Charles R. Vinson, Ph. D.

Center for Cancer Research, National Cancer Institute, National Institutes of Health

Research

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 form an a-helical coiled coil structure with the basic region, extending of the leucine zipper. The stabilization that occurs through the interaction of the acidic extension with the basic region of the B-ZIP domain prevents DNA binding of B-ZIP proteins. The pathology of activated stress pathways caused by B-ZIP proteins can be examined using these A-ZIPs. Ultimately, we hope to use these gene-based A-ZIPs as adjuvants with other medical approaches to cure human disease, particularly chemotherapy resistant cancers. 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.

This laboratory has systematically analyzed the contribution of individual amino acids to coiled coil stability and dimerization specificity. These studies have helped us generate designs for dominant negatives to B-ZIP proteins by replacing the B-ZIP basic region with an acidic amphipathic protein sequence N-terminus of the leucine zipper. The acidic extension of the leucine zipper heterodimerizes with the B-ZIP basic region to produce a coiled coil extension of the leucine zipper. This zippering up of the leucine zipper into the basic region by the acidic extension stabilizes the heterodimer 2.5 to 5 kcal mol-1. In a competition assay containing an equal concentration of the B-ZIP and A-ZIP protein, DNA binding of the B-ZIP protein is abolished. The acidic extension interacts with all the B-ZIP basic regions we have examined.

Thus, we have developed a general strategy for developing A-ZIPs that inhibit B-ZIP DNA binding in a leucine zipper dependent manner. We have demonstrated this for the mammalian B-ZIP proteins CEBP, JUN, TFE, CREB, and ATF2. A similar strategy of replacing the basic region with an acidic protein sequence to produce A-HLH-ZIP dominant negatives works for the B-HLH-ZIP proteins examined, MYC/MAX, USF, and MITF.

The utility of these reagents to alter living systems was examined by producing transgenic mice expressing an A-ZIP dominant negative in only fat cells. The transgenic mice have 20 copies of a construct consisting of 7.6 kb of the fat specific aP2 promoter driving expression of an A-ZIP dominant negative that inhibits the DNA binding of both CEBP and FOS/JUN B-ZIP domains. The resultant mouse has essentially no fat. The metabolic consequences are profound, mimicking the human disease lipodystrophy. The mouse eats 1.7 fold more food and the liver is 2.3 fold larger than normal. The diabetic nature of the metabolic confusion is seen in insulin levels that are 100 fold higher than normal and glucose levels that are 3 fold elevated. The mice die prematurely, starting at 5 months. Troglitazone, a thiazolidinedione agonist of the transcription factor PPRg, reverses the diabetes.

Future plans

Aim 1) Dimerization specificity of B-ZIP domains. We will analyze the specificity of dimerization between B-ZIP domains. This will be determined by monitoring thermal stability using circular dichroism spectroscopy. This information is essential to determine B-ZIP dimerization partners and the possible targets for A-ZIPs in vivo. Preferential heterodimerization is easily determined by mixing B-ZIP domains and determining if the thermal stability increases. The energetic difference favoring homodimerization vs. heterodimerization between two B-ZIP domains is harder to determine. We propose to entropically constrain different B-ZIP domains thus forcing heterodimerization. This will allow us to quantify the interaction between B-ZIP domains that do not prefer to heterodimerize. These experiments will address the structural rules governing leucine zipper dimerization specificity and potential partners of the A-ZIPs.

Aim 2). Yeast screen to identify proteins that interact with B-ZIP dimers bound to DNA. We will identify proteins that bind to dimeric B-ZIP proteins bound to DNA using a yeast interaction screen. B-ZIP proteins undergo a dramatic conformational change upon dimerization. The monomeric leucine zipper becomes a-helical upon dimerization and the basic region becomes a-helical upon DNA binding. The B-ZIP dimer bound to DNA creates new surfaces available for interaction with new proteins. We want to identify proteins that interact with this new structural surface. This novel "bait" may allow for interactions missed previously.

Aim 3) Characterization of a transgenic mouse with no fat. We have generated a transgenic mouse with no fat tissue that is a model for the human disease lipodystrophy. Using 7.6 kb of the aP2 promoter, which is only active in white and brown fat, we have expressed an A-ZIP which we refer to as A-ZIP(F). The A-ZIP(F) zipper interacts with both the CEBP and JUN family of leucine zippers and inhibits DNA binding of CEBP and AP1 (FOS/JUN). The physiological consequence of having no fat is profound. The liver is 2.3 fold larger than normal. Insulin levels are elevated 100 fold indicative of the extreme diabetes of this animal. We plan to produce transgenic mice expressing A-CEBP or A-FOS to determine if inhibition of CEBP and/or FOS/JUN activity is critical for inhibiting fat growth and differentiation. Mice expressing these A-ZIPs under the control of tetracycline inducible promoters will allow us to examine thereversibility of the onset of lipodystrophy. A two mouse system consisting of one mouse that is transgenic for a tissue specific promoter driving expression of the tetracycline repressor-activator and a second mouse expressing the tetracycline dependent promoter driving expression of an A-ZIP dominant negative is being developed. An additional bonus is that the drug tetracycline can be added to the water to prevent A-ZIP expression This inducible feature will enable us to examine the development of any phenotypes observed in the crosses between the mice and identify potential transcriptional targets.

Aim 4) Adenovirus delivery of A-ZIPs to identify direct transcription targets. To identify direct transcriptional targets for B-ZIP proteins, cells will be infected with replication deficient adenoviruses containing A-ZIP DNs. Adenovirus can infect 100% of a growing or non-growing cell culture. RNA will be isolated from infected cell cultures over a time course of several days to produce fluorescent cDNA. Using the new chip technologies being developed, which monitors mRNAs concentration for thousands of genes, we will identify transcriptional targets. The sequence of the promoters will be assessed for common cis elements as potential binding sites for the B-ZIP transactivator. In vivo footprinting techniques will be used to test the hypothesis that following A-ZIP expression, a B-ZIP protein cannot bind DNA. We plan on using these adenoviral vectors to determine which B-ZIP proteins mediate stress responses. Ultimately, we hope to use these adenovirus to deliver A-ZIP's as adjuvants to chemotherapy treatments, expecting that incapacitation of the stress response pathways by the A-ZIP's may make human cancer cells more sensitive to chemotherapy. A present problem with chemotherapy is that cells become resistant to drug killing. In the case of cisplatinum resistance, cells have elevated DNA repair activity because of leveled activity of the transcriptional B-ZIP complex AP1. Presently, we are studying an ovarian cell line, CP70, that is resistant to elevated levels of cisplatinum. We find that expressing the A-FOS DN, using adenovirus delivery to 100% of the cells in a culture, both obliterated the AP1 gel shift and kills the cells at 5 fold lower drug concentrations. Other A-ZIP's, e.g. A-CREB and A-CEBP do not change the killing properties of the cisplatinum. WE plan the examine the universality of this result and proceed toward human trials of this reagent as an adjuvant to human chemotherapy.

Microarray Data

BMC genomics data

Representative Publications:

Vinson, C., Hai, T., and Boyd, S. Dimerization specificity upon DNA binding of the leucine zipper containing bZIP motif: prediction and rational design. Genes Dev. 7:1047- 1058, 1993.

Baxevanis, A. and Vinson, C. Interactions of coiled coils in transcription factors: Where is the specificity? Current Opinions in Genetics and Development 3:278-285, 1993.

Thompson, K., Vinson, C. and Freire, E. Thermodynamic characterization of the structural ability of the coiled-coil region of the bZIP transcription factor GCN4. .Biochemistry 32:5491-5496, 1993.

Krylov, D., Mikhailenko, I. and Vinson, C. A thermodynamic scale for leucine zipper stability and specificity: e and g interhelical interactions. EMBO 13:2849-2861, 1994.

Krylov, D., Olive, M. and Vinson, C. Extending dimerization interfaces: the bZIP basic region can form a coiled coil. EMBO 14:5329-5337, 1995.

Olive, M., Williams, S., Dezan, C., Johnson, P. and Vinson, C.: Design of a bZIP protein that has both dominant negative and gain-of-function properties acting via C/EBP. J. Biol. Chem., JBC 271:2040-2047, 1996.

Roesler, W., Crosson, S., Vinson, C., and McFie, P. The a-isoform of the CCAAT/Enhancer-binding protein is required for mediating cAMP responsiveness of the phosphoenolpyruvate carboxykinase promoter in hepatoma cells. J. Biol. Chem. 271: 8068-8074 1996.

Vinson, C., Olive, O., Mikhailenko, I., and Krylov, D. Stability and specificity of coiled coils: extending the interface into the basic region. Biological Strucuture and Dynamics. 225-232, 1996.

Szilak, L., Moitra, J, Krylov, D. and Vinson, C. Phosphorylation destabilizes a-helices. Nature Str Biol 4: 112-114, 1997.

Olive, M, Krylov, D, Echlin, D., Taparowsky, B., Powers, C., Gardner, K., and Vinson C. A dominant negative to AP1 that abolishes DNA binding and oncogenesis; J. Biol. Chem. 272: 18586-18594, 1997.

Szilak, L., Moitra, J., and Vinson, C. Stabilization of a leucine zipper by an engineered phosphorylation of a serine in the e position. Protein Science 6: 1273-1283, 1997.

Krylov, D., Echlin, D., Kasai, K., Arnheiter H., Taparowsky, E., and Vinson, C. A general method to design dominant negatives to bHLHZip proteins that abolish DNA binding. PNAS. 94 12274-12279, 1997.

Moitra, J., Szilák, L., Krylov, D., and Vinson, C. Leucine is the most stabilizing aliphatic amino acid in the d position of a dimeric leucine zipper coiled coil. Biochemistry 36 12567-12573, 1997.

Dmitry Krylov, Debra R. Echlin, B. J. Taparowsky, and Charles Vinson. Abolishing Myc/Max DNA binding and transformation: design of a dominant negative. Current Topics in Microbiology and Immunology: C-Myc in B-Cell Neoplasia. Ed. M. Potter and F. Melchers 169-177 1997.

Butscher, W., Powers, C., Olive, M., Vinson, C., and Gardner, K. Coordinate transactivation of the interleukin-2 CD28 response element by c-Rel and ATF-1/CREB2. J. Biol. Chem. 273 552-560, 1998.

Ahn, S., Olive, M., Aggarwal, S., Krylov, M., Ginty, D., and Vinson, C. A novel Dominant-Negative to CREB reveals it is a general Mediator of Stimulus-Dependent Transcription of c-fos.. Mol Cell Biol. 18 967-977, 1998.

Krylov, D., Barchi, J., and Vinson C. Inter-helical interactions in the leucine zipper coiled coil dimer: pH and salt dependence of coupling energy between charged amino acids. J Mol Biol. 279, 959-972,1998.
 
 

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