GLUCOCORTICOIDS REPRESS TRANSCRIPTION FROM A NEGATIVE GLUCOCORTICOID RESPONSE ELEMENT RECOGNIZED BY 2 HOMEODOMAIN-CONTAINING PROTEINS, PBX AND OCT-1

Several studies have established that the prolactin (PRL) gene is expr essed not only in lactotrophs and somatotrophs of the anterior pituita ry but, albeit to a lesser extent, in non-pituitary cells like human t hymocytes, decidualized endometrium, mammary glands during lactation, and some human non-pituitary cell lines. Despite the requirement in th e pituitary for the pituitary-specific transcription factor Pit-1/GHF- 1 for PRL expression, the expression in non-pituitary cells occurs in the absence of Pit-1/GHF-1 and can be repressed by glucocorticoids. Th is prompted us to investigate the transcription factors in non-pituita ry cells which are involved in controlling expression and glucocortico id repression of a previously characterized negative glucocorticoid re sponse element from the bovine prolactin gene (PRL3 nGRE). Here we hav e demonstrated that non-pituitary cells (COS-7 and mouse hepatoma Hepa 1c1c7 cells) conferred increased expression via the PRL3 nGRE mainly b ecause of the binding of the ubiquitously expressed POU-homeodomain-co ntaining octamer transcription factor-1 (Oct-1), to an AT-rich sequenc e present in the PRL3 sequence. However, full transcriptional activity required the binding of a second ubiquitously expressed homeodomain-c ontaining protein, Pbx, previously shown to bind cooperatively with se veral homeotic selector proteins. The Pbx binding site in the PRL3 nGR E, located just upstream of the Oct-1 binding site, showed a strong se quence similarity with known Pbx binding sites and bound Pbx with an a ffinity similar to that of other established Pbx target sequences. Int erestingly, both Oct-1 and Pbx binding to the PRL3 nGRE were found to be required for glucocorticoid repression. Addition of in vitro transl ated glucocorticoid receptor DNA binding domain to the nuclear extract prevented Oct-1 and Pbx from binding to the PRL element. The involvem ent of the homeobox protein Pbx in glucocorticoid repression via an nG RE identifies a new role for this protein.