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Proc Natl Acad Sci U S A. Apr 7, 2009; 106(14): 5819–5824.
Published online Mar 23, 2009. doi:  10.1073/pnas.0901676106
PMCID: PMC2667001
Medical Sciences

COUP-TFII acts downstream of Wnt/β-catenin signal to silence PPARγ gene expression and repress adipogenesis

Abstract

Wnt signaling through β-catenin and TCF maintains preadipocytes in an un-differentiated proliferative state; however, the molecular pathway has not been completely defined. By integrating gene expression microarray, chromatin immunoprecipitation-chip, and cell-based experimental approaches, we show that Wnt/β-catenin signaling activates the expression of COUP-TFII which recruits the SMRT corepressor complex to the first introns located downstream from the first exons of both PPARγ1 and γ2 mRNAs. This maintains the local chromatin in a hypoacetylated state and represses PPARγ gene expression to inhibit adipogenesis. Our experiments define the COUP-TFII/SMRT complex as a previously unappreciated component of the linear pathway that directly links Wnt/β-catenin signaling to repression of PPARγ gene expression and the inhibition of adipogenesis.

Keywords: ChIP-chip, epigenome, histone modification, obesity, Wnt

The physiological differentiation program converting preadipocytes into adipocytes has been well characterized, predominantly in cultured mouse cell lines (1). This process, called adipogenesis, is orchestrated by an elaborate cascade of sequentially acting transcription factors and chromatin modifying coregulators that shape the differentiation through the actions of hormones and other signaling pathways. During the very early stages of adipogenesis, there is a rapid and transient induction of CCAAT/enhancer-binding protein β (C/EBPβ) and C/EBPδ, followed by the activation of 2 critical pro-adipogenic transcription factors, C/EBPα and peroxisome proliferator-activated receptor γ (PPARγ). These 2 factors are key activators for the global changes in gene expression that cause the loss of preadipocyte characteristics and the acquisition of the mature fat-laden adipocyte phenotype.

The decision process used by preadipocytes to either remain proliferative or enter the differentiation pathway is influenced by both inhibitory and stimulatory factors (1), and one class of endogenous factors proposed to repress adipogenesis includes the Wnt proteins (2). There are several individual Wnt proteins in the family and they influence a diverse array of developmental and differentiation processes, including the fate of mesenchymal progenitors. The endogenous Wnt isoform involved in maintaining preadipocytes in their quiescent state has been proposed to be Wnt10b (2). The canonical Wnt signaling pathway is initiated by the binding of a specific Wnt as a ligand to the cell surface LRP5/Frizzled receptor complex. Signaling through Frizzled results in the stabilization and nuclear translocation of β-catenin where it associates with members of the T cell factor/lymphoid enhancer factor (TCF/LEF) family, and the resulting complex targets key genes to mediate the Wnt response (3, 4).

Inhibition of endogenous Wnt signaling by a dominant negative TCF7L2 (dnTCF7L2) results in spontaneous adipogenesis. Furthermore, an inhibitor of Wnt signaling in preadipocytes, harmine, has pro-adipogenic actions that target PPARγ (5). Expression of Wnts does not influence the rapid and transient induction of C/EBPβ and C/EBPδ but completely prevent the downstream induction of C/EBPα and PPARγ (2). However, the mechanism by which Wnt/β-catenin blocks C/EBPα and PPARγ gene expression has not been defined.

Despite the fact that Wnts are clearly key negative regulators of adipocyte differentiation, no β-catenin target gene(s) that account for the inhibition of adipogenesis has been identified, and the downstream transcriptional cascade of β-catenin in the adipocyte lineage remains largely unexplored. To gain insight into the molecular mechanisms that underlie the functions of β-catenin in regulating adipogenesis, we undertook a genome-wide identification of direct target genes regulated by β-catenin in 3T3-L1 preadipocytes using a combination of gene expression profiling, oligonucleotide microarray, and chromatin immunoprecipitation-chip (ChIP-chip) analyses. Our current results identify β-catenin as a direct regulator of chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), a potent antiadipogenic factor (ref. 6 and the current study). Furthermore, we show that COUP-TFII recruits the silencing mediator for retinoic acid receptor and thyroid hormone receptor (SMRT) corepressor complex to binding sites close to both promoters for the PPARγ gene where the complex maintains the chromatin in a hypoacetylated and repressed state that is reversed upon differentiation.

Results

Wnt/β-catenin Signaling Directly Regulates COUP-TFs Expression.

To address the regulatory program governing inhibition of adipogenesis by canonical Wnt/β-catenin signaling; we first analyzed global gene expression profiles in 3T3-L1 preadipocytes compared with companion cultures that were induced to differentiate for 8 days with a hormonal cocktail containing 3-isobutyl-1-methylxanthine, dexamethasone, and insulin (MDI) in the absence or presence of exogenous Wnt3a protein as described in SI Materials and Methods. Total RNA was isolated and global gene expression profiles were compared using Affymetrix microarray mouse gene chip platform M430A2. There are 48 nuclear receptors genes are encoded and 43 genes of them are represented on the chip. Of these, 23 were expressed at detectable levels on either day 0 or day 8 of differentiation (here “expressed” is defined as transcripts with a detection call of “present” on the microarray for these time points). This chip-microarray analysis revealed that the mRNA for the orphan receptor COUP-TFII was induced to the highest level (8-fold) relative to all of the expressed nuclear receptors in response to Wnt3a addition during the 8 day differentiation program [supporting information (SI) Fig. S1A]. Immunoblotting analysis demonstrated that COUP-TFII protein declined and was almost undetectable at day 8 of MDI treatment in cells cultured without Wnt and this was accompanied by a similar decline in nuclear β-catenin. However when recombinant Wnt3a was added during the differentiation process, the levels of both COUP-TFII and nuclear β-catenin remained high (Fig. S1B). Wnt3a also prevented the induction of C/EBPα, PPARγ, and liver X receptor α (LXRα) as expected for these genes that normally increased during adipogenesis (2).

Wnt3a also induced COUP-TFII and nuclear β-catenin when added directly to preadipocytes (Fig. 1A, Left). Because Wnt signals through β-catenin, we hypothesized that COUP-TFII might be a direct Wnt/β-catenin target gene. To test this possibility, we knocked down β-catenin by the application of siRNA and analyzed whether Wnt dependent COUP-TFII expression was altered. Two independent siRNA oligonucleotide sequences that target β-catenin were transfected into Wnt3a treated 3T3-L1 cells and both decreased β-catenin and COUP-TFII expression (Fig. 1A, Middle). Similar results were obtained when the Wnt pathway was inhibited by a separate method using a retrovirus expressing a dnTCF7L2 (7). This dominant negative TCF proteins, binds normally to TCF/LEF consensus binding sites but cannot be activated by β-catenin (8) (Fig. 1A, Right). Similar results were obtained in mouse mesenchymal stem ST2 cells [Fig. S1C].

Fig. 1.
β-catenin associates with COUP-TFII promoter and regulates COUP-TFII mRNA and protein expression. (A) Immunoblot analysis showing up-regulation of COUP-TFII upon Wnt3a treatment (Left) in 3T3-L1 preadipocytes and down-regulation of COUP-TFII in ...

Identification of COUP-TFII as a Direct Target of Wnt/β-catenin.

The results presented above suggest that COUP-TFII might be a direct target of the canonical Wnt signaling pathway through β-catenin. To determine if the β-catenin containing TCF complex directly controls COUP-TFII gene expression; we performed ChIP-chip analysis using a genome wide promoter tiling array platform. In this experiment, chromatin was prepared from 2 day postconfluent 3T3-L1 cells that were cultured for 16 h in the presence of the MDI mixture and recombinant Wnt3a followed by a standard procedure for ChIP analyses. Duplicate chromatin immunoprecipitations were performed with 3T3-L1 preadipocyte chromatin that was enriched after incubation with an antibody against C-terminal domain of β-catenin. The enriched DNA was then hybridized to an Affymetrix array platform containing tiled oligonucleotide probes that span between −6.0 to + 2.5 kb relative to the transcription initiation site of all annotated 28,000 mouse proximal promoter sequences. This analysis identified highly significant β-catenin binding (P < 10−5) to the promoter region of COUP-TFII 4.7 kb upstream relative to translation initiation site (Fig. 1B). In agreement with this result, a perfect TCF binding motif (CTTTGAA) that is conserved among species was found at this position (Fig. 1B). A quantitative gene specific ChIP analysis confirmed that the COUP-TFII promoter fragment from −4 kb region was enriched by the β-catenin antibody relative to the signal derived from an irrelevant genomic region used for normalization (Fig. 1C, Top). As a positive control, we also showed that β-catenin was recruited to the well-known β-catenin target gene cyclin D1 (CCND1) by the β-catenin antibody (Fig. 1C, Bottom).

Up-Regulation of COUP-TFII Promoter Activity by a Complex of β-catenin and TCF7L2.

To determine whether the β-catenin/TCF complex directly activates the COUP-TFII promoter, we subcloned a DNA fragment containing the 5′-franking region (FR) of the mouse COUP-TFII gene, which contained one TCF/LEF binding motif into a luciferase reporter construct, and transfected it into 3T3-L1 preadipocytes with or without expression vectors for an activated form of β-catenin and/or wild-type TCF7L2 (Fig. 1D). The reporter activity of the COUP-TFII promoter reporter construct was significantly enhanced (3.7-fold) by the combination of an activated form of β-catenin and wild-type TCF7L2, which was abrogated by the cotransfection of an expression vector for the Wnt signaling inhibitory protein, ICAT, (inhibitor of β-catenin and TCF4) (9) that interacts with β-catenin and prevents it from translocating into the nucleus (10). Taken together, these results all demonstrate that COUP-TFII is a bona fide target of β-catenin and Wnt signaling in 3T3-L1 preadipocytes.

COUP-TFII Inhibits Adipocyte Differentiation.

To specifically evaluate a potential role for COUP-TFII in adipogenesis, we examined the effects of overexpression of COUP-TFII by retroviral transduction during adipocyte differentiation. In this experiment, COUP-TFII overexpression blocked the morphological changes associated with adipogenesis, most notably through a marked decrease in accumulation of Oil Red O (ORO) (Fig. S2A). These morphological effects were accompanied by a decrease in the induction of the key adipogenic transcription factors C/EBPα and PPARγ. However, C/EBPβ and C/EBPδ were induced normally (Fig. S2B), suggesting that COUP-TFII acts upstream of PPARγ and C/EBPα but independent of C/EBPβ and C/EBPδ during adipogenesis.

Ectopic Expression of PPARγ or C/EBPα Overcomes COUP-TFII Inhibition of Adipogenesis.

To further assess the epistatic relationship between COUP-TFII and PPARγ or C/EBPα; we infected 3T3-L1 cells with retroviruses carrying cDNAs for each one and tested whether they could rescue differentiation in COUP-TFII expressing preadipocyte cells that fail to differentiate upon MDI treatment. After stable selection, cells were induced to differentiate with the treatment of the standard MDI adipogenic mixture and 8 days after the induction, cells expressing COUP-TFII alone remained fibroblastic and failed to accumulate significant amounts of lipid as expected; however, when either PPARγ or C/EBPα was coexpressed, almost all of the cells differentiated into adipocytes (Fig. S2C). Increased expression of LXRα, a known PPARγ target gene and another marker for adipocyte differentiation (11), was also observed and this was also decreased by COUP-TFII overexpression (Figs. S1B and S2D). These results suggest that COUP-TFII represses differentiation by inhibiting the expression of C/EBPα and PPARγ transcription factors, but not C/EBPβ nor C/EBPδ, which is also consistent with a key role downstream of Wnt-mediated inhibition of adipogenesis (2).

To complement the sufficiency results, we undertook necessity tests by predicting that a decrease in COUP-TFII expression would enhance differentiation of 3T3-L1 preadipocytes (Fig. 1E). Because the MDI mixture is very efficient in converting 3T3-L1 preadipocytes into mature adipocytes, we reasoned that it would be difficult to assess an increase in adipogenesis using MDI. Therefore, we only used dexamethasone treatment to induce differentiation, which is much more inefficient by itself at inducing differentiation than the complete mixture (compare Fig. S2A and Fig. 1E). A control siRNA or 2 independent siRNA oligonucleotide sequences that target COUP-TFII were separately transfected into 3T3-L1 cells which were then treated with dexamethasone and cultured for 8 days. Treatment with either COUP-TFII siRNA decreased COUP-TFII protein levels and increased lipid accumulation (Figs. 1E and Fig. S2E) consistent with the hypothesis that COUP-TFII is a negative regulator of adipogenesis. Knockdown of COUP-TFII in ST2 cells also resulted in an increase in lipid accumulation (Fig. S2F).

If COUP-TFII acts as a downstream mediator of Wnt signaling then theoretically, reduction of COUP-TFII should prevent Wnt from inhibiting differentiation. We tested this prediction by using siRNA. In control cells, 20 ng/well of recombinant Wnt3a completely inhibited adipogenesis as measured by ORO accumulation (Fig. 1F). However, in cells treated with the COUP-TFII siRNA, Wnt exhibited a diminished ability to repress adipocyte differentiation, indicating that COUP-TFII contributes at least in part to Wnt-induced suppression of adipogenesis (Figs. 1F and S2G).

Tri-methylation of Histone H3-K4 Within the PPARγ1 and PPARγ2 Loci Is Altered by the Binding of the COUP-TFII/RXRα Heterodimer.

The above results showed that the down-regulation of COUP-TFII is essential for adipogenesis. To identify COUP-TFII candidate genes with the potential to modulate adipogenesis, ChIP-chip analysis was conducted with chromatin from 3T3-L1 preadipocytes using an antibody directed against COUP-TFII. Because COUP-TFII is a nuclear receptor that forms a heterodimer with retinoid X receptor α (RXRα) to bind to response elements (12), we also used a RXRα antibody in a separate ChIP-chip experiment. This screen led to the identification of highly significant COUP-TFII binding (P < 10−4) to sites downstream of the transcription start sites for both PPARγ1 and PPARγ2 mRNAs (Fig. 2A). The separate analysis for RXRα binding revealed significant overlap between COUP-TFII and RXRα in both regions. Validation of COUP-TFII and RXRα sites obtained by direct gene specific ChIP analysis that showed that the 2 nuclear receptors were indeed binding to the respective sites identified by ChIP-chip (Fig. 2A). Inspection of the DNA sequence within each region shows there are conserved nuclear receptor half-sites (AGGTCA) in both. These results suggest that COUP-TFII binds to sites in the first intron of both PPARγ1 and γ2 mRNA isoforms to suppress their expression and thereby prevent adipogenesis. Intriguingly, independent ChIP-chip analysis with an antibody that detects H3-K4 tri-methylation (H3K4me3) revealed that patterns of H3-K4 tri-methylation close to the COUP-TFII sites increases during differentiation (Fig. 2B). Since H3-K4 tri-methylation is a mark of histone chromatin modification associated with gene activation, these results also suggest that PPARγ gene expression is regulated through changes in histone modification close to both COUP-TFII binding sites.

Fig. 2.
COUP-TFII/RXRα heterodimer binds to PPARγ1 and PPARγ2 loci and induces changes in the pattern histone H3-K4 tri-methylation (H3K4me3) within each region. A schematic diagram shows the mouse Pparγ1 and γ2 genes and ...

COUP-TFII Mediates Inactivation of PPARγ Gene Expression by Repressing Histone Modification.

To further analyze the mechanisms by which COUP-TFII suppresses PPARγ1 and γ2, we performed a ChIP of endogenous histone modifications within their first introns of both PPARγ1 and γ2 mRNAs close to the COUP-TFII binding regions we identified above (Fig. 3). We used antibodies that detect acetylated forms of both histone H3 and H4 that are known to correlate positively with high levels of gene expression (13, 14). Before differentiation, we observed low levels of mono-acetylation of histones H3 and H4 at the COUP-TFII binding regions from the PPARγ2 locus in both control empty vector transduced and COUP-TFII retrovirus transduced cells and very low level of tri-methylation of H3-K4 at PPARγ2 locus (Fig. 3 D-F, lanes 3 and 6). In contrast, we found hyperacetylation of histones H3 and H4 together with a striking increase in tri-methylation of histone H3-K4 in the first intron of PPARγ2 at day 8 of differentiation in control empty vector transduced cells (Fig. 3 D-F, lane 9). These changes were completely abrogated in COUP-TFII overexpressing cells (Fig. 3 D-F, compare lane 9 and lane 12). A very similar pattern was also observed for the PPARγ1 COUP-TFII binding region with the exception that a significant level of tri-methylation of H3-K4 was already observed together with increased acetylation of histone H3 before the induction of differentiation (Fig. 3 A, lane 3, and C, lane 3). These results suggest that changes in COUP-TFII binding to the first introns located downstream from the first exons of both PPARγ1 and γ2 mRNAs is required for alterations in histone acetylation that accompany gene induction during 3T3-L1 differentiation.

Fig. 3.
COUP-TFII regulates the histone modifications of PPARγ1 and PPARγ2 genes. 3T3-L1 cells transduced with retrovirus carrying empty or COUP-TFII vector were subjected to ChIP with anti-AcH3 (A and D), anti-AcH4 (B and E), and anti-H3K4me3 ...

siRNA-mediated Knockdown of SMRT Reverses COUP-TFII Induced Suppression of Adipogenesis.

A recent report indicates that DNA bound COUP-TF represses gene expression by using a domain within the ligand-binding region that recruits a corepressor to the DNA (i.e., nuclear receptor corepressor (NCoR) and SMRT; ref. 15). This results in a repressed chromatin structure that has been proposed to block transcriptional activation of target genes (12). Taken together with our results, this suggests that COUP-TFII might recruit an inhibitory complex to the first intron in each PPARγ locus to inhibit transcription. To test this hypothesis, we performed ChIP analysis on both PPARγ loci using anti-NCoR and anti-SMRT antibodies. Before differentiation, we observed the association of both proteins at the COUP-TFII binding regions from the PPARγ2 locus in COUP-TFII retrovirus transduced cells (Fig. 4A) indicating that COUP-TFII recruits inhibitory complex containing these repressors to the PPARγ2 locus. To test the relevance of NCoR and SMRT binding here, we predicted that knockdown of NCoR or SMRT would rescue COUP-TFII-induced suppression of PPARγ gene expression and adipogenesis. Accordingly, we transfected a siRNA designed to inactivate SMRT into COUP-TFII transduced cells and examined adipocyte differentiation induced by MDI. Similar to the results in Fig. S2A, transduction with a retroviral expression vector for COUP-TFII significantly inhibited the ability of the MDI mixture to induce differentiation and, consistent with our hypothesis, specific knockdown of SMRT reversed the inhibition of COUP-TFII on adipogenesis (Fig. 4 B and C).

Fig. 4.
Knockdown of SMRT restores the COUP-TFII mediated suppression of adipogenesis. (A) Cells were subjected to ChIP with anti-NCoR or anti-SMRT antibodies. qPCR analysis of PPARγ2 promoter sequences normalized to cyclophilin levels in control DNA ...

Expression of COUP-TFI and -TFII Are Down-Regulated in Diet and Genetic Mouse Models of Obesity.

COUP-TFI is related to COUP-TFII and both are expressed in adipose tissues of rats as shown by immunostaining (Fig. 5A). Because COUP-TFII expression is down-regulated during adipocyte differentiation, it is possible that a low level of COUP-TFII expression is associated with obesity. To investigate this possibility, we examined potential changes in expression of both COUP-TF family members in adipose tissue of 2 different mouse models of obesity; one that occurs as a result of high fat feeding in C57BL/6J mouse (DIO) and the other is the genetically predisposed obese ob/ob mouse. For DIO, we randomized 6-week old C57BL/6J littermates and fed them either normal or high fat chow for 8 weeks, extracted RNA from epididymal fat depots, and examined expression of COUP-TFs by quantitative PCR (qPCR). We found that DIO significantly decreased mRNA levels for both COUP-TFI and TFII in white adipose tissue (Fig. 5B). A similar down-regulation of COUP-TFI and TFII expression was observed in adipose tissues from 10-week old ob/ob genetically obese mice compared to matched controls (Fig. 5B). The concordant expression patterns in these 2 different obesity models support the idea that COUP-TFI and TFII signals may be germane to fat differentiation and possibly pathophysiology.

Fig. 5.
COUP-TFI and -TFII are expressed in white adipose tissue of rats and down regulated in diet-induced and genetically predisposed obese ob/ob mice. (A) Immunohistochemistry of paraffin-embedded section from rat using anti-COUP-TFI and -TFII antibodies. ...

Because COUP-TFI is also a negative regulator of gene expression, we analyzed its role in over/under expression and ChIP-chip analyses similar to those described here for COUP-TFII. We were able to confirm that COUP-TFI has a similar role as COUP-TFII in regulating PPARγ gene expression downstream of Wnt (Fig. S3).

Discussion

PPARγ expression is necessary and over-expression is sufficient for adipogenesis (16), and it was previously known that Wnt signaling inhibits adipogenesis by silencing PPARγ gene expression (2). However, the molecular pathway for this regulation was incompletely defined and a big challenge in the field of Wnt signaling and adipogenesis has been to integrate known components of the Wnt signaling pathway with the downstream effectors controlling adipogenesis and PPARγ activity.

In the current study, by combining global gene expression analyses with a ChIP-chip microarray approach, we demonstrate that COUP-TFII provides a direct link between the canonical Wnt signaling pathway and PPARγ gene activation. Further studies demonstrated that histone acetylation patterns predictive of gene activation were increased in the proximity of the COUP-TFII binding sites at the PPARγ locus during adipocyte differentiation, and over-expression of COUP-TFII not only prevented adipocyte conversion but also blunted the increase in histone acetylation as well.

Consistent with an important role for COUP-TFII in histone hypoacetylation and gene repression in preadipocytes, we also showed that its binding resulted in the corecruitment of NCoR and SMRT deacetylase containing corepressor complexes and that a knockdown of SMRT reversed the repressive effects of COUP-TFII on adipogenesis.

Since COUP-TFII is down-regulated during conditions associated with excess nutrition, it is tempting to speculate that down-regulation of COUP-TFII, in response to over nutrition, induces adipogenesis and hence links the development of obesity and the metabolic syndrome. We and others have reported that proteins involved in the Wnt signal cascade including the Wnt coreceptors LRP5 and LRP6 and its nuclear effector protein TCF7L2 are linked to type 2 diabetes (3, 17). Thus, the Wnt signaling cascade acting through COUP-TF to modulate PPARγ expression may be a key pathway in the development of obesity, insulin resistance, and the metabolic syndrome.

It was recently reported that COUP-TFII has an important role downstream of Hedgehog signaling to inhibit adipogenesis acting at the C/EBPα promoter (6). When combined with our study, the results define COUP-TFII as a molecular hub that integrates the input through 2 key developmental signaling pathways, Wnt and Hedgehog, with PPARγ gene expression and adipocyte differentiation.

Materials and Methods

The complete methods are described in detail in SI Text. All the PCR primers used in this article are listed in Tables S1 and S2 .

ChIP-Chip.

ChIP DNA samples were amplified by 2 cycles of in vitro translation (IVT) as described (18). ChIP and input DNA samples were hybridized on arrays according to the manufacture's instruction. After scanning and data extraction, enrichment values (ChIP/input DNA) were calculated by using the model-based analysis of tiling-array (MAT) algorithm (19). The details are described in SI Text.

Statistics.

For multiple comparison, one-way analysis of variance, followed by Turkey's honestly significant difference test was used.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank Drs. Takeshi Inagaki, Naoko Nishikawa, Naoko Kamimura, Shuying Jiang, and Sigeo Ihara for helpful discussions; Dr. Toshio Kitamura for a retroviral packaging cell line and pMX plasmids; Drs. Tetsu Akiyama, Bert Vogelstein, Ming-Jer Tsai, and Masahiro Takiguchi for plasmid constructs. This study was supported in part by Translational Systems Biology and Medicine Initiative, Grants-in-Aid for Scientific Research, and Technology, a grant of the Genome Network Project from the Ministry of Education, Culture, Sports, Science and Technology, Japan, by the grants from the Program of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation, by NFAT project of New Energy and Industrial Technology Development Organization. M.Y. is an investigator at the Howard Hughes Medical Institute.

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0901676106/DCSupplemental.

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