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-bind- ing 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 tran- scription 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