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（由申请人提供）：AF9（MLLT3）首先被确定为急性髓样白血病中混合谱系白血病（MLL）基因的融合伴侣。当时，注意到与酵母SWI/SNF复合物TFG3/ANC1的组成部分共享序列同源性，这表明在转录中起作用。 AF9显然在造血中的特定血统命运决策中起着关键作用。此外，其在HSC中的高表达强烈表明在维持HSC所需的基因表达程序中起着关键作用。尚未建立这些AF9这些角色的机械基础。该赠款的主要目标之一是为AF9的功能提供分子基础，并特别强调关键的N和C末端域的作用及其与基因调节机制的关系。我们建议，AF9 N末端Yeats结构域与组蛋白H3的结合以及AF9 C末端结构域与AF4的结合对于正常的红细胞和巨核细胞分化至关重要。此外，我们建议MLL-AF9与AF4的相互作用对于肿瘤转化至关重要。本赠款申请中提出的实验被组织为两个具体目标。 AIM 1专注于N末端Yeats域，AIM 2专注于AF9 C末端域。 AIM 1：AF9 Yeats域的结构和功能。我们的初步数据表明，AF9 Yeats域特异性与组蛋白H3区域结合。我们提出了三个子-AIM，以探测这种相互作用在AF9函数中的作用。首先，我们将解决与H3肽结合的Af9 Yeats结构域的结构。基于结构，我们将开发特定的点突变，以破坏这种相互作用，该相互作用将引入全长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节中的作用。第三，我们将研究Yeats域-H3相互作用在AF9在驱动红细胞/巨核细胞开发中的作用中的作用。 AIM 2：AF9 C末端域的结构和功能 - AF4复合物。我们提出了6个子AIM，以检查AF9-AF4相互作用在AF9和MLL-AF9函数中的作用。首先，我们将确定AF9 C末端域-AF4复合物的结构，并使用它来开发突变以破坏相互作用。这些突变将用于测试这种相互作用在正常AF9功能中的作用，包括对AF9复合物组成的影响，编码造血转录因子的特定基因的调节以及红细胞/巨核细胞的发展。由于这是在MLL-AF9融合中融合的域，因此这些突变也将用于测试对HOXA9和MEIS1的效果，由MLL-AF9和MLL-AF9和MLL-AF9白血病发生。