Structure/Function of AF9 and MLL-AF9

AF9和MLL-AF9的结构/功能

• 批准号: 8974717
• 负责人: JOHN Hackett BUSHWELLER
• 金额: $ 0.83万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-15 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8974717
• 关键词: Acute Myelocytic Leukemia; Applications Grants; Automobile Driving; Binding; Blood; Blood Cells; C-terminal; Chimeric Proteins; Complement; Complex; DNA; Data; Development; Disease; Erythrocytes; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Goals; Grant; HOXA9 gene; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Histone H3; Length; MEIS1 gene; MLL gene; MLLT2 gene; MLLT3 gene; Megakaryocytes; Methylation; Mixed-Lineage Leukemia; Molecular; Mono-S; Mutation; Myeloid-Lymphoid Leukemia Protein; N-terminal; Neoplastic Cell Transformation; Peptides; Play; Point Mutation; Proteins; Recruitment Activity; Regulation; Regulator Genes; Regulatory Element; Role; SWP29; Sequence Homology; Spectrum Analysis; Structure; Testing; Time; Transcription Elongation; Yeasts; base; drug development; leukemia; leukemogenesis; novel; outcome forecast; prevent; programs; research study; self-renewal; transcription factor

DESCRIPTION (provided by applicant): AF9 (MLLT3) was first identified as a fusion partner of the Mixed Lineage Leukemia (MLL) gene in acute myeloid leukemia. At the time it was noted to share sequence homology to a component of the yeast SWI/SNF complex TFG3/ANC1, suggesting a role in transcription. AF9 clearly plays a key role in specific lineage fate decisions in hematopoiesis. In addition, its high level of expression in HSCs strongly suggests a critical role in maintaining the gene expression program necessary for HSCs. The mechanistic basis for these roles of AF9 has not been established. One of the primary goals of this grant is to provide a molecular basis for the function of AF9 with special emphasis on the role of the critical N- and C-terminal domains and their relationship to the gene regulatory machinery. We propose that binding of the AF9 N-terminal YEATS domain to histone H3 as well as binding of the AF9 C-terminal domain to AF4 are essential for normal erythrocyte and megakaryocyte differentiation. In addition, we propose that the interaction of MLL-AF9 with AF4 is essential for neoplastic transformation. The experiments that are proposed in this grant application are organized into two specific aims. Aim 1 focuses on the N-terminal YEATS domain and Aim 2 focuses on the AF9 C-terminal domain. Aim 1: Structure and function of the AF9 YEATS domain. Our Preliminary Data shows that the AF9 YEATS domain binds specifically to a region of histone H3. We are proposing three Sub-Aims to probe the role of this interaction in AF9 function. First, we will solve the structure of the AF9 YEATS domain bound to an H3 peptide. Based on the structure, we will develop specific point mutations to disrupt this interaction which will be introduced into full-length AF9 for functional testing. Second, we will examine the role of this interaction in the regulation of specific genes encoding hematopoietic transcription factors. Third, we will examine the role of the YEATS domain - H3 interaction in AF9's role in driving erythrocyte/megakaryocyte development. Aim 2: Structure and function of the AF9 C-terminal domain - AF4 complex. We are proposing 6 Sub-Aims to examine the role of the AF9-AF4 interaction in AF9 and MLL-AF9 function. First, we will determine a structure of the AF9 C-terminal domain - AF4 complex and use this to develop mutations to disrupt the interaction. These mutations will be used to test the role of this interaction in normal AF9 function, including effects on composition of the AF9 complex, the regulation of specific genes encoding hematopoietic transcription factors, and erythrocyte/megakaryocyte development. As this is the domain fused to MLL in MLL-AF9 fusions, these mutations will also be used to test effects on regulation of HOXA9 and MEIS1 by MLL-AF9 and MLL-AF9 leukemogenesis.

描述(申请人提供)：AF9(MLLT3)首次被鉴定为急性髓系白血病混合系白血病(MLL)基因的融合伙伴。当时注意到它与酵母SWI/SNF复合体TFG3/ANC1的一个组分有序列同源性，表明在转录中发挥了作用。显然，AF9在决定特定血统命运的造血过程中起着关键作用。此外，它在HSCs中的高水平表达强烈地表明了它在维持HSCs所需的基因表达程序中的关键作用。AF9这些作用的机制基础还没有建立起来。这项资助的主要目标之一是为AF9的功能提供分子基础，特别强调关键的N-末端和C-末端结构域的作用及其与基因调控机制的关系。我们认为AF9 N端的Yeats结构域与组蛋白H3的结合以及AF9 C端的结构域与AF4的结合对于正常的红细胞和巨核细胞的分化是必不可少的。此外，我们认为MLL-AF9与AF4的相互作用是肿瘤转化所必需的。在这项拨款申请中提出的实验被组织成两个特定的目标。目的1侧重于N-末端的Yeats结构域，而目的2侧重于AF9的C-末端结构域。目的1：AF9叶芝结构域的结构和功能。我们的初步数据表明，AF9Yeats结构域特异性地结合到组蛋白H3的一个区域。我们提出了三个子目标来探讨这种相互作用在AF9功能中的作用。首先，我们将解决AF9叶芝结构域与H3肽结合的结构。基于这种结构，我们将开发特定的点突变来破坏这种相互作用，并将其引入全长AF9进行功能测试。其次，我们将研究这种相互作用在编码造血转录因子的特定基因调控中的作用。第三，我们将研究叶芝结构域-H3相互作用在AF9的S在驱动红细胞/巨核细胞发育中的作用。目的2：AF9C-末端结构域-AF4复合体的结构和功能。我们提出了6个亚目标来研究AF9-AF4相互作用在AF9和MLL-AF9功能中的作用。首先，我们将确定AF9 C-末端结构域-AF4复合体的结构，并用它来产生突变来破坏相互作用。这些突变将被用来测试这种相互作用在正常AF9功能中的作用，包括对AF9复合体组成的影响，对编码造血转录因子的特定基因的调节，以及对红细胞/巨核细胞发育的影响。由于这是MLL-AF9融合中与MLL融合的结构域，这些突变也将用于测试MLL-AF9和MLL-AF9白血病发生对HOXA9和Meis1调控的影响。