Identification of a 30-base pair regulatory element and novel DNA binding protein that regulates the human GLUT4 promoter in transgenic mice.
We have previously demonstrated that the important cis-acting elements regulating transcription of the human GLUT4 gene reside within 895 base pairs (bp) upstream of the transcription initiation site (Thai, M. V., Guruswamy, S., Cao, K. T., Pessin, J. E., and Olson, A. L. (1998) J. Biol. Chem. 273, 14285-14292). Our studies demonstrated that an MEF2 binding site within this region was necessary, but not sufficient, for GLUT4 promoter function in transgenic mice. We have identified a second regulatory element (Domain I) that functions cooperatively with the MEF2 domain in regulating GLUT4 transcription. Using a yeast-one hybrid screen, we obtained a partial cDNA and generated an antibody directed against a protein binding specifically to Domain I. Sequence analysis of the partial cDNA indicates that the protein binding to Domain I is a novel protein. The antibody specifically labels two proteins of approximately 70 and 50 kDa in Western blot analysis. These molecular masses correspond to Domain I binding proteins identified by UV-cross-linking nuclear extracts to a Domain I probe. The antibody raised against the Domain I binding protein inhibited formation of a Domain I-protein complex in electrophoretic mobility shift assays. We conclude that we have identified an authentic, novel, Domain I binding protein required for transcriptional regulation of the human GLUT4 promoter.
Oshel
K M
KM
Department of Biochemistry and Molecular Biology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.
Knight
J B
JB
Cao
K T
KT
Thai
M V
MV
Olson
A L
AL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D051275:Glucose Transporter Type 4; D006801:Humans; D007328:Insulin / Q000172:deficiency; D064326:MEF2 Transcription Factors; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D009124:Muscle Proteins; D018006:Myogenic Regulatory Factors; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D013311:Streptozocin / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D051275:Glucose Transporter Type 4; D007328:Insulin; D064326:MEF2 Transcription Factors; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D018006:Myogenic Regulatory Factors; C495692:SLC2A4 protein, human; C495693:Slc2a4 protein, mouse; D014157:Transcription Factors; D013311:Streptozocin