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The Homeodomain Protein, Pit-1/GHF-1, Is Capable of Binding to and Activating Cell-Specific Elements of Both the Growth Hormone and Prolactin Gene Promoters

Departments of Medicine, New York University School of Medicine New York, New York 10016
Departments of Cell Biology, New York University School of Medicine New York, New York 10016
Departments of Pharmacology, New York University School of Medicine New York, New York 10016

Address requests for reprints to: Dr. Herbert H. Samuels, Department of Medicine, TH-454, New York University Medical Center, 550 First Avenue, New York, New York 10016.

Abstract

Studies were conducted to determine whether the trans-acting protein Pit-1/GHF-1 can bind to and activate promoter elements in both the GH and PRL genes that are necessary for cell-specific expression. Four pituitary cell lines that differentially express the endogenous GH and PRL genes were examined for their ability to activate GH and PRL promoter constructs containing sequences necessary for cell-specific expression (CSEs). Plasmids containing one CSE, –96 PRL and –104 GH, were similarly expressed in each of the four cell lines. Of the plasmids containing two CSEs, –173 PRL was always activated to a greater extent than –145 GH, with this relative activation being stronger in GC and GH¹ cells than in 235-1 and GH⁴¹cells. Protein-DNA binding assays were used to show that the GH and PRL CSEs specifically bound two highly abundant nuclear proteins (31 and 33 kDa). The two proteins were present at similar levels in all four pituitary cell lines and were recognized by a Pit-1/GHF-1 antibody. In contrast, HeLa and Rat2 cells did not activate transfected GH or PRL plasmids and did not contain nuclear proteins that specifically bound to the GH and PRL CSEs. However, cotransfection of these cells with the expression vector RSV-Pit-1/GHF-1 resulted in the activation of –173 PRL and –145 GH (PRL > GH). HeLa cells transfected with RSV-Pit-1/GHF-1 also contained 31- and 33-kDa nuclear proteins that bound to the GH and PRL CSEs. These results show that Pit-1/GHF-1 is present at levels in pituitary cell lines that are sufficient to activate the minimal elements in both the GH and PRL promoters necessary for cell-specific expression of these genes.

FOOTNOTES

This work was supported by NIH Grants DK-16636 (to H.H.S.), DK-36615 (to F.S.), and BRSG-RR5399-27 (to S.R.F.); the New York University program in Molecular and Cell Biology (M.T.C.J.), and the Sackler Institute of Graduate Medical Studies (Z.-S.Y.).

Received for publication February 2, 1990. Revision received March 27, 1990. Accepted for publication March 27, 1990.

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