Structure/Function of AF9 and MLL-AF9

描述（由申请人提供）：AF 9（MLLT 3）首次被鉴定为急性髓性白血病中混合谱系白血病（MLL）基因的融合伴侣。当时注意到它与酵母SWI/SNF复合物TFG 3/ANC 1的组分共享序列同源性，表明在转录中的作用。AF 9显然在造血中的特定谱系命运决定中起关键作用。此外，其在HSC中的高水平表达强烈表明其在维持HSC所需的基因表达程序中起关键作用。AF 9的这些作用的机制基础尚未建立。该基金的主要目标之一是为AF 9的功能提供分子基础，特别强调关键N-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AF 9 N-末端YEATS结构域与组蛋白H3的结合以及AF 9 C-末端结构域与AF 4的结合对于正常红细胞和巨核细胞的分化是必不可少的。此外，我们提出MLL-AF 9与AF 4的相互作用对于肿瘤转化是必不可少的。在这项拨款申请中提出的实验分为两个具体目标。目的1关注N-末端YEATS结构域，目的2关注AF 9 C-末端结构域。目的1：AF 9 YEATS结构域的结构和功能。我们的初步数据表明，AF 9 YEATS结构域特异性结合组蛋白H3的一个区域。我们提出了三个子目标来探索这种相互作用在AF 9功能中的作用。首先，我们将解决与H3肽结合的AF 9 YEATS结构域的结构。基于该结构，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AF 9进行功能测试。其次，我们将研究这种相互作用在造血转录因子编码的特定基因的调节中的作用。第三，我们将研究YEATS结构域- H3相互作用在AF 9驱动红细胞/巨核细胞发育中的作用。目的2：AF 9 C端结构域-AF 4复合物的结构和功能。我们提出了6个子目标，以检查AF 9-AF 4相互作用在AF 9和MLL-AF 9功能中的作用。首先，我们将确定AF 9 C-末端结构域-AF 4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AF 9功能中的作用，包括对AF 9复合物组成的影响，对编码造血转录因子的特定基因的调节，以及红细胞/巨核细胞发育。由于这是MLL-AF 9融合体中与MLL融合的结构域，因此这些突变也将用于测试MLL-AF 9和MLL-AF 9白血病发生对HOXA 9和MEIS 1调节的影响。

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.