26 The 5′ proximal gene region of CD133 containing promoter-1 and -2 are located in a CpG island, indicating that they may be sensitive to DNA methylation. Given that inhibition of DNMTs induced CD133 expression, we investigated whether TGFβ1 regulated CD133 transcription through

CD133 promoter demethylation. Using human CD133 promoter-1 driving luciferase vectors, we performed in vitro methylation using M. SssI followed by restriction enzyme HpaII cleavage to verify methylation status (Supporting Information Fig. 2). The luciferase activity of CD133 promoter-1 was significantly reduced by 53-fold following in vitro methylation, indicating that CD133 transcription was silenced by promoter methylation (Supporting Information Fig. 3). To examine if DNMTs regulate CD133 expression through promoter methylation, we examined DNMT1, learn more DNMT3α, and DNMT3β expression in CD133+ and CD133− cells. As demonstrated, there was no

significant difference in DNMT1 expression between CD133+ and CD133− cells at baseline, and TGFβ1 stimulation reduced DNMT1 expression (Fig. 6A, upper panel). DNMT3α expression was significantly higher in CD133− cells compared to CD133+ cells, and TGFβ1 was not able to significantly alter DNMT3α expression (Fig. 6A, middle panel). DNMT3β expression is elevated in CD133− cells compared to CD133+ cells, and, DNMT3β expression was significantly suppressed by TGFβ1 stimulation in CD133− cells (Fig. 6A, lower panel).

Given that DNMT1 and DNMT3β expression appeared PLX4032 order to be regulated by TGFβ, we used inhibitory Smads to examine if this effect was promoted by a Smad-dependent pathway. Huh7 cells were transfected with Smad6 or Smad7 vectors followed by 5 ng/mL TGFβ1 stimulation. TGFβ1 suppression of DNMT1 expression was not rescued by inhibitory Smads (data not shown). However, inhibitory Smads were capable of attenuating the effect of TGFβ1 on DNMT3β expression (Fig. 6B). This expression pattern was confirmed using qPCR (Fig. 6C). These results indicate that TGFβ1 is involved in the regulation of DNMT expression. Next, we examined nuclear DNA methyltransferase activity in CD133− Bay 11-7085 cells. After TGFβ stimulation, global DNMT activity in CD133− cell nuclei was significantly reduced, from 7.8 ± 2.5 units/hr/mg in untreated cells to 4.4 ± 0.8 units/hour/mg (n = 3, mean ± SD, P < 0.05). Because TGFβ1 suppresses DNMT activity and increases CD133 expression, we investigated if TGFβ1 induces CD133 expression through promoter demethylation. CD133− cells were stimulated with 5 ng/mL TGFβ1 for 48 hours and genomic DNA was extracted and subjected to pyrosequencing analysis. As shown in Fig. 7A, two DNA fragments containing seven potential CpG methylation sites within CD133 promoter-1 were analyzed. TGFβ1 was able to significantly reduce the methylation percentage across multiple CpG sites after 48 hours incubation (Fig. 7B, Supporting Information Fig. 4).