CCAAT/enhancer binding protein gene promoter: binding of nuclear factors during differentiation of 3T3-L1 preadipocytes.

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 nudeotides -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.Differentiation of 3T3-L1 preadipocytes into adipocytes in culture is accompanied by a dramatic rise in the rate of transcription of adipose-specific genes (1, 2). The isolation of a number of these genes (3-5) and characterization of their respective promoters prompted a search for transcription factors that coordinately activate their expression. Recently, it was discovered (6-8) that the CCAAT/enhancer binding protein (C/EBP) exhibits many of the characteristics of such a transcriptional activator.C/EBP was first isolated from rat liver nuclei as a sequence-specific DNA binding protein with preference for binding to CCAAT boxes and to the enhancer regions of several viral promoters (7). High levels of expression of C/EBP were subsequently found to be limited to terminally differentiated cell types-e.g., hepatocytes and adipocytesthat play a central role in energy metabolism, particularly in the synthesis and mobilization of glycogen and fat (6, 9, 10). Most recently, C/EBP was identified as the differentiationinduced nuclear factor that binds specifically to homologous regulatory elements within the promoters of three genes that are coordinately expressed when 3T3-L1 preadipocytes differentiate into adipocytes (6, 11). It was further demonstrated that C/EBP can function as a trans-activator ofthe promoters of these genes (6,11,12) and that mutation of the C/EBP binding site obliterates trans-activation (6, 12).Given that C/EBP may play a pleiotropic role in coordinating gene expression in this system, it wa...

CCAAT/enhancer-binding proteins are required for granulopoiesis independent of their induction of the granulocyte colony-stimulating factor receptor

“…[53][54][55] Optimal transcription of the G-CSFR, LF, and C/EBP␣ genes apparently requires factors not already present in uninduced 32D cl3 cells. Similarly, MPO RNA is rapidly induced by C/EBP␣WT-ER but not in the presence of cycloheximide, a finding that may reflect regulation of the MPO distal enhancer by both C/EBPs and PU.1 and the ability of C/EBP␣-ER to induce PU.1 expression.…”

Adenovirus-mediated Transfer of CCAAT/Enhancer-binding Protein-α Identifies a Dominant Antiproliferative Role for This Isoform in Hepatocytes

“…A 2200-base pair BamHI-HindIII fragment containing the entire coding sequence for the C/EBP␣ gene was excised from pBluescript (24) and subcloned into the vector pE1RSV (25), a modified version of p⌬E1sp1B (obtained from F. Graham, McMaster University, Hamilton, Ontario, Canada) that incorporated the Rous sarcoma virus long terminal repeat promoter, a multiple cloning site, and the polyadenylation signal from bovine growth hormone between adenovirus E1 recombination sequences. This plasmid, denoted pCEBP␣, was cotransfected with pJM17 (26), containing the full human adenovirus serotype 5 genome, into HEK293 cells using Lipofectamine (Life Technologies, Inc.).…”

Conjugated linoleic acid and obesity control: efficacy and mechanisms

“…52 One possible target gene of CLA is C/EBPa, which is not expressed during proliferation, 55 but is highly induced by the onset of differentiation of 3T3-L1 preadipocytes. 56 Reduced expression of C/EBPa in 3T3-L1 preadipocytes after CLA supplementation has been observed. 57 Dietary CLA has also been shown to suppress the expression and activity of PPARg.…”