Snail, a transcriptional regulator, represses adiponectin expression by directly binding to an E-box motif in the promoter

Abstract

Objective. Adiponectin is a hormone that modulates many metabolic processes and is exclusively expressed in adipose tissue. However, complete understanding of the factors that regulate adiponectin expression is lacking. The following were investigated: (1) functional analysis of the human adiponectin promoter, (2) putative adiponectin repressor sequence activity in 3T3-L1 adipocytes using promoter mutagenesis, (3) whether Snail, an E-box binding transcription factor, binds this repressor sequence, (4) if Snail regulates adiponectin expression in 3T3-L1 pre-adipocytes. Materials/Methods. To further understand how adiponectin expression is regulated, we isolated the human adiponectin promoter and analyzed its activity after serial deletions. Results. We found a negative cis-regulatory element located in the adiponectin proximal promoter sequence (-174 to -152 bp), which contained an E-box site (CAACTG). The DNA binding activity of this putative negative regulatory factor was found to be sequence-specific and the binding activity is decreased during adipocyte differentiation time-dependently. Affinity chromatography identified the zinc-finger transcription factor Snail (SNAI1) as the putative negative regulatory factor. Chromatin immunoprecipitation assay and electrophoretic mobility shift assay confirmed that Snail binds to this negative cis-regulatory element in pre-adipocytes, exclusively. Inhibition of Snail expression using small interfering RNA techniques increased adiponectin expression in 3T3-L1 adipocytes, while overexpression of Snail reduced adiponectin expression. Furthermore, we observed an inverse relation between the expression of Snail and the expression of CCAAT-enhancer-binding protein alpha and peroxisome proliferator-activated receptor gamma, which are transcription factors that regulate adipogenesis. Conclusions. Snail is a novel regulator of adiponectin expression and probably has a role in regulating adipogenesis. (C) 2012 Elsevier Inc. All rights reserved.