Inhibiting DNA methylation switches adipogenesis to osteoblastogenesis by activating Wnt10a

Both adipocytes and osteoblasts share the mesodermal lineage that derives from mesenchymal stem cells. Most studies investigating the mechanisms underlying the regulation of adipogenic or osteoblastogenic development focus on transcriptional pathways; little is known about the epigenetic mechanisms in this process. We thus determined the role of 5-aza-2′-deoxycytidine (5-Aza-dC), an inhibitor of DNA methylation, in the lineage determination between adipogenesis and osteoblastogenesis. Inhibiting DNA methylation in 3T3-L1 preadipocytes by 5-Aza-dC significantly inhibited adipogenesis whereas promoted osteoblastogenesis. This dual effect of 5-Aza-dC was associated with up-regulation of Wnt10a, a key factor determining the fate of the mesenchymal lineage towards osteoblasts. Consistently, IWP-2, an inhibitor of Wnt proteins, was found to prevent the anti-adipogenic effect of 5-Aza-dC in 3T3-L1 preadipocytes and block the osteoblastogenic effect of 5-Aza-dC in ST2 mesenchymal stem cell line. Finally, the Wnt10a 5′-region is enriched with CpG sites, whose methylation levels were markedly reduced by 5-Aza-dC. Thus we conclude that inhibiting DNA methylation by 5-Aza-dC mutual-exclusively regulates the lineage determination of adipogenesis and osteoblastogenesis by demethylating Wnt10a gene and upregulating its expression. Our study defines DNA methylation as a novel mechanism underlying adipocyte and bone cell development.

proliferation and development 8 . Without the presence of Wnt proteins (i.e., Wnt10a), β -catenin, a component of a repressing complex comprised of Axin, adenomatosis polyposis coli (APC), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α ), is subjected to phosphorylation by the kinases GSK3 and CK1α within the complex, and is subsequently programmed for ubiquitin-associated proteosomal degradation 9 . With the presence of the Wnt proteins, β -catenin can be stabilized in the cytosol by dissociation from the repressing complex, translocate into the nucleus, and co-activate the transcriptional factors TCF/LEF, which in turn promotes transcription of target genes 9 . Wnt signaling pathways are highly conserved and have been extensively investigated organismal development, cancer, and stem cell biology 8 . Research has established the Wnt/β -catenin signaling as a key determinant of the fate between adipogenic and osteobalstogenic lineages 5,10 . For example, Activation of Wnt signaling suppresses adipogenesis by inhibiting PPARγ and C/EBPα 5 . Wnt family proteins have also been shown to exert a coordinated control over inhibition of adipogenesis and stimulation of osteoblastogenesis via a Wnt/β -catenin-dependent mechanism 10 .
Most studies investigating the mechanisms underlying the regulation of adipogenesis and osteoblastogenesis focus on transcriptional pathways; little is known about the epigenetic mechanisms in this process. Epigenetic regulation, including DNA methylation, is a molecular link between environmental factors (e.g. diets) and complex diseases, including obesity and diabetes. DNA methylation of cytosines at primarily CpG dinucleotides is the most common epigenetic modification. CpGs are often enriched in the promoter and the first exon/5′ -untranslated region of genes 11 . De novo methylation is mediated by DNA methyltransferase (DNMT) 3a and 3b. Once established, DNA methylation is then maintained through mitosis primarily by the maintenance enzyme DNMT1 12 . Promoters of transcriptionally active genes are typically hypo-methylated 13 , whereas DNA hyper-methylation can result in gene silencing by affecting the binding of methylation-sensitive DNA binding proteins and/or by interacting with various histone modifications and co-repressors that alter DNA accessibility to transcriptional factors 13 . Evidence converges to suggest that epigenetic events figure prominently in adipogenesis 14,15 . This is a new emerging research area; however, much remains to be discovered on how epigenetic mechanisms regulate lineage determination of adipogenesis vs. osteoblastogenesis.
5-Aza-dC is a nucleoside-based DNA methyltransferase inhibitor widely used to study the role of DNA methylation in regulation of cell development and cancer 16,17 . In the present study, we aimed to determine the role of 5-aza-2′ -deoxycytidine (5-Aza-dC), an inhibitor of DNA methylation, in regulation of adipogenesis and osteoblastogenesis. We characterized the adipogenic and osteoblastogenic phenotypes in 3T3-L1 preadipocytes and ST2 mesenchymal stem cells where the DNA methylation was pharmacologically inhibited by 5-Aza-dC. We then explored the molecular mechanism underlying the phenotypic changes of these cells by inhibition of DNA methylation.

Results
Inhibition of DNA methylation by 5-Aza-dC at the early stage of differentiation suppresses 3T3-L1 adipogenesis. We employed a pharmacological approach to inhibit DNA methylation during 3T3-L1 differentiation. 5-Aza-dC is a nucleoside-based DNA methyltransferase inhibitor widely used to study the role of DNA methylation in the regulation of cell development and cancer 16,17 . 3T3-L1 preadipocytes were induced to differentiate with a differentiation medium and were concurrently treated with either PBS or 5-Aza-dC at day 1-2, 3-5, or 6-8, and cells were harvested at day 8 of the differentiation. We found that inhibiting DNA methylation by 5-Aza-dC at the early differentiation stage from day 1-5 inhibited adipogenesis, evident by decreased mRNA expression of adipogenic markers such as PPARγ 2, CEBPα , and aP2, (Fig. 1A). However, the inhibitory effect of 5-Aza-dC disappeared when cells were treated with 5-Aza-dC at the late stage of differentiation (day 6-8) (Fig. 1A). Similar inhibition of PPARγ at protein levels was also observed in 3T3-L1 cells treated with 5-Aza-dC at the early stage of differentiation at day 1-2 ( Fig. 1B). Oil Red O staining revealed less lipid droplet accumulations in 3T3-L1 cells treated with 5-Aza-dC at the early differentiation stage of day 1-2 ( Fig. 1C). These data suggest that inhibition of DNA methylation by 5-Aza-dC at the early stage of differentiation suppresses adipogenesis.
The Wnt10a expression is up-regulated by 5-Aza-dC in 3T3-L1 cells. To further explore the mechanism underlying the suppression of adipogenesis by the early inhibition of DNA methylation with 5-Aza-dC, we surveyed the expression of a number of key transcriptional factors that control the adipogenic program. Since demethylation of gene promoters by 5-Aza-dC activates gene expression and the early treatment of 5-Aza-dC led to inhibition of adipogenesis in our study, we reckoned that the anti-adipogenic transcriptional factors might be 5-Aza-dC's potential targets, activation of which may be responsible for the suppression of adipogenesis by the treatment of 5-Aza-dC during early differentiation. Among the adipogenic repressors we screened using a group of pooled samples (n = 3-6 samples pooled for one real-time PCR measurement), Wnt10a mRNA expression was dramatically induced by early treatment of 5-Aza-dC, with a peak level reached at day 1-2 ( Fig. 2A). We confirmed this result by measuring Wnt10a expression in individual samples (Fig. 2B). This evidence suggests that Wnt10a may play a key role in inhibiting adipocyte differentiation by 5-Aza-dC treatment.
The Wnt10a 5′ untranslated region including part of the first exon is enriched with CpG sites, raising the possibility that Wnt10a may be regulated by DNA methylation. Among approximate 46 CpG sites in the 5′ region, we chose to measure the methylation status of the 33 CpG sites located on the first exon region before the start codon (Supplemental Fig. 1). Using pyrosequencing analysis, we found that DNA methylation levels were significantly reduced on all 33 CpG sites measured in 3T3-L1 cells treated with 5-Aza-dC at day 0-2 of differentiation (Fig. 2C). These data suggest that demethylation of Wnt10a gene by 5-Aza-dC may cause Wnt10a overexpression, leading to suppression of adipogenesis during the early stage of differentiation.
We further examined phospho-GSK3 and β -catenin levels, and found that both phospho-GSK3β and β -catenin protein content were up-regulated in 3T3-L1 cells treated with 5-Aza-dC at the early differentiation stage (Fig. 3A,B). Since Wnt/β -catenin signaling is important in the determination of adipogenic vs. osteobalstogenic fate 5,10 , we tested whether activation of Wnt/ β -catenin signaling would increase osteoblastogenesis in the 3T3-L1 cells treated with 5-Aza-dC. Indeed, the early treatment of 5-Aza-dC enhanced the expression of osteoblastogenic markers such as Bone Gamma-Carboxyglutamate (Gla) Protein/ Osteocalcin (BGLAP) and Distal-Less Homeobox 5 (Dlx5) (Fig. 3C). These data suggest that inhibiting DNA methylation by 5-Aza-dC increases Wnt10a expression by demethylating the 5′ untranslated region of the gene, which in turn activates Wnt/ β -catenin signaling, leading to reciprocal down-regulation of adipogenesis and up-regulation of osteoblastogenesis.

Inhibition of DNA methylation by 5-Aza-dC enhances osteobalstogenesis in ST2 stem cells.
To further determine the role of DNA methylation in regulation of osteoblastogenesis, we used ST2 cells, a mesenchymal stem cell model that has a pluripotent capacity to develop into either adipocytes or osteoblasts depending upon differentiation conditions. ST2 cells were induced to differentiate into osteoblasts in the presence of either PBS or 5-Aza-dC. We found that treatment of 5-Aza-dC markedly induced mRNA expression of Wnt10a during osteoblastogenesis (Fig. 4A). This was associated with up-regulation of the expression of osteoblastogenic markers such as alkaline phosphatase (Alpl), BGLAP, Basic Helix-Loop-Helix Transcription Factor 1 (Twist1) (an early osteoblastogenic marker 18 ), and Dlx5 ( Fig. 4B-E). We further confirmed osteoblastogenic phenotype in differentiated ST2 cells by measuring calcium content and Alkaline Phosphatase activity. Treating ST2 cells with 5-Aza-dC for 21 days during osteoblastogenesis significantly increased calcium content, as measured by Alizarin red staining and quantitation (Fig. 5A,B). Similar results were observed on alkaline phosphatase activity in ST2 cells treated with 5-Aza-dC (Fig. 5C). 3T3-L1 preadipocyte were induced to differentiate with a differentiation medium as described in the Materials and Methods, and were concurrently treated with either PBS or 5-Aza-dC at day 1-2, 3-5 and 6-8 as indicated in the figure and cells were harvested at day 8 of the differentiation for the measurement of gene expression. mRNA and protein levels were measured by real-time RT-PCR and immunoblotting, respectively. Oil Red O staining were performed as described in Materials and Methods. Representative blots and images are shown in (B,C), respectively. All data are expressed as mean ± SEM (n = 3-6); *p < 0.05; **p < 0.01 vs. control.

Activation of the Wnt signaling mediates the anti-adipogenic effect of 5-Aa-dC in 3T3-L1 cells.
To determine whether activation of Wnt signaling mediates the anti-adipogenic effect of 5-Aza-dC, we blocked Wnt signaling with a pharmacological approach in 3T3-L1 cells. We chose to use IWP-2, an inhibitor of Wnt protein production 19 . 3T3-L1 cells were induced to differentiate with an adipogenic cocktail and were concurrently treated with either PBS or 5-Aa-dC in the presence or absence of IWP-2 at day 1-2 of the differentiation period. Interestingly, treatment of IWP-2 completely reversed the inhibitory effect of 5-Aza-dC on the expression of PPARγ , aP2 and fatty acid synthase (FAS) mRNA levels and partially blocked the inhibitory effect of 5-Aza-dC on the expression of C/EBPα mRNA level (Fig. 6A). Moreover, Oil Red O staining revealed less lipid droplet accumulation in 3T3-L1 cells treated with 5-Aza-dC at day 1-2 of the early differentiation stage, which was completely reversed by IWP-2 treatment (Fig. 6B). These data suggest that inhibition of the Wnt signaling by IWP-2 prevents the anti-adipogenic effect of 5-Aa-dC in 3T3-L1 cells. Activation of the Wnt signaling mediates the pro-osteoblastogenic effect of 5-Aa-dC in ST2 cells. We further determined whether activation of Wnt signaling mediates the pro-osteoblastogenic effect of 5-Aza-dC in ST2 cells. ST2 stem cells were induced to differentiate with an osteoblastogenic regimen and were concurrently treated with either PBS or 5-Aza-dC in the presence or absence of IWP-2 in the differentiation period. We found that treatment of IWP-2 completely reversed the inhibitory effect of 5-Aza-dC on the expression of the osteoblastogenic marker BGLAP (Fig. 7). These data suggest that inhibition of the Wnt signaling by IWP-2 prevents the osteoblastogenic effect of 5-Aa-dC in ST2 cells.
Inhibition of DNA methylation by 5-Aza-dC promotes osteoblastogenic conversion in the adipocyte lineage 3T3-L1 cells. 3T3-L1 fibroblasts are committed preadipocytes belonging to adipocyte lineage cells 20 , which are presumably difficult to be differentiated into osteoblasts via lineage conversion. To determine the ability of 5-Aza-dC to promote osteoblastogenesis, we adopted the adipocyte lineage cells 3T3-L1 to study osteoblastogenic process. 3T3-L1 cells were induced to differentiate with an osteoblastogenic regimen and were concurrently treated with either PBS or 5-Aa-dC during the differentiation period. As expected, treatment of 5-Aza-dC stimulated the expression of Wnt10a in 3T3-L1 cells subjected to osteoblastogenic     IWP-2 prevents the stimulatory effect of 5-Aza-dC on the expression of the osteoblastogenic marker BGLAP. ST2 stem cells were induced to differentiate with an osteoblastogenic regimen and were concurrently treated with either PBS or 5-Aza-dC in the presence or absence of IWP-2 in the differentiation period. ST2 cells were harvested at day 21 after fully differentiated into osteoblasts. Gene expression was measured by real-time RT-PCR and normalized to cyclophilin. All data are expressed as mean ± SEM (n = 3-6); groups labeled with different letters are statistically different from each other.

Discussion
Both Adipocytes and osteoblasts originate from the mesenchymal stem cells 1,2 . The signaling pathways regulating lineage determination of the mesenchymal stem cells have received intense investigation. Recent data suggest that Wnt/β -catenin mutual-exclusively determines adipogenic or osteoblastogenic lineage 5,10 . In the present study, we demonstrate that DNA methylation plays an important role in determining adipogenic versus osteoblastogenic lineage through regulating Wnt10a gene. Our data indicate that inhibiting DNA methylation by 5-Aza-dC at the early stage of differentiation suppresses adipogenesis, while promoting osteoblastogenesis presumably through stimulating Wnt10a expression by demethylating its promoter.
We demonstrate in this study that DNA methylation orchestrates the adipogenic or osteoblastogenic program through Wnt10a. We actually observed a transient but profound induction of DNA methyltransferase 1 (DNMT1), a key enzyme for maintaining DNA methylation, within 48 hours of the early adipogenic stage (data not shown and will be reported elsewhere). The unique expression pattern of DNMT1 during early adipogenesis is extremely interesting and may dictate the terminal fate of adipogenesis distinguished from osteoblastogenesis. Methylation of gene promoter by DNMTs typically results in gene silencing. We reasoned that the surge of DNMT1 expression at the beginning of adipogenesis may silence the early adipogenic repressor Wnt10a, which may be critical in determining adipogenic fate 10 . Indeed, we found that inhibiting DNA methylation by 5-Aza-dC demethylates the Wnt10a promoter and subsequently induces its expression, leading to the suppression of adipogenic program and the simultaneous promotion of osteoblastogenic program. The anti-adipogenic and pro-osteoblastogenic effects of 5-Aa-dC can be largely reversed by a pan-Wnt inhibitor IWP-2. It is noteworthy that several Wnt family proteins such as Wnt10a, Wnt10b, and Wnt6 have been shown to mutual-exclusively regulate the terminal fate of osteoblastogenesis versus adipogenesis 10 . Interestingly, a number of Wnt ligands such as Wnt4, Wnt5a, Wnt 5b, Wnt 7b, Wnt 9b and Wnt11 are subjected to DNA methylation 21 . We found that only Wnt10a was significantly increased in 5-Aza-dC treated 3T3-L1 and ST2 cells, but not other Wnt members. Therefore, inhibition of DNA methylation by 5-Aza-dC treatment may mainly target Wnt10a during 3T3-L1 adipogenesis and ST2 osteoblastogenesis. Nonetheless, additional studies involving genetic approaches are required to identify and confirm 1) which DNMTs are responsible for the anti-adipogenic and osteoblastogenic effects of 5-Aa-dC; 2) which Wnt ligand(s) mediates these effects of 5-Aa-dC. On the other hand, it would be interesting to know whether DNMTs also act on other gene promoters to regulate transcription during adipogenesis and osteoblastogenesis.
Most studies investigating the mechanisms underlying the regulation of adipogenesis and osteoblastogenesis focus on transcriptional pathways; little is known about the epigenetic mechanisms in this process. Epigenetic regulation, including DNA methylation, is a molecular link between environmental factors (e.g. diets) and complex diseases, including obesity and diabetes. This is a new emerging research area. However, evidence converges to suggest that epigenetic events figure prominently in adipogenesis 14,15 . Using genome-wide epigenetic mapping, Mikkelsen et al. have found regulated changes of histone methylation/acetylation marks during adipogenesis 22 . These dynamic changes of histone marks may actively regulate adipogenesis through interaction with key transcriptional activators or repressors that control adipogenic program. Indeed, the transcriptional active mark H3K4me has been shown to promote adipogenesis through interacting with the promoter of PPARγ 14 . In addition, Wnt proteins have been identified as epigenetic targets during adipogenesis. For example, histone methyltransferase G9a deletion decreases the methylation at the repressive histone mark histone 3 lysine 9 (H3K9) at Wnt10a promoter, leading to Wnt10a overexpression and subsequent inhibition of adipogenesis 23 . Further, evidence suggests that histone 3 lysine 27(H3K27) methylation inhibits adipogenesis through de-repressing the Wnt family proteins 14 . Changes in DNA methylation have been shown to modulate histone methylation, which may act cooperatively to influence chromatin structure and thereby regulate gene expression 24 . This raises another interesting question as to whether DNA methylation also regulates adipogenesis and osteoblastogenesis through modulating histone methylation. Additional studies are warranted to investigate the potential crosstalk between DNA methylation and histone modification in the reciprocal regulation of adipogenesis and osteoblastogenesis.
It is noteworthy that the DNA methylation inhibitor 5-Aza-dC has been used to convert embryonic stem cells C3H10T1/2 into mesodermal stem cell lineages that can further differentiate into myocytes, chondrocytes and adipocytes 25,26 . Further studies from Dr. Daniel Lane's group demonstrated that a subpopulation of 5-Aza-dC-induced C3H10T1/2 cells have a strong propensity to differentiate into adipocyte upon treatment with an adipogenic differentiation regimen 27 . The discrepancy between their observations and our findings are not clear. One possibility might be the different cell models at different developmental stages that are used for differentiation in these published studies. C3H10T1/2 cells are a kind of embryonic stem cells that presumably have pluripotent potentials to develop into diverse cell types 1 , while either 3T3-L1 preadipocytes or bone marrow-derived ST2 cells 28 are more committed cell types in the realm of the mesodermal lineage. Therefore, the ability of 5-Aza-dC to suppress adipogenesis and promote osteoblastogenesis may only occur in more committed cell types. Nonetheless, a more recent report that investigated the role of 5-Aza-dC in osteogenesis in adipose-derived stem cells is consistent with our findings 29 . Yan et al. recently demonstrated that 5-Aza-dC promotes osteogenesis in human adipose-derived mesenchymal cells by DNA demethylation 29 .
Adipogenesis contributes to the development of obesity 30,31 , which was thought be protective against osteoporosis due to the beneficial effect of enhanced mechanical loading from the excessive weights [32][33][34] . The positive effect of obesity on bone mineral density 35 was questioned by other large population studies showing no positive correlation between bone mass and BMI 34,36,37 . Instead, recent epidemiological studies demonstrated that increased fat mass particularly visceral fat is associated with osteoporosis 32,33 . The underlying mechanisms linking increased adiposity to bone loss involve the endocrine and immune functions of adipose tissue, which secretes numerous adipokines and cytokines 32,33 . These fat-derived factors during the development of obesity are detrimental to the bone formation 32 . Another potential link between obesity and bone loss may reside on the bone marrow mesenchymal stem cells (MSCs), the common precursors capable of becoming both adipocytes and osteoblasts 32,38 . It is noteworthy that the fate of MSCs is regulated by signaling pathways such as Wnt/β -catenin that mutual-exclusively determines adipogenic or osteoblastogenic lineage 5,10 . Our study demonstrates an epigenetic mechanism controlling the commitment of MSCs into either adipocytes or osteoblasts via modulation of DNA methylation at the Wnt10a promoter. Future studies are warranted to determine whether obesity-related factors such as pro-inflammatory cytokines can increase the promoter methylation of Wnt10a, resulting in diminished capacity of MSCs to become osteoblasts and leading to osteoporosis.
In summary, we demonstrate that inhibiting DNA methylation by 5-Aza-dC at the early stage of differentiation significantly inhibits adipogenesis in 3T3-L1 cells whereas promoting osteoblastogenesis. 5-Aza-dC demethylates Wnt10a gene, leading to enhanced Wnt10a expression, which likely mediates the anti-adipogenic and pro-osteoblastogenic effect of 5-Aza-dC in 3T3-L1 and ST2 cells. We conclude that DNA methylation plays a significant role in lineage determination of adipogenesis versus osteoblastogenesis. Our study defines DNA methylation as a novel mechanism underlying adipocyte and bone cell development. The findings could also help guide the development of epigenetic regulation as new therapeutic targets in the prevention and treatment of obesity and/or osteoporosis.
Scientific RepoRts | 6:25283 | DOI: 10.1038/srep25283 Methods Adipogenesis. 3T3-L1 preadipocytes (ATCC, Manassas, VA) were grown in a DMEM medium containing 10% new born calf serum (NBCS) and 5% penicillin. Two days after confluence, 3T3-L1 preadipocytes were induced to differentiate with DMEM containing 10% fetal bovine serum (FBS), 0.5 mM 3-isobutyl-1-methylxanthine, 1 μ M dexamethasone, and 400 nM insulin. Cells were incubated in this medium for two days and then cultured in growth medium containing 400 nM insulin for another 2 days. Subsequently, cells were maintained in growth medium (without insulin) throughout the adipocyte stage and cultures were refed every two days. Cells were treated with either PBS or 5-Aza-dC (0.5 μ M) at different time points indicated in the figures and were harvested at day 8 of the differentiation.
Osteoblastogenesis. To induce osteoblastogenesis, 3T3-L1 or ST2 cells were grown and incubated in a DMEM low glucose medium containing 10% FBS and 5% Penicillin solution. Two days after confluence (post-confluence), 3T3-L1 preadipocytes or ST2 stem cells (designated as day 0) were cultured with a DMEM containing 10% FBS, 10 mM β -glycerophosphate and 20 μ g/ mL ascorbic acid-2-phosphate. The osteoblastogenic medium was replaced every 2 days afterwards. Cells were treated with either PBS or 5-Aza-dC throughout the differentiation process and were harvested at different points indicated in the figures. In some experiments, ST2 cells were treated with either PBS or 5-Aza-dC in the presence or absence of 10 mM IWP-2 and were harvested at different time points indicated in the figures.
Oil red O staining. Accumulation of lipids in adipocytes was assessed by Oil Red O staining. Oil Red O was purchased from Sigma-Aldrich (St. Louis, MO) and was prepared as 0.5% solution in isopropanol. Cells were fixed with 10% formaldehyde for at least 1 hour and washed twice with water. Oil red O working solution was prepared in the ratio of 40% water and 60% Oil red O, left at least 20 minutes, filtered and then added to fixed cells. Cells were incubated at room temperature for 10 minutes and then washed 4 times with water.
Alizarin Red staining. The degree of mineralization in osteoblasts was determined using Alizarin Red staining 39 . Alizarin Red S (catalog # A5533) was purchased from Sigma-Aldrich (St. Louis, MO) and was prepared as 2% solution in water. The solution was filtered and added to fixed cells. Cells were incubated with the Alizarin Red solution at room temperature for 30 minutes. The Cells were then de-stained for quantification by adding 1 mL 5% perchloric acid and the absorbance was read in a 96 wells plate reader at 490 nm.
Measurement of alkaline phosphatase activity. The activity of alkaline phosphatase (Alp) in osteoblasts was measured using an Acid Phosphatase Assay Kit (catalog # CS0740) purchased from Sigma-Aldrich (St. Louis, MO). Cells were lysed by using a lysis buffer (1% v/v Igepal ® CA-630; 10 mM Tris-HCl; 1 mM MgCl 2 , pH 7.5) for overnight at − 80 °C. Lysates then were centrifuged at 12,000 rpm for 10 minutes and 50 μ l of supernatant was then used to measure Alp activity using the Acid Phosphatase Assay Kit according to manufacturer's instruction.
Total RNA extraction and quantitative RT-PCR. Total RNA of the 3T3-L1 cells and ST2 cells was extracted using the Tri Reagent kit (Molecular Research Center, Cincinnati, OH), according to the manufacturer's protocol. The expression of genes of interest was assessed by quantitative RT-PCR (ABI Universal PCR Master Mix, Applied Biosystems, Foster City, CA) using a Stratagene Mx3005p thermocycler (Stratagene, La Jolla, CA), as we previously described 40,41 . The primer and probe pairs used in the assays were purchased from Applied Biosystems (Foster City, CA).
Bisulfite conversion and pyrosequencing. Genomic DNA was prepared by phenol/chloroform extraction. Bisulfite conversion was performed using EpiTech Bisulfite Kit (Qiagen, Valencia, CA). The converted DNA was used as template to amplify DNA sequence covering the putative CpG sites at the Wnt10A or SREBP1C promoter regions. Approximately 1μ g of bisulfite converted DNA was amplified by PCR and pyrosequencing was performed by EpiGenDx (Hopkinton, MA). The PCR and pyrosequencing primers that were used to measure the DNA methylation on the Wnt10a 5′ region covering CpG sites are described in Supplemental Table 1.