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Glucocorticoid Inhibition of Transcription from Adenovirus Major Late Promoter

Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch Galveston, Texas 77550

Address requests for reprints to: Pramod B. Mahajan, Department of Human Biological Chemistry and Genetics, F45, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77550.

Abstract

Glucocorticoids inhibit proliferation of lymphosarcoma P1798 cells in culture. This is accompanied by inhibition of expression of class I as well as some class II genes. We have used the technique of transcription in vitro and the adenovirus major late promoter (AdMLP) to investigate molecular mechanisms of glucocorticoid inhibition of transcription of class II genes. Nuclear extracts from exponentially growing P1798 cells support faithful transcription from AdMLP. However, treatment of P1798 cells with 10^–7 M dexamethasone (a synthetic glucocorticoid) for 24 h impairs the ability of nuclear extracts prepared from such cells to support faithful transcription from AdMLP. Transcription from the human histone H₄ gene promoter is unaffected by dexamethasone treatment. Gel mobility shift assays using synthetic oligonucleotide probe indicate no difference in the CAAT box binding activity of control and dexaethasone-treated cell extracts. Similarly, dexamethasone treatment does not affect the upstream stimulatory factor activity of P1798 cells. Furthermore, up-stream stimulatory factor purified from control cell extracts is unable to reconstitute the glucocorticoid-treated cell extracts for transcription from AdMLP. Fractionation of the control cell extracts on Sepharose S yields two protein fractions, neither of which supports transcription from AdMLP. However, one of these fractions, S-II, confers upon glucocorticoid-treated cell extracts the ability to support faithful transcription from AdMLP. We conclude that glucocorticoids regulate the transcription from AdMLP by regulating the activity of a frans-acting transcription factor which copurifies with fraction SII.

FOOTNOTES

This work was supported by Institutional Grant DHHS 2507-RR-07205-08 (to P.B.M) and NIH Grants CA-24347 and CA-22394 (to E.A.T).

^* Recipient of Faculty Research Award from the American Cancer Society (FRA 299).

Received for publication May 25, 1990. Revision received July 16, 1990. Accepted for publication July 20, 1990.