The object of this proposal is to elucidate the mechanisms for the earliest stages of gene induction in activated or differentiating monocytes. The primary focus will be on gene regulation, paying particular attention to inducible transcription factors responsible for mediating differentiation and cell activation. The approach is to use prointerleukin 1-beta (IL1B) and a small number of other inducible monocyte-specific genes as examples for investigating the mechanisms of immediate early gene induction in monocytes. Evaluation of transiently transfected portions of these genes will be studied along with the binding of specific protein factors in order to gain insight into the regulatory mechanisms. The cDNA sequences for several transcription factors which appear to be important for monocyte activation and differentiation will be used in cotransfection studies aimed at overexpression in order to evaluate specific effects. Similarly, dominant-negative mutants of these factors will also be investigated.
Specific Aims are: (I) to elucidate the functional mechanism of action for the IL1B Upstream Induction Sequence (UIS) which targets cell type-independent induction of the IL1B gene; (2) to examine the function of the tissue-restricted Spi-I/PU.I transcription factor, which binds to the promoter proximal sequence in the IL1B gene and other monocyte-specific genes to confer tissue-specific expression. Our previous studies demonstrated that human cytomegalovirus (HCMV) IE1 protein trans-activation of the IL1B gene requires an intact Spi-I/PU.I binding site. Therefore, the relationship between Spi-I/PU.I and HCMV IE1 will be investigated using these two proteins in functional and structural studies.
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