Two different gene elements are required for glucose regulation of S14 transcription.
Carbohydrate feeding increases the transcriptional activity of the hepatic S14 gene. The region of the S14 promoter between -1384/-1275 contributes to the transcriptional regulation by carbohydrate. A previously identified element (-1303/-1289) within this region is required but is not sufficient for the carbohydrate effect. Therefore, we ligated -1384/-1275 to a heterologous promoter and created mutants in this region to identify other potential responsive sequences. We found that mutation within -1365/-1350 eliminated the response to high glucose (27.5 mM). However, three copies of this element ligated to a mouse mammary tumor virus-luciferase vector did not respond to glucose indicating the -1365/-1350 element is insufficient to confer a glucose response in isolation. Nevertheless. mutating the -1365/-1350 element in the native promoter led to a loss of response to glucose, proving this element is necessary. Electrophoretic mobility shift assays (EMSA) using three copies of the element showed significant binding to rat hepatic nuclear extracts, but no difference between the dietary states. Competition EMSA studies showed that the previously identified element at -1303/-1289 was unable to compete for proteins that bind to the -1365/-1350 element. Therefore, we have demonstrated two separate elements within the -1384/-1275 region of the S14 gene that bind different proteins and interact to elicit the carbohydrate effect.
Liu
B
B
Department of Medicine, University of Minnesota, Minneapolis 55455, USA.
Li
W
W
Mariash
C N
CN
D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D005260:Female; D005947:Glucose / Q000494:pharmacology*; D008156:Luciferases / Q000235:genetics; D008297:Male; D008325:Mammary Neoplasms, Experimental; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014157:Transcription Factors; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D011993:Recombinant Fusion Proteins; C051065:THRSP protein, human; C501477:Thrsp protein, mouse; C501478:Thrsp protein, rat; D014157:Transcription Factors; D008156:Luciferases; D005947:Glucose