Cell-free transcription directed by the 422 adipose P2 gene promoter: activation by the CCAAT/enhancer binding protein.
Previous investigations have shown that CCAAT/enhancer binding protein (C/EBP) can function as a trans-activator of the promoters of several adipocyte-specific genes--i.e., the 422 adipose P2 (422/aP2), stearoyl-CoA desaturase 1 (SCD1), and glucose transporter 4 (GLUT4) genes, in 3T3-L1 mouse preadipocytes. We now describe a cell-free system prepared from nuclei of 3T3-L1 cells that carries out transcription directed by these promoters. To measure transcript formation, we employed a polymerase chain reaction-assisted analysis. Nuclear extract from 3T3-L1 adipocytes that express C/EBP supports a higher rate of transcription of chimeric 422(aP2) promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs than nuclear extract from preadipocytes that lack C/EBP. A competitor oligonucleotide containing the C/EBP binding site sequence and antibodies raised against C/EBP inhibit transcription directed by the 422(aP2) promoter. The factor limiting transcription by nuclear extract from preadipocytes appears to be C/EBP, since recombinant C/EBP (rC/EBP) markedly activates transcription of the 422(aP2) promoter-CAT gene with preadipocyte extract but not with adipocyte extract. rC/EBP also activates cell-free transcription of SCD1 promoter-CAT and GLUT4 promoter-CAT chimeric genes. Point mutations within the C/EBP binding site in the 422(aP2) promoter markedly decrease transcription activated by rC/EBP. Consistent with activation by cAMP of the 422(aP2) promoter in intact preadipocytes, cAMP-dependent protein kinase activates transcription through this promoter with the cell-free system, this effect being independent of C/EBP. Thus, regulation of transcription directed by the 422(aP2) promoter in the cell-free system resembles that which occurs in intact 3T3-L1 cells.
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
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D
Cheneval
R J
RJ
Christy
D
D
Geiman
P
P
Cornelius
M D
MD
Lane
D000273:Adipose Tissue / Q000502:physiology*; D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D002474:Cell-Free System; D004268:DNA-Binding Proteins / Q000502:physiology*; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D005796:Genes; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D009687:Nuclear Proteins / Q000502:physiology*; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics; D013230:Stearoyl-CoA Desaturase / Q000235:genetics*; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009687:Nuclear Proteins; D009841:Oligonucleotides; D012333:RNA, Messenger; D014157:Transcription Factors; D013230:Stearoyl-CoA Desaturase; D011494:Protein Kinases