TRANSCRIPTIONAL REGULATION OF ALPHA(1B) ADRENERGIC-RECEPTORS (ALPHA(1B)AR) BY NUCLEAR FACTOR-1 (NF1) - A DECLINE IN THE CONCENTRATION OF NF1 CORRELATES WITH THE DOWN-REGULATION OF ALPHA(1B)AR GENE-EXPRESSION IN REGENERATING LIVER

The 5' upstream region from -490 to -540 (footprint II) within the dom inant P2 promoter of the rat alpha(1b) adrenergic receptor (alpha(1b)A R) gene is recognized by a sequence-specific DNA-binding protein (B. G ao, M, S, Spector, and G. Kunos, J. Biol. Chem. 270:5614-5619, 1995). This protein, detectable in Southwestern (DNA-protein) blots of crude nuclear extracts as 32- and 34-kDa bands, has been purified 6,000-fold from rat livers by DEAE-Sepharose, heparin-Sepharose, and DNA affinit y chromatography. Sodium dodecyl sulfate-polyacrylamide gel electropho resis and UV cross-linking of the purified protein indicated the same molecular mass as that in crude extracts, Methylation interference ana lysis revealed strong contact with a TTGGCT hexamer and weak contact w ith a TGGCGT hexamer in the 3' and 5' portions of footprint II, respec tively. Nucleotide substitutions within these hexamers significantly r educed protein binding to footprint II and the promoter activity of P2 in Hep3B cells. The purified protein also bound to the nuclear factor 1 (NF1)/CTF consensus sequence, albeit with lower affinity, Gel mobil ity supershift and Western blotting (immunoblotting) analyses using an antibody against the NF1/CTF protein identified the purified 32- and 34-kDa polypeptides as NF1 or a related protein. Cotransfection into H ep3B cells or primary rat hepatocytes of cDNAs of the NF1-like protein s NF1/L, NF1/X, and NF1/Red1 resulted in a three- to fivefold increase in transcription directed by wild-type P2 but not by the mutated P2, Partial hepatectomy markedly decreased the levels of NF1 in the remnan t liver and its binding to P2, which paralleled declines in the rate o f transcription of the alpha(1b)AR gene and in the steady-state levels of its mRNA. These observations indicate that NF1 activates transcrip tion of the rat alpha(1b)AR gene via interacting with its P2 promoter and that a decline in the expression of NF1 is one of the mechanisms r esponsible for the reduced expression of the alpha(1b)AM gene during l iver regeneration.