Abstract
Differentiation of 3T3-L1 preadipocytes into adipocytes is accompanied by increased expression of the nuclear protein C/EBP (CCAAT/enhancer binding protein) and by transcriptional activation of a group of adipose-specific genes. We report here the isolation of the murine C/EBP gene and the characterization of its promoter. Consistent with its proposed role in coordinating transcription during preadipocyte differentiation, an increase in the rate of transcription of the C/EBP gene precedes that of several adipose-specific genes whose promoters are transactivated by C/EBP. DNase I cleavage-inhibition patterns (footprinting) of the C/EBP gene promoter by nuclear factors from differentiated and undifferentiated 3T3-L1 cells identified two sites of differential factor binding. One site in the C/EBP gene promoter between nucleotides -252 and -239 binds a nuclear factor(s) present in preadipocytes that is lost or modified upon differentiation. Another site, between nucleotides -203 and -176, exhibits different but overlapping footprints by nuclear factors present in differentiated and undifferentiated cells. Gel retardation analysis with oligonucleotides corresponding to these sites revealed protein-oligonucleotide complexes containing these differentially expressed nuclear factors. The factor present in differentiated cells that binds at this site was identified as C/EBP (possibly in heterodimeric form with a homologous leucine-zipper protein), suggesting that C/EBP may regulate expression of its own gene.
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Selected References
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- Akira S., Isshiki H., Sugita T., Tanabe O., Kinoshita S., Nishio Y., Nakajima T., Hirano T., Kishimoto T. A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J. 1990 Jun;9(6):1897–1906. doi: 10.1002/j.1460-2075.1990.tb08316.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bernlohr D. A., Angus C. W., Lane M. D., Bolanowski M. A., Kelly T. J., Jr Expression of specific mRNAs during adipose differentiation: identification of an mRNA encoding a homologue of myelin P2 protein. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5468–5472. doi: 10.1073/pnas.81.17.5468. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Blackwell T. K., Kretzner L., Blackwood E. M., Eisenman R. N., Weintraub H. Sequence-specific DNA binding by the c-Myc protein. Science. 1990 Nov 23;250(4984):1149–1151. doi: 10.1126/science.2251503. [DOI] [PubMed] [Google Scholar]
- Bohmann D., Bos T. J., Admon A., Nishimura T., Vogt P. K., Tjian R. Human proto-oncogene c-jun encodes a DNA binding protein with structural and functional properties of transcription factor AP-1. Science. 1987 Dec 4;238(4832):1386–1392. doi: 10.1126/science.2825349. [DOI] [PubMed] [Google Scholar]
- Christy B., Nathans D. DNA binding site of the growth factor-inducible protein Zif268. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8737–8741. doi: 10.1073/pnas.86.22.8737. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Descombes P., Chojkier M., Lichtsteiner S., Falvey E., Schibler U. LAP, a novel member of the C/EBP gene family, encodes a liver-enriched transcriptional activator protein. Genes Dev. 1990 Sep;4(9):1541–1551. doi: 10.1101/gad.4.9.1541. [DOI] [PubMed] [Google Scholar]
- Freytag S. O. Enforced expression of the c-myc oncogene inhibits cell differentiation by precluding entry into a distinct predifferentiation state in G0/G1. Mol Cell Biol. 1988 Apr;8(4):1614–1624. doi: 10.1128/mcb.8.4.1614. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Friedman A. D., Landschulz W. H., McKnight S. L. CCAAT/enhancer binding protein activates the promoter of the serum albumin gene in cultured hepatoma cells. Genes Dev. 1989 Sep;3(9):1314–1322. doi: 10.1101/gad.3.9.1314. [DOI] [PubMed] [Google Scholar]
- 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]
- Herrera R., Ro H. S., Robinson G. S., Xanthopoulos K. G., Spiegelman B. M. A direct role for C/EBP and the AP-I-binding site in gene expression linked to adipocyte differentiation. Mol Cell Biol. 1989 Dec;9(12):5331–5339. doi: 10.1128/mcb.9.12.5331. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kaestner K. H., Christy R. J., Lane M. D. Mouse insulin-responsive glucose transporter gene: characterization of the gene and trans-activation by the CCAAT/enhancer binding protein. Proc Natl Acad Sci U S A. 1990 Jan;87(1):251–255. doi: 10.1073/pnas.87.1.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kaestner K. H., Flores-Riveros J. R., McLenithan J. C., Janicot M., Lane M. D. Transcriptional repression of the mouse insulin-responsive glucose transporter (GLUT4) gene by cAMP. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1933–1937. doi: 10.1073/pnas.88.5.1933. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Landschulz W. H., Johnson P. F., Adashi E. Y., Graves B. J., McKnight S. L. Isolation of a recombinant copy of the gene encoding C/EBP. Genes Dev. 1988 Jul;2(7):786–800. doi: 10.1101/gad.2.7.786. [DOI] [PubMed] [Google Scholar]
- McKnight S. L., Lane M. D., Gluecksohn-Waelsch S. Is CCAAT/enhancer-binding protein a central regulator of energy metabolism? Genes Dev. 1989 Dec;3(12B):2021–2024. doi: 10.1101/gad.3.12b.2021. [DOI] [PubMed] [Google Scholar]
- 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]
- Roman C., Platero J. S., Shuman J., Calame K. Ig/EBP-1: a ubiquitously expressed immunoglobulin enhancer binding protein that is similar to C/EBP and heterodimerizes with C/EBP. Genes Dev. 1990 Aug;4(8):1404–1415. doi: 10.1101/gad.4.8.1404. [DOI] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
- 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]
- Wingender E. Compilation of transcription regulating proteins. Nucleic Acids Res. 1988 Mar 25;16(5):1879–1902. doi: 10.1093/nar/16.5.1879. [DOI] [PMC free article] [PubMed] [Google Scholar]