Hpych4iv digestion

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).

Zfp423 promoter (−1037/−1002) was amplified by PCR and cloned into the firefly luciferase reporter pCpGfree-promoter-Lucia vector (InvivoGen). A one-step PCR-based mutagenesis technique was used to generate site-specific mutation [28 (link)] and produce a mutated construct. One complementary pair of primers was designed that contained the desired mutation, replacing the cytosine at position −1016 with adenine. The wild-type and mutated constructs were transformed into E. coli GT115 cells. These cells were purchased from InvivoGen and are mycoplasma-free. In vitro methylation was performed using M.SssI methyltransferase following the manufacturer’s protocol (New England BioLabs). Unmethylated wild-type and mutated constructs were obtained in the absence of M.SssI. Methylation was confirmed by resistance to HpyCH4IV digestion (New England Biolabs). After 48 h, firefly and Renilla luciferase activity were assayed using a luciferase reporter assay kit, as reported in the previous paragraph.