The p16-Specific Reactivation and Inhibition of Cell Migration Through Demethylation of CpG Islands by Engineered Transcription Factors

Abstract

Methylation of CpG islands inactivates transcription of tumor suppressor genes including p16 (CDKN2A). Inhibitors of DNA methylation and histone deacylation are recognized as useful cancer therapeutic chemicals through reactivation of the expression of methylated genes. However, these inhibitors are not target genespecific, so that they lead to serious side effects as regular cytotoxic chemotherapy agents. To explore the feasibility of methylated gene-specific reactivation by artificial transcription factors, we engineered a set of Sp1-like seven-finger zinc-finger proteins (7ZFPs) targeted to a 21-bp sequence of the p16 promoter and found that these 7ZFPs could bind specifically to the target p16 promoter probe. Then the p16-specific artificial transcription factors (p16ATFs) were made from these 7ZFPs and the transcription activator VP64. Results showed that transient transfection of some p16ATFs selectively up-regulated the endogenous p16 expression in the p16-active 293T cells. Moreover, the transient transfection of the representative p16ATF-6I specifically reactivated p16 expression in the p16-methylated H1299 and AGS cells pretreated with a nontoxic amount of 5'-azadeoxycytidine (20 and 80 nM, respectively). In addition, stable transfection of the p16ATF induced demethylation of p16 CpG island and trimethylation of histone H3K4, and inhibited recruitment of DNA methyltransferase 1 and trimethylation of H3K9 and H3K27 in the p16 promoter in H1299 cells without 5'-aza-deoxycytidine pretreatment. Notably, inhibition of cell migration and invasion was observed in these p16-reactivated cells induced by transient and stable p16ATF transfection. These results demonstrate that p16ATF not only specifically reactivates p16 expression through demethylation of CpG islands, but also restores methylated p16 function.

Emerging Concept in DNA Methylation: Role of Transcription Factors in Shaping DNA Methylation Patterns

“…The characterization of the factors underlying the dynamic nature of DNA methylation and its plasticity with regards to environmental cues highlight potential pathways leading to changes in DNA methylation and how this information might be used for translational applications. For instance, in cell‐based assays, it is possible to reprogram the methylation level of a CpG island by targeting a TET‐fusion to the given CpG island (Zhang et al, ; Maeder et al, ). With the development of sophisticated systems of genome engineering, one can imagine in the future to use such strategies to revert aberrant methylation at a specific locus and treat a disease (with all the ethical issues such approaches will raised).…”

Prolonged re-expression of the hypermethylated geneEPB41L3using artificial transcription factors and epigenetic drugs

“…Artificial transcription factor (ATF)-mediated expressional changes, however, have shown to be only transient, as targeted promoters are incompletely reprogrammed and genes tend to return to the 'normal' state after the ATF is removed. 7,8 Recently, a number of TSGs were successfully reactivated using ZFPs linked to VP64, including maspin, 7,9,10 CDKN2A, 11 and C13ORF18, 12 and re-activation of these 3 TSGs was associated with site-specific DNA demethylation, while re-expression of the latter two was also shown to be accompanied with a decreased repressive histone methylation status. Despite these epigenetic changes, ATF-induced effects are transient, and more stable epigenetic reprogramming is required to prolong the effects.…”

Hydroxymethylated-P16 Allele Is Transcription-Inactive

“…The sequences of the universal primers used to amplify these fragments are listed in Table S1. 5 To construct the P16-specific DNA dioxygenase (P16-TET) expression vector, an SP1-like engineered seven-zinc finger protein (7ZFP-6I) [19] that can specifically bind to the 21-bp fragment (5'-gaggaaggaaacggggcgggg-3', including an Sp1-binding site) within the human P16 core promoter [28,29], was fused with the catalytic domain (CD: 1418-2136 aa) of human TET1 (NM_030625.2) [30] and inserted into a pcDNA3.1b vector and then used in transient transfection assays. An inactive P16-TET mutant containing an H1671Y mutation in the CD domain vector was also constructed and used as a negative control vector ( Figure S2A).…”

“…To study whether P16H affects gene transcription, an expression controllable P16-specific dioxygenase pcDNA3.1-vector (P16-TET) and its inactive mutant control vector were constructed through fusing an engineered P16 promoter-specific seven zinc finger protein (7ZFP-6I) [28] with the catalytic domain of human TET1 ( Figure S2A). H1299 cells were chosen because epigenetic editing of the methylated P16 CGIs by the P16-specific transcription factor (P16-ATF; 7ZFP-6I-VP64) has been optimized in this cell type [28]. As expected, the results of both qRT-PCR and immunofluorescence staining showed that the methylated P16 alleles were re-activated in H1299 cells 6 days after transient transfection with the P16-TET pcDNA3.1-vector ( Figure 2).…”