Differentiation-induced gene expression in 3T3-L1 preadipocytes: CCAAT/enhancer binding protein interacts with and activates the promoters of two adipocyte-specific genes.

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Abstract
Previous studies have shown that differentiation of 3T3-L1 preadipocytes leads to the transcriptional activation of a group of adipose-specific genes. As an approach to defining the mechanism responsible for activating the expression of these genes, we investigated the binding of nuclear factors to the promoters of two differentiation-induced genes, the 422(aP2) and stearoyl-CoA desaturase 1 (SCD1) genes. DNase I footprinting and gel retardation analysis identified two binding regions within the promoters of each gene that interact with nuclear factors present in differentiated 3T3-L1 adipocytes. One differentiation-induced nuclear factor interacts specifically with a single binding site in the promoter of each gene. Competition experiments showed that the interaction of this nuclear factor with the SCD1 promoter was prevented specifically by a synthetic oligonucleotide corresponding to the site footprinted in the 422(aP2) promoter. Several lines of evidence indicate that the differentiation-induced nuclear factor is CCAAT/enhancer binding protein (C/EBP), a DNA-binding protein first isolated from rat liver. Bacterially expressed recombinant C/EBP binds to the same site at which the differentiation-specific nuclear factor interacts within the promoter of each gene. Northern analysis with RNA from 3T3-L1 cells shows that C/EBP mRNA abundance increases markedly during differentiation. Transient cotransfection studies using a C/EBP expression vector demonstrate that C/EBP can function as a trans-activator of both the 422(aP2) and SCD1 gene promoters.