Biochemical and Biophysical Research Communications
Transcription factor TFIIH components enhance the GR coactivator activity but not the cell cycle-arresting activity of the human immunodeficiency virus type-1 protein Vpr
Section snippets
Materials and methods
Plasmids. pCDNA3-VPR, which expresses the wild-type Vpr protein under the control of the Cytomegalovirus (CMV) promoter, was described previously [11]. pCDNA3-CDK7, pCDNA3-Cyclin H, and pCDNA3-MAT1 were constructed by inserting cDNAs of each protein amplified from MO15-3M, CyclH-HA, and pHT7-MAT1, which are gifts from Dr. R. Fisher (University of California, San Francisco, CA) and Dr. R. Roeder (The Rockefeller University, New York, NY), respectively, into pCDNA3 (Invitrogen, Palo Alto, CA).
Effect of CDK7, Cyclin H, and MAT1 on the GR coactivator activity of Vpr
To examine whether TFIIH contributes to Vpr’s GR coactivator activity, we examined overexpression of the three core components of TFIIH, CDK7, Cyclin H, and MAT1, separately or altogether, with and without Vpr, on the glucocorticoid-responsive MMTV promoter in HeLa and A204 cells (Fig. 1). CDK7, Cyclin H, and MAT1, when examined individually, did not alter the activity of the dexamethasone-stimulated MMTV promoter, while coexpression of all three proteins enhanced the activity of this promoter
Discussion
We demonstrated that overexpression of all three major TFIIH core components enhanced Vpr’s GR coactivator activity. When only each protein was overexpressed, however, CDK7 and MAT1 enhanced this activity, while Cyclin H suppressed it. Also, CDK7 S170A, which is defective in CTD phosphorylating activity, failed to enhance Vpr’s GR coactivator activity. These results indicate that the TFIIH core complex may participate in Vpr’s GR coactivator function. On the other hand, the TFIIH core proteins
Acknowledgements
We thank Drs. R. Fisher, R. Roeder, D.M. Livingston, and G.L. Hager for kindly providing us their plasmids, Drs. G.N. Pavlakis and A. Gragerov for helpful discussion, and Mr. K. Zachman and Ms. A. Vervalis for excellent technical assistance.
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