Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1992 Sep 1;89(17):7895–7899. doi: 10.1073/pnas.89.17.7895

Identification of a transcriptional repressor down-regulated during preadipocyte differentiation.

A G Swick 1, M D Lane 1
PMCID: PMC49821  PMID: 1355601

Abstract

During differentiation of 3T3-L1 preadipocytes into adipocytes, the transcription of adipocyte genes, including the stearoyl-CoA desaturase 2 (SCD2) gene, is activated. Transfection experiments with chimeric SCD2 promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs revealed a preadipocyte repressor element (PRE) capable of repressing transcription of the reporter gene in preadipocytes but not in adipocytes. DNase I protection and gel retardation analyses were used to localize the PRE site between nucleotides -435 and -410 of the SCD2 promoter and to identify a nuclear PRE binding protein present at high levels in preadipocytes and HeLa cells but lacking or inactive in adipocytes. Southwestern blot analysis indicated that the PRE binding protein has an apparent molecular mass of approximately 58 kDa. A single copy of the PRE site, inserted upstream of the simian virus 40 enhancer/promoter of pSV2CAT, was capable of strongly repressing transcription of the reporter gene in preadipocytes and HeLa cells but not in adipocytes. Taken together these results suggest that the PRE site and binding protein may regulate transcription of SCD2 and possibly other adipocyte genes by inhibiting their transcription in preadipocytes.

Full text

PDF
7895

Images in this article

7897Image
on p.7897
7898Image
on p.7898
7898Image
on p.7898

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bernlohr D. A., Bolanowski M. A., Kelly T. J., Jr, Lane M. D. Evidence for an increase in transcription of specific mRNAs during differentiation of 3T3-L1 preadipocytes. J Biol Chem. 1985 May 10;260(9):5563–5567. [PubMed] [Google Scholar]
  2. Birkenmeier E. H., Gwynn B., Howard S., Jerry J., Gordon J. I., Landschulz W. H., McKnight S. L. Tissue-specific expression, developmental regulation, and genetic mapping of the gene encoding CCAAT/enhancer binding protein. Genes Dev. 1989 Aug;3(8):1146–1156. doi: 10.1101/gad.3.8.1146. [DOI] [PubMed] [Google Scholar]
  3. Chapman A. B., Knight D. M., Dieckmann B. S., Ringold G. M. Analysis of gene expression during differentiation of adipogenic cells in culture and hormonal control of the developmental program. J Biol Chem. 1984 Dec 25;259(24):15548–15555. [PubMed] [Google Scholar]
  4. Christy R. J., Kaestner K. H., Geiman D. E., Lane M. D. CCAAT/enhancer binding protein gene promoter: binding of nuclear factors during differentiation of 3T3-L1 preadipocytes. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2593–2597. doi: 10.1073/pnas.88.6.2593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Christy R. J., Yang V. W., Ntambi J. M., Geiman D. E., Landschulz W. H., Friedman A. D., Nakabeppu Y., Kelly T. J., Lane M. D. Differentiation-induced gene expression in 3T3-L1 preadipocytes: CCAAT/enhancer binding protein interacts with and activates the promoters of two adipocyte-specific genes. Genes Dev. 1989 Sep;3(9):1323–1335. doi: 10.1101/gad.3.9.1323. [DOI] [PubMed] [Google Scholar]
  6. Cook J. S., Lucas J. J., Sibley E., Bolanowski M. A., Christy R. J., Kelly T. J., Lane M. D. Expression of the differentiation-induced gene for fatty acid-binding protein is activated by glucocorticoid and cAMP. Proc Natl Acad Sci U S A. 1988 May;85(9):2949–2953. doi: 10.1073/pnas.85.9.2949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cook K. S., Hunt C. R., Spiegelman B. M. Developmentally regulated mRNAs in 3T3-adipocytes: analysis of transcriptional control. J Cell Biol. 1985 Feb;100(2):514–520. doi: 10.1083/jcb.100.2.514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Distel R. J., Ro H. S., Rosen B. S., Groves D. L., Spiegelman B. M. Nucleoprotein complexes that regulate gene expression in adipocyte differentiation: direct participation of c-fos. Cell. 1987 Jun 19;49(6):835–844. doi: 10.1016/0092-8674(87)90621-0. [DOI] [PubMed] [Google Scholar]
  9. Enoch H. G., Catalá A., Strittmatter P. Mechanism of rat liver microsomal stearyl-CoA desaturase. Studies of the substrate specificity, enzyme-substrate interactions, and the function of lipid. J Biol Chem. 1976 Aug 25;251(16):5095–5103. [PubMed] [Google Scholar]
  10. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  12. Graves B. J., Johnson P. F., McKnight S. L. Homologous recognition of a promoter domain common to the MSV LTR and the HSV tk gene. Cell. 1986 Feb 28;44(4):565–576. doi: 10.1016/0092-8674(86)90266-7. [DOI] [PubMed] [Google Scholar]
  13. Graves R. A., Tontonoz P., Spiegelman B. M. Analysis of a tissue-specific enhancer: ARF6 regulates adipogenic gene expression. Mol Cell Biol. 1992 Mar;12(3):1202–1208. doi: 10.1128/mcb.12.3.1202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kaestner K. H., Ntambi J. M., Kelly T. J., Jr, Lane M. D. Differentiation-induced gene expression in 3T3-L1 preadipocytes. A second differentially expressed gene encoding stearoyl-CoA desaturase. J Biol Chem. 1989 Sep 5;264(25):14755–14761. [PubMed] [Google Scholar]
  15. Keller A. D., Maniatis T. Identification and characterization of a novel repressor of beta-interferon gene expression. Genes Dev. 1991 May;5(5):868–879. doi: 10.1101/gad.5.5.868. [DOI] [PubMed] [Google Scholar]
  16. Lin F. T., Lane M. D. Antisense CCAAT/enhancer-binding protein RNA suppresses coordinate gene expression and triglyceride accumulation during differentiation of 3T3-L1 preadipocytes. Genes Dev. 1992 Apr;6(4):533–544. doi: 10.1101/gad.6.4.533. [DOI] [PubMed] [Google Scholar]
  17. Ntambi J. M., Buhrow S. A., Kaestner K. H., Christy R. J., Sibley E., Kelly T. J., Jr, Lane M. D. Differentiation-induced gene expression in 3T3-L1 preadipocytes. Characterization of a differentially expressed gene encoding stearoyl-CoA desaturase. J Biol Chem. 1988 Nov 25;263(33):17291–17300. [PubMed] [Google Scholar]
  18. Samuelsson L., Strömberg K., Vikman K., Bjursell G., Enerbäck S. The CCAAT/enhancer binding protein and its role in adipocyte differentiation: evidence for direct involvement in terminal adipocyte development. EMBO J. 1991 Dec;10(12):3787–3793. doi: 10.1002/j.1460-2075.1991.tb04948.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Spiegelman B. M., Frank M., Green H. Molecular cloning of mRNA from 3T3 adipocytes. Regulation of mRNA content for glycerophosphate dehydrogenase and other differentiation-dependent proteins during adipocyte development. J Biol Chem. 1983 Aug 25;258(16):10083–10089. [PubMed] [Google Scholar]
  20. Student A. K., Hsu R. Y., Lane M. D. Induction of fatty acid synthetase synthesis in differentiating 3T3-L1 preadipocytes. J Biol Chem. 1980 May 25;255(10):4745–4750. [PubMed] [Google Scholar]
  21. Swick A. G., Blake M. C., Kahn J. W., Azizkhan J. C. Functional analysis of GC element binding and transcription in the hamster dihydrofolate reductase gene promoter. Nucleic Acids Res. 1989 Nov 25;17(22):9291–9304. doi: 10.1093/nar/17.22.9291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Umek R. M., Friedman A. D., McKnight S. L. CCAAT-enhancer binding protein: a component of a differentiation switch. Science. 1991 Jan 18;251(4991):288–292. doi: 10.1126/science.1987644. [DOI] [PubMed] [Google Scholar]
  23. Vinson C. R., LaMarco K. L., Johnson P. F., Landschulz W. H., McKnight S. L. In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage. Genes Dev. 1988 Jul;2(7):801–806. doi: 10.1101/gad.2.7.801. [DOI] [PubMed] [Google Scholar]
  24. Wilkison W. O., Min H. Y., Claffey K. P., Satterberg B. L., Spiegelman B. M. Control of the adipsin gene in adipocyte differentiation. Identification of distinct nuclear factors binding to single- and double-stranded DNA. J Biol Chem. 1990 Jan 5;265(1):477–482. [PubMed] [Google Scholar]
  25. Yang V. W., Christy R. J., Cook J. S., Kelly T. J., Lane M. D. Mechanism of regulation of the 422(aP2) gene by cAMP during preadipocyte differentiation. Proc Natl Acad Sci U S A. 1989 May;86(10):3629–3633. doi: 10.1073/pnas.86.10.3629. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES