This proposal is focused on a newly discovered group of transcription factors that are likely to play important roles in the differentiation and proliferation of hematopoietic stem cells. The myc family of protooncogenes has been shown to be involved in neoplasia, apoptosis, differentiation and proliferation. The proteins encoded by this gene family (c-, N-, and L-Myc) are members of the basic-helix-loop-helix- zipper (bHLHZ) class of transcription factors. Evidence indicates that they all function through interaction with the bHLHZ protein Max. Myc:Max heterodimers bind to DNA in a sequence-specific manner and activate transcription at their DNA binding site through the transcriptional activation domain of Myc. Importantly other proteins also interact with Max. One of these proteins, Mad, is also a member of the bHLHZ group, and appears to """"""""oppose"""""""" the function of Myc by competing for binding with Max and acting as a transcriptional repressor. Mad expression is induced upon differentiation of hematopoietic cells during a period when Myc levels decline. These changes lead to a switch from Myc:Max heterodimers to Mad:Max heterodimers as an early event following the induction of differentiation and is thought to reflect a transcriptional switch in expression of growth regulatory genes. Very recently we have identified four other novel bHLHZ proteins (MIP1-4) that specifically interact with Max. Two of these are highly related to Mad. In this application we propose to study potential roles for Mad and MIP1-4 specifically in the biology of hematopoietic stem cells. We first plan to examine the expression of Mad and MIP 1-4 during differentiation of hematopoietic cell lines and in normal bone marrow cells. Once we have established the pattern of expression of these proteins we plan to ectopically express them in hematopoietic cells using several inducible vector systems. This will permit us to assess the effects of their expression on the proliferating cell cycle and on differentiation. Lastly we plan to prepare a series of protein expression libraries from different hematopoietic cell types and stages in order to identify, by interaction cloning strategies, additional Max binding proteins whose expression may be cell-type restricted. These experiments are likely to lead to a better understanding of the transcription factors and molecular mechanisms that regulate normal hematopoiesis.
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