Hironori Waki 1 , 2 , Masahiro Nakamura 1 , Toshimasa Yamauchi 1 , * , Ken-ichi Wakabayashi 3 , Jing Yu 1 , Lisa Hirose-Yotsuya 1 , Kazumi Take 1 , Wei Sun 1 , Masato Iwabu 1 , 4 , Miki Okada-Iwabu 1 , 5 , Takanori Fujita 3 , Tomohisa Aoyama 1 , Shuichi Tsutsumi 3 , Kohjiro Ueki 1 , Tatsuhiko Kodama 6 , Juro Sakai 7 , * , Hiroyuki Aburatani 3 , * , Takashi Kadowaki 1 , *
20 October 2011
Identification of regulatory elements within the genome is crucial for understanding the mechanisms that govern cell type–specific gene expression. We generated genome-wide maps of open chromatin sites in 3T3-L1 adipocytes (on day 0 and day 8 of differentiation) and NIH-3T3 fibroblasts using formaldehyde-assisted isolation of regulatory elements coupled with high-throughput sequencing (FAIRE-seq). FAIRE peaks at the promoter were associated with active transcription and histone modifications of H3K4me3 and H3K27ac. Non-promoter FAIRE peaks were characterized by H3K4me1+/me3-, the signature of enhancers, and were largely located in distal regions. The non-promoter FAIRE peaks showed dynamic change during differentiation, while the promoter FAIRE peaks were relatively constant. Functionally, the adipocyte- and preadipocyte-specific non-promoter FAIRE peaks were, respectively, associated with genes up-regulated and down-regulated by differentiation. Genes highly up-regulated during differentiation were associated with multiple clustered adipocyte-specific FAIRE peaks. Among the adipocyte-specific FAIRE peaks, 45.3% and 11.7% overlapped binding sites for, respectively, PPARγ and C/EBPα, the master regulators of adipocyte differentiation. Computational motif analyses of the adipocyte-specific FAIRE peaks revealed enrichment of a binding motif for nuclear family I (NFI) transcription factors. Indeed, ChIP assay showed that NFI occupy the adipocyte-specific FAIRE peaks and/or the PPARγ binding sites near PPARγ, C/EBPα, and aP2 genes. Overexpression of NFIA in 3T3-L1 cells resulted in robust induction of these genes and lipid droplet formation without differentiation stimulus. Overexpression of dominant-negative NFIA or siRNA–mediated knockdown of NFIA or NFIB significantly suppressed both induction of genes and lipid accumulation during differentiation, suggesting a physiological function of these factors in the adipogenic program. Together, our study demonstrates the utility of FAIRE-seq in providing a global view of cell type–specific regulatory elements in the genome and in identifying transcriptional regulators of adipocyte differentiation.
Humans consist of a few hundred types of specialized-function cells. Spatial and temporal transcriptional regulation of genes is essential for manifestation of cellular phenotypes. Identification of regulatory regions in the genome is central to understanding the mechanism of cell type–specific gene regulation. Recently developed high-throughput sequencing technology and computational analyses allow genome-wide investigation of the genome's chromatin structure. Using the FAIRE-seq technique, we identified the genome's open chromatin regions, which harbor regulatory elements in adipocytes. Open chromatin regions distal to genes' transcription start sites significantly differ among cell types. Multiple cell type–specific open chromatin regions exist near genes regulated during adipocyte differentiation. Computational motif analysis of adipocyte-specific open chromatin regions revealed enrichment of a binding motif for the NFI transcription factor family. These factors bind to the regulatory elements near adipogenic PPARγ, C/EBPα, and aP2 genes and regulate their expression. Overexpression of NFIA in 3T3-L1 cells resulted in robust induction of these genes and lipid droplet formation without differentiation stimulus and knockdown of NFIA or NFIB significantly suppressed both induction of genes and lipid accumulation during differentiation. Our study demonstrates the utility of FAIRE-seq in providing a global view of regulatory elements and in identifying transcriptional regulators of cellular functions.