Regulation and Function of the TAL1/SCL Gene

描述(申请人提供)：TAL1(或SCL)基因的异常表达是T细胞急性淋巴细胞白血病最常见的功能获得突变之一。这种螺旋-环-螺旋(HLH)转录因子在胚胎发育和出生后红系和巨核系分化的造血规范中也是重要的。TAL1参与DNA结合复合体的作用，该复合体包含HLHDNA结合伙伴、GATA-1转录因子、仅LIM蛋白和LIM结构域结合蛋白LDB1，并识别E盒-GATA DNA序列基序。在本供资期间的工作确定了该复合体的其他成员，包括SWI/SNF蛋白BRG1、辅阻遏物ETO2和MTGR-1以及单链DNA结合蛋白-2和-3。虽然关于TAL1和GATA-1的信息很多，但对该复合体的非DNA结合成员的功能知之甚少。这一新的应用将检验这样的假设，即LDB1对TAL1-和GATA-1靶基因的转录起重要作用，以及它的同源寡聚能力对于红系基因表达的远程控制是重要的。第一个特定目的是确定Ldb1表达对E box-GATA DNA结合活性、基因表达和小鼠红系祖细胞分化的重要性。这些研究将确定在两种体外红系细胞分化模型中，降低LDB1的表达对E盒-GATA DNA结合复合体的丰度及其与DNA的亲和力、TAL1-和GATA-1复合体的选定目标基因的转录以及这些基因的转录因子占据、RNA聚合酶II募集和组蛋白乙酰化的影响。第二个具体目的是确定LDB1同源二聚体的形成对E盒-GATA DNA结合活性、基因表达的短期调控和细胞分化的贡献。这些研究将确定LDB1同源二聚化所需的最小结构域，确定LDB1同源二聚化对E盒-GATA DNA结合活性的重要性，开发一种特异性的LDB1二聚多肽抑制剂，并测试该抑制剂对E盒-GATA DNA结合活性、红系基因表达和终末分化的影响。第三个具体目的是确定LDB1同源二聚化在基因表达的远程控制中的重要性。这些研究将探讨Ldb1是否介导了小鼠β-珠蛋白(Maj)基因上游调节区和启动子的远程相互作用，并确定了Ldb1在小鼠基因组中占据的其他位点，以阐明其在转录调控中的作用。这些研究结果将促进对红系分化的基本了解，对受LIM结构域和HLH蛋白调控的其他细胞程序具有相关性，并为转录调控和白血病发生的基本机制提供见解。 项目简介：本申请中提出的研究与公共卫生高度相关。除了进一步了解红细胞是如何产生的，这适用于被称为贫血的红细胞制造障碍，这项工作还可能导致T细胞急性淋巴细胞性白血病以及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.