The studies on lead (Pb) exposure linking to epigenetic modulations are caused by its differential actions on global DNA methylation and histone modifications. These epigenetic changes may result in increased accessibility of the transcription factors to promoter DNA-binding elements leading to activation and expression of the gene. The protein arginine methyltransferase 5 (PRMT5) and its partner methylosome protein 50 (MEP50) together catalyze the mono- and symmetric dimethylation of arginine residues in many histone and non-histone protein substrates. Moreover, it is overexpressed in many forms of cancer. In the present study, the effects of Pb on the PRMT5 and MEP50 expression and formation of the symmetrically dimethylated arginine (SDMA), the catalytic product of the PRMT5–MEP50 complex were analyzed in vitro after exposing the A549 and MCF-7 cells. The results show that exposure to 0.1 and 1 µM of Pb strongly enhanced the expression of both PRMT5 and MEP50 transcript and protein leading to increased SDMA levels globally with H4R3 being increasingly symmetrically dimethylated in a dose-dependent manner after 48 h of Pb exposure in both cell types. The methylation-specific PCR also revealed that the CpG island present on the PRMT5 promoter proximal region was increasingly demethylated as the dose of Pb increased in a 48-h exposure window in both cells, with MCF-7 being more responsive to Pb-mediated PRMT5 promoter demethylation. The bisulfite sequencing confirmed this effect. The findings therefore indicate that Pb exposure increasing the PRMT5 expression might be one of the contributing epigenetic factors in the lead-mediated disease processes as PRMT5 has a versatile role in cellular functions and oncogenesis.

Introduction

Lead (Pb) and other heavy metals have contaminated our environment since the advent of industrialization. Apart from the petroleum fuels, paints, canned foods, plastic pipes used for drinking water supply, cosmetics, ayurvedic herbal medicines, and the battery/plastic/e-waste recycling industry are reported as the primary source of human exposure [13]. The several regulatory steps taken in the last three decades have succeeded in curbing its deleterious exposure globally [4,5]. However, due to its environmentally persistent chemical nature and a multitude of exposure routes, high levels of Pb in human blood have been reported in multiple studies [6,7]. Moreover, the human reference mean blood lead level (BLL) >10 µg/dl or even lower BLL has been significantly associated with various human diseases including cardiovascular diseases, cognitive impairment, neurodegenerative diseases, chronic kidney disease, and cancer in several populations worldwide [710].

The toxicity of Pb is relatively well studied. However, recent studies on the Pb-mediated epigenetic mechanisms have drawn attention as Pb exposure has been linked with altered DNA methylation pattern, globally and locally in a dynamic and sex-dependent manner [1115]. The methylation of cytosine residues at the CpG dinucleotide clusters, called as CpG islands (CGIs), majorly located on the gene promoter elements in DNA throughout the genome, is catalyzed by a family of DNA methyltransferases (DNMTs): DNMT1, DNMT3A, and DNMT3B; these enzymes are involved in the regulation of gene expression, and their anomalies are highly correlated with tumorigenesis [16]. Commonly, the gene promoter region flanking the transcriptional start sites is typically surrounded by CGIs on the DNA, and their methylation is linked with their transcriptional silencing, while demethylation results in transcriptional activation due to alterations in the binding of methylation-sensitive transcription factors [17,18]. Generally, promoter hypomethylation results in increased accessibility of the transcription factors to the promoter DNA-binding elements leading to activation and expression of the gene. However, it cannot be ruled out that the presence of Pb might alter the expression of a responsive gene possibly through other, previously unknown epigenetic mechanisms.

In addition to DNA methylation, covalent post-translational modifications of histone residues, including methylation and acetylation, catalyzed by an array of epigenetic enzymes, result in significant reversible epigenetic marks (‘histone codes hypothesis’), which collaboratively control chromatin structure and gene expression [19]. Methylation of arginine residues in histone and non-histone proteins is catalyzed by a family of nine protein arginine methyltransferase (PRMT) enzymes [20]. Protein arginine methyltransferase 5 (PRMT5) along with its activity partner methylosome protein 50 (MEP50) is the predominant member of the type II PRMTs responsible for monomethylation as well as symmetric dimethylation of arginine residues in histones (H2A, H3, and H4) and non-histone protein substrates [Sm proteins, p53, E2F-1, eIF4E, C-RAF, EGFR, NF-κB p65, programmed cell death 4 (PDCD4), cyclin E1, E-cadherin, and IL-2] [21]. A growing body of evidence suggests that PRMT5 is an oncogene and plays a critical role in cancer progression by promoting cell proliferation and inhibition of apoptosis; moreover, it is overexpressed in many forms of human cancers [2225]. However, the exact mechanism by which PRMT5 is overexpressed is not precise and whether Pb could affect its expression especially by its DNMT modulatory effect, including alterations in its promoter CGIs, requires further investigations.

In this study, the aim was to detect alterations in PRMT5/MEP50 expression and function using human lung and breast cancer cell lines, A549 and MCF-7, respectively, as in vitro systems after exposing them to physiologically relevant Pb levels. The investigations involve the analysis of Pb-induced global DNA hypomethylation followed by detection of the methylation status of PRMT5 promoter CGI in a Pb concentration-dependent manner. Understanding more of the epigenetic changes due to Pb exposure will reveal novel molecular mechanisms of Pb-mediated cellular effects and will also have implications for the prevention of Pb-toxicity.

Experimental methods

Cell culture

The human lung and breast cancer cell lines, A549 and MCF-7, respectively, were procured from the cell repository at the National Centre for Cell Science, Pune, India. The A549 cell line was originated from a Caucasian male, while the MCF-7 cell line was established from a Caucasian female. Both the cells were maintained in Dulbecco's modified Eagle medium (DMEM) (Himedia, India) supplemented with 10% fetal bovine serum (FBS) and 1× antibiotic–antimycotic solution (Himedia) in a humidified CO2 incubator (Thermo Fisher Scientific) maintaining 5% CO2 at 37°C. For treatment, cells were cultured in phenol red-free DMEM (Himedia) containing 2.5% FBS (charcoal-stripped) and 1× antibiotic–antimycotic solution under experimental conditions involving exposure to indicated doses of Pb. The Pb [lead (II) chloride, #203572, Sigma–Aldrich, U.S.A.] stock was prepared by dissolving an appropriate amount of Pb chloride in sterile double distilled water, and the working stocks were diluted in phenol red-free DMEM containing 2.5% FBS (charcoal-stripped) and 1× antibiotic–antimycotic solution for treatment. Following experimental doses of Pb exposure for respective periods, cells were washed with PBS and subjected to following experiments.

Cell viability assays

For determination of cell viability, direct cell counting (using trypan blue), MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and neutral red uptake assays were performed for both A549 and MCF-7 cells. For trypan blue dye exclusion assay, ∼10 000 cells were allowed to grow in 35 mm cell culture dishes. After confirming their attachment, cells were treated by adding the above-mentioned media containing Pb (0, 0.001, 0.01, 0.1, 1, 5, and 10 µM) for 24 or 48 h followed by preparation of a suspension of the dislodged cells. The cell suspension (10 µl) was then mixed with 40 µl of 0.4% trypan blue solution (Sigma–Aldrich) and allowed to stand for 5 min. The non-viable (blue) cells and viable cells were observed under a microscope and counted. The total viable cells were obtained from the following formula: the average count (viable cells only)/square × dilution factor × 104 × the original volume of cell suspension from which cell sample was taken. The MTT assay was performed by seeding 5000 cells/well in a 96-well plate and post attachment, treated with either DMEM alone (vehicle control) or DMEM containing various concentrations of Pb taking triplicates in each group. After 24 and 48 h of exposure, 5 µl of MTT (5 mg/ml) was added to each well and incubated for 3 h. Then, the cells were lysed, and the formazan crystals were solubilized with 100 µl of DMSO. The optical density was measured at 570 nm in a microplate reader (PerkinElmer, U.S.A.) against a reagent blank. The viability (%) of each cell line in response to respective Pb dose was calculated. The experiment was repeated at least three times. The neutral red uptake assay was performed according to Repetto et al. [26]. Briefly, 200 µl of cell suspension (containing 5000 cells) per well was allowed to attach to a 96-well plate followed by treatment in the log phase with either DMEM alone (vehicle control) or DMEM containing various concentrations of Pb with triplicates for each treatment group. The blank group contained complete medium without cells. Following the desired incubation period, 10 µl of freshly prepared neutral red (Sigma–Aldrich) reagent (0.33%) was added to each well, and after 2 h incubation at 37°C, the cells were quickly and gently rinsed with PBS. To solubilize the dye, 150 µl of destain solution (50% ethanol, 49% deionized water, and 1% glacial acetic acid) was added per well and mixed by shaking the plate on a shaker for 10 min, and the color developed was read at 540 nm (with 690 nm for background) in a microplate reader (Enspire, PerkinElmer, Inc.) using blanks without cells as a reference. All analyses were performed for at least three independent triplicate experiments.

Total RNA isolation, synthesis of cDNA, and real-time PCR

After 24 and 48 h of incubation, total RNA from the Pb (0.1 and 1 µM) treated or untreated (0 µM) cells (triplicate samples for each group) was isolated according to the TRIzol method (Invitrogen, San Diego, CA). The RNA (1 µg) was reverse-transcribed to obtain first-strand cDNAs by using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, India). The resulting cDNA was stored at −20°C and appropriate dilutions were used in the real-time PCR step using the LightCycler® 480 SYBR Green I Master mix (Roche, India), 0.5 µM forward and reverse primers, and nuclease- free water in a LightCycler® 480 Instrument (Roche Molecular Diagnostics, U.S.A.). The details of the primer pairs used to perform qPCR experiments are shown in Supplementary Table S1. Each sample (10 µl) in triplicates was subjected to real-time PCR analysis. The fold change of the target gene mRNA expression was normalized to the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) mRNA (endogenous control) expression with the help of the method. The data of the Pb-treated samples were compared with those from the untreated control.

Immunoblotting

For detecting the expression of proteins under investigation, cells were lysed using cOmplete Lysis-M buffer (Roche) supplemented with protease inhibitor cocktail tablets (Roche). The lysate was cleared by brief sonication and vortexing for 5 min, and the supernatant was obtained by centrifugation at 14 000g for 10 min at 4°C. The total protein was measured by the Bradford method, and 30 µg of total protein was subjected to sodium dodecyl sulfate electrophoresis on 10% acrylamide gels and transferred onto polyvinylidene difluoride membranes. The nonspecific binding was blocked by incubating the membrane with 5% skimmed milk/bovine serum albumin solution in Tris-buffered saline with 0.1% Tween-20 at room temperature for 1 h and subjected to overnight incubation with appropriate primary antibody at 1 : 1000 dilutions with gentle shaking at 4°C. The next day, the membrane was washed and incubated for 1 h with the HRP-conjugated secondary antibody (either antimouse or antirabbit) at 1 : 10 000 dilutions. The protein band image was developed using the Clarity western ECL substrate (Bio-Rad, U.S.A.). The details of antibodies used are listed in Supplementary Table S2. The ColorBurst™ Electrophoresis Marker (#C1992 Sigma) of the molecular mass range of 8–220 kDa was used to detect the molecular mass on Western blots. The β-actin was used as loading control. The density of each band was quantified using the ImageJ software (NIH, U.S.A.).

Global DNA methylation analysis

The level of global DNA methylation in control and Pb-exposed cells (in duplicates) was analyzed by the imprint methylated DNA quantification kit (Sigma–Aldrich) as per the manufacturer's instructions. Briefly, the isolated DNA samples along with the methylated DNA standard were first diluted with DNA-binding solution and allowed to bind to the wells of duplicated samples. The DNA blank controls (negative control) were also run in parallel. After a blocking step, methylated DNA was then incubated with capture and detection antibodies with intermediate washing steps. A developing solution was added which upon incubation turns blue and after 10 min, the stop solution was added which turned the solution yellow. The absorbance was read at 450 nm. The replicates were averaged, and the mean ± SEM value was used for further analyses. Quantification of percentage global DNA methylation was performed from the number of methylated cytosines (5 mC) in the sample relative to that in the methylated positive control.

Bisulfite conversion, methylation-specific PCR, and bisulfite sequencing

The in silico analysis by the MethPrimer online program [27] revealed the presence of at least two CGIs spanning the very proximal promoter and first exon of the PRMT5 gene. The CpG dinucleotide islands were predicted based on the universally accepted criteria for CGI, which includes >200 bp DNA region containing above 50% GC and an observed/expected frequency of CpG >0.6. For the analysis of methylation status of these CGIs on the PRMT5 promoter DNA, the A549 and MCF-7 cells were treated with either DMEM or Pb (0.1 and 1 µM) for 48 h. To perform bisulfite conversion of genomic DNA (in which unmethylated but not methylated cytosines are converted to uracil), after the exposure period, the cells were directly lysed and the genomic DNA was bisulfite treated overnight in a thermal cycler followed by purification and elution of the converted DNA according to the EpiTect Fast Lyse All Bisulfite Kit (Qiagen). The bisulfite-converted genomic DNA was then amplified using the EpiTect Whole Bisulfitome kit (Qiagen) using a thermal cycler for 8 h maintaining 28°C followed by a denaturation step at 95°C for 5 min.

Next, for rapid analysis of the methylation status of the CGIs located on the PRMT5 promoter and the first exon, methylation-specific PCR (MSP) was performed. The MethPrimer online program designed the primer pairs (Supplementary Table S3) to detect only methylated DNA (M primers) or only unmethylated DNA (U primers). Although the island at −80 to −372 (illustrated as set 1) spanning the proximal promoter region matches the criteria most, another CGI flanking the transcription start site (TSS) and the first exon within the region −70 and +231 (set 2) were selected to analyze the 5-mC level. Bisulfite-converted, but unmethylated and methylated human control DNA (Qiagen) was used as a positive control for the unmethylated and methylated MSP reactions, respectively. Water alone was used as a negative PCR control in both reactions. For performing the PCR, the EpiMark Hot Start TaqDNA Polymerase (NEB) along with the supplied buffer was used to amplify the specific regions on the PRMT5 promoter flanking from −326 to −204 bp (set 1) and +50 to +185 (set 2) respective to the TSS. The following PCR conditions were used: initial denaturation at 94°C for 30 s followed by 35 cycles of 94°C for 20 s, 51°C for 20 s, and 68°C for 20 s; and a final extension for 5 min at 68°C. The specificity of the PCR was confirmed by 2% agarose gel electrophoresis for 30 min at 100 V. The intensity of the bands on the gel was quantified by densitometry using the image acquisition software image laboratory (Bio-Rad). The following formula calculated the percentage of unmethylation DNA in each group: unmethylation intensity/(methylation intensity + unmethylation intensity) × 100. The experiments were repeated at least three times to obtain the methylation status of the PRMT5 promoter and the first exon.

For bisulfite sequencing, the bisulfite-converted and amplified DNA prepared from either the DMEM or Pb (1 µM)-treated MCF-7 cells, as mentioned above was subjected to the bisulfite PCR using the bisulfite PCR primers (Supplementary Table S4) designed by the Methprimer program. For this experiment, duplicates of each of the DMEM or Pb-treated cells were taken in an individual experiment and the experiment was repeated three times. For performing the PCR, the EpiMark Hot Start TaqDNA Polymerase (NEB) along with the supplied buffer was used to amplify a 209 bp region on the PRMT5 promoter flanking from −254 to −46 bp respective to the TSS. The following PCR conditions were used: initial denaturation at 94°C for 30 s followed by 35 cycles of 94°C for 20 s, 51°C for 30 s and 68°C for 20 s, and a final extension for 5 min at 68°C. The specificity of the PCR was confirmed by 2% agarose gel electrophoresis. The PCR product was then column purified using the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer's protocol. The purified PCR products from both the control and treated group were (in duplicates) ligated into a pCR® 2.1vector using a TA cloning kit (Invitrogen) and transformed into competent DH5α cells and grown on agar plates containing ampicillin, X-gal, and IPTG. The bacterial cells containing the vector with inserted PCR product were then selected from the white colonies. About three isolated colonies from each of the duplicated plates of DMEM or Pb-treated group were picked (i.e. total of six colonies/group) and individually grown overnight in LB broths containing ampicillin to extract the plasmid containing the insert using the QIAprep Spin Miniprep Kit (Qiagen) according to the provided protocols. The plasmid insert of all 12 samples (six/group) was then sequenced from an outsourcing laboratory in an ABI 3500xL Genetic Analyzer. The sequence data from each of the clones were aligned and analyzed for DNA methylation status within the sub-cloned amplicon. The overall methylation status at the amplified region of the PRMT5 promoter DNA of DMEM-treated and Pb-treated cell group was presented taking together the data from six clones of each group.

Statistical analysis

To test the statistically significant differences between the two treatment groups, one-way ANOVA was performed with Tukey post hoc means comparison test set at P < 0.05 level.

Results

Effect of Pb on human A549 and MCF-7 cancer cell viability

To check the potential cytotoxicity of Pb, its effects on the viability of both A549 and MCF-7 cells were assayed by the trypan blue dye exclusion assay (cell counts), MTT (a function of mitochondria), and neutral red uptake (a function of lysosomes) assays. As shown in Figure 1, Pb exposure resulted in a significant increase in the viability of both A549 and MCF-7 cells up to a concentration of 1 µM. However, it turned cytotoxic at ≥5 µM of Pb after 48 h of exposure. The apparent increase in viability was observed in all three assays which confirm the Pb concentration-dependent response in vitro. Therefore, 0.1 and 1 µM of Pb exposure for 24 and 48 h, which considerably affected the cell viability, was considered for further studies.

Effect of Pb exposure on the viability of human A549 and MCF-7 cell lines.

Figure 1.
Effect of Pb exposure on the viability of human A549 and MCF-7 cell lines.

(A) Trypan blue exclusion assay was performed when both the cell lines treated with various concentrations of Pb for 24 and 48 h. The data shown here represent mean ± SE (n = 6, duplicates from three repeated experiments against each treatment condition). The P-value from significance analysis is indicated as **P < 0.05; ***P < 0.001 compared with untreated control. (B) The cell viability was measured by the MTT assay after exposure to the above-indicated concentrations of Pb for 24 and 48 h. The viability is presented as percentage values. The data are expressed as the mean ± standard deviation (SD), and the 0 µM group was treated as the control group (0). The mean value of control group was set at 100%. *P < 0.05, **P < 0.05 and ***P < 0.001 versus the control group (n = 4). (C) The neutral red uptake assay was performed by exposing the A549 and MCF-7 cells adhered to the indicated concentrations of Pb for 24 and 48 h. Post-exposure, the cells were incubated with the neutral red dye for 2 h at 37°C, rinsed in DPBS and the color was quantified at 540 nm using blanks which contain no cells as a reference. The percentage of viable cells relative to the cells in the PBS-treated controls was calculated, and the data represent mean ± SE of three individual experiments under identical conditions (n = 6; **P < 0.05; ***P < 0.001 compared with untreated control).

Figure 1.
Effect of Pb exposure on the viability of human A549 and MCF-7 cell lines.

(A) Trypan blue exclusion assay was performed when both the cell lines treated with various concentrations of Pb for 24 and 48 h. The data shown here represent mean ± SE (n = 6, duplicates from three repeated experiments against each treatment condition). The P-value from significance analysis is indicated as **P < 0.05; ***P < 0.001 compared with untreated control. (B) The cell viability was measured by the MTT assay after exposure to the above-indicated concentrations of Pb for 24 and 48 h. The viability is presented as percentage values. The data are expressed as the mean ± standard deviation (SD), and the 0 µM group was treated as the control group (0). The mean value of control group was set at 100%. *P < 0.05, **P < 0.05 and ***P < 0.001 versus the control group (n = 4). (C) The neutral red uptake assay was performed by exposing the A549 and MCF-7 cells adhered to the indicated concentrations of Pb for 24 and 48 h. Post-exposure, the cells were incubated with the neutral red dye for 2 h at 37°C, rinsed in DPBS and the color was quantified at 540 nm using blanks which contain no cells as a reference. The percentage of viable cells relative to the cells in the PBS-treated controls was calculated, and the data represent mean ± SE of three individual experiments under identical conditions (n = 6; **P < 0.05; ***P < 0.001 compared with untreated control).

Lead exposure overexpressed PRMT5/MEP50 and enhanced the formation of the H4R3me2s epigenetic mark

The study investigated the alteration in the expression and function of PRMT5 after exposure to 0.1 and 1 µM of Pb in vitro. It was observed that Pb exposure significantly induced PRMT5 mRNA and protein overexpression that also coincided with increased symmetrically dimethylated arginine (SDMA) formation and H4R3me2s generation in both cell lines when treated with Pb for 48 h (Figure 2). It was also found that Pb exposure can strongly influence the expression of MEP50 in both cells under investigation (Figure 2B,C). Therefore, our findings indicate that Pb can influence the expression as well as the catalytic product formation of the PRMT5–MEP50 arginine methylation machinery within the cell leading to an enhanced level of post-translational modification at arginine residues in a multitude of target proteins (SDMA) and histone H4. These modifications can potentially affect multiple cellular pathways and regulatory systems as reported previously [21,25] and could serve as a mechanism of Pb-mediated alterations in cells.

Impact of Pb exposure on PRMT5 expression and its function.

Figure 2.
Impact of Pb exposure on PRMT5 expression and its function.

(A) Enhanced expression of PRMT5 mRNA as obtained from real-time PCR analysis from A549 and MCF-7 cells treated with Pb for 24 and 48 h. Data were obtained from triplicate samples, represented as mean ± SEM; **P < 0.01 and ***P < 0.001. (B) Increased level of PRMT5 protein, symmetrically dimethylated arginine motifs in global proteins and H4R3me2s, and MEP50, respectively (top to bottom panels), as detected by immunoblotting after Pb exposure for 48 h. Pan-histone H4 and β-actin were used as an endogenous and loading control, respectively. (C) Relative band intensity of the immunoblots. The intensity of each blot was measured using ImageJ software. The band density in each lane of the immunoblot was normalized against the density of the β-actin band in the same lane. ‘**’ and ‘***’ represents significance at P < 0.01 and P < 0.001 levels when compared with control.

Figure 2.
Impact of Pb exposure on PRMT5 expression and its function.

(A) Enhanced expression of PRMT5 mRNA as obtained from real-time PCR analysis from A549 and MCF-7 cells treated with Pb for 24 and 48 h. Data were obtained from triplicate samples, represented as mean ± SEM; **P < 0.01 and ***P < 0.001. (B) Increased level of PRMT5 protein, symmetrically dimethylated arginine motifs in global proteins and H4R3me2s, and MEP50, respectively (top to bottom panels), as detected by immunoblotting after Pb exposure for 48 h. Pan-histone H4 and β-actin were used as an endogenous and loading control, respectively. (C) Relative band intensity of the immunoblots. The intensity of each blot was measured using ImageJ software. The band density in each lane of the immunoblot was normalized against the density of the β-actin band in the same lane. ‘**’ and ‘***’ represents significance at P < 0.01 and P < 0.001 levels when compared with control.

Lead induced global DNA hypomethylation and PRMT5 promoter CpG demethylation as the DNMT levels were decreased

As observed, Pb exposure significantly decreased the gene and protein expression of all three DNMTs (Figure 3A–C), which was also correlated with the reduced percentage in the global DNA methylation level in A549 and more significantly in MCF-7 cells (Figure 3D). Recalling the DNA hypomethylating action of Pb from previous studies [1115], subsequent investigation was initiated to find whether Pb induced any alteration in the CGIs located in the promoter region of the human PRMT5 gene on chromosome 14. The initial in silico search for CGIs revealed that the PRMT5 promoter harbors two CGIs in the proximal region spanning the TSS (Figure 4A). The results from the MSP experiments suggest that the extent of the amplicon formation by the set 1 methylation-specific primers was significantly decreased in a dose-dependent manner when compared with the untreated control in both cell lines (Figure 4B,C). A minimum level of methylation was observed in the control cells, which in contrast with unmethylation-specific PCR product is less, but Pb treatment, however, decreased the methylation-specific PCR product formation with maximum impact at 1 µM dose after 48 h of exposure. On the other hand, the methylation-specific set 2 primers, unlike set 1 primers, did not yield any product in the unexposed or Pb-exposed cells irrespective of the cell line used. Therefore, our results show that Pb introduced either complete or partial demethylation of only the upstream PRMT5 promoter CGI located within −372 to −80 bp corresponding to the TSS.

Impact of Pb on the expression of DNMTs and global DNA methylation in vitro.

Figure 3.
Impact of Pb on the expression of DNMTs and global DNA methylation in vitro.

Alterations in the gene (A) and protein (B) expressions of DNMTs in A549 and MCF-7 cells exposed to the indicated concentrations of Pb for 48 h. (C) Corresponding densitometric analysis of the representative immunoblot in (B) showing relative intensity as mean ± SEM of the expressed protein level of DNMTs in untreated control and lead-treated cells (**P < 0.01 and ***P < 0.001; n = 6). (D) Level of global DNA methylation in the A549 and MCF-7 cells following exposure to 1 µM of Pb compared with unexposed cells. Data represented as mean ± SEM (n = 6) and significance at P < 0.001 level shown as ***, compared with the untreated control group.

Figure 3.
Impact of Pb on the expression of DNMTs and global DNA methylation in vitro.

Alterations in the gene (A) and protein (B) expressions of DNMTs in A549 and MCF-7 cells exposed to the indicated concentrations of Pb for 48 h. (C) Corresponding densitometric analysis of the representative immunoblot in (B) showing relative intensity as mean ± SEM of the expressed protein level of DNMTs in untreated control and lead-treated cells (**P < 0.01 and ***P < 0.001; n = 6). (D) Level of global DNA methylation in the A549 and MCF-7 cells following exposure to 1 µM of Pb compared with unexposed cells. Data represented as mean ± SEM (n = 6) and significance at P < 0.001 level shown as ***, compared with the untreated control group.

Pb exposure alters the PRMT5 promoter methylation status.

Figure 4.
Pb exposure alters the PRMT5 promoter methylation status.

(A) The schematics of the human PRMT5 promoter region with two CGIs corresponding to the TSS and the regions amplified by methylated-specific and unmethylated-specific primer pairs for respective CGIs designed using the MethPrimer online service. Set 1 primers MF/UF and MR/UR indicate forward and reverse primers amplifying the methylated or unmethylated DNA located within the CGI flanking −370 to −80, while set 2 primers amplify the methylated or unmethylated DNA located within the CGI flanking −70 to + 231 region within the PRMT5 promoter. (B) Gel image of the amplified products obtained from the methylation-specific PCR of the PRMT5 promoter. The bisulfite-converted DNA isolated from cells treated with or without Pb. Top two panels indicate results using set 1 primer pairs for MSP analysis of DNA from A549 and MCF-7 cells and the bottom panel indicates that obtained using set 2 primer pairs. L, DNA ladder; −ve, no template control; UC, unmethylated and converted human control DNA amplified with unmethylation-specific primers; MC, methylated and converted human control DNA amplified with methylation-specific primers; U, unmethylated DNA product obtained from unmethylation-specific primer pairs; M, methylated DNA product amplified using methylation-specific primer pairs. (C) Percentages of the unmethylated DNA were calculated from the band intensities of unmethylated and methylated DNA from each group. Bars represent percentage mean ± SEM of the unmethylated DNA from three individual experiments; ***P < 0.001 compared with the untreated group.

Figure 4.
Pb exposure alters the PRMT5 promoter methylation status.

(A) The schematics of the human PRMT5 promoter region with two CGIs corresponding to the TSS and the regions amplified by methylated-specific and unmethylated-specific primer pairs for respective CGIs designed using the MethPrimer online service. Set 1 primers MF/UF and MR/UR indicate forward and reverse primers amplifying the methylated or unmethylated DNA located within the CGI flanking −370 to −80, while set 2 primers amplify the methylated or unmethylated DNA located within the CGI flanking −70 to + 231 region within the PRMT5 promoter. (B) Gel image of the amplified products obtained from the methylation-specific PCR of the PRMT5 promoter. The bisulfite-converted DNA isolated from cells treated with or without Pb. Top two panels indicate results using set 1 primer pairs for MSP analysis of DNA from A549 and MCF-7 cells and the bottom panel indicates that obtained using set 2 primer pairs. L, DNA ladder; −ve, no template control; UC, unmethylated and converted human control DNA amplified with unmethylation-specific primers; MC, methylated and converted human control DNA amplified with methylation-specific primers; U, unmethylated DNA product obtained from unmethylation-specific primer pairs; M, methylated DNA product amplified using methylation-specific primer pairs. (C) Percentages of the unmethylated DNA were calculated from the band intensities of unmethylated and methylated DNA from each group. Bars represent percentage mean ± SEM of the unmethylated DNA from three individual experiments; ***P < 0.001 compared with the untreated group.

The methylation status at the CpG dinucleotides located on the proximal region of the promoter was investigated by bisulfite sequencing PCR of the bisulfite converted DNA followed by subcloning and sequencing. For performing the PCR reaction the EpiMark Hot Start Taq DNA Polymerase (NEB) along with the supplied buffer was used to amplify a 209 bp region on the PRMT5 promoter flanking from −254 to −46 bp respective to the TSS. The following PCR conditions were used: initial denaturation at 94°C for 30 s followed by 35 cycles of 94°C for 20 s, 51°C for 30 s and 68°C for 20 s, and a final extension for 5 min at 68°C. The specificity of the PCR was confirmed by 2% agarose gel electrophoresis. The sequencing of at least six individual clones from either DMEM-treated or Pb-treated MCF-7 cells revealed that out of nine CpG dinucleotides, five were methylated, while four were unmethylated in the unexposed cells. But, exposure to 1 µM of Pb for 48 h demethylated four of the five methylated CpGs, leaving only one methylated cytosine within the −254 to −46 bp upstream element in the PRMT5 promoter (Figure 5). Among the nine CpG dinucleotides, the percentage of methylation reduced more than five-fold when the cells were exposed to Pb. Interestingly, the bisulfite sequencing results largely coincided with the MSP outcomes, confirming that the Pb-induced demethylation might be a determining step that leads to increased PRMT5 level in the exposed cells.

Impact of Pb exposure on the CpG methylation at the PRMT5 promoter as determined by bisulfite sequencing.

Figure 5.
Impact of Pb exposure on the CpG methylation at the PRMT5 promoter as determined by bisulfite sequencing.

(A) The 209 bp region (−254 to −46) of the proximal promoter of PRMT5 was amplified from the bisulfite-converted DNA obtained from the DMEM or Pb (1 µM)-treated MCF-7 cells. The PCR product was then ligated into a TA cloning vector, transformed into DH5α cells, the positive colonies were picked and six clones from each group were sequenced using the Sanger sequencing. The sequence of each clone was aligned with the reference bisulfite-converted genomic sequence, and finally, the average methylation status is schematically shown here. The transcription start site is indicated in arrow, and the nine CpG dinucleotides located on this region are depicted below the promoter DNA. To mention the methylation status of these CpGs, white (unmethylated) or black (methylated) circles were used. The upper panel represents the bisulfite sequencing output of the DMEM-treated sample as retrieved from the bisulfite sequencing of six clones from each treatment group. (B) The bottom panel indicates the overall methylation percentage of the treated groups mentioned in the figure.

Figure 5.
Impact of Pb exposure on the CpG methylation at the PRMT5 promoter as determined by bisulfite sequencing.

(A) The 209 bp region (−254 to −46) of the proximal promoter of PRMT5 was amplified from the bisulfite-converted DNA obtained from the DMEM or Pb (1 µM)-treated MCF-7 cells. The PCR product was then ligated into a TA cloning vector, transformed into DH5α cells, the positive colonies were picked and six clones from each group were sequenced using the Sanger sequencing. The sequence of each clone was aligned with the reference bisulfite-converted genomic sequence, and finally, the average methylation status is schematically shown here. The transcription start site is indicated in arrow, and the nine CpG dinucleotides located on this region are depicted below the promoter DNA. To mention the methylation status of these CpGs, white (unmethylated) or black (methylated) circles were used. The upper panel represents the bisulfite sequencing output of the DMEM-treated sample as retrieved from the bisulfite sequencing of six clones from each treatment group. (B) The bottom panel indicates the overall methylation percentage of the treated groups mentioned in the figure.

Discussion

Human exposure to Pb compounds (organic or inorganic) occurs through multiple routes. Leaded petrol or gasoline was the primary source of environmental Pb contamination until 1996 [28]. Although its use was phased out by 2000, it cannot be ruled out that its exposure-associated epigenetic alterations might have at least partially contributed towards Pb-associated diseases, including cancers. Moreover, Pb exposure or blood Pb level has been correlated with increased risk of lung cancer, stomach cancer, prostate cancer, gliomas, and other cancers [29,30]. The International Agency for Research on Cancer (IARC) in 2006 has classified inorganic Pb compounds as probable human carcinogens (group 2A) due to limited human and animal research data [31]. Although occupational Pb exposure has been shown to be genotoxic to cells, as it causes DNA damage [3234], it might also have the potential to derail normal cellular functioning by altering epigenetics (in addition to DNA methylation) in the exposed cells.

The present study revealed that Pb increased the viability of both human lung and breast cancer cell lines with an increase in Pb concentration up to 1 µM within 48 h, with MCF-7 being more responsive to Pb. We selected the MCF-7 and A549 cell lines based on our initial screening where Pb showed most prominent alterations regarding cell viability and PRMT5 transcript level. Moreover, these two cell lines generally represent the breast and lung cancer — the most predominant form of human cancers worldwide, and more interestingly, overexpression of PRMT5 has been reported in these two cancers. Another vital rationale to use these two cell lines obtained from male (A549) and female (MCF-7) rightfully address the sex-specific actions of Pb as the recent literature also suggests [13]. Taken together, we have considered these cell lines for this study as a model. However, similar effects were observed in another non-cancerous cell line (HEK-293) as shown in Supplementary Figure S1. Existing studies from multiple countries have found mean blood Pb level in the range of 5–30 µg/dl across the populations including underage children [1,3537]. In our study, the effective concentration of Pb (i.e. 1 µM Pb which is roughly equivalent to 20.7 µg/dl), however, falls within this range, which suggests that the findings from this study are correlated with the environmentally relevant dose of human Pb exposure levels.

Our primary objective was to investigate whether Pb can alter the PRMT5–MEP50-mediated arginine methylation events in these cells. In our findings, Pb-induced increased cell viability also coincided with significant up-regulation of PRMT5 expression after 1 µM Pb exposure in both A549 and MCF-7 cells. Mono- and/or dimethylation of guanidyl-side chain of arginine residues present in histones and non-histone proteins catalyzed by the PRMT family play a crucial role in normal cellular functioning as well as in diseases [38]. PRMT5 forms dimer with MEP50 that aids its activity and recruitment of substrates for methylation [39]; together they can symmetrically demethylate histones (forming H3R8 and H4R3me2s ‘marks’) as well as non-histone substrates including tumor suppressors that can significantly contribute to tumorigenesis and increased cell proliferation [21,22,25]. The H4R3me2s mark has been reported to be enriched at several repressed genes where it serves as a loading site for DNMT3A, bridging the methylation of histone and DNA events that leads to gene silencing [20]. The existing knowledge largely suggests that the PRMT5–MEP50 is essential for promoting the transcriptional regulation of cancer cell [40]. Knockdown of PRMT5 triggered cell cycle arrest in G1 phase that was strongly related to p53 expression and function [41] and nuclear transcription factor Y (NFYA) function [42]. Recently, Sheng and Wang [43] have shown that the PRMT5/MEP50 complex actively regulates multiple genes related to cell growth of prostate and lung tumorigenesis. PRMT5 and MEP50 expression is a marker of tumor aggressiveness and grade in astrocytomas [24]. Although earlier reports from animal studies showed that Pb can differentially alter the global levels of activating H3K9Ac, H3K14Ac, and H3K4me3 and repressive H3K9me2 and H3K27me3 mark at different developmental stages [4447]; however, alterations in H4R3me2s were not investigated. Taken together, our findings demonstrate that Pb can influence histone post-translational modifications in the form of elevated level of SDMA formation globally in several proteins (which need to be identified in future) as well as in H4R3me2s repressive marks through modulation of the expression of PRMT5–MEP50 methyltransferases.

Generally, the promoters of genes, when methylated, become inactive, but the demethylation by the demethylases reactivates the promoters as they become accessible to transcription factors [17]. Hypomethylation of these gene regulatory regions and overexpression of those genes have been implicated in the formation and progression of tumors [17,48]. The decrease in DNMT isoforms is one of the mechanisms leading to DNA hypomethylation. In a recent finding from a Zebrafish model, Sanchez et al. [49] have shown that Pb can bring about a significant alteration in global DNA methylation levels by lowering the activity of all DNMTs through a noncompetitive inhibitory mechanism. The lead was previously shown to alter the action of DNMTs [13] and induced hypomethylation in the CGIs of long interspersed nuclear element 1 [50]. This study also shows a micromolar Pb exposure significantly decreased the expression of all three DNMTs with DNMT1 and DNMT3B being affected mostly which was also correlated with the reduced percentage in the global DNA methylation level in A549 and more significantly in MCF-7 cells. Therefore, we hypothesized that the Pb-induced decrease in DNMT expression and global hypomethylation might be involved in activating the PRMT5 promoter resulting in its overexpression. Despite the involvement of transcription factors as implicated in other studies [42], it needs to be explored to elucidate the epigenetic alterations by Pb.

DNA hypomethylation is one such prominent mechanism, which can promote carcinogenesis by directly and indirectly altering the expression of genes [17]. Being an oncogene, PRMT5 is overexpressed in many cancer cells [2225]. Thus, it was detectably expressed in both cell lines in the control group; however, its expression was significantly increased with Pb exposure (Figure 2). This led to investigate our hypothesis about Pb-mediated demethylation of the PRMT5 promoter proximal region as we observed decreased DNMT expression (Figure 3). Among the two CGIs (located in the −372 to −80 bp and −70 to +231 bp regions corresponding to the TSS) analyzed by the MSP, only the region spanning the −372 to −80 bp is significantly demethylated after Pb exposure (Figure 4). Thus, the very proximal upstream region (∼300 bp) of the PRMT5 promoter was found to be increasingly demethylated as the dose of Pb was increased within 48 h of exposure. However, the CGI located within the region −70 and +231 bp which flanks the TSS and the first exon was already demethylated in the unexposed cells, and thus, Pb exposure had little or no effect on this CGI. For better resolution of the methylation of each of the CpG sites, the bisulfite-converted DNA after Pb exposure was subjected to bisulfite sequencing following bacterial cloning. The outcomes of the bisulfite sequencing also supported the results obtained in the MSP experiments. Lead (1 µM) exposure demethylated four CpG sites which were found methylated in the unexposed cells within the region −254 to −46 (relative to TSS) of the PRMT5 promoter. Given the fact that a notable level of PRMT5 was expressed in the unexposed control cells, it probably supports the generalized hypothesis that promoter DNA demethylation has the potential role in regulating nucleosome dynamics, transcription factor binding, and activation of specific genes [51]. The demethylated promoter element (along with other factors like increased binding of transcription factors) might have resulted in the overactive promoter and thus, increased expression of PRMT5 in the Pb-exposed cells.

The DNMTs ensure the methylation of unmethylated as well as hemimethylated DNA, while the ten-eleven translocation (TET) enzymes bind to CpG-rich sequences in transcriptionally active promoters to prevent DNMT activity and trigger demethylation and gene activation [52,53]. Although the results show decreased DNMT expression, the involvement of TET enzymes in Pb-mediated global hypomethylation, and local demethylation of the PRMT5 promoter cannot be ruled out and requires further investigations.

Toxic heavy metals like nickel (Ni), arsenic (As), mercury (Hg), hexavalent chromium [Cr(VI)], Cd, and Pb are present in the environment, and their chronic human exposure has attracted many authors to link them with cancer [54]. Although estrogenicity of cadmium (Cd) has been reported earlier [55], no such clear evidence of estrogenicity is available for Pb. Interestingly, MCF-7 cells are known to harbor a higher level of estrogen receptor α (ERα), and although not clearly understood, ERα promotes the proliferation of MCF-7 cells [56]. However, as the increased viability of the A549 cells was also observed, an ERα-independent mechanism of Pb-mediated induction of cell viability cannot be ruled out, which requires further investigations. In conclusion, the findings of this study indicate that Pb-mediated epigenetic alterations that include augmented PRMT5 expression from its demethylated promoter due to decreased DNMT expression result in increased level of repressive H4R3me2s marks. We hypothesize that these modifications could potentially alter cellular functions (Figure 6), which might contribute to the Pb-responsive pathologies.

Graphical scheme of the key findings of the study.

Figure 6.
Graphical scheme of the key findings of the study.

We hypothesize that the alterations in DNMT and PRMT5 expression due to increased demethylation of the PRMT5 promoter resulting in increased level of SDMA and H4R3me2s marks could possibly alter the cellular functions.

Figure 6.
Graphical scheme of the key findings of the study.

We hypothesize that the alterations in DNMT and PRMT5 expression due to increased demethylation of the PRMT5 promoter resulting in increased level of SDMA and H4R3me2s marks could possibly alter the cellular functions.

Environmental and occupational exposure, chemical persistence, bioaccumulation in the body and decreased excretion in the urine/sweat are significant concerns of Pb regarding human pathologies and cancer. Alteration of epigenetics by environmental chemicals in any stage of life is a significant concern as it directly affects the gene transcription and protein function. A plethora of epigenetic modifications that occur within DNA and histone/non-histone proteins have already been linked to various diseases including cancer as evidenced by increasing interest in the discovery of anticancer epigenetic therapeutics and clinical trials [40,57]. A growing body of evidence have linked adverse environmental and industrial exposure to heavy metals to epigenetic changes (mostly, DNA hypomethylation) in association with human diseases including neurodevelopmental problems and cancer [58]. This study, therefore, is the first report on Pb-mediated alteration of the PRMT5 enzyme in cell line models. Recalling that the human lung and breast cancer cell lines as the model were used in this investigation to resemble the fact that lungs are the primary organs to which aerial exposure to environmental toxicants occurs, while the MCF-7 cells were used as they are highly sensitive cells ubiquitously used to identify estrogenicity of toxicants. However, the findings of this in vitro study can be used to validate in animal models or in Pb-exposed human subjects in further studies. Although a limited but at first attempt, this study has addressed the promoter demethylation as one among the likely factors leading to PRMT5 gene transcription, the exact mechanisms need to be investigated in future.

Abbreviations

     
  • DMEM

    Dulbecco's modified Eagle medium

  •  
  • DMSO

    dimethyl sulfoxide

  •  
  • DNMT

    DNA methyltransferase

  •  
  • FBS

    fetal bovine serum

  •  
  • MEP50

    methylosome protein 50

  •  
  • MSP

    methylation-specific PCR

  •  
  • MTT

    3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

  •  
  • PRMT5

    protein arginine methyltransferase 5

  •  
  • SDMA

    symmetrically dimethylated arginine

  •  
  • TSS

    transcription start site

Author Contribution

K.G. contributed in designing and performing the experiments, acquisition of data, writing the draft manuscript. B.C. performed the experiment and S.R.K. planned, designed, approved, and revised the manuscript critically.

Funding

This work was supported by the DST/SERB Government of India, New Delhi [SB/YS/LS-152/2013].

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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Author notes

Present address: Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Central University P.O., Hyderabad-500046, Telangana, India.