENL) 在造血干细胞和祖细胞中具有非冗余作用 细胞（HSPC）基因调控需要其 YEATS 结构域和 C 端 AHD 功能。使用野生型 MLLT3 和 MLLT1 的点突变形式可以选择性破坏组蛋白或 RNA 结合，我们 将通过 ChIP-seq、RNA-seq 探讨 H3Kcr 和 RNA 相互作用的功能作用以及对体外的影响 和体内 HSPC 功能。野生型和突变型 MLLT3 和 MLLT1 特异性破坏与 AF4 的结合， DOT1L、BCOR 和 CBX8 将探讨这些相互作用对基因表达和 HSPC 功能的作用。