Misexpression of the TAL1/SCL gene is the most frequent gain-of-function mutation observed in T-cell acute lymphoblastic leukemia. Gene knockout and overexpression studies have demonstrated that this basic helix-loop-helix transcription factor is essential for specification of blood cell formation and vascular remodeling during embryogenesis, generation of all hematopoietic cell types in the adult, and terminal differentiation of the erythroid and megakaryocytic lineages. Studies from our laboratory have demonstrated that TAL1 can interact in a reciprocal manner with corepressor and coactivator complexes in differentiating murine erythroleukemia (MEL) cells, that posttranslational modification can modify TAL1 interaction with the nuclear corepressor mSin3A, and that the Protein 4.2 gene is a physiologic target subject to sequential repression and activation by TAL1-containing complexes in differentiating MEL cells. This renewal application will investigate the hypothesis that TAL1 has experimentally separable functions determined by its interaction with specific coregulator complexes and will test a model of TAL1 action in erythroid cells that has TAL1-mediated transcriptional repression inhibiting differentiation and/or stimulating proliferation and TAL1-directed transactivation promoting terminal differentiation.
The first aim i s to determine the structural requirements for select TAL1-coregulator interactions. Residues critical for TAL1 interaction with the LIM-only protein LMO2 and corepressor mSin3A will be determined through mutagenesis, and specific interaction-defective alleles of TAL1 will be identified.
The second aim i s to determine the importance of specific TAL1 -coregulator interactions in the expression of a TAL1 target gene. TAL1's interaction with components of corepressor and coactivator complexes will be investigated on the Protein 4.2 promoter in living cells, and the effects of trans-dominant TAL1 and coregulator mutants will be determined on Protein 4.2 gene expression.
The third aim i s to determine the importance of TAL1-coregulator interactions in erythroid differentiation. The effects of interaction-defective mutants of TAL1, a dominant negative mutant of a coregulator present in TAL1-containing complexes (L.dbl), and experimental chimeras of TAL1 and potent activation and repression domains will be tested on erythroid proliferation and differentiation in two experimental models and in primary murine hematopoietic cells. The results of these studies will advance basic understanding of hematopoietic differentiation and provide insights into mechanisms of leukemogenesis.
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