Regulation and Function of the TAL1/SCL Gene

描述（由申请人提供）：TAL 1（或SCL）基因的异常表达是T细胞急性淋巴细胞白血病中最常见的功能获得性突变之一。这种螺旋-环-螺旋（HLH）转录因子在胚胎发生和出生后红细胞和巨核细胞谱系分化期间的造血特化中也很重要。TAL 1形成DNA结合复合物，该复合物含有HLH DNA结合配偶体、加塔-1转录因子、LIM-only蛋白和LIM结构域结合蛋白Ldb 1，并识别E盒-加塔DNA序列基序。在当前的资助期内，工作确定了该复合物的其他成员，包括SWI/SNF蛋白Brg 1，辅阻遏物ETO 2和MTGR-1，以及单链DNA结合蛋白-2和-3。虽然关于TAL 1和加塔-1的信息相当多，但关于该复合物的非DNA结合成员的功能知之甚少。该更新申请将测试Ldb 1对TAL 1-和加塔-1靶基因的转录有重要贡献以及其同源寡聚化能力对红系基因表达的远程控制很重要的假设。第一个具体的目的是确定Ldb 1表达的重要性E盒GATA DNA结合活性，基因表达，和分化的小鼠红系祖细胞。这些研究将确定减少Ldb 1表达对E盒-加塔DNA结合复合物的丰度及其对DNA的亲和力、含TAL 1和加塔-1复合物的选择靶基因的转录、转录因子占有率、RNA聚合酶II募集和在红系细胞分化的两种体外模型中这些基因的启动子处的组蛋白乙酰化的影响。第二个具体的目的是确定的贡献Ldb 1同源二聚体形成E盒GATA DNA结合活性，基因表达的短程控制，和细胞分化。这些研究将定义Ldb 1同源二聚化所需的最小结构域，确定Ldb 1同源二聚化对E盒GATA DNA结合活性的重要性，开发Ldb 1二聚化的特异性多肽抑制剂，并测试该抑制剂对E盒GATA DNA结合活性、红细胞基因表达和终末分化的影响。第三个具体目标是确定Ldb 1同源二聚化在基因表达的远程控制中的重要性。这些研究将解决Ldb 1是否介导的上游调控区和启动子的小鼠β-珠蛋白（maj）基因的远程相互作用，并确定其他位点在小鼠基因组中占据Ldb 1阐明其在调节其转录的作用。这些研究的结果将推进红细胞分化的基本理解，与LIM结构域和HLH蛋白调控的其他细胞程序相关，并提供对转录调控和白血病发生的基本机制的见解。 项目叙述：本申请中提出的研究与公共卫生高度相关。除了推进对红细胞是如何产生的理解，这适用于红细胞产生障碍，称为贫血，这项工作可能会导致T细胞急性淋巴细胞白血病和乳腺癌和口腔癌的新治疗方法。

DESCRIPTION (provided by applicant): Aberrant expression of the TAL1 (or SCL) gene is one of the most frequent gain-of-function mutations in T-cell acute lymphoblastic leukemia. This helix-loop-helix (HLH) transcription factor is also important in hematopoietic specification during embryogenesis and differentiation of the erythroid and megakaryocytic lineages postnatally. TAL1 contributes to a DNA-binding complex that contains an HLH DNA-binding partner, the GATA- 1 transcription factor, a LIM-only protein, and the LIM domain-binding protein Ldb1 and recognizes an E box- GATA DNA sequence motif. Work in the current funding period identified additional members of this complex, including the SWI/SNF protein Brg1, corepressors ETO2 and MTGR-1, and Single-Stranded DNA-Binding Protein-2 and -3. While considerable information is available about TAL1 and GATA-1, much less is known about the functions of the non-DNA-binding members of this complex. This renewal application will test the hypotheses that Ldb1 contributes importantly to the transcription of TAL1- and GATA-1 target genes and that its ability to homo-oligomerize is important for long-range control of erythroid gene expression. The first specific aim is to determine the importance of Ldb1 expression for E box-GATA DNA-binding activity, gene expression, and differentiation of murine erythroid progenitors. These studies will determine the effects of reducing Ldb1 expression on the abundance of the E box-GATA DNA-binding complex and its affinity for DNA, transcription of select target genes of the TAL1- and GATA-1-containing complex, and transcription factor occupancy, RNA polymerase II recruitment, and histone acetylation at the promoters of these genes in two in vitro models of erythroid cell differentiation. The second specific aim is to determine the contribution of Ldb1 homodimer formation to E box-GATA DNA-binding activity, short-range control of gene expression, and cellular differentiation. These studies will define the minimal domain required in Ldb1 homodimerization, determine the importance of Ldb1 homodimerization for E box-GATA DNA-binding activity, develop a specific polypeptide inhibitor of Ldb1 dimerization, and test the effect of this inhibitor on E box-GATA DNA-binding activity, erythroid gene expression, and terminal differentiation. The third specific aim is to determine the importance of Ldb1 homodimerization in long-range control of gene expression. These studies will address whether Ldb1 mediates long-range interaction of the upstream regulatory regions and promoter of the mouse beta-globin (maj) gene and identify additional loci in the mouse genome occupied by Ldb1 to elucidate its role in regulation of their transcription. The results of these studies will advance basic understanding of erythroid differentiation, have relevance to other cellular programs regulated by LIM domain and HLH proteins, and provide insights into fundamental mechanisms of transcriptional regulation and leukemogenesis. Project Narrative: The studies proposed in this application are highly relevant to public health. In addition to advancing understanding of how red blood cells are made, which is applicable to the disorder of red cell production known as anemia, this work could lead to new treatment approaches for T-cell acute lymphoblastic leukemia and cancers of the breast and oral cavity.