Virion protein 16 induces demethylation of DNA integrated within chromatin in a novel mammalian cell model

Abstract

DNA methylation and demethylation play important roles in mediating epigenetic regulation. So far, the mechanism of DNA demethylation remains elusive and controversial. Here, we constructed a plasmid, named with pCBS-luc, that contained an artificial CpG island, eight Gal4 DNA-binding domain binding site, an SV40 promoter, and a firefly luciferase reporter gene. The linearized pCBS-luc plasmid was methylated in vitro by DNA methyltransferase, and transfected into the HEK293 cells. The stable HEK293 transfectants with methylated pCBS-luc (me-pCBS-luc) were selected and obtained. The methylation status of the selected stable cell lines were confirmed by bisulfite sequencing polymerase chain reaction amplification. The methylation status could be maintained even after 15 passages. The virion protein 16 (VP16) was reported to enhance DNA demethylation around its binding sites of the promoter region in Xenopus fertilized eggs. Using our me-pCBSluc model, we found that VP16 also had the ability to activate the expression of methylated luciferase reporter gene and induce DNA demethylation in chromatin DNA in mammalian cells. Altogether, we constructed a cell model stably integrated with the me-pCBS-luc reporter plasmid, and in this model we found that VP16 could lead to DNA demethylation. We believe that this cell model will have many potential applications in the future research on DNA demethylation and dynamic process of chromatin modification.

Components from the Human c-myb Transcriptional Regulation System Reactivate Epigenetically Repressed Transgenes

“…To determine AAP activity within silenced chromatin, we targeted AAP fusion proteins (Supplementary Figure S2) to a chromosomal luciferase reporter that had been previously targeted by Polycomb repressive complexes (PRCs). The AAP open reading frames (ORFs) encode catalytic subunits or full length proteins (Supplementary Figure S2) that have been shown to support an epigenetically active state in several prior studies [39,40,44,46,[58][59][60][61][62][63][64]. All of these ORFs exclude DNA binding and histone binding domains, except for the ORF encoding FOXA1, which has a catalytic domain that requires histone interactions.…”

5-Hydroxymethylcytosine and disease

“…Numerous studies have shown that many proteins are involved in DNA active demethylation, including Gadd45a [9], Elp3/KAT9 [10], AID/APOBEC [11][12][13][14], MBD4 [15,16], virion protein16 (Vp16) [17], SMUG1 [18], TGF-b [19], and FGF [20]. TETs and thymine DNA glycosylase-mediated (TDG-mediated) active demethylation are considered a classic pathway [21][22][23][24][25][26][27][28][29][30][31].…”

Components from the human c-myb transcriptional regulation system reactivate epigenetically repressed transgenes

“…to a chromosomal luciferase reporter that had been previously targeted by Polycomb repressive complexes (PRCs). The AAP open reading frames (ORFs) encode catalytic subunits or full length proteins (SupplementaryFigure S2) that have been shown to support an epigenetically active state in several prior studies(42,43,47,49,(61)(62)(63)(64)(65)(66)(67) . All of these ORFs exclude DNA binding and histone binding domains, except for the ORF encoding FOXA1 which has a catalytic domain that requires histone interactions.…”