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Binding Specificity of Cyclic Adenosine 3',5'-Monophosphate- Responsive Element (CRE)-Binding Proteins and Activating Transcription Factors to Naturally Occurring CRE Sequence Variants

Thyroid Unit, Massachusetts General Hospital, Harvard Medical School Boston, Massachusetts 02114

Address requests for reprints to: J. Larry Jameson, Thyroid Unit, Jackson 1021, Massachusetts General Hospital, Boston, Massachusetts 02114.

Abstract

The human glycoprotein hormone gene contains a palindromic cAMP-responsive element (CRE), TGACGTCA, which is critical for both basal and cAMP-stimulated expression. Members of the CREbinding protein (CREB)/activating transcription factor transcription family as well as members of the Jun/Fos family have been shown to bind to this sequence. In nonprimate species, the gene contains a variant of the CRE sequence TGAtGTCA, which differs by a single base substitution. The functional properties of promoters containing variant CREs were examined in transient expression assays in JEG-3 cells (human placental cells) and GH₃ cells (rat pituitary cells). Relative to the palindromic CRE, the basal level of expression was reduced with the variant CRE constructions. However, 8-bromo-cAMP stimulated the expression of constructions containing either the palindromic or the variant CREs. Protein interactions with the palindromic and variant CREs were also examined. By Southwestern blot analyses in which proteins were initally separated on a denaturing sodium dodecyl sulfate-gel, transferred to a membrane, and then probed with radiolabeled DNA, several different proteins were bound by either the palindromic or the variant CRE sequences. However, the binding of the variant CRE to CREB was markedly reduced. In nondenaturing gel shift assays, multiple protein-ONA complexes were also formed with both types of CREs, but the variant CRE lacked a major complex that may correspond to homodimers of CREB. To determine the protein composition of these complexes, the gel shift bands were eluted and subjected to Southwestern blot analysis. CREB was identified as one of the protein components in several of the gel shift complexes formed with the variant CRE. We conclude that although the variant CRE binds CREB homodimers poorly, CREB can still interact with this element when associated with other proteins. Thus, variants of the palindromic CRE sequence may provide a mechanism for specifying interactions with different combinations of CREBs.

FOOTNOTES

This work was supported in part by USPHS Grants HD- 23262andHD-23519.

Received for publication January 24, 1991. Revision received July 17, 1991. Accepted for publication July 29, 1991.

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