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 (DN's) that inhibit B-ZIP DNA binding. These dominant negatives will inhibit the DNA binding of whole families of structurally related B-ZIP proteins. This may be important as related proteins often have redundant properties. We have designed D-N, termed A-ZIPs, that inhibit B-Zip DNA binding in an equimolar competition. 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. The acidic extension heterodimerizes with the basic region of the B-ZIP motif to stabilize the complex up to 5 kcal/mol. The ZIP represents the leucine zipper that determines the specific of A-ZIP action. A related strategy also has been devised for B-HLH-ZIP proteins We are delivering these A-ZIP's into human cells using adenovirus and into mice using tet regulable promoters. A clinical problem with cancer chemotherapy is the acquisition of drug resistance, which has been attributed to activation of MAPK pathways, including the AP-1 transcription factor. Adenovirus delivery of A-FOS, a DN that inhibits AP-1 DNA binding lowers the cisplatin concentration needed to kill a drug resistance human ovarian cell line. To examine the general utility of this observation, we infected the NCI 60 cell line screen. Several unexpected observations were made. The empty virus control is able kill all of the cells, expect the blood cells which it is not able to infect at concentrations that do not kill normal cells. The p53 expressing virus, the one being used in clinical trials is not better than the empty virus, even under conditions of genotoxic stress. AdA-FOS is better than either of these viruses. WE are exploring these issues more, trying to understand what molecular properties of the cells correlate with the ease of adenovirus killing. We have recently succeeded in the regulated expression of A-CREB, A-FOS, and A-C/EBP in mice. Using either a liver or skin promoter, expression of these DNs causes death during development but not in the adult. We plan on using this paradigm to identify B-ZIP transcriptional targets in response 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.
Showing the most recent 10 out of 36 publications
