Identification of cis- and trans-acting factors regulating the expression of rat salivary-specific RP4 gene.
The molecular basis of tissue-specific and cyclic AMP (cAMP)-inducible gene expression in salivary glands is not well understood. Previously, we cloned a salivary-specific proline-rich protein gene, RP4. To analyze the cis-regulatory element(s) that mediates the regulation of this rat salivary RP4 gene, chimeric pRP4CAT constructs containing up to 1.7 kb of the 5'-flanking region of RP4 fused to a reporter gene were transiently transfected into salivary cells. Deletion studies suggest that a 159 bp (-147/+12) fragment of the RP4 5'-flanking region is sufficient to confer salivary-specific induction by agents that can raise intracellular cAMP concentration. Further delineation of this essential sequence revealed that a segment from -136 to -109 is necessary and sufficient to confer cAMP responsiveness in a salivary-specific manner when linked to a heterologous promoter. However, this 28 bp fragment (-136/-109) does not contain an identical match to the consensus cAMP response element (CRE). DNA mobility shift binding assays establish that a sequence-specific DNA-protein complex is formed between this DNA fragment and nuclear proteins from salivary cells, but not with nuclear proteins from HeLa cells, which contain canonical CRE binding proteins (CREBs). Taken together, these data demonstrate that we have identified a 28 bp cis-regulatory element in the RP4 gene that mediates salivary-specific cAMP-inducible gene expression. We propose that the novel salivary-specific CRE binding protein (SCBP) is a key regulator for salivary cAMP-inducible gene expression.
Lin
H H
HH
Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455.
Ann
D K
DK
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D016384:Consensus Sequence; D000242:Cyclic AMP / Q000502:physiology; D004790:Enzyme Induction; D005786:Gene Expression Regulation / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010452:Peptide Biosynthesis; D010455:Peptides / Q000235:genetics*; D055232:Proline-Rich Protein Domains; D011433:Propranolol / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D012469:Salivary Glands / Q000166:cytology / Q000378:metabolism*; D012471:Salivary Proteins and Peptides / Q000096:biosynthesis / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D013806:Theophylline / Q000494:pharmacology; D014157:Transcription Factors / Q000502:physiology*
D010455:Peptides; D011993:Recombinant Fusion Proteins; D012471:Salivary Proteins and Peptides; D014157:Transcription Factors; D005576:Colforsin; D011433:Propranolol; D013806:Theophylline; D000242:Cyclic AMP; D011494:Protein Kinases; D007545:Isoproterenol