Figure 3: The Mir29b2c gene is silenced by methylation in keratinocytes.

(a) Location of CpGs at the promoter region of the mouse A330023F24Rik gene harbouring the Mir29b2c cluster and of the human C1ORF132 gene containing the MIR29B2C cluster. Positions of AREs within the last exon of both genes (bound by Nrf2 in mouse keratinocytes, but not in human keratinocytes) are indicated; single CpGs chosen for validation by bisulfite conversion are indicated (CpG1,2 for A330023F24Rik and CpG1-25 for C1ORF132). The murine CpG region also contains two AREs not bound by Nrf2 (AREs). (b) MeDIP analysis of DNA from mouse epidermis showing enrichment of methylated DNA in intron 1 of the A330023F24Rik gene compared with the region >2 kb away from the CpGs (ns-CpG). Levels of DNA methylation of control genes used for MeDIP are shown in Supplementary Fig. 2C. (c) Bisulfite conversion and pyrosequencing analysis of the DNA from mouse epidermis shows highly methylated single CpGs at positions 1 and 2 indicated in a. (d) The promoter region (2.5 kb) of the A330023F24Rik lincRNA was cloned into a CpG-free pCpGL reporter vector, subjected to in vitro methylation, and luciferase activity was measured in cells transfected with the plasmid harbouring the methylated or non-methylated promoter. The basic CpG-free vector without promoter was used as negative control, the pCpGL vector with a CMV promoter served as positive control. N=4. (e) HaCaT cells and primary human keratinocytes (HPKs) were treated with DMSO (−AZA) or 5-aza-2′-deoxyuridine (+AZA), and levels of primary miR29B2C transcripts were measured by qRT-PCR. HaCaT cells were analysed in triplicates; two independent batches of human primary keratinocytes were analysed. (f) MeDIP analysis of the DNA isolated from primary human keratinocytes shows significant enrichment of methylated DNA in exon 1–intron 2 of the C1ORF132 gene. DNA methylation of control genes used for MeDIP are shown in Supplementary Fig. 2H. The NRF2-bound ARE of MIR29AB1 was used as negative control. (g) Bisulfite sequencing of the DNA from HPKs shows highly methylated CpGs at positions 1–25 (also indicated in a inside intron 2). Error bars represent s.d., t-test P value *<0.05; **<0.01. RT–PCR, reverse transcriptase-PCR.