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Journal of Immunotoxicology

ISSN: 1547-691X (Print) 1547-6901 (Online) Journal homepage: http://www.tandfonline.com/loi/iimt20

CpG promoter methylation status is not aprognostic indicator of gene expression inberyllium challenge

Brian C. Tooker, Katherine Ozawa & Lee S. Newman

To cite this article: Brian C. Tooker, Katherine Ozawa & Lee S. Newman (2015): CpG promotermethylation status is not a prognostic indicator of gene expression in beryllium challenge,Journal of Immunotoxicology, DOI: 10.3109/1547691X.2015.1115447

To link to this article: http://dx.doi.org/10.3109/1547691X.2015.1115447

Published online: 16 Dec 2015.

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JOURNAL OF IMMUNOTOXICOLOGY, 2015http://dx.doi.org/10.3109/1547691X.2015.1115447

RESEARCH ARTICLE

CpG promoter methylation status is not a prognostic indicator of geneexpression in beryllium challenge

Brian C. Tookera,b, Katherine Ozawaa and Lee S. Newmana,b

aDivision of Allergy and Clinical Immunology, Department of Medicine, School of Medicine, Aurora, CO, USA; bCenter for Health, Work andEnvironment, Department of Environmental and Occupational Health, Colorado School of Public Health, Aurora, CO, USA

ABSTRACTIndividuals exposed to beryllium (Be) may develop Be sensitization (BeS) and progress to chronicberyllium disease (CBD). Recent studies with other metal antigens suggest epigenetic mechanismsmay be involved in inflammatory disease processes, including granulomatous lung disorders andthat a number of metal cations alter gene methylation. The objective of this study was todetermine if Be can exert an epigenetic effect on gene expression by altering methylation in thepromoter region of specific genes known to be involved in Be antigen-mediated gene expression.To investigate this objective, three macrophage tumor mouse cell lines known to differentiallyproduce tumor necrosis factor (TNF)-�, but not interferon (IFN)-�, in response to Be antigen werecultured with Be or controls. Following challenges, ELISA were performed to quantify inducedTNF� and IFN� expression. Bisulfate-converted DNA was evaluated by pyrosequencing to quantifyCpG methylation within the promoters of TNF� and IFN�. Be-challenged H36.12J cells expressedhigher levels of TNF� compared to either H36.12E cells or P388D.1 cells. However, there were novariations in TNF� promoter CpG methylation levels between cell lines at the six CpG sites tested.H36.12J cell TNF� expression was shown to be metal-specific by the induction of significantly moreTNF� when exposed to Be than when exposed to aluminum sulfate, or nickel (II) chloride, but notwhen exposed to cobalt (II) chloride. However, H36.12J cell methylation levels at the six CpG sitesexamined in the TNF� promoter did not correlate with cytokine expression differences.Nonetheless, all three cell lines had significantly more promoter methylation at the six CpG sitesinvestigated within the IFN� promoter (a gene that is not expressed) when compared to the sixCpG sites investigated in the TNF� promoter, regardless of treatment condition (p51.17� 10�9).These findings suggest that, in this cell system, promoter hypo-methylation may be necessary toallow expression of metal-induced TNF� and that promoter hyper-methylation in the IFN�promoter may interfere with expression. Also, at the dozen CpG sites investigated in the promoterregions of both genes, beryllium had no impact on promoter methylation status, despite its abilityto induce pro-inflammatory cytokine expression.

ARTICLE HISTORY

Received 23 July 2015Revised 16 October 2015Accepted 27 October 2015Published online15 December 2015

KEYWORDS

Beryllium; epigenetics;chronic beryllium disease;metal antigen; CpG; DNAmethylation

Introduction

Inhalation of particulate forms of beryllium (Be) metal,beryllium oxide ceramics or Be-containing alloys canlead to beryllium sensitization (BeS) (Baggerly et al.2004), an adaptive immune response to Be (Newman &Kreiss 1992; Kelleher et al. 2001; Infante & Newman2004; Maier et al. 2008) in a proportion of exposedindividuals. BeS has been shown to presage the devel-opment of chronic beryllium disease (CBD) at a rate of6–8% per year (Newman et al. 2005). In CBD, individualsdemonstrate an inflammatory process in the lungcharacterized by non-caseating granulomas and/or mono-nuclear cell infiltrates in lung tissue (Newman et al. 1989).A substantial body of literature has demonstrated differ-ences in pro-inflammatory cytokine levels such as

interferon (IFN)-�, interleukin (IL)-2, (IL)-6 and tumornecrosis factor (TNF)-� (Bost et al. 1994; Tinkle et al.1997, 1999; Kelleher et al. 2001) between patients with BeSand CBD when either white blood cells or lung lavagecells, especially CD4+ T-lymphocytes and macrophages,are incubated ex vivo the presence of Be salts. However, wehave only a limited understanding of the underlyingmechanisms by which Be may affect the expression ofthese pro-inflammatory cytokines.

Two lines of evidence have led us to investigate thehypothesis that variations in DNA promoter regionmethylation may explain variations in gene expressionand that Be, a metal cation, may be able to alter DNAmethylation states. First, although there have been nopublished studies in CBD to date, preliminary data from

CONTACT Brian C. Tooker brian.tooker@ucdenver.edu University of Colorado Denver, Division of Allergy and Clinical Immunology, Building RC2, Room:10420D, Mail Stop B164, 12700 E. 19th Ave, Aurora, CO 80045, USA

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a recent abstract suggests differential methylationbetween patients with BeS and CBD in bronchoalveolarlavage (BAL)-derived cell populations. In these cells,lower levels of methylation (hypo-methylation) wereobserved in TNF� promoters of patients with CBD whencompared to methylation levels of BAL-derived cellsfrom patients with BeS (Silveira et al. 2013). Further,Maeda et al. (2009) demonstrated gene-associated hypo-methylation in patients with sarcoidosis, a granuloma-tous disorder immuno-pathogenically similar to CBD.Liu et al. showed that epigenetics might play a role inimmune-mediated pulmonary diseases (He et al. 2013).Second, an emerging body of literature demonstratesthat certain metal cations, i.e. nickel, lead, chromium,arsenic and cadmium, can induce epigenetic alterations,although Be has not yet been studied (Lee et al. 1995;Baggerly et al. 2004; Baccarelli and Bollati 2009; Hannaet al. 2012).

To investigate the hypothesis that Be can affect geneexpression by modulating promoter methylation, ourgroup utilized three related macrophage mouse tumorcell lines, H36.12J, H36.12E and P388D.1, that areknown to differentially express TNF� when challengedwith Be (Hamada et al. 2000; Sawyer et al. 2000b).In previous studies, P388D.1 (parental cell line) andH36.12E (daughter line) both failed to express high levelsof TNF� when challenged with beryllium sulfate(BeSO4), cobalt sulfate (CoSO4) or aluminum sulfate(Al2[SO4]3). However, H36.12J, a daughter cell linederived from P388D.1, expressed high levels of TNF�when challenged with BeSO4, but not Al2(SO4)3 norCoSO4 (Sawyer et al. 2000b).

In the studies reported here, these three cell lines wereexposed to either Be, other multivalent metal salts asmetal controls, PBS as a volume control and a no-addition as an additional negative control to confirmdifferential TNF� expression and a lack of IFN�expression. DNA from challenged cells was then isolated,subjected to sodium bisulfite treatment and evaluatedusing pyrosequencing to assess specific CpG methylationin both the IFN� and TNF� promoter regions.

Materials and methods

Cell culture and challenge

Three mouse macrophage tumor cell lines, H36.12J,H36.12E and P388D.1 which have been shown to bedifferentially responsive to Be (Sawyer et al. 2000a, b),were obtained from the American Type CultureCollection (ATCC, Manassas, VA) and cultured at37 �C under 5% CO2 in HyClone DMEM (ThermoFisher, Pittsburgh, PA) with the addition of 10%

fetal bovine sera (Sigma, St. Louis, MO), 100 U penicil-lin/ml, 100 mg streptomycin/ml, 100 mM HEPES and10 mM sodium pyruvate (all Thermo Fisher, Pittsburgh,PA).

For challenges, cells were removed from cultureflasks and centrifuged (400 � g, 5 min) to pellet cells.Supernatants were decanted and cells suspended at 106

cells/ml media and transferred to 24-well plates (CorningInc. Corning, NY) at 106 cells/well and cells were allowedto recover for 2 h at 37 �C, under 5% CO2. Cells werethen challenged with 100mM BeSO4, 100 mM CoCl2,100mM Ni(II)Cl2, 100mM Al2(SO4)3, 100 ml phosphate-buffered saline (PBS, pH 7.4) or non-addition control(NC) and then incubated at 37 �C under 5% CO2 for18 h. In separate experiments, it was confirmed by trypanblue exclusion studies that none of the metal salts used inthese experiments induced cell death (data not shown).Following challenges, there were mixtures of suspensionand adherent cells in culture wells. Well supernatantswere transferred to 15-ml tubes and centrifuged (400�g,5 min) to pellet suspension cells. Supernatants wereremoved to 96-well plates and stored at �80 �C untilutilized for ELISA.

Adherent cells were removed from treatment wells byfirst washing wells twice with 2 ml PBS pH 7.4 to removetrace amounts of FBS and residual growth media.These and subsequent washes were added to corres-ponding 15-ml tubes holding the suspension cell pellets.Treatment wells were then incubated with 500ml of0.025% trypsin (Thermo Fisher) for 3 min at 37 �C.Trypsin was quenched by the addition of 2 ml growthmedia and cells dislodged from wells by scraping andrepeated pipetting. Previously adherent cells weretransferred to 15-ml tubes holding corresponding sus-pension cells and wells washed twice with 2 ml PBS pH7.4. Tubes containing suspension and adherent cells werecentrifuged (400�g, 5 min) to pellet cells. Supernatantswere decanted and the cell pellets immediately subjectedto DNA isolation (see below).

ELISA

Cell culture supernatants were analyzed for the presenceof both TNF� and IFN� according to a standardeBioscience (San Diego, CA) ELISA protocol. Briefly,capture antibody was diluted in eBioscience CoatingBuffer and 100 ml added to each well of Costar 96-wellhigh binding EIA/RIA plates (Corning). Wells werethen sealed with Nunc Aluminum sealing tape (NalgeNunc, Rochester, NY) and incubated overnight at 4 �C.Following incubation, capture antibody solutions weredecanted and wells washed 5�with 250 ml PBS-0.5%Tween 20 solution (PBST). Wells were then blocked

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for 60 min at room temperature, with shaking on aMicroMix 5 system (Siemens, Washington, DC) atsettings of Form: 20 and Amplitude: 7, with the additionof 100ml of the assay diluent to limit non-specificbinding. Blocking solution was then removed and plateswashed 5� as described above.

Culture supernatants were diluted 1:1 in assay diluentand 100ml added to wells of ELISA plates. Standardswere serially diluted as per manufacturer instructionsand 100ml also added to ELISA plates. Plates containingsamples/standards were incubated for 2 h at roomtemperature with shaking on the MicroMix 5 as above.Following incubation, well contents were discarded andwells washed 5� as above. Secondary biotin-labeleddetection antibodies were diluted in assay diluent asper manufacturer instructions and 100ml added to eachwell; plates were then incubated for 60 min at roomtemperature with gentle shaking. Supernatants contain-ing unbound detection antibodies were then discardedand wells washed 5� as above. Avidin-horseradishperoxidase (HRP) solution was diluted to manufacturerspecifications and 100ml added to each well for a 30 minincubation at room temperature, with gentle shaking.Avidin-HRP was removed and wells washed 7� asabove. To each well, 100 ml substrate solution (TMB;3,30,5,50-tetramethylbenzidine) was added and platesincubated at room temperature in the dark for up to15 min. Substrate color changes were halted by additionof 100 ml 2M H2SO4. Absorbances at 450 and 570 nmwere then determined on a Powerwave HT plate reader(BioTek, Winooski, VT). OD values at 570 nm weresubtracted from the OD at 450 nm to obtain a normal-ized OD 450 nm and sample cytokine concentrationswere determined by extrapolation from the standardcurves generated in parallel.

DNA isolation and bisulfate conversion

Cell culture pellets were moved to 1.5 ml Eppendorftubes, washed in 500 ml PBS pH 7.4 and centrifuged for10 s at 14 000�g. Resulting supernatants were aspirated,leaving �50ml supernatant atop each cell pellet. Pelletswere suspended and DNA isolated utilizing a WizardGenomic DNA purification kit (Promega, Madison, WI).DNA quantity and quality were determined by absorb-ance at 260 and 280 nm on the Powerwave HT platereader. DNA was stored under ethanol (2.5 vol of 100%ethanol and 1/10 vol 3 M sodium acetate) at�80 �C untilutilized.

DNA bisulfite conversion was performed using anEpiTect Bisulfite Kit (Qiagen, Hiden, Germany) as permanufacturer protocols. Briefly, up to 2 mg DNA wassubjected to the bisulfite conversion and converted DNAfinally suspended at 10 ng/ml and aliquoted into individ-ual tubes at 100 ng/tube. Aliquoting was performed toavoid freeze/thaw cycles that the manufacturer statesmay interfere with downstream bisulfite sequencing.Aliquoted bisulfite converted DNA was stored at�20 �Cuntil utilized.

Pyrosequencing

In duplicate, bisulfite-converted DNA was amplifiedutilizing PyroMark PCR kits (Qiagen) following manu-facturer protocols with some minor modifications. Fiveprimer sets for amplification and six correspondingsequencing primers were designed utilizing PyroMarkAssay Design 2.0 Software (Qiagen), to investigate CpGmethylation in both IFN� and TNF� promoter regions(Table 1). Amplicon generation, evaluated by agarose gelelectrophoresis and DNA Sanger sequencing (data notshown), was performed using the following reaction

Table 1. Amplification and pyrosequencing primer sets.

Primer Direction Sequence Bases Tm Annealing Tm (C) Amplification Primer Set

IFN1-1(2)-F Forward AGTAGTGAATTATAGTTTAAGGGTTGAA 28 52.4 60.2IFN1-1(2)-BIOTIN-R Reverse biotin-CTCTCAACAACACCCTAAC 19 50.3IFN1-4(2)-1-BIOTIN-F Forward biotin-GAGGAAAAGTTAGGGTGTTGTTGAGAG 27 57.4 58.1IFN1-4(2)-1-R Reverse ACCAATCATACCCTATCAAACTTCTTCT 28 55.9TNF1-1F Forward TGGAGGAGTAGTTGGAGTG 19 53.2 59.8TNF1-1R Reverse ACAACCCATCCACTAACACCACTAA 25 58.0TNF1-2F Forward AGTAGTTGGAGTGGTTGAGTTA 22 53.1 59.8TNF1-2R Reverse CAACCTTATCCCTTAAAAAAAACCTAAA 28 52.7TNF1-3F Forward TTTAGGTTTTTTTTAAGGGATAAGGT 26 51.3 59.2TNF1-3R Reverse ATTAATATCCTTAAAACAAAAACTCT 26 47.7IFN1-1(2)-SEQ ND TTTTATAGTAATTGAAATTTTAAGA 25 43.5 IFN1-1(2)F/RIFN1-2(2)-SEQ ND GTTAATGAAGTGTGTTTATTGTGAA 25 50.3 IFN1-1(2)F/RIFN1-4(2)-SEQ ND CCTACAATATCAAATTTACAAAC 23 46.0 IFN1-4(2)F/RTNF1-1-SEQ ND TTGGAGTGGTTGAGT 15 46.2 TNF1-1F/RTNF1-2-SEQ ND ATTAATTAGTGGTGTTAG 18 40.5 TNF1-2F/RTNF1-4-SEQ ND GTTATTTTATATTAGGAGAAAGTTA 25 44.8 TNF1-3F/R

Primers and conditions used for amplification and pyosequencing of mouse CpG in TNFa and IFNg promoter regions. Each sequencing primer (-SEQ at the endof the primer name) was paired with a set of amplification primers. These primers were designed using PyroMark Assay Design 2.0 and DNA sequences NCBIaccession number NC_8000083.5 and NC_000076.5 for TNFa and IFNg, respectively.

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mixture: 10 ml of 2�Master Mix, 2 ml of 5�Q Solution,1.6 ml of a mixed Forward/Reverse primer set (3 mMbiotinylated primer and 5 mM non-biotinylated primer)(Table 1), 10 ng of bisulfite converted DNA and H2O to20ml. Amplification with primers TNF1-3 F/R had allthe constituents above, but used 20 ng converted DNAand had an additional 3.75 mM MgCl2 in each reaction.Amplifications were preformed using the conditions:95 �C, 15 min (94 �C, 1 min; Tm (Table 1), 1 min; 72 �C,1 min)40; 72 �C, 10 min. Amplifications were held at22 �C until subjected to pyrosequencing.

Pyrosequencing was performed on the ampliconsusing a Pryomark Q96 according to manufacturerprotocols (Qiagen) and involved four steps: sequencing

primer (Table 1) hybridization, dNTP addition, ATPsulfurylase addition for conversion of pyrophosphate(PPi) to ATP and addition of apyrase to degrade non-incorporated nucleotides. These steps were repeated untilthe program reached the end of the sequence to analyze.Incorporation of dNTP and PPi release revealed thepercentage of cytosine methylation at the specific sitesinvestigated in the amplicon, namely the CpG sites.

Statistical analysis

All statistical analysis was preformed utilizing a Student’st-test with Bonferroni corrections for multiplecomparisons.

Figure 1. TNFa expression (mean ± standard deviation) of cells treated with 100 mM BeSO4, 100 mM CoCl2, 100 mM Ni(II)Cl2, 100 mMAl2(SO4)3, 100 ml phosphate-buffered saline (PBS, pH 7.4) or non-addition control (NC); n¼ 3 experiments. H36.12J cells treated withBe expressed 446.90 [± 110.98] pg TNFa/ml, significantly more than produced by H36.12J cells treated with either Al2(SO4)3 239.78[± 10.85] pg TNFa/ml, Ni(II)Cl2 170.38 [± 19.12] pg TNFa/ml, PBS 122.41 [± 23.23] pg TNFa/ml or NC 152.95 [± 22.01] pg TNFa/ml(p50.01). This amount of TNFa was also significantly greater than amounts made by either the H36.12E or P388D.1 cells (p50.03)treated with Be.

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Results

ELISA

TNF� and IFN� ELISA were performed on the cellculture supernatants of cells exposed to Be or controlsolutions to confirm that H36.12J, H36.12E and P388D.1cells differentially-expressed cytokines. Further, thisstudy sought to confirm this TNF� production was Be-specific within the H36.12J Be-responsive cells, asprevious studies have shown (Sawyer et al. 2000b).

None of the conditions tested induced any of the threecell lines to produce IFN�. However, when H36.12J (Be-responsive) cells were treated with 100 mM BeSO4, theyexpressed significantly more TNF� (446.93 [± 110.98]pg/ml) than when treated with either Al2(SO4)3,Ni(II)Cl2, PBS or NC (p50.008) (Figure 1). When Be-

responsive cells were treated with 100 mM cobalt chlor-ide, they produced levels of TNF� that were notsignificantly different from the amount of TNF�produced with 100 mM BeSO4 treatment (p50.068).Further, when comparing levels of TNF� produced bythe three lines challenged with 100 mM BeSO4, theH36.12J cells (Be-responsive) produced significantlymore TNF� than H36.12E cells (Be-non-responsive) orP388D.1 (parental) cells (p50.003, p50.034),confirming previous study results using these lines(Sawyer et al. 2000b).

Pyrosequencing

By performing pyrosequencing on the sodium bisulfiteconverted DNA of treated cells, percentage methylation

Table 2. Percentage CpG methylation.

TNF1-1 TNF1-2 TNF1-3 TNF1-4 TNF2-1 TNF4-1

Cell Line Stim Avg. Stdev Avg. Stdev Avg. Stdev Avg. Stdev Avg. Stdev Avg. Stdev

TNF-alpha

H36.12J PBS 5.50 3.12 2.17 1.15 2.17 1.61 6.33 1.53 2.17 1.04 17.75 3.18BeSO4 4.50 1.80 2.17 0.76 2.00 1.00 6.17 0.76 2.67 1.15 19.50 2.50CoCl2 5.67 3.21 2.00 1.00 2.50 1.50 7.50 1.80 1.67 0.76 17.33 2.02Ni(II)Cl2 5.00 1.73 2.83 1.61 3.17 1.89 8.00 1.32 2.83 1.04 19.17 4.54Al2(SO4)3 5.83 2.25 2.67 1.44 3.00 1.32 6.17 1.44 2.83 1.04 26.17 5.30NC 5.33 1.61 2.33 1.26 2.67 1.53 6.67 1.26 2.17 1.04 23.67 2.36

H36.12E PBS 5.50 2.65 2.67 2.08 3.17 2.08 6.83 1.89 2.17 0.29 22.67 1.61BeSO4 3.83 1.04 1.83 1.04 2.00 1.32 8.00 1.80 2.17 0.29 22.83 2.08CoCl2 5.83 3.01 2.83 1.26 2.50 1.32 8.50 2.78 2.17 0.76 24.83 3.21Ni(II)Cl2 3.33 0.76 2.00 0.87 2.50 1.50 8.17 3.25 2.83 2.36 27.00 0.00Al2(SO4)3 4.67 1.26 2.67 1.04 2.83 0.76 7.17 0.58 2.50 0.87 30.25 5.30NC 4.83 1.76 2.83 1.61 2.67 1.53 6.83 0.76 2.50 0.50 22.50 2.29

P388D.1 PBS 2.67 0.29 1.17 0.58 2.00 0.50 8.00 3.50 1.67 0.76 23.67 1.04BeSO4 3.67 0.76 1.83 0.58 2.17 2.02 5.83 3.69 3.33 1.44 29.17 1.76CoCl2 4.00 2.65 1.83 1.44 2.00 1.32 5.67 2.75 3.67 2.31 26.33 0.58Ni(II)Cl2 4.33 1.53 2.33 2.75 1.83 1.04 7.17 2.47 2.67 0.76 22.00 4.00Al2(SO4)3 6.17 3.33 3.50 2.50 2.67 1.76 6.17 2.25 2.83 1.44 27.50 0.87NC 3.50 1.80 2.17 1.61 2.50 2.12 7.00 1.41 2.50 0.50 25.33 2.02

IFN1-1 IFN1-2 IFN2-1 IFN2-2 IFN2-3 IFN4-1

IFN-gamma

H36.12J PBS 90.50 1.32 90.50 3.97 95.17 0.29 98.67 0.76 96.33 2.08 94.33 1.76BeSO4 90.00 1.73 90.33 1.04 95.17 0.76 97.67 0.29 96.33 1.76 93.33 2.02CoCl2 91.67 1.04 93.33 1.44 96.50 0.50 98.17 0.29 97.17 0.29 88.17 11.03Ni(II)Cl2 90.67 3.21 89.83 2.93 95.50 1.50 97.50 0.50 96.33 1.76 90.00 12.62Al2(SO4)3 92.83 1.89 91.67 2.84 94.00 0.87 98.17 0.29 94.83 1.61 87.33 13.29NC 90.83 2.02 89.50 4.44 94.50 1.73 97.17 1.89 95.50 1.32 92.50 2.65

H36.12E PBS 90.50 0.50 86.67 2.84 90.50 0.50 97.50 0.50 95.17 0.58 86.17 3.51BeSO4 92.33 1.04 90.00 4.09 91.00 4.50 98.17 0.58 96.00 1.32 86.17 3.33CoCl2 92.17 1.89 90.83 2.02 92.00 1.80 97.83 0.76 95.33 1.44 88.00 3.12Ni(II)Cl2 90.50 1.00 87.00 3.04 92.33 3.06 96.67 1.89 92.33 3.62 83.17 6.01Al2(SO4)3 88.33 2.89 85.83 3.21 90.17 1.53 97.83 0.58 95.33 1.26 86.50 0.50NC 89.67 1.44 87.83 3.33 90.67 2.75 96.50 1.32 94.33 1.53 92.67 3.18

P388D.1 PBS 91.17 1.44 92.17 1.04 96.00 0.87 98.33 0.29 96.00 2.29 86.83 15.89BeSO4 90.17 1.26 91.67 3.33 95.83 1.44 97.83 1.04 97.00 1.00 89.33 11.56CoCl2 92.33 0.76 94.00 2.60 96.00 1.73 96.50 2.18 96.33 2.02 93.33 2.52Ni(II)Cl2 90.67 0.58 93.00 1.00 96.67 0.29 97.33 0.76 95.67 1.04 92.67 4.48Al2(SO4)3 89.50 3.91 91.67 3.18 96.33 0.76 97.83 0.76 96.67 1.15 93.50 4.36NC 90.33 1.04 91.17 3.69 94.00 2.78 97.50 1.50 97.00 1.32 89.67 3.01

Percentage methylation found at the 12 CpG in the TNFa and IFNg promoter regions. TNFa CpG sites TNF1-1: 35 337 466, TNF1-2: 35 337 464, TNF1-3: 35 337458, TNF1-4: 35 337 443, TNF2-1: 35 337 403 and TNF4-1: 35 337 274 of NCBI Accessions Number NC_8000083.5. IFNg CpG sites IFN1-1: 117 883 310, IFN1-2:117 883 029, IFN2-1: 117 883 285, IFN2-2: 117 883 283, IFN2-3: 117 883 274 and IFN4-1: 117 883 231 of NCBI Accessions Number NC_000076.5.

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was determined for 12 CpG sites within the promoterregions for TNF� (six CpG sites at positions: 35 337 466,35 337 464, 35 337 458, 35 337 443, 35 337 403 and 35337 274 of NCBI Accessions Number NC_8000083.5)and IFN� (six CpG sites at positions: 117 883 310, 117883 029, 117 883 285, 117 883 283, 117 883 274 and 117883 231 of NCBI Accessions Number NC_000076.5),with notably different results (Table 2). The percentagemethylation of the six CpG sites assayed in theTNF� promoter region varied from 0.29–29.17%across cell lines and treatment (Figure 2a and Table 2).In contrast, percentage methylation of the six CpG sitesassayed in the IFN� promoter region varied from83.17–98.67% across cell lines and treatment (Figure 2band Table 2).

To investigate treatment differences in levels of TNF�CpG promoter methylation in the Be-responsive cell line,

H36.12J, methylation levels in Be-treated cells werecompared to methylation levels in aluminum-treated,cobalt-treated, nickel-treated, PBS-treated or NC-cells(Figure 3). There was no significant difference inmethylation levels across treatment in the H36.12J atthe six TNF� promoter CpG sites investigated.

These sites were further investigated for possiblemethylation differences across the three cell typestreated with Be. Although TNF� production variedacross the three cell types with Be treatment (H36.12J:446.93 [± 110.98], H36.12E: 35.37 [± 6.20] and P388D.1:242.83 [± 9.50] ng/ml) comparing CpG percentagemethylation levels between Be-responsive cells vs par-ental cells or Be-responsive cells vs Be-non-responsivecells at the six TNF� promoter CpG investigated,revealed no significant variation, after Bonferroni cor-rection, which required a p50.004 for significance (see

Figure 2. Representative CpG methylation for TNFa (a) and IFNg (b) (mean ± standard deviation) of cells treated with 100 mM BeSO4,100 mM CoCl2, 100 mM Ni(II)Cl2, 100 mM Al2(SO4)3, 100 ml phosphate-buffered saline (PBS, pH 7.4) or a non-addition control (NC); n¼ 3experiments. (a) Percentage methylation of TNF1-1 CpG by treatment for each of the three cell types is representative of CpGmethylation seen at this site within TNFa promoter region. (b) Percentage methylation observed for the three cell types (bytreatment) for the IFN1-1 CpG site is representative of methylation levels observed in the IFNg promoter region.

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Figure 4). However, between the Be-responsive cells andthe parental cells, the level of TNF� promoter CpGmethylation at site TNF4-1 approached significance(p50.005).

Finally, overall promoter methylation levels at the twogenes of interest, TNF� and IFN�, was investigated.Within each of the three cell lines, means and standarddeviations of percentage methylation, regardless oftreatment, were calculated using the 12 CpG (six inTNFa and six in IFN�) methylation percentagesdetermined in this study. As shown in Figure 5, ineach of the three cell lines, overall promoterpercentage CpG methylation was significantly higher inthe IFN� promoter than in the TNF� promoter

(H36.12J: p50.001, H36.12E: p50.001 and P388D.1:p50.001).

Discussion

The goals of this set of experiments were two-fold. First,to determine if beryllium (Be) could modulate geneexpression in an epigenetic fashion by altering promoterCpG DNA methylation in a gene-specific fashion and,second, to determine if differences in CpG promotermethylation may contribute to controlling gene expressionin a model system with beryllium stimulation. Here, weutilized a mouse macrophage tumor cell line known toexpress TNF� but not IFN� in response to Be-stimulation

Figure 3. H36.12J percentage TNFa promoter CpG methylation (mean ± standard deviation) of cells treated with 100 mM BeSO4, 100mM CoCl2, 100 mM Ni(II)Cl2, 100 mM Al2(SO4)3, 100 ml phosphate-buffered saline (PBS, pH 7.4) or a non-addition control (NC); n¼ 3experiments. Percentage methylation observed with each treatment at each of the six TNFa promoter CpG sites investigated showedno significant difference between treatments.

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(H36.12J). As controls we utilized a sister mouse macro-phage tumor cell line that does not respond to Bestimulation (H36.12E) and the parental mouse macro-phage tumor cell line (P388D.1) that also fails to respondto Be stimulation. This study determined there was anoverall state of hypo-methylation present at all investigatedCpG in the TNF� promoter and an overall state of hyper-methylation present at all investigated CpG in the IFN�promoter for all three cell lines. Furthermore, the studyestablishes that Be, unlike a number of other metal cations,had no impact on the promoter methylation status at the12 CpG sites investigated in these three cell lines, despitean ability to induce pro-inflammatory cytokine expression.Although we observed no detrimental effects to culturingthe cells in the presence of these metal salts, we did not

exhaustively investigate metal toxicity, but did observemetal salts failed to induce cell death by trypan blueexclusion testing (data not shown).

Be-responsive H36.12J cells treated with Be did expresshigh levels of TNF� and this cell line was shown to have aTNF� promoter CpG sites with methylation levels from2.17–17.75% (Table 2). This correlates well with humandata from a genome wide methylation study (Silveira et al.2013) that showed less DNA methylation in the TNF�promoter of BAL cells from CBD patients than BAL cellsfrom BeS patients. However when treated with Be, theTNF� promoter CpG sites in both the Be-non-responsiveH36.12E cell line and the parental P388D.1 cell line alsorevealed very low states of methylation (0.29–22.83% and0.58–29.17%, respectively). Also, unlike other metal

Figure 4. Percentage TNFa promoter CpG methylation (mean ± standard deviation) of cells treated with 100 mM BeSO4, 100 mM CoCl2,100 mM Ni(II)Cl2, 100 mM Al2(SO4)3, 100 ml phosphate-buffered saline (PBS, pH 7.4) or a non-addition control (NC); n¼ 3 experiments.Comparing the levels of CpG promoter methylation between the three mouse cell lines cultured with 100 mM BeSO4 overnightrevealed significantly less (p50.005) methylation at CpG site TNF 4-1 in H36.12J cells when compared to in P388D.1 cells, the parentalcell line.

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cations such as Cr, As, Ni, Pb or Cd (Lee et al. 1995;Chanda et al. 2006; Jiang et al. 2008; Rozhon et al. 2008;Verstraeten et al. 2008) that have been shown in othersystems to change CpG methylation states in other genes,this hypo-methylation was not a phenomenon of Betreatment. The state of hypo-methylation was observed atall TNF� promoter CpG, regardless of treatment(Table 2).

These studies, thus far, investigated the levels of CpGmethylation between three cell lines that differentiallyexpressed TNF�, when challenged with Be. To deter-mine if this promoter CpG hypo-methylation wasspecific only to expressed genes, as expected in anepigenetically controlled gene (Bird 1986; Meehan et al.1992) or an overall genome-wide phenomenon in these

cell lines, pyrosequencing was performed on several CpGin the IFN� promoter, a gene whose expression was notinducible with treatments investigated in these studies. Itwas expected that this set of promoter CpG should behighly methylated.

Of the six IFN� promoter CpG methylation sitestested, all had levels of methylation that were at least 4-times higher, in most cases 16–20-times higher, than thesix TNF� promoter CpG methylation sites examined inthese studies. When we compared the overall promotermethylation states within each of the cell lines, weobtained highly significant differences (p50.001; seeFigure 5) in the levels of CpG methylation between theexpressed (TNF�) and non-expressed cytokine (IFN�)in each of the three cell lines.

Figure 5. Comparisons of TNFa and IFNg CpG percentage methylation (mean ± standard deviation) of cells treated with 100 mMBeSO4, 100 mM CoCl2, 100 mM Ni(II)Cl2, 100 mM Al2(SO4)3, 100 ml phosphate-buffered saline (PBS, pH 7.4) or non-addition control (NC);n¼ 3 experiments. Comparison of overall percentage CpG methylation for each promoter region separated by cell type, regardless ofcell treatment, shows percentage CpG methylation of the TNFa promoter was much lower than that of the IFNg promoter within eachcell type.

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In this set of experiments we attempted to determineif there was epigenetic control of gene expression atthe level of DNA promoter CpG methylation. Theseobservations, that the TNF� promoter is in a state ofhypo-methylation corresponding to gene expression(Bird 1986; Antequera 2003) and the IFN� promoter isin a state of hyper-methylation corresponding to genesilencing (Meehan et al. 1992), correlates well with themajority of the epigenetic literature (Alam et al. 2010;Huang et al. 2011; Hanna et al. 2012; Lou et al. 2013;Niedzwiecki et al. 2013; Sanders et al. 2014). Although,in the six TNF� promoter CpG investigated in thesethree mouse macrophage tumor cell lines, these studiessuggest that CpG methylation does not correlate withpro-inflammatory gene expression in response to Be. It ispossible that Be may interact with other CpG locatedwithin the promoter regions of either the TNF� gene (21CpG total). It is also possible that there are alternativemechanisms, beyond the purview of this study, whichmay control pro-inflammatory gene expression in thiscell system. Future research may be warranted toexamine these other epigenetic mechanisms – such asmicroRNA modulation or histone modifications – whichhave been implicated in the response to cations such asnickel, cobalt, lead and chromium (Chen et al. 2006; Liet al. 2009; Bollati et al. 2010; He et al. 2013).

Acknowledgements

The authors would like to thank Dr Ivana Yang and MissCorinne Hennessy for their help in the design of pyrosequen-cing primers and Dr Ivana Yang and Dr David Schwartz forallowing us to utilize their pyrosequencing machine andlaboratory space.

Declaration of interest

The authors report no conflicts of interest. The authors aloneare responsible for the content and writing of the paper.

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