Role of specificity protein-1, PPARgamma, and pituitary protein transcription factor-1 in transcriptional regulation of the murine CORS-26 promoter.
The collagenous repeat-containing sequence of 26-kDa protein (CORS-26) was recently described as a new gene that is induced during adipocyte differentiation. Since the transcription factors specificity protein-1 (SP-1) and PPARgamma have been demonstrated to modulate transcriptional activation of adipocytic genes, we investigated the putative role of SP-1 and PPARgamma in the regulation of the murine CORS-26 promoter. Computer-based sequence analysis revealed two putative SP-1 binding sites and binding sites for PPARgamma and Pit-1 within the TATA-box containing promoter. Electrophoretic mobility shift assays (EMSA) with nuclear extracts from 3T3-L1 adipocytes and appropriate promoter fragments demonstrated that SP-1 binds specifically to both SP-1 binding sites. Specificity was demonstrated by (i) the appearance of supershift bands, (ii) competition experiments and, (iii) by using oligonucleotides carrying mutated SP-1 binding sites. Functional promoter activity was analyzed by Luciferase reporter gene assays and SP-1 was shown to exert inhibitory effects on the transcriptional activation of the murine CORS-26 gene. Additionally, specific binding activity of PPARgamma and Pit-1 to the CORS-26 promoter was demonstrated. Taken together, the present data demonstrate the functionality of the proximal murine CORS-26 promoter, which is regulated specifically by two SP-1 binding sites via SP-3-independent repressive effects of SP-1 on transcriptional activation. Pit-1 and PPARgamma can bind specifically to the promoter and might play an additive functional role in gene regulation of murine CORS-26.
Schäffler
A
A
Department of Internal Medicine I, University of Regensburg, Franz-Josef-Strauss-Allee 11, D-93042 Regensburg, Germany. andreas.schaeffler@klinik.uni-regensburg.de
Ehling
A
A
Neumann
E
E
Herfarth
H
H
Paul
G
G
Tarner
I
I
Gay
S
S
Buechler
C
C
Schölmerich
J
J
Müller-Ladner
U
U
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism; D054392:Adipokines; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000502:physiology*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D009154:Mutation; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000502:physiology*; D018160:Receptors, Cytoplasmic and Nuclear / Q000502:physiology*; D012984:Software; D016329:Sp1 Transcription Factor / Q000502:physiology*; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000502:physiology*; D015533:Transcriptional Activation
D054392:Adipokines; D004268:DNA-Binding Proteins; D008156:Luciferases; D011506:Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D016329:Sp1 Transcription Factor; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; C428135:CORS26 protein, mouse; C494185:Pou1f1 protein, mouse