Enrichment of Cxcl12 promoter with TET2: A possible link between promoter demethylation and enhanced gene expression in the absence of PARP-1

Abstract

Previously, we described the link between C-X-C motif chemokine 12 (Cxcl12) gene induction and DNA hypomethylation in the absence of poly(ADP-ribose) polymerase 1 (PARP-1). We have now firmly established that demethylation is the primary cause of gene induction on the basis of Cxcl12 gene upregulation upon treatment with the demethylating agent 5-azacytidine (5-aza). Since the demethylation state of Cxcl12 is favored by PARP-1 absence, we investigated the presence of ten-eleven translocation (TET) proteins on the Cxcl12 promoter in order to corroborate the relationship between the demethylation process and increased gene expression that occurs in the absence of PARP-1. Analysis was performed on the promoter region within CpG islands of Cxcl12 from control mouse embryonic fibroblasts (NIH3T3) and PARP-1 knockout mouse embryonic fibroblasts (PARP1-/-). The lack of PARP-1 increased the abundance of TET2 on the Cxcl12 promoter, suggesting that TET-mediated demethylation provoked by the absence of PARP-1 could account for the observed increased expression of this chemokine. Deciphering the regulation of DNA (de)methylation factors that control Cxcl12 expression may provide an additional therapeutic approach in pharmacological interventions where gene switching on or off based on targeted stimulation or inhibition is necessary.

TET-mediated DNA hydroxymethylation is negatively influenced by the PARP-dependent PARylation

“…Though PARP-1 and PARylation can influence DNA (de)methylation by modulating DNA methyltransferase expression and activity [ 14 , 41 ], a detected increase in hydroxymethylation indicates that in this study DNA demethylation is achieved via TET activity induced by the lack of PARP-1 or inhibited PARylation. This is in line with and expands on our previous research showing that the inhibitory role of PARP-1 in the local regulation of Cxcl12 gene expression is in part achieved via the negative influence of PARP-1 on local TET-mediated DNA demethylation [ 20 , 21 ]. The activating effects on TET activity shown in our study could be the base for additional evaluation of the efficacy of PARP inhibitors in the treatment of cancers that are characterised by the diminishing level of 5hmC.…”

“…Consistent with a previous observation [ 17 ], we show that the recombinant murine TET1 catalytic domain is PARylated by PARP-1 in vitro. Furthermore, we show that the catalytic domain of TET2 is also PARylated in vitro, which may indicate the functional significance of already established PARP-1/TET2 interaction [ 19 , 21 ]. To enable purification of the TET2 catalytic domain, used in our in vitro experiments, its long unstructured region had to be removed [ 29 ], thus implying that PARylation target sites are within the structured parts of the TET2 catalytic domain.…”

“…Previously, we have discovered a connection between PARP-1 and TET enzymes on a local genomic level. We observed the inhibitory effect of PARP-1 on the demethylation of the Cxcl12 gene [ 20 , 21 ]. This study further explored the influence of PARP-dependent PARylation on TET hydroxylase activity at the genome level.…”

“…Besides, the interaction of endogenous TET1 and PARP-1 has been detected in HEK293T cells and it has been confirmed in vitro that this interaction can be established directly, without the mediation of other proteins [ 17 ]. In addition, we have previously demonstrated that TET2 can likewise interact with PARP-1 in NIH3T3 cells [ 21 ].…”

“…Our previous work indicated that PARP-1 affects TET activity on the local level of a single gene coding for the CXCL12 chemokine [ 20 , 21 ], propelling us to further explore PARP/TET cross-talk on a global level by specifically looking into the influence that PARP activity exerts on TET-mediated DNA hydroxymethylation/demethylation. It has been proposed that excessive TET activity can induce DNA damage as active DNA demethylation involves forming abasic sites in the last steps of the process [ 22 , 23 ].…”