The focus of this project is the characterization of a new transcription factor, PU.l which is expressed in macrophages and B cells. The DNA binding site for PU.l is a purine rich sequence, 5' -GAGGAA-3' (PU box). PU.l is a transcriptional activator that may be an important regulator of gene expression in macrophages and B cells. The carboxy terminal amino acid sequence of PU.1 has significant identity (41 percent) with a region of amino acid sequence in the ets oncogene family. This region of identity contains the DNA binding domain of PU.l and is probably the DNA binding domain of ets proteins. The first specific aim is to characterize the DNA binding domain and putative activation domain of PU.l. By deletion analysis and in vitro mutagenesis. These regions will be defined in detail. The information from this study is hoped to provide a better understanding of how the PU.l protein interacts with DNA and is able to activate transcription. The second specific aim is to study the function of PU.l. Experiments have been designed to isolate genes that may be regulated by PU.l. A number of gene promoters which contain PU.l binding sites will be evaluated and a determination will be made as to whether PU.l binding site is necessary for transcription. Other approaches to identify the function of PU.l include the over-expression of sense and antisense constructions of PU.l. The modulation of PU.l may have dramatic effects on cell growth or activation. The level of PU.l protein will be examined in macrophages and B cells treated with cytokines or other activating agents. These results may link PU.l with specific functions of macrophages and B cells. The long-term objectives of this project are to gain a better understanding of transcriptional regulation in the immune system. The cell type specific transcription factor PU.l may provide an opportunity to achieve this objective.
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