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.
Liu
Mei
M
Key Laboratory of Molecular Medicine, Ministry of Education, Department of Biochemistry and Molecular Biology, Fudan University Shanghai Medical College, Shanghai 200032, China.
Huang
Hai-yan
HY
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