Identification and validation of novel C/EBPbeta-regulated genes in preadipocyte proliferation.
BACKGROUND
CCAAT/enhancer-binding protein beta (C/EBPbeta) is required for mitotic clonal expansion (MCE) during adipogenesis. It is still unclear how C/EBPbeta regulates MCE in the earlier differentiation programs of 3T3-L1 preadipocytes. The purpose of this paper was to understand why C/EBPbeta is required for preadipocyte proliferation, and identify new target genes of C/EBPbeta with chromatin immunoprecipitation (ChIP)-on-chip.


METHODS
Postconfluent growth-arrested 3T3-L1 preadipocytes were induced to differentiation using a standard differentiation protocol. ChIP was performed at 20 hours after induction with specific anti-C/EBPbeta antibodies. The precipitated DNA was amplified, labeled and hybridized with a mouse promoter microarray. Compared with the control in which the ChIP experiment was performed with non-specific antibody, only the genes with a signal increasing more than 2 fold were considered as candidate genes.


RESULTS
A total of 110 candidate genes were identified. BTG3 associated nuclear protein (SMAR1, Banp) and tripartite motif-containing 35 (Hls5, trim35) were two target genes among the 110 candidate genes which are involved in cell cycle regulation; the binding of C/EBPbeta to the promoter of banp and trim35 was verified by ChIP-PCR.


CONCLUSIONS
C/EBPbeta may regulate preadipocyte proliferation through activation of banp and trim35.
Key Laboratory of Molecular Medicine, Ministry of Education, Department of Biochemistry and Molecular Biology, Fudan University Shanghai Medical College, Shanghai 200032, China.
Mei
M
Liu
Hai-yan
HY
Huang
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology*; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology*; D049109:Cell Proliferation; D047369:Chromatin Immunoprecipitation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D011485:Protein Binding
D051017:Apoptosis Regulatory Proteins; C485952:Banp protein, mouse; D022782:CCAAT-Enhancer-Binding Protein-beta; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins