Absence of PARP‐1 affects Cxcl12 expression by increasing DNA demethylation

Abstract Poly [ADP‐ribose] polymerase 1 (PARP‐1) has an inhibitory effect on C‐X‐C motif chemokine 12 gene (Cxcl12) transcription. We examined whether PARP‐1 affects the epigenetic control of Cxcl12 expression by changing its DNA methylation pattern. We observed increased expression of Cxcl12 in PARP‐1 knock‐out mouse embryonic fibroblasts (PARP1−/−) in comparison to wild‐type mouse embryonic fibroblasts (NIH3T3). In the Cxcl12 gene, a CpG island is present in the promoter, the 5′ untranslated region (5′ UTR), the first exon and in the first intron. The methylation state of Cxcl12 in each cell line was investigated by methylation‐specific PCR (MSP) and high resolution melting analysis (HRM). Both methods revealed strong demethylation in PARP1−/− compared to NIH3T3 cells in all four DNA regions. Increased expression of the Ten‐eleven translocation (Tet) genes in PARP1−/− cells indicated that TETs could be important factors in Cxcl12 demethylation in the absence of PARP‐1, accounting for its increased expression. Our results showed that PARP‐1 was a potential upstream player in (de)methylation events that modulated Cxcl12 expression.


| INTRODUC TI ON
Poly [ADP-ribose] polymerase 1 (PARP-1) is the founding member of the PARP family of enzymes which promotes the formation of ADP-ribose polymers (PARs) and their addition to PARP-1 itself and other acceptor proteins in a process referred to as PARylation. 1 PARP-1 is an abundant nuclear chromatin-associated protein involved in a plethora of functions such as DNA repair, recombination, cell proliferation and death, inflammation and gene transcription.
The regulatory functions of PARP-1 were established after its discovery four decades ago, and the initially described role in DNA repair was followed by confirmation of its involvement in transcriptional regulation. The link between PARP-1 and epigenetic events was hypothesised in light of its role related to genome stability and histone PARylation that leads to chromatin opening resembling the outcome of histone acetylation. The regulation of DNA demethylation is a newly discovered housekeeping role of PARP-1, which is realized through interaction with ten-eleven translocation enzymes 1 (TET1) and the ability of PARP-1 to PARylate TET1 both covalently and noncovalently. 2  converts 5mC to 5-hydroxymethylcyosine (5hmC) and then to 5formylcytosine (5fc) and 5-carboxylcytosine (5caC) by the action of the TET family of dioxygenases (TET1, TET2 and TET3). The DNA repair pathways remove 5fc and 5caC, rendering the cytosine unmethylated, with these sequential modifications of 5mC comprising the active DNA demethylation processes. 3,4 5hmC is mainly associated with promoter proximal regions or distal regulatory elements within CpG islands, which indicates its involvement in transcriptional regulation of gene expression. 2 We previously reported that PARP-1 has a pivotal role in suppressing the Cxcl12 gene promoter 5 as a transcriptional regulator with a strong binding affinity for the Cxcl12 promoter. CXCL12 is a chemokine produced in stromal tissues in multiple organs. CXCL12 is a potent chemoattractant involved in angiogenesis, leucocyte trafficking, stem cell homing and in processes including development, cell survival, tissue repair and regeneration. 6 CXCL12 plays an important role in β-cell differentiation, pancreatic islet genesis and in anti-apoptotic/anti-necrotic protection of β-cells from diabetogenic agents. 7,8 Moreover, CXCL12 is as an important player in various diseases (including cancer, inflammatory disorders, atherosclerosis, HIV pathology and diabetes), 9,10 hence the biological significance of methylation-dependent regulation of the Cxcl12 gene.
Our previous results regarding PARP-1-related suppression of Cxcl12 raised the question whether this regulatory role of PARP-1 controls Cxcl12 expression via an epigenetic mechanism. To address this possibility, we examined whether epigenetic events such as primary DNA de/methylation drive PARP-1-mediated suppression of Cxcl12 gene expression.
These concentrations correspond to the EC 50 for the two cell lines.

| Immunoblot analysis
Secreted proteins were precipitated with 13% trichloroacetic acid from the serum-free culture media in which NIH3T3 and PARP1−/− cells were cultivated for 24 hours. These samples were separated by 15% tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis (tricine-SDS-PAGE) and electrotransferred onto a polyvinylidene difluoride membrane. Immunoblotting was performed using the anti-CXCL12 primary antibody (FL-93, Santa Cruz Biotechnology, Santa Cruz, CA, USA) incubated overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated anti-rabbit secondary antibody at room temperature for 1 hour.
Staining was performed by the chemiluminescent technique according to the manufacturer's instructions (Amersham Pharmacia Biotech). The intensities of the signals were quantified using TotalLab electrophoresis software, ver. 1.10 (Phoretix, Newcastle upon Tyne, UK). Statistical significance was estimated by the t test.

| RNA isolation and real-time quantitative PCR (RT-qPCR)
The GeneJET RNA Purification Kit (Thermo Fisher Scientific, USA) was used to isolate total RNA from NIH3T3 and PARP1−/− cells, were used for RT-qPCR at the following thermal cycles: initial denaturation at 95°C for 10 minutes and 40 cycles of two-step PCR at 95°C for 15 seconds and at 60°C for 60 seconds. The relative expression of target genes was calculated relative to GAPDH (as an internal control) by the delta Ct method (2 dCt ). Statistical tests were performed using log2 transformed data and mean values, and error bars were back transformed to linear scale for graphs.
Statistical significance was estimated using paired t test by pairing NIH3T3 and PARP1−/− samples that were isolated simultaneously. Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/ primer-blast/) was used to design the primers (Table S1)

| Isolation of high molecular weight DNA
Cells were lysed in buffer (2 mmol/L EDTA, 10 mmol/L Tris HCl pH 7.5, 10 mmol/L NaCl, 0.5% SDS) supplemented with 0.04 µg/mL proteinase K and the lysate was incubated at 55°C overnight. High molecular weight DNA was isolated by ethanol precipitation (with cold 75 mmol/L sodium acetate diluted in absolute ethanol) and dissolved in water.

| P CR-BA S ED ME THYL ATION ANALYS IS
For DNA methylation analysis, four sets of primers were designed in MethPrimer (http://www.urogene.org/methprimer2/) which encompass four regions of the Cxcl12 gene: part of the promoter (1MU), the TSS, the exon-intron boundary (2MU) and part of the intron (3MU).
Each set of primers consists of two primer pairs, one specific for methylated (M) and the other for unmethylated (U) bisulphite-converted sequence. The same primers were used for both methylationspecific PCR (MSP) and high resolution melting analysis (HRM) ( Table   S2). In MSP, each primer pair was used in separate reactions while for HRM, both M and U primers from the same set were combined in a single reaction in order to cover all possible variants in methylation status. Both MSP and HRM runs were performed on the QuantStudio 3 Real-Time PCR system (Applied Biosystems). For 1MU, 2MU and 3MU primer sets, PCR was initiated with initial denaturation at 95°C for 10 minutes and 40 cycles of two-step PCR at 95°C for 15 seconds and 58°C for 60 seconds. For TSS, the primer set touchdown PCR approach was used with initial denaturation at 95°C for 10 minutes and each cycle starting with denaturation at 95°C for 30 seconds followed by a 30 seconds annealing step at 61°C for the first five cycles, at 58°C for the next five cycles and at 55°C for the final 35 cycles, with each cycle ending with a 60 seconds elongation step at 72°C. For HRM analysis, after amplification, the additional melt curve stage consisted of temperature ramping from 60-95°C by 0.025°C/s with florescence acquisition at each temperature increment. HRM Statistical tests were performed using log2 transformed data; the mean values and error bars were back transformed to linear scale for graphs. Statistical significance was estimated using paired t test by pairing NIH3T3 and PARP1−/− samples that were isolated simultaneously. In HRM analysis, the melting temperatures were determined from derivative melting curves and these temperatures were used for assessing and comparing overall methylation levels of the target regions.

| In situ nuclear HALO preparation and immunostaining
Nuclear HALOs were prepared as previously described. 13 In brief, nuclei were pelleted onto microscope slides, permeabilized and his-

| Assessment of global levels of DNA methylation
Global methylation levels were measured by the 5-mC DNA ELISA kit (Zymo Research, California, USA) according to the manufacturer's protocol and guidelines. Statistical significance was estimated using one-way ANOVA with blocking, treating each ELISA plate as a block.

| The level of Cxcl12 expression in PARP1−/− cells compared to NIH3T3
Cxcl12 expression in NIH3T3 and PARP1−/− cells is presented in

| Examining the global level of DNA methylation in NIH3T3 and PARP1−/− cells
To better understand the local differential methylation patterns be-

| D ISCUSS I ON
Our previous results based on transfection experiments revealed that PARP-1 plays a role in suppression of the Cxcl12 promoter. 5 Findings presented herein strongly support an inhibitory role of PARP-1 in the regulation of Cxcl12 gene expression. Namely, we detected a significantly higher level of Cxcl12 gene expression in PARP1−/− cells than in control NIH3T3 cells. This was accompanied by increased protein abundance in the PARP1−/− cell medium, confirming previously obtained results regarding the ability of PARP-1 to down-regulate Cxcl12 promoter activity. Thus, we extended our research to determine whether DNA methylation is integrated in It is well established that Cxcl12 is epigenetically regulated by the methylation of cytosine in CpG dinucleotides located in the promoter sequence. 14 We suggest that epigenetic regulation of Cxcl12 gene expression mediated by PARP-1 could serve as a therapeutic approach in diseases associated with CXCL12 down-regulation or in disease where CXCL12 was shown to exert a protective effect. Namely, studies have shown protective effects of CXCL12 in atherosclerosis and in myocardial infarction-ischaemia-reperfusion injury, based on increased recruitment of progenitor cells and neo-angiogenesis. [16][17][18] Also, CXCL12 possesses an anti-diabetogenic potential due to promotion of beta-cell survival and its involvement in the regulation of beta-cell mass in pancreas, suggesting that manipulation of Cxcl12 gene expression could be used in a potential diabetes treatment. 6,[19][20][21][22] Furthermore, we recently showed that the DNA methylation profile of Cxcl12 gene played an important role in progression of periodontitis. 23 It was reported that epigenetic down-regulation of Cxcl12 is involved in breast carcinoma, higher proliferation rates of breast cancer cells, non-small cell lung cancer and lymph node metastasis development. 24 Epigenetic down-regulation of Cxcl12 expression by hypermethylation mediated by DNMT1 was documented in osteosarcoma. 25 Additionally, the observation that DNMT1 inhibition restored CXCL12 secretion, which consequently suppressed tumour growth and retained osteosarcoma progression, was in accordance with the overall survival effect connected with increased Cxcl12 expression. 25 Hence, due to the potential antitumor effect of elevated Cxcl12 expression, the epigenetic targeting of Cxcl12 gene expression by a demethylating treatment could have therapeutic relevance. Our results revealed that PARP-1 serves as a potential upstream regulator of (de)methylation events that modulate Cxcl12 F I G U R E 5 Comparison of relative expresssion levels of Cxcl12 in control conditions vs treatment (Vit C or DMOG) in NIH3T3 and PARP−/− cell lines (n = 3). Data presented as mean ± standard error of the mean, *P ≤ 0.05, **P ≤ 0.01, n-number of independent experiments expression. According to literature data, depletion of PAR leads to silencing of Dnmt1 by hypermethylation, which accounts for defective methylation activity and consequently demethylation processes. 3,26 Furthermore, site-specific demethylation has also been documented for gene promoters as a result of PARP-1 depletion. 3,27 According to our results, changes in Dnmts expression were not statistically significant. We assumed that the detected decrease in the global level of DNA methylation in PARP1−/− cells was primarily due to increased expression of Tet genes. It is more likely that promotion of TET-dependent active demethylation takes place in PARP1−/− cells rather than DNMT-related suppressed methylation.
The observed hypomethylation of mouse Cxcl12 in PARP1−/− cells pointed to the involvement of PARP-1 in the promotion of DNA demethylation, and it is tempting to speculate the inhibitory role of PARP-1 on the expression of Tet genes. This is in disagreement with the observation that PARP activity positively regulates Tet1 expression, which consequently results in initiation of active demethylation processes. 28,29 This discrepancy may be due to the different experimental approach, including different cells and methods for evaluating the effect of PARP-1 on Tet1 transcriptional regulation. Namely, the cited authors used HEK293T Parp-1-silenced (siPARP-1) cells where PARP-1 was present in a low amount but was not completely absent. 28 Also, the authors showed that PARP-1-dependent regula- The role of PARP in the control of active demethylation mediated by TET enzymes has emerged, implying a high level of complexity of PARP/TET cross-talk. Also, TET is capable of stimulating PARP-1 activity in vitro, even in the absence of DNA damage; TET1 could be a target of PARP-1 activity by covalent and non-covalent PARylation which affects TET activity differently, activating or inhibiting it respectively. 3 Namely, non-covalent PARylation of TET1 resulted in negative regulation of TET1 activity while covalent PARylation had a stimulatory effect on TET1 activity in vitro. The result obtained from an experiment with overexpressed, engineered TET1, and a specific DNA binding domain showed that PARylation impaired TET1 activity in vivo. 2 In our study, besides increased expression of Tet genes, an increased level of 5hmC as a first intermediary product in TET-mediated demethylation in PARP1−/− cells was detected.
Immunofluorescence analysis of 5hmC level revealed strong staining in PARP1−/− cells. This reflected the activities of TETs in mediating oxidation of 5mC to 5hmC, which was assumed to be activated in PARP1−/− cells, suggesting a potential inhibitory role of PARP-1 on the activities of TETs.
Considering the potential involvement of TET-dependent demethylation of Cxcl12, which could be responsible for its elevated expression in PARP1−/− cells, we performed experiments with TET enzyme activator (VitC) and inhibitor (DMOG) in order to verify whether TET activity is involved, at least in part, in the modulation of Cxcl12 gene expression. Vitamin C promotes TET-dependent DNA demethylation in embryonic stem cells and increases 5hmC levels through enhanced Fe 2+ recycling. [30][31][32][33] On the other hand, DMOG is a small-molecule, an analogue of 2-oxoglutarate which inhibits members of 2-oxoglutarate-dependent dioxygenases, and is known to impede the enzymatic activity of TET enzymes. 34,35 In NIH3T3 cells, both treatments did not significantly influence Cxcl12 expression. This could be explained by the extremely low rate of Cxcl12 expression, which is insufficient to allow for the detection of changes in expression. Also, the low