Global mapping of cell type-specific open chromatin by FAIRE-seq reveals the regulatory role of the NFI family in adipocyte differentiation.
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.
Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
Hironori
H
Waki
Masahiro
M
Nakamura
Toshimasa
T
Yamauchi
Ken-ichi
K
Wakabayashi
Jing
J
Yu
Lisa
L
Hirose-Yotsuya
Kazumi
K
Take
Wei
W
Sun
Masato
M
Iwabu
Miki
M
Okada-Iwabu
Takanori
T
Fujita
Tomohisa
T
Aoyama
Shuichi
S
Tsutsumi
Kohjiro
K
Ueki
Tatsuhiko
T
Kodama
Juro
J
Sakai
Hiroyuki
H
Aburatani
Takashi
T
Kadowaki
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology* / Q000378:metabolism; D050156:Adipogenesis / Q000235:genetics*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002843:Chromatin / Q000235:genetics / Q000378:metabolism*; D042002:Chromatin Assembly and Disassembly; D047369:Chromatin Immunoprecipitation; D002874:Chromosome Mapping; D005786:Gene Expression Regulation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D011495:Histone-Lysine N-Methyltransferase; D050356:Lipid Metabolism / Q000235:genetics*; D051379:Mice; D051839:NFI Transcription Factors / Q000235:genetics / Q000378:metabolism*; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000235:genetics; D054730:Protein Interaction Domains and Motifs; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D002843:Chromatin; D051839:NFI Transcription Factors; D011495:Histone-Lysine N-Methyltransferase