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. 2008 Jan;7(1):86-94.
doi: 10.1016/j.cmet.2007.11.002.

Interferon regulatory factors are transcriptional regulators of adipogenesis

Jun Eguchi  1 , Qing-Wu Yan, Dustin E Schones, Michael Kamal, Chung-Hsin Hsu, Michael Q Zhang, Gregory E Crawford, Evan D Rosen
Affiliations

Interferon regulatory factors are transcriptional regulators of adipogenesis

Jun Eguchi et al. Cell Metab. 2008 Jan.

Abstract

We have sought to identify transcriptional pathways in adipogenesis using an integrated experimental and computational approach. Here, we employ high-throughput DNase hypersensitivity analysis to find regions of altered chromatin structure surrounding key adipocyte genes. Regions that display differentiation-dependent changes in hypersensitivity were used to predict binding sites for proteins involved in adipogenesis. A high-scoring example was a binding motif for interferon regulatory factor (IRF) family members. Expression of all nine mammalian IRF mRNAs is regulated during adipogenesis, and several bind to the identified motifs in a differentiation-dependent manner. Furthermore, several IRF proteins repress differentiation. This analysis suggests an important role for IRF proteins in adipocyte biology and demonstrates the utility of this approach in identifying cis- and trans-acting factors not previously suspected to participate in adipogenesis.

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Figures

Figure 1
Figure 1. DNase hypersensitive regions can be identified flanking adipocyte-selective genes
A, DNase hypersensitivity results for Day 7 adipocytes. The X axis represents the difference in cycle threshold (ΔCT) between DNase digested and undigested samples. The Y axis represents the percent of all primer sets that displayed any given ΔCT. The red line corresponds to random primers (negative control). The blue line corresponds to primers that amplify known DHS in other cell types (positive control). The green line describes results using primer pairs derived from the adipocyte selective gene set. See text for details. B, Top motifs identified by DME in differentiation-dependent DHS ranked by Error Rate. The information displayed includes the sequence logo, the Error Rate, and the best matching TRANSFAC profile including the accession identifier, the name of the TRANSFAC profile, and the KL divergence score of the discovered motif and the TRANSFAC motif.
Figure 2
Figure 2. IRFs are expressed in adipose tissue in vivo and in vitro
A, Q-PCR-based expression of IRF1-9 in tissues from male FVB mice. Br, brain; H, heart; Lu, lung; Liv, liver; K, kidney; Sp, spleen; Int, intestine; WAT, white adipose tissue; BAT, brown adipose tissue. Data are expressed as fold induction relative to IRF mRNA expression in WAT; all values are normalized to 36B4 expression. Mean ± SEM, n= 3. B, IRF expression was assessed in samples of fractionated fat pads from C57Bl/6 mice. SVF, stromal-vascular fraction; Macs, F4/80+ macrophages; Ads, adipocytes. Data are expressed as fold induction relative to IRFs mRNA expression in SVF. Mean ± SD, n= 6, **=p<0.01 vs. SVF, n.d. = not detectable. C, IRF expression during 3T3-L1 differentiation. Note that the time scale is not linear; the shaded area indicates timepoints < 24 hours. Data are normalized to 36B4 expression and are expressed as fold induction relative to IRF mRNA at Day 0.
Figure 3
Figure 3. IRFs bind to regions identified by DME
A, Binding of IRFs to DHS in adipocytes using ChIP. Data are representative of 3 experiments. B, EMSA using Cd36 I3 as the probe. Extract from Day 0 3T3-L1 pre-adipocytes (Left) was co-incubated with radiolabeled Cd36 I3 probe and an IRF-specific antibody (or IgG). The right panel shows a similar experiment using cells transfected with IRF-expressing plasmids. C, EMSA using Slc2a4 I2 as the probe. Extract from Day 0 cells was co-incubated with labeled Slc2a4 I2 probe and an IRF-specific antibody (or IgG)(Left). The right panel shows a similar experiment using cells transfected with IRF-expressing plasmids. For B and C, arrows denote complexes containing IRF isoforms, while arrowheads denote an antibody-mediated supershift. The asterisk indicates a complex of uncertain provenance. D, The murine Slc2a4 promoter is shown with the IRF-RE from −801 to −788 (top). Constructs with and without the IRF-RE were co-transfected into Day 5 3T3-L1 adipocytes with plasmids expressing IRF2, IRF4, or empty vector, and luciferase expression was determined 24 hrs later. Results are expressed as mean ± SD, n=3, *p<0.05 relative to vector control, *p<0.05 relative to Wt promoter.
Figure 4
Figure 4. IRF proteins repress adipogenesis
A, 3T3-L1 pre-adipocytes were transduced with retroviruses expressing IRFs and differentiated with DMI. Cells were stained with Oil red O 7 days post-DMI. B, Cells shown in A were harvested at Day 7 post-DMI and Q-PCR was used to determine levels of adipocyte genes relative to control cells. All samples are normalized to 36B4. Results are expressed as mean ± SD, n=3, * p<0.05, ** p<0.01 vs. vector control. C, 3T3-L1 pre-adipocytes were transduced with lentiviruses expressing shRNAs directed against the indicated IRF isoforms. Oil red O staining was performed at Days 3, 5, and 7 post-DMI. D, Cells shown in C were harvested at Day 7 post-DMI and Q-PCR was used to determine levels of adipocyte genes relative to control cells. All samples are normalized to 36B4. Results are expressed as mean ± SD, n=3, * p<0.05, ** p<0.01 vs. vector control.
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