CAMP-DEPENDENT TRANSACTIVATION INVOLVING THE HOMEODOMAIN PROTEIN PBX1

Pbx1 is a DNA-binding homeodomain protein originally discovered in the t(1;19) chromosomal translocation associated with pediatric pre-B acu te lymphoblastic leukemia. Previously we reported a cAMP-regulatory se quence (CRS1) in the promoter region of the bovine CYP17 gene encoding steroid 17 alpha-hydroxylase cytochrome P450 (P450c17) to be the firs t endogenous Pbx1 binding site and that overexpression of Pbx1 in mous e adrenal Y1 tumor cells enhances cAMP-dependent transcription mediate d by this element. Here we report further characterization of Pbx1 bin ding site in CRS1 and role of Pbx1 in cAMP-dependent, CRS1-mediated tr anscription. By gel shift analysis utilizing nuclear extracts from Y1 cells, a high-affinity Pbx-binding sequence has been determined to be TTGAT(T/ G)GA(T/C)A which represents the 5' portion of CRS1. An artifi cial Pbx-binding sequence (PRS), previously determined by random PCR a nalysis, is similar to the Pbx1-binding sequence in CRS1 and by both g el shift analysis and transfection studies shows characteristics very similar to CRS1. Upon overexpression, Pbx1 is found capable of enhanci ng CRS1-mediated transcription in both steroidogenic (Y1, JEG3) and no nsteroidogenic (HepG2 and S194) cells when coexpressed with the cataly tic subunit of cAMP-dependent protein kinase A. Thus even though Pbx1 has been found to be involved only in cAMP-dependent transcription of a gene involved in steroidogenesis (CYP17), Pbx1 is capable of partici pating in cAMP-dependent transcription of target genes without complex formation with steroidogenic tissue-specific nuclear factors. (C) 199 7 Academic Press.