Nutr Res Pract.  2007 Mar;1(1):19-28.

Transcriptome analysis and promoter sequence studies on early adipogenesis in 3T3-L1 cells

• Kim SJ
• Lee KH
• Lee YS
• Mun EG
• Kwon DY
• Cha YS

Abstract

To identify regulatory molecules which play key roles in the development of obesity, we investigated the transcriptional profiles in 3T3-L1 cells at early stage of differentiation and analyzed the promoter sequences of differentially regulated genes. One hundred and sixty-one (161) genes were found to have significant changes in expression at the 2nd day following treatment with differentiation cocktail. Among them, 86 transcripts were up-regulated and 75 transcripts were down-regulated. The 161 transcripts were classified into 10 categories according to their functional roles; cytoskeleton, cell adhesion, immune, defense response, metabolism, protein modification, protein metabolism, regulation of transcription, signal transduction and transporter. To identify transcription factors likely involved in regulating these differentially expressed genes, we analyzed the promoter sequences of up- or -down regulated genes for the presence of transcription factor binding sites (TFBSs). Based on coincidence of regulatory sites, we have identified candidate transcription factors (TFs), which include those previously known to be involved in adipogenesis (CREB, OCT-1 and c-Myc). Among them, c-Myc was also identified by our microarray data. Our approach to take advantage of the resource of the human genome sequences and the results from our microarray experiments should be validated by further studies of promoter occupancy and TF perturbation.

Keyword

Microarray analysis; promoter analysis; adipocyte differentiation; 3T3-L1 cells; transcription factor binding sites

MeSH Terms

3T3-L1 Cells*
Adipogenesis*
Binding Sites
Cell Adhesion
Cytoskeleton
Gene Expression Profiling*
Genome, Human
Humans
Metabolism
Microarray Analysis
Obesity
Signal Transduction
Transcription Factors
Transcriptome*
Transcription Factors

Figure

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  Fig. 1 Histological and biochemical analysis of adipocyte differentation Post-confluent 3T3-L1 cells were hormonally treated with differentiation cocktail (1µ M dexamethasone, 5 µg/ml insulin, and 0.5 mM IBMX) for 6 days as described in the Methods section. (a) The cells were fixed at the indicated time points and stained with Oil Red O to assess lipid accumulation (100× magnification). (b) (c) Data of triglycerides and protein are expressed as mean ± S.D (n = 3). Asterisks denote significant difference (ANOVA) between the control and differentiation cocktail treatment on days 2, 4 and 6 (p<0.05).

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  Fig. 2 Representative MA plots and SAM plot (a) Representative MA plots comparing the 3T3-L1 preadpocytes vs. the differentiated cells. M represents the log ratio of the two fluorescent dyes used to label probes, and A represents averaged logarithmic intensity. Broken line represents a 2-fold change. (b) SAM scatter plot of observed relative difference versus the expected relative difference. The genes showing significant difference in expression between the 3T3-L1 preadpocytes and the differentiated cells were identified. Broken line represents δ = 0.66.

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  Fig. 3 Global gene expression profile in functional categories Black bars and white bars represent the percentage of induced and repressed genes, respectively.

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  Fig. 4 Comparison between cDNA microarray analysis and real time RT-PCR 3T3-L1 cells were treated with differentiation cocktail for two days and total RNA was isolated. The relative mRNA expression levels of genes were measured by real-time RT-PCR analysis with SYBRO green. Data are expressed as fold changes (means ± SD), normalized to β-actin mRNA expression, where the values for the 3T3-L1 preadipocytes were set at 1.00. The analyses were performed in triplicate.

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  Fig. 5 Matrix of genes differentially expressed during adipogenesis and TF binding sites statistically over-represented in their promoters.

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