Repressor Transcription Factor 7-Like 1 Promotes Adipogenic Competency in Precursor Cells

Repressor transcription factor 7-like 1 promotes adipogenic competency in precursor cells

Ana G. Cristanchoa, Michael Schuppa, Martina I. Lefterovaa, Shengya Caoa, Daniel M. Cohenb, Christopher S. Chenb, David J. Stegera, and Mitchell A. Lazara,1

aDivision of Endocrinology, Diabetes, and Metabolism, Departments of Medicine and Genetics and Institute for Diabetes, Obesity, and Metabolism, Perelman School of Medicine at the University of Pennsylvania; and bDepartment of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104

Edited by Bruce M. Spiegelman, Dana-Farber Cancer Institute/Harvard Medical School, Boston, MA, and approved August 15, 2011 (received for review June 10, 2011)

The identification of factors that define adipocyte precursor poten- found that cell shape regulation is essential for determining line- tial has important implications for obesity. Preadipocytes are age decisions in mesenchymal stem cells (MSCs) (22, 24, 25). In- fibroblastoid cells committed to becoming round lipid-laden adipo- terestingly, many of the genes repressed early after the addition of cytes. In vitro, this differentiation process is facilitated by conflu- adipogenic stimuli to confluent preadipocytes are regulators of cell ency, followed by adipogenic stimuli. During adipogenesis, a large structure (26–28). The repressed cell structure genes are not γ α number of cytostructural genes are repressed before adipocyte enriched as genomic targets for PPAR or C/EBP (8, 9), sug- gene induction. Here we report that the transcriptional repressor gesting a role for an as-yet unknown transcriptional repressor in transcription factor 7-like 1 (TCF7L1) binds and directly regulates the regulation of cell shape during adipocyte differentiation. expression of cell structure genes. Depletion of TCF7L1 inhibits Transcription factor 7-like 1 (TCF7L1, formerly known as differentiation, because TCF7L1 indirectly induces the adipogenic TCF3) is an intriguing candidate for such a repressor. Tran- scription factor proteins play a role in the canonical Wnt path- transcription factor peroxisome proliferator-activated receptor γ in way that regulates adipogenesis (29), MSC lineage commitment a manner that can be replaced by inhibition of myosin II activity. (30), and expression of cell structure genes (31). A dominant TCF7L1 is induced by cell contact in adipogenic cell lines, and ectopic fl negative form of TCF7L2 promotes adipogenesis (29), and the expression of TCF7L1 alleviates the con uency requirement for adi- transcription factor 7 family member motif is enriched at sites of pocytic differentiation of precursor cells. In contrast, TCF7L1 is not histone modification in preadipocytes (26). TCF7L1 is of par- fl fi induced during con uency of non-adipogenic broblasts, and, re- ticular interest because it has been genetically linked to type 2 fi markably, forced expression of TCF7L1 is suf cient to commit non- diabetes (32) and shown to be an important transcriptional re- adipogenic fibroblasts to an adipogenic fate. These results establish pressor of canonical Wnt signaling targets (33–36). TCF7L1 TCF7L1 as a transcriptional hub coordinating cell–cell contact with regulates cell fate decisions in mouse embryonic stem cells (36, the transcriptional repression required for adipogenic competency. 37) and is a key regulator of terminal differentiation of other tissues (34, 38, 39). However, the extent to which TCF7L1 is dipose tissue is a highly specialized compartment of cells important for mammalian cell differentiation remains unknown, Aactively involved in maintaining global metabolic homeo- because TCF7L1 null mice are early embryonic lethal (33). stasis through lipid synthesis and storage, adipokine secretion, Here we show that TCF7L1 represses structure-related genes and insulin responsiveness (1). Adipocytes compose the majority during adipogenesis. Intriguingly, TCF7L1 is induced in a cell of cells in adipose tissue and play a critical role in normal contact–dependent manner by confluency in preadipocytes and is physiology, but their dysfunction is also at the center of a diverse required for adipocyte differentiation by repressing transcription range of diseases, including obesity, diabetes, and lipodystrophies of cell structure genes. TCF7L1 also is sufficient to bestow adi- (2). Furthermore, primary preadipocytes and adipose-derived pogenic potential on non-adipogenic cells. These results impli- stem cells have shown promise in treating multiple conditions cate TCF7L1 as an adipogenic competency factor that uniquely (3–5). Therefore, it is critical to understand the process by which determines adipogenic fate through cell structure organization spindly fibroblastic precursor cells undergo conversion into round required for adipocyte gene activation. lipid-laden fat cells. In vitro models of adipogenesis, such as the extensively studied Results committed preadipocyte cell line 3T3-L1 cells, have elucidated TCF7L1 Represses Cytostructural Genes During Adipocyte Differen- two major phases of adipogenesis: commitment and terminal tiation. To determine whether TCF7L1 was directly targeting the differentiation (6, 7). Terminal differentiation is characterized by cell structure genes rapidly repressed on the addition of adipogenic the induction of metabolic genes, many of which are the direct stimuli to confluent cells (26, 27), we used ChIP early in adipo- targets of the transcription factors peroxisome proliferator-acti- genesis with a validated TCF7L1 antibody (Fig. S1A), followed by vated receptor γ (PPARγ) and C/CAAT-binding protein (C/ deep sequencing (ChIP-seq). This analysis identified 556 high- EBP) α and β (8–14). Recent efforts have focused on identifying confidence [i.e., 2% false discovery rate (FDR)] binding sites in the committed preadipocyte populations in vivo (15, 16), as well as 3T3-L1 genome. A representative sample of these sites was con- on determining molecular factors that define the committed firmed by TCF7L1 ChIP-qPCR in control cells, with a loss of en- preadipocytes phenotype. Zinc finger protein 423 (Zfp423) is a critical preadipocyte factor upstream of PPARγ that is not present in non-adipogenic fibroblasts (17). However, Zfp423 also Author contributions: A.G.C. and M.A.L. designed research; A.G.C., M.S., S.C., and D.M.C. has been identified as a regulator of neurologic development performed research; A.G.C., M.S., M.I.L., C.S.C., and D.J.S. contributed new reagents/ana- (18), suggesting that other factors also may be involved in lytic tools; A.G.C., M.I.L., S.C., D.M.C., D.J.S., and M.A.L. analyzed data; and A.G.C. and specifying adipogenic competency and commitment of precursor M.A.L. wrote the paper. cells upstream of PPARγ. The authors declare no conflict of interest. Confluency could provide insight into other factors that confer This article is a PNAS Direct Submission. adipogenic competency, because it promotes adipogenesis in many Data deposition: The data reported in this paper have been deposited in the Gene Ex- model systems (19, 20). This cell–cell contact is associated with pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE31867). substantial reorganization of the actomyosin as well as the mi- 1To whom correspondence should be addressed. E-mail: [email protected]. crotubule cytoskeleton, providing permissive conditions for adi- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. CELL BIOLOGY pocyte differentiation (21–23). Moreover, several studies have 1073/pnas.1109409108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1109409108 PNAS | September 27, 2011 | vol. 108 | no. 39 | 16271–16276 Downloaded by guest on September 24, 2021 richment detected in TCF7L1-depleted cells (Fig. 1A). TCF7L1 target genes or independent of this pathway, as has been described binding was detected primarily in intergenic regions (Fig. 1B), and previously for skin stem cells (39). Using PANTHER to group binding sites demonstrated marked conservation with other species related genes in pathways (43), we found that cell structure was the (Fig. S1B), as has been found for other transcription factors (8, 9, most enriched biological process in the 833 genes within 100 kb of 40). Furthermore, these binding sites are enriched in TCF7L1-re- TCF7L1-binding sites (Fig. 1C). lated motifs (Table S1). Interestingly, previously described Wnt TCF family members repress transcription by recruiting histone pathway targets in adipogenesis, such as cyclin D1, COUP-TFII, deacetyase 1 (HDAC1) (44), a chromatin-modifying enzyme and FABP4 (41, 42), did not have any TCF7L1-binding sites (data shown to play critical roles in determining progression of the early adipogenic cascade (45–47). HDAC1 was signiﬁcantly enriched at available at the GEO Web site, www.ncbi.nlm.nih.gov/geo), sug- D gesting that TCF7L1 may be binding at a subset of classic Wnt TCF7L1-binding sites relative to negative control sites (Fig. 1 ) and to a greater extent than at enhancers without TCF7L1-binding sites, where HDAC1 nevertheless can be recruited by other transcription factors (Fig. S1C). Histone 3 lysine 9 acetylation (H3K9ac) positively correlates with active transcription at both transcription start sites and enhancer regions (48), and previous reports validated this histone mark as a marker of active transcription during adipogenesis and in mature adipocytes (8, 49). Indeed, H3K9ac enrichment was diminished at TCF7L1-binding sites during adipogenesis (Fig. 1E), speciﬁcally at genes repressed during differentiation (Fig. S1D). Approximately one-quarter of TCF7L1-binding sites were found within 100 kb of genes down-regulated during adipogenesis, much more than at random genes (Fig. 1F). In comparison, a similar enrichment of PPARγ-binding sites was noted within 100 kb of genes induced during adipogenesis (Fig. 1F), consistent with the known role of PPARγ as a transcriptional activator (50, 51). TCF7L1 binding also was enriched at induced genes (Fig. 1F), at a similar number of average binding sites per gene (Fig. S1E), suggesting that TCF7L1 may have a function at those sites as well. However, H3K9ac did not increase at these sites during early adipogenesis, and thus it is unlikely that TCF7L1 functioned as a transcriptional activator (Fig. S1F). Taken together, the enrichment of TCF7L1 binding and reduced H3K9ac at sites neighboring down-regulated genes suggests that TCF7L1 indeed functions as a transcriptional repressor in early differentiation.

TCF7L1 Is Required for Adipogenesis. Because TCF7L1 was found to bind near cytostructure-related genes, and cell structure regulation is critical for adipogenesis, we next asked whether TCF7L1 is required for adipogenesis by using two independent siRNA to deplete TCF7L1 from 3T3-L1 preadipocytes. Adipo- cyte differentiation was impaired in the TCF7L1-depleted cells but not in cells treated with nontargeting siRNA, as assessed by Oil Red O (ORO) staining for neutral lipids (Fig. 2A) and accumulation of adipocyte proteins (Fig. 2B). Moreover, adipocyte gene expression was reduced (Fig. 2C) and preadipocyte gene expression was increased (Fig. 2D) in TCF7L1-depleted cells subjected to the adipocyte differentiation protocol, and de- Fig. 1. TCF7L1 represses cytostructural genes during adipocyte differentiation. (A) Validation of TCF7L1-binding sites from ChIP-seq. TCF7L1 ChIP-qPCR in pletion of TCF7L1 in 3T3-F442A cells, an alternate adipogenic control cells and TCF7L1-depleted 3T3-L1 cells at 24 h after the addition of model, also led to a substantial reduction in lipid accumulation adipogenic stimull (DMI; see SI Materials and Methods). (B) Distribution of (Fig. S2), suggesting that TCF7L1 is required for adipogenesis. TCF7L1-binding sites throughout the genome. (C) TCF7L1 binds near genes in cell structure pathways. PANTHER biological processes enriched with an FDR TCF7L1 Indirectly Activates the PPARγ Gene Locus via Effects on Cell <5% for all genes within 100 kb of TCF7L1-binding sites. (D) HDAC1 colocalizes Structure. We next sought to determine whether TCF7L1 regu- with TCF7L1. HDAC1 ChIP-qPCR in 3T3-L1 cells during early adipogenesis. (E) lates early adipogenesis. C/EBPβ is induced within 24 h of adi- Reduced H3K9Ac at TCF7L1-binding sites. Average H3K9ac profile at TCF7L1- pogenic stimulation of 3T3-L1 cells (52), and PPARγ2 also was binding sites in day 0 preadipocytes, at 24 h after addition of DMI, and in day 10 induced at this time point (Fig. 3A), although this induction mature adipocytes. The profile of input for each time point was determined as represented only a fraction of the eventual induction of PPARγ2 well. The one-tailed Wilcoxon rank-sum test was used to compare the difference in mature adipocytes (Fig. S3A). Depletion of TCF7L1 had little in acetylation at TCF7L1-binding sites with acetylation at matched control effect on C/EBPβ induction (Fig. S3B), but markedly attenuated regions in a 1-kb region. P < 0.05, preadipocyte > 24 h after DMI > adipocytes. early PPARγ2 induction (Fig. 3A). Consistent with a role for (F) TCF7L1 binding is increased at genes repressed during adipogenesis. Per- γ γ TCF7L1 in the upstream regulation of PPAR , ectopic expres- centage of TCF7L1 and PPAR -binding sites within 100 kb of genes repressed sion of PPARγ was able to rescue the adipogenic defect in (1,406 genes) or induced during adipogenesis (1,011 genes) or a random set of B C genes (1,366 genes). Fisher’s exact test was used to compare the percentage of TCF7L1-depleted preadipocytes (Fig. 3 and ). Evaluation by TCF7L1-binding sites near repressed or induced genes with the percentage near FAIRE (Formaldehyde-Assisted Isolation of Response Ele- χ2 γ ments) (53) of an enhancer located ∼182 kb upstream of the random genes. The test was used to compare the percentage of PPAR - PPARγ2 binding sites near repressed or induced genes with the percentage near random promoter that is functional early in adipogenesis and genes. ***P < 0.001; *P < 0.05. For A and D, 20 TCF7L1-binding sites and five whose activation is correlated with PPARγ2 expression (49) negative control sites were interrogated. Each point represents the percent revealed that depletion of endogenous TCF7L1 in preadipocytes input of one site. The lines represent mean ± SEM for TCF7L1 or control sites in led to a more repressive, closed chromatin structure (Fig. 3D), each cell population, *** P < 0.001. which may contribute to the reduced activation of the PPARγ

16272 | www.pnas.org/cgi/doi/10.1073/pnas.1109409108 Cristancho et al. Downloaded by guest on September 24, 2021 Fig. 2. TCF7L1 depletion abrogates adipogenesis. (A) Reduced lipid accumulation. ORO staining (Upper) and phase-contrast microscopy (Lower) of siControl, siTCF7L1 #1, and siTCF7L1 #2 3T3-L1 electroporated cells differentiated for 7 d. (B) Reduced adipocyte protein expression. TCF7L1 and RAN protein levels in preadipocytes before DMI treatment (Upper)andPPARγ, FABP4, and RAN protein levels in day 7 adipocytes (Lower). (C) Reduced expression of adipocyte genes. siControl, siTCF7L1 #1, and siTCF7L1 #2 cells at day 7. (D) Increased expression of preadipocyte genes. siControl, siTCF7L1 #1, and siTCF7L1 #2 cells at day 7. Graphed values represent mean ± SEM (n =3).*P < 0.05; **P < 0.01; ***P < 0.001.

gene. Of note, the −182-kb enhancer was not bound by TCF7L1 early in adipogenesis (Fig. S3C), suggesting that the positive effect of TCF7L1 on enhancer loci accessibility is indirect. Al- though TCF7L1-binding sites were found neighboring PPARγ start sites, H3K9ac did not change at these sites during early adipogenesis (Fig. S3D), and the binding was markedly weaker than that at other TCF7L1 sites validated by ChIP-qPCR (Fig. S3E). We first considered whether TCF7L1 regulated Dlk1/Pref-1, whose down-regulation is required for adipogenesis (54, 55). However, our ChIP-seq experiment did not identify a TCF7L1- binding site near the Dlk1 gene (Fig. S4A; and complete dataset at GEO, www.ncbi.nlm.nih.gov/geo), and Dlk1/Pref-1 down- γ regulation during early adipogenesis was unaffected by TCF7L1 Fig. 3. TCF7L1 indirectly regulates PPAR enhancer in early adipogenesis. E (A) TCF7L1 depletion prevents PPARγ2 induction. PPARγ2 mRNA levels in depletion (Fig. 3 ), indicating that the effects of TCF7L1 are siControl and siTCF7L1 3T3-L1 cells at 0 h and 24 h after DMI addition. (B) independent of Dlk1/Pref-1. In contrast, during early adipo- Ectopic PPARγ2 rescues adipogenic defects in TCF7L1-depleted cells. ORO genesis, TCF7L1 depletion did induce several cell structure genes (Upper) and phase-contrast microscopy (Lower) at 7 d after the addition of with nearby TCF7L1-binding sites (Fig. 3E and Fig. S4 B and C). adipogenic stimuli to 3T3-L1 preadipocytes infected with Empty or PPARγ2 virus and electroporated with siTCF7L1 or siControl. (C) Ectopic PPARγ2res- Adipogenesis in TCF7L1-Depleted Cells Is Rescued by Inhibiting cues adipocyte-specific protein expression in TCF7L1-depleted cells. TCF7L1, Myosin Activity. Because cytostructure has been shown to regu- PPARγ, and RAN protein levels in 3T3-L1 cells before DMI treatment (Upper) late adipogenesis and adipogenic gene expression (21–25), we and PPARγ, FABP4, and RAN protein levels at 7 d after adipogenic stimuli hypothesized that TCF7L1-dependent induction of PPARγ2is (Lower). (D) TCF7L1 depletion decreases PPARγ2 gene enhancer accessibility. an indirect result of cytostructural regulation. TCF7L1-depleted FAIRE-qPCR at the 36b4 and PPARγ2 −182-kb enhancer in siControl and cells demonstrated an increase in myosin fiber formation (Fig. siTCF7L1 3T3-L1 cells treated for 24 h with DMI. (E) TCF7L1 depletion increases 4A). To test whether TCF7L1 regulation of myosin is required levels of cell structure–related genes that neighbor TCF7L1-binding sites, but α for adipogenesis, we treated TCF7L1-depleted preadipocytes not Dlk1, which lacks TCF7L1-binding sites. Col1 2, Dcn, Dlk1, Dpt, Ptn, Tes, with compounds that inhibit specific cytoskeletal components Thbs1, Thbs2, and Tiam2 mRNA levels in siControl and siTCF7L1 3T3-L1 cells were treated for 24 h with DMI. In A, D, and E, graphed values represent and have been shown to promote adipogenesis (21, 22, 25). mean ± SEM (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. Blebbistatin, a myosin II ATPase inhibitor, rescued PPARγ2 levels in TCF7L1-depleted cells during the first 24 h of differentiation (Fig. 4B). In contrast, disrupters of actin filaments gest that the TCF7L1-dependent induction of PPARγ2is (cytochalasin D) or microtubules (nocododazole) did not rescue mediated by myosin-dependent changes of cell organization. CELL BIOLOGY PPARγ2 in TCF7L1-depleted cells (Fig. S5). These results sug- Furthermore, blebbistatin rescued adipogenesis in TCF7L1-

Cristancho et al. PNAS | September 27, 2011 | vol. 108 | no. 39 | 16273 Downloaded by guest on September 24, 2021 Fig. 4. Myosin inhibition rescues TCF7L1-depleted cells. (A) TCF7L1-depleted cells display an increase in myosin fiber formation. Myosin IIa im- munofluorescence in siControl and siTCF7L1 3T3-L1 cells treated with DMI for 24 h. Nuclei are counterstained with DAPI. (Scale bar: 20 μm.) Graphed Fig. 5. TCF7L1 promotes adipogenesis in preconfluent cells. (A) TCF7L1 values represent mean ± SEM (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. (B) during adipogenesis. TCF7L1, PPARγ, FABP4, and RAN protein levels during Inhibition of myosin contraction rescues PPARγ2 expression in TCF7L1-de- 3T3-L1 adipogenesis after addition of DMI at day 0. (B) TCF7L1 is induced by pleted preadipocytes. PPARγ2 expression levels in siControl and siTCF7L1 the confluency of preadipocytes. TCF7L1, TCF7L2, and RAN protein levels in 3T3-L1 cells were treated for 24 h with DMI and DMSO or 50 μM blebbistatin. preconfluent and confluent 3T3-L1 preadipocytes, 3T3-F442A preadipocytes, The one-tailed Student t test was used to determine significance. (C)In- and primary MEFs. (C) TCF7L1 induction by confluency is calcium-dependent. creased lipid accumulation in blebbistatin-treated, TCF7L1-depleted cells. TCF7L1 and RAN protein levels in confluent 3T3-L1 preadipocytes were cul-

ORO (Upper) and phase-contrast microscopy (Lower) at 7 d after addition of tured in the presence of 2 mM EGTA, 2 mM CaCl2, or 2 mM MgCl2 for 16 h. adipogenic stimuli to 3T3-L1 preadipocytes electroporated with siTCF7L1 or (D) TCF7L1 induction is cadherin-dependent. Confluent preadipocytes were fi siControl. During the rst 4 d of the differentiation protocol, cells were treated for 3 d with vehicle (H2O), 500 μg/mL of control cyclic peptide, or 500 treated with DMSO or 50 μM blebbistatin as indicated. μg/mL of pan type 1 cadherin blocking cyclic peptide. (E) Increased lipid accumulation. Phase-contrast microscopy of preconfluent preadipocytes infected with empty or TCF7L1 virus before addition of DMI (Left). ORO depleted cells (Fig. 4C). Together, these data suggest that (Center) and phase-contrast (Right) microscopy of same cells 7 d after ad- TCF7L1-dependent cell shape regulation indirectly remodels the dition of adipogenic stimuli. (F) Increased adipocyte protein expression. chromatin conformation of an adipogenic enhancer of PPARγ2 TCF7L1 and RAN protein levels in preconfluent and confluent preadipocytes expression, promoting adipocyte differentiation. before DMI treatment (Upper) and PPARγ, FABP4, and RAN protein levels at 7 d after addition of adipogenic stimuli (Lower). TCF7L1 Is Permissive for Adipogenesis of Preconfluent Preadipocytes. fl Because con uency is associated with important cytostructural fi changes in adipogenesis (22, 28), we hypothesized that the the same conditions, ectopic expression of TCF7L1 was suf cient to support robust adipogenesis of preconfluent preadipocytes transcriptional repressor of cell structure genes must be present E F in early adipogenesis and may be induced by confluency. (Fig. 5 and ). Cell structure and motility pathways were the fl TCF7L1 was present in confluent preadipocytes and early adi- only categories of repressed genes enriched in precon uent fi pogenesis, but diminished as terminal differentiation progressed preadipocytes expressing TCF7L1 (Fig. S7), con rming the fi (Fig. 5A). However, TCF7L1 was induced by confluency in speci c role of TCF7L1 in repressing cytostructural genes. Thus, fi fl multiple models of adipogenesis before the addition of adipo- TCF7L1 acts speci cally as a mediator of the con uency re- genic stimuli (Fig. 5B). The confluency-dependent induction of quirement for adipogenesis. Although TCF7L1 overcame the TCF7L1 was specific; neither isoform of the related TCF7L2 requirement for confluency, adipogenesis did not occur without changed on confluency (Fig. 5B). Induction of TCF7L1 mRNA exposure to adipogenic stimuli (Fig. S8 A and B), suggesting that with confluence required cell contact, and mRNA levels were not induction of TCF7L1 functions specifically as a mediator of affected by the treatment of preconfluent cells with conditional adipogenic competency, but not terminal differentiation. media from confluent cells (Fig. S6). TCF7L1 protein induction during confluency was blocked by the calcium chelator EGTA, TCF7L1 Is Sufficient to Confer Adipogenic Competency. Although which was rescued by addition of calcium chloride, but not NIH 3T3 cells are non-adipogenic, ectopic expression of such magnesium chloride, to the cells (Fig. 5C). This suggests a role factors as PPARγ (58) and Zfp423 (17) is sufficient to convert for cadherins, calcium-dependent cell junction proteins impli- these cells to adipocytes in the presence of differentiation me- cated in a wide variety of developmental processes (56). Indeed, dium. NIH 3T3 cells lack robust TCF7L1 expression (Fig. 6A). treatment of confluent cells with a peptide that inhibits type 1 Remarkably, ectopic expression of TCF7L1 in NIH 3T3 cells cadherins (57), but not a control peptide, decreased levels of rendered these cells competent to undergo nearly complete TCF7L1 in confluent cells (Fig. 5D). Therefore, induction of adipogenesis at the levels of lipid accumulation and morphology TCF7L1 by confluency likely is cadherin-dependent. (Fig. 6B) as well as gene expression (Fig. 6 C–E). Consistent with We next addressed the importance of TCF7L1 in mediating the hypothesis that TCF7L1 promotes a preadipocyte-like phe- the permissive effect of confluency on adipogenesis. Pre- notype, ectopic expression of TCF7L1 in NIH 3T3 cells dra- adipocytes transduced with empty retrovirus did not undergo matically induced PPARγ2 expression (Fig. 6F) and increased adipogenesis when exposed to differentiation medium before accessibility of the PPARγ gene enhancer after adipogenic confluency (Fig. 5 E and F), although they eventually become stimulation (Fig. 6G). TCF7L1 promotion of adipogenic com- confluent during the differentiation process. In contrast, under petency did not require induction of Zfp423 (Fig. 6H), implying

16274 | www.pnas.org/cgi/doi/10.1073/pnas.1109409108 Cristancho et al. Downloaded by guest on September 24, 2021 that TCF7L1 may be downstream of Zfp423 or can promote adipogenesis in a parallel pathway. Discussion We have shown that confluency of committed preadipocytes induces the transcriptional repressor TCF7L1, which binds to specific sites in chromatin to silence genes involved in cell structure after addition of adipogenic stimuli. TCF7L1-mediated cell structure changes contribute to PPARγ2 induction, providing synchrony between adipogenic commitment and terminal differentiation. This critical role of TCF7L1 is reflected in its requirement for adipocyte differentiation and its sufficiency as an adipogenic competency factor for non-adipogenic fibroblasts. To date, mammalian studies of TCF7L1 have focused on its role in regulating pluripotency (37) and skin stem cell differentiation (34, 39). The present study identifies a role for TCF7L1 in adipogenesis, where it likely functions as a transcriptional repressor of cell structure–related genes. This study also provides evidence that a downstream transcription factor is directly involved in critically regulating cell structure during the transition from adipogenic commitment to terminal differentiation, and illustrates the predictive power of ChIP-seq data to discover novel cell type–specific functions for transcription factors during differentiation. Nevertheless, it is possible that TCF7L1 also may have indirect effects on other factors affecting adipogenesis. In addition, this study has identified a molecular mechanism by which confluency signals transcriptional events, mediated by TCF7L1, allowing adipogenic stimuli-dependent regulation of cytostructural reorganization. Thus, TCF7L1 acts as a hub between two well-known but unexplained phenomena that occur during adipocyte differentiation: the requirement for cell–cell contact and the subsequent repression of genes controlling cell structure during adipogenesis. Elevated PPARγ level is a hall- mark of in vivo adipocyte precursor populations (16); thus, the cytostructural regulator TCF7L1 confers a preadipocyte-like phenotype by promoting PPARγ enhancer accessibility and expression during early adipogenesis. Importantly, in TCF7L1-depleted cells, PPARγ2 levels and adipocyte differentiation could be rescued by chemical inhibition of myosin ATPase activity. Thus, these data also suggest that TCF7L1 provides a link between structural changes during adipogenic commitment and subsequent gene induction during terminal differentiation. Our findings indicate that TCF7L1 induction by preadipocyte confluency may be dependent on cadherin-mediated cell contact. Cadherins, functioning as calcium-dependent cell–cell junctions, have been previously associated with a wide variety of developmental processes (56), including cell fate decisions (24), but their role in adipocyte differentiation has not been explored extensively. Cadherin composition or function may be a dis- tinguishing feature of adipogenic and non-adipogenic fibroblasts, given that cell contact does not induce TCF7L1 in NIH 3T3 cells. Recently, Zfp423 was reported to be present in preadipocytes, but not non-adipogenic fibroblasts, and to be required for adipogenesis (17). However, TCF7L1 does not require robust Fig. 6. TCF7L1 confers adipogenic competency to NIH 3T3 cells. (A) TCF7L1 fl Zfp423 expression in NIH 3T3 cells to bestow adipogenic com- is not induced by con uency of non-adipogenic NIH 3T3 cells. TCF7L1 and petency to these cells, demonstrating that induction of TCF7L1 RAN protein levels in confluent 3T3-L1 cells and preconfluent, confluent, and DMI plus rosiglitazone (TZD) treated NIH 3T3 cells. (B) Adipogenesis of NIH is also a major determinant of the adipocyte precursor pheno- 3T3 cells expressing TCF7L1. ORO (Upper) and phase-contrast microscopy type. Other stem cell populations have been shown to require a (Lower) of NIH 3T3 cells infected with Empty or TCF7L1 virus 7 d after ad- core group of transcription factors that cooperatively maintain dition of adipogenic stimuli. (C) Adipocyte protein expression in NIH 3T3 precursor characteristics, such that disruption of any single cells expressing TCF7L1. TCF7L1 and RAN protein levels in NIH 3T3 cells be- component may alter the differentiation potential (37). Because fore DMI plus TZD treatment (Upper) and PPARγ, FABP4, and RAN protein both TCF7L1 and Zfp423 are capable of promoting a commit- levels at 7 d after adipogenic stimuli (Lower). (D) Increased adipocyte gene ment to adipogenesis in NIH 3T3 cells, it seems likely that expression in NIH 3T3 cells expressing TCF7L1. (E) Decreased preadipocyte preadipocyte populations subscribe to this paradigm. Moreover, gene expression in NIH 3T3 cells expressing TCF7L1. (F) TCF7L1 promotes the role of TCF7L1 in adipogenesis provides general mechanistic PPARγ2 expression in NIH 3T3 cells. Empty and TCF7L1 virus-infected NIH 3T3 cells at 0 h and 24 h after DMI plus TZD addition. (G) TCF7L1 promotes PPARγ2 gene enhancer accessibility in NIH 3T3 cells. Empty and TCF7L1 NIH 3T3 cells treated for 24 h with DMI plus TZD. (H) Zfp423 expression levels. For D–H, graphed values represent mean ± SEM (n =3).**P < 0.01; **P < 3T3-L1 preadipocytes and NIH 3T3 cells infected with TCF7L1 or empty virus, 0.01; ***P < 0.001. ns, not significant. CELL BIOLOGY

Cristancho et al. PNAS | September 27, 2011 | vol. 108 | no. 39 | 16275 Downloaded by guest on September 24, 2021 insights into how cells can coordinate cell contact, structure, and ACKNOWLEDGMENTS. We thank P. Seale for a critical read of the transcriptional regulation during the process of differentiation. manuscript, R. Wells and members of the M.A.L. laboratory for valuable discussions, L. Everett for help with biostatistics, X. S. Liu for allowing access to Cistrome for bioinformatic analysis, and X. Liang for technical Materials and Methods assistance. We also thank the Functional Genomics Core of the Penn Further details on the materials and methods used in this study are provided Diabetes and Endocrinology Research Center (J. Schug) [supported by in SI Materials and Methods. National Institutes of Health (NIH) Grant DK19525] for deep sequencing, the Penn Bioinformatics Core (D. Baldwin and J. Tobias) for microarray analysis, and S. Soleimanpour, G. P. Swain, and the Morphology Core (sup- Cell Culture. Murine 3T3-L1, 3T3-F442A, NIH 3T3, MEFs, and BOSC cells were ported by NIH Grant DK49210) for assistance with imaging. This work was maintained in DMEM (Invitrogen) supplemented with 10% FBS (Tissue Bio- supported by NIH DK49780 (to M.A.L.) and GM074048 (to C.S.C.). A.G.C. logicals) and 1% penicillin/streptomycin (Invitrogen). 3T3-L1, 3T3-F442A, and was supported by a Gilliam Fellowship from the Howard Hughes Medical- NIH 3T3 were differentiated as described in SI Materials and Methods. Institute.

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