Molecular analysis of the differential hepatic expression of rat kininogen family genes.
Serum concentration of rat T1 kininogen increases 20- to 30-fold in response to acute inflammation, an induced hepatic synthesis regulated primarily at the transcriptional level. We have demonstrated by transient transfection analyses that rat T1 kininogen gene/chloramphenicol acetyltransferase (T1K/CAT) constructs are highly responsive to interleukin-6 and dexamethasone. In these studies we examined the regulation of a highly homologous K kininogen gene promoter and showed that it is minimally induced under identical conditions. The basal expression of the KK/CAT construct was, however, five- to sevenfold higher than that of the analogous T1K/CAT construct. Promoter-swapping experiments to examine the molecular basis of this differentially regulated basal expression showed that at least two K kininogen promoter regions are important for conferring its high basal expression: a distal 19-bp region (C box) constituted a binding site for C/EBP family proteins, and a proximal 66-bp region contained two adjacent binding sites for hepatocyte nuclear factor 3 (HNF-3). While the C box in the K kininogen promoter was able to interact with C/EBP transcription factors, the T1 kininogen promoter C box could not. In addition, HNF-3 binding sites of the K kininogen promoter demonstrated stronger affinities than those of the T1 kininogen promoter. Since C/EBP and HNF-3 are highly enriched in the liver and are known to enhance transcription of liver-specific genes, these differences in their binding activities thus accounted for the K kininogen gene's higher basal expression. Our studies demonstrated that evolutionary divergence of a few critical nucleotides may lead to subtle changes in the binding affinities of a transcription factor to its recognition site, profoundly altering expression of the downstream gene.
Chen
H M
HM
Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Houston 77030.
Liao
W S
WS
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D016384:Consensus Sequence; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D007704:Kininogens / Q000096:biosynthesis* / Q000235:genetics*; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection; D014407:Tumor Cells, Cultured
D007704:Kininogens; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase