My laboratory studies the structure-function relationships of the B-ZIP class of sequence-specific DNA binding dimeric proteins. Over 50 B-ZIP genes have been identified in the mammalian genome. In the most general terms, B-ZIP proteins both activate and repress gene expression in response to physiological changes, growth factors (FOS), stress (ATF2), neuronal signaling (CREB), or metabolic changes (CEBP). We want to study B-ZIP transcriptional function using dominant-negatives (DNs) that inhibit B-ZIP DNA binding. A problem with the design of such reagents is that B-ZIP proteins become stabilized by binding DNA. We have overcome this problem by extending the dimerization domain into the basic region to produce A-ZIPs. The A represents an N-terminal Acidic amphipathic extension of the leucine zipper that replaces the basic region critical for sequence-specific DNA binding of the B-ZIP dimer. These A-ZIP proteins act as D-Ns by inhibiting the DNA binding of B-ZIP proteins because of the stabilization that occurs through the interaction of the acidic extension with the basic region of the B-ZIP domain. They form an alpha-helical coiled coil extension of the leucine zipper. The pathology of excited stress pathways caused by B-ZIP proteins can be examined using these A-ZIPs. We are delivering these A- ZIPs into human cells using adenovirus and into mice using tet regulable promoters. Adenovirus delivery of A-FOS allows us to kill drug resistance cells at lower drug doses. We have recently succeded in the regulated expression of several A-ZIPs in mice. Expression of these causes death during development but noit in the adult. We plan on using this paradigm to identify B-ZIP transcriptional targets in responce to different stress agents. The hypothesis driving this work is that direct transcriptional targets of a B-ZIP protein can be identified by expression of the corresponding A-ZIP protein. - Dominant negatives, Oncogenes, tetracycline control, Adrenovirus, B-ZIP protein, - Neither Human Subjects nor Human Tissues
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