CHARACTERIZATION OF A CORTICOTROPIN-RELEASING HORMONE-RESPONSIVE ELEMENT IN THE RAT PROOPIOMELANOCORTIN GENE PROMOTER AND MOLECULAR-CLONINGOF ITS BINDING-PROTEIN

A corticotropin-releasing hormone (CRH) and cAMP-responsive region (-2 36/-133) in the rat POMC gene promoter previously reported to confer C RH/cAMP responsiveness to heterologous reporter constructs has been ch aracterized. DNAse footprint analysis revealed that multiple elements in this region were bound by nuclear proteins from the POMC expressing AtT20 cells. When these individual DNA elements were separately teste d in heterologous reporter constructs for CRH induction, only one elem ent, designated PCRH-RE (POMC CRH responsive element, -171/-160) was f ound to give strong CRH stimulation (5- to 7-fold). This element appea rs novel as to the possible binding factors, although it has homology to the mouse metallothionein metal regulatory element. Gel shift analy ses of the PCRH-RE with AtT20 cell nuclear extracts showed marked stim ulation of retarded nucleoproteins following CRH stimulation, suggesti ng that the possible binding factor(s) may mediate transcriptional reg ulation at this site. The activity of PCRH-RE binding protein was inhi bited by divalent cations, with Cu2+ and Cd2+ being most effective; Zn 2+ had no effect, indicating that this binding factor(s) is functional ly distinct from the metallothionein metal regulatory element binding protein. A 2.6 kilobase cDNA clone encoding a protein (PCRH-REB-1) bin ding to this element was isolated by Southwestern screening of an AtT2 0 expression library with radiolabeled PCRH-RE oligonucleotides. This clone was used to isolate several other cDNA clones to determine the s equence corresponding to the entire coding region of the protein (PCRH -REB), which proved to be identical to a recently described DNA bindin g protein of the replication factor C complex, mRFC140/Mouse Southwest ern. Primer extension and Northern blot analysis revealed that the siz e of the full length mRNA is about 4.9 kilobases. PCRH-REB mRNA expres sion is not restricted to corticotrophs but is present in a broad tiss ue distribution as evaluated by reverse transcription polymerase chain reaction analysis. A bacterially expressed beta-galactosidase-PCRH-RE B-1 fusion protein was shown to bind PCRH-RE efficiently. Furthermore, binding of the PCRH-REB-1 fusion protein to the POMC CRH-responsive e lement was inhibited by divalent cations with similar sensitivities to those observed using AtT20 nuclear extracts. The predicted PCRH-REB p rotein sequence presents several interesting motifs: one p-loop motif (ATP binding site), nine protein kinase A phosphorylation sites (imply ing a possible role in responding to the CRH-induced cAMP signal), and regions of homology to proteins involved in DNA replication and repai r. PCRH-REB is, therefore, a potential transacting factor binding to a major CRH-responsive element in the POMC promoter.