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.
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