E coli gt115 cells

ANKRD26 promoter (− 716/− 597 bp) was amplified by PCR. The purified PCR fragment was cloned into the firefly luciferase reporter pCpG-free-promoter-Lucia vector (Invivogen, Toulouse, France). This vector is completely void of CpG dinucleotides in all the elements required for replication and selection of the plasmid in E. coli and gene expression in mammalian cells. Also, it is devoid of all Dam methylation sites (GATC) to prevent prokaryotic methylation [51 ]. The pCpG-Ankrd26 vector was amplified in E. coli GT115 cells (Invivogen). In vitro methylation was performed using the M.SssI CpG methyltransferase following manufacturer’s protocol (New England BioLabs, Ipswich, MA). Un-methylated DNA was obtained in the absence of M.SssI. Methylation was confirmed by digestion with MspJI (New England BioLabs). HEK-293 cells were transfected with the CpG methylated or un-methylated pCpG-ANKRD26 vector and Renilla control vector (Promega, Madison WI) by lipofectamine (Life Technologies), following manufacturer’s instructions. Firefly luciferase activity of each transfection was normalized for transfection efficiency against Renilla luciferase activity.

Ankrd26 promoter (−733 bp/−344 bp) was amplified by PCR. The purified PCR fragment was cloned into the firefly luciferase reporter pCpGfree-promoter-Lucia vector (Invivogen, Toulouse, France). The following site-specific mutated constructs were generated by PCR-based mutagenesis: pCpG-Ankrd26-436, pCpG-Ankrd26-431, pCpG-Ankrd26-391. The wilde type (Wt) pCpG-Ankrd26 vector, used as template, was removed from the PCR reaction by DpnI digestion (New England BioLabs, Ipswich, MA). Wt and mutated (mut) vectors were amplified into E. coli GT115 cells (Invivogen). Site-specific mutagenesis of each construct was validated by sequencing. In vitro methylation was performed using the M.SsI CpG methyltransferase following manufacturer’s protocol (New England BioLabs). Un-methylated DNA was obtained in the absence of M.SsI. Methylation was confirmed by resistance to HpyCH4IV digestion (New England BioLabs).