bioRxiv preprint doi: https://doi.org/10.1101/365809; this version posted July 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Thy1 (CD90) expression is regulated by DNA methylation during adipogenesis E’Lissa M. Flores1, Collynn F. Woeller*2, Megan L. Falsetta2, Martha Susiarjo2, Richard P. Phipps1,2,3 Clinical & Translational Science Institute1, Department of Environmental Medicine2, Department of Microbiology and Immunology3, University of Rochester School of Medicine and Dentistry. Rochester, NY. *Corresponding author, [email protected] Corresponding author (to whom all correspondence should be addressed): Collynn Woeller, Ph.D. Research Associate Professor University of Rochester Department of Environmental Medicine 601 Elmwood Ave, Box 850, Room 3-11001 Rochester, NY 14642 Phone: 585-275-7836 The authors have no conflict of interest. Work funded by F31ES027767, TL1-TR000096, ES001247, and ES0023032 Running title: Thy1 methylation and adipogenesis Key words: adipogenesis, Thy1 (CD90), methylation, 5-aza-dC, epigenetics 1 bioRxiv preprint doi: https://doi.org/10.1101/365809; this version posted July 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Abstract 2 The obesity epidemic is developing into the most costly health problem facing the 3 world. Obesity, characterized by excessive adipogenesis and enlarged adipocytes, 4 promotes morbidities such as diabetes, cardiovascular disease and cancer. Regulation 5 of adipogenesis is critical to our understanding of how fat cell formation causes obesity 6 and associated health problems. Thy1 (also called CD90), a widely used stem cell 7 marker, blocks adipogenesis and reduces lipid accumulation. Thy1 knockout-mice are 8 prone to diet-induced obesity. While the importance of Thy1 in adipogenesis and 9 obesity is now evident, how its expression is regulated is not. We hypothesized that 10 DNA methylation plays a role in promoting adipogenesis and affects Thy1 expression. 11 Using the methylation inhibitor 5-aza-2’-deoxycytidine (5-aza-dC), we investigated 12 whether DNA methylation alters Thy1 expression during adipogenesis in both mouse 13 3T3-L1 pre-adipocytes and mouse mesenchymal stem cells. Thy1 protein and mRNA 14 levels were decreased dramatically during adipogenesis. However, 5-aza-dC treatment 15 prevented this phenomenon. Pyrosequencing analysis shows that the CpG sites at the 16 Thy1 locus are methylated during adipogenesis. These new findings highlight the 17 potential role of Thy1 and DNA methylation in adipogenesis and obesity. 18 19 Introduction 20 Obesity rates have risen markedly in the last 30 years. More than 700 million 21 people worldwide are clinically obese1,2. Obesity promotes type 2 diabetes, fatty liver 22 disease, and cardiovascular disease, and is linked with certain cancers3-5. Health care 23 costs associated with obesity and its comorbidities are enormous and will continue to 2 bioRxiv preprint doi: https://doi.org/10.1101/365809; this version posted July 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 24 rise as currents trends continue and the population ages6. Thus, a further understanding 25 of obesity and its underlying mechanisms and causes are urgently needed. 26 Obesity results from a positive energy balance when more calories are 27 consumed than are used. The surplus energy is packaged into lipid-based storage 28 molecules and sent to fat storage cells called adipocytes. In obesity, there is both an 29 increase in adipocyte size and an increase in adipocyte number to accommodate the 30 lipid7. Adipocytes are formed during the process of adipogenesis and arise from stem 31 cells, fibroblasts, or other progenitor cells when appropriately programmed8. 32 Adipogenesis is a highly regulated process that requires the activation of several key 33 signaling pathways, including STAT5 and Fyn and activation of the transcription factors 34 PPARγ and C/EBPα. Numerous genes involved in fatty acid transport and storage, such 35 as fatty acid binding protein 4 (Fabp4) are induced during adipogenesis to promote lipid 36 accumulation in adipocytes. 37 Several proteins including Pref-1, Wnt, TGFβ, and Thy1 (formally called CD90) 38 have been shown to inhibit adipogenesis by blocking pro-adipogenic signaling9-13. Our 39 recent study showed that Thy1 blocked the activity of the Src family kinase, Fyn, in pre- 40 adipocytes14. Thy1 mediated inhibition of Fyn activity prevented adipocyte formation. 41 Interestingly, while pre-adipocytes expressed high levels of Thy1, its expression was 42 lost during adipogenesis, and mature adipocytes expressed almost no Thy1. Thy1 is a 43 member of the immunoglobulin supergene family and is a glycophosphatidyl inositol 44 linked surface protein. While Thy1 is expressed on pre-adipocytes and subsets of 45 fibroblasts, neurons, and stem cells, little is known about how its expression is 46 controlled. We recently showed that Thy1 levels can be regulated by microRNAs. 3 bioRxiv preprint doi: https://doi.org/10.1101/365809; this version posted July 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 47 Specifically, the miR-103/107 family of miRNAs can target Thy1 mRNA and reduce its 48 expression15. Furthermore, the Thy1 gene contains several CpG rich elements termed 49 CpG islands, which are hotspots for cytosine methylation and gene regulation16,17. To 50 date, there have been no reports studying DNA methylation of Thy1 during 51 adipogenesis. However, Thy1 methylation has been studied in context of fibrosis and in 52 T cells, where they show an increase in Thy1 methylation correlated with a decrease in 53 Thy1 expression18-20. Therefore, we investigated the same CG rich region within intron 1 54 (termed Thy1-CGI1), which is part of the promoter17,20-23. 55 While changes in DNA methylation patterns at the Thy1 locus have not been 56 characterized in the context of adipogenesis, recent reports have shown that DNA 57 methylation changes are an integral part of adipocyte formation24,25. In the most widely 58 used and well accepted model of adipogenesis, the murine 3T3-L1 pre-adipocyte line, 59 global DNA methylation has been shown to increase during adipogenesis26,27. 60 Interestingly, addition of the DNA methylation inhibitor 5-aza-2’-deoxycytidine (5-aza- 61 dC) at the onset of adipogenic differentiation can completely block adipocyte formation, 62 suggesting that DNA methylation changes are essential for adipogenesis27,28. Specific 63 changes in regional DNA methylation are also critical for adipogenesis, and blocking 64 these patterns can alter the differentiation pathway of precursor cells towards 65 osteoblastogenesis rather than adipogenesis24,29,30. For example, when blocking DNA 66 methylation using 5-aza-dC in 3T3-L1 cells, Wnt10a expression increases through the 67 hypomethylation of several of its CpG sites. The increase in Wnt10a expression helps 68 steer the differentiation program towards osteoblastogenesis and away from 69 adipogenesis30. 4 bioRxiv preprint doi: https://doi.org/10.1101/365809; this version posted July 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 70 Because Thy1 protein and mRNA levels are rapidly suppressed during 71 adipogenic differentiation, we hypothesized that its reduced expression is linked to 72 hypermethylation of the Thy1 locus. Excitingly, we are the first to show methylation 73 sensitive pyrosequencing data of the Thy1-CGI1 region and report herein that DNA 74 methylation is one of the essential regulators of Thy1 expression during adipogenesis. 75 76 Results 77 Reduced Thy1 expression during adipogenesis is partially attenuated by 5-aza-dC in 78 bone marrow-derived mouse mesenchymal stem cells. 79 Previously, we demonstrated that pre-adipocytes and mesenchymal stem cells 80 must down-regulate Thy1 expression to differentiate into adipocytes14. To determine if 81 DNA methylation regulates Thy1 expression during adipogenesis, we first 82 pharmacologically inhibited DNA methyltransferase in bone-marrow derived mouse 83 mesenchymal stem cells (mMSCs) before differentiation into adipocytes28. mMSCs 84 were treated daily with either DMSO (vehicle) or the DNA methyltransferase inhibitor, 5- 85 aza-dC for 12 days and were concurrently treated with either media alone or with the 86 adipogenic cocktail medium (ACT), starting at day 3 (Fig. 1A). mMSCs typically 87 differentiate into adipocytes in 2 weeks when exposed to ACT31. Therefore, we 88 examined an intermediate time point to examine Thy1 levels. As expected, when given 89 ACT, Thy1 levels decreased while fatty acid binding protein 4 (Fabp4) levels were 90 increased compared to media alone (Fig. 1B-D); Fabp4 is highly expressed by 91 adipocytes and serves as an adipogenic marker. However, inhibiting DNA methylation 92 with 5-aza-dC increased Thy1 protein and mRNA levels versus media alone and 5 bioRxiv preprint doi: https://doi.org/10.1101/365809; this version posted July 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 93 attenuated the down-regulation of Thy1 in ACT samples (Fig. 1B-C). We also 94 determined that protein and mRNA levels of Fabp4, a protein highly expressed by 95 adipocytes that serves as an adipogenic marker, were decreased in mMSCs treated 96 with 5-aza-dC (Fig. 1B,D). Immunofluorescence staining revealed that compared to 97 cells not treated with ACT, 5-aza-dC-treated mMSCs had increased surface expression 98 of Thy1 and decreased Fabp4 levels (Fig. 1E). These results indicate that inhibition of 99 DNA methylation impairs adipogenesis.