 Professor and Chair of Biochemistry
Telephone: 215 762-4424
jane.clifford@drexelmed.edu
Education:
Ph.D. (1978), Developmental & Cellular Biology
University of Maryland
Research Program
We have studied the dihydrofolate reductase (DHFR) gene, which is required for DNA replication and cell growth. Transcription of the DHFR gene appears to be very tightly coupled to the growth state of the cell. DHFR transcription is shut down in quiescent cells (G0) and is stimulated in late G1 prior to entry into S phase. DHFR transcription, both at the basal level and in response to a stimulus, is mediated through changes in the activities of two transcription factors, Sp1 and E2F. E2F has been clearly shown to modulate cell cycle regulated transcription, both through repression in quiescent cells and induction of transcription in mid-G1. Sp1 binding sites alone can confer late G1 expression on a promoter1; however, E2F1 and Sp1 synergistically activate transcription of several promoters containing binding sites for either or both factors. E2F1 and Sp1 not only interact functionally, but also physically2. Both E2F1 and Sp1 interact with the transcriptional co-activators CBP/p300, which may be involved in their synergism. Sp1 is also acetylated by p/CAF and the sites of acetylation will be mapped to determine function. Phosphorylation of Sp1 is increased in mid to late G1, coincident with activation of DHFR transcription3. The site of Sp1 that is phosphorylated in mid-G1 is a serine residue (S654) in the center of the middle zinc finger. We have mutated this serine residue and found that extracts obtained from mid-G1 cells are unable to phosphorylate the mutated protein. Current studies are directed at understanding the mechanism of phosphorylation control and its effect on the properties and activity of Sp1. Continuing studies will address the role of the interactions in cell cycle regulation of transcription and how phosphorylation of Sp1 affects its activity. Efforts to identify the kinase that phosphorylates Sp1 S654 include biochemical purification based on its tight association with Sp1, as well as a yeast two-hybrid screen.
A controlled program of viral gene expression is executed upon virus infection through cellular transcription factors that are activated by the virus. Cytomegalovirus is a herpes virus that is usually asymptomatic, but when acquired by an immunocompromised host, such as a transplant recipient or an HIV patient or a pregnant woman has profound consequences. The CMV immediate early gene (HCMV-IE) products affect the activity of several cellular transcription factors that in turn stimulate transcription of both viral and cellular genes. The transcription factor E2F is a target of HCMV-IE, specifically the 72 kDa HCMV-IE1 gene product (IE72) by dissociation from inhibitory proteins, association with stimulatory proteins, and changes in phosphorylation. E2F1 and IE72 interact in cells and in cell free systems (in vitro). That this interaction is essential for viral infection is suggested by the finding that a mutated version of IE72 that can interact with E2Fs but cannot trans-activate has a dominant negative effect on activation by wildtype IE72 and on viral infection. In contrast to the other viral immediate early oncoproteins, IE72 is a kinase that auto phosphorylates and phosphorylates E2F1-3, p107 and p130, but not E2F4 or 5 or pRB4. Auto phosphorylation of IE72 or phosphorylation by cellular kinase(s) is required for its interaction with E2F. Moreover, when the kinase function is abolished it cannot activate transcription.
We will design and test peptides to block the kinase function of IE72 and test the antiviral properties of these compounds. The significance of these phosphorylation events in the activation of E2F-dependent transcription and in inter-S phase growth arrest during HCMV infection will also be explored.
Selected Publications
1. Jensen, D. E., Black, A. R., Swick, A.G. and Azizkhan, J. C., J. Cell. Biochem. 67:1-8, 1997.
2. Lin, S.-Y., Black, A.R., Kostic, A., Pajovic, S., Hoover, C. N. and Azizkhan, J. C. Mol. Cell. Biol. 16:1668-1675, 1996.
3. Black, A.R., Jensen, D.E., Lin, S.-Y., and Azizkhan, J.C. 1998. J. Biol. Chem. 274:1207-1215, 1999.
4. Pajovic, S., Wong, E.L., Black, A.R. and Azizkhan, J.C. Mol. & Cell. Biol. 17:6459-6464, 1997. |