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

Figures & data

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

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

Figure 1. TNFα expression (mean ± standard deviation) of cells treated with 100 μM BeSO₄, 100 μM CoCl₂, 100 μM Ni(II)Cl₂, 100 μM Al₂(SO₄)₃, 100 μl phosphate-buffered saline (PBS, pH 7.4) or non-addition control (NC); n = 3 experiments. H36.12J cells treated with Be expressed 446.90 [± 110.98] pg TNFα/ml, significantly more than produced by H36.12J cells treated with either Al₂(SO₄)₃ 239.78 [± 10.85] pg TNFα/ml, Ni(II)Cl₂ 170.38 [± 19.12] pg TNFα/ml, PBS 122.41 [± 23.23] pg TNFα/ml or NC 152.95 [± 22.01] pg TNFα/ml (p < 0.01). This amount of TNFα was also significantly greater than amounts made by either the H36.12E or P388D.1 cells (p < 0.03) treated with Be.

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

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	3.18
	BeSO4	4.50	1.80	2.17	0.76	2.00	1.00	6.17	0.76	2.67	1.15	19.50	2.50
	CoCl2	5.67	3.21	2.00	1.00	2.50	1.50	7.50	1.80	1.67	0.76	17.33	2.02
	Ni(II)Cl2	5.00	1.73	2.83	1.61	3.17	1.89	8.00	1.32	2.83	1.04	19.17	4.54
	Al2(SO4)^3	5.83	2.25	2.67	1.44	3.00	1.32	6.17	1.44	2.83	1.04	26.17	5.30
	NC	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	1.61
	BeSO4	3.83	1.04	1.83	1.04	2.00	1.32	8.00	1.80	2.17	0.29	22.83	2.08
	CoCl2	5.83	3.01	2.83	1.26	2.50	1.32	8.50	2.78	2.17	0.76	24.83	3.21
	Ni(II)Cl2	3.33	0.76	2.00	0.87	2.50	1.50	8.17	3.25	2.83	2.36	27.00	0.00
	Al2(SO4)^3	4.67	1.26	2.67	1.04	2.83	0.76	7.17	0.58	2.50	0.87	30.25	5.30
	NC	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	1.04
	BeSO4	3.67	0.76	1.83	0.58	2.17	2.02	5.83	3.69	3.33	1.44	29.17	1.76
	CoCl2	4.00	2.65	1.83	1.44	2.00	1.32	5.67	2.75	3.67	2.31	26.33	0.58
	Ni(II)Cl2	4.33	1.53	2.33	2.75	1.83	1.04	7.17	2.47	2.67	0.76	22.00	4.00
	Al2(SO4)^3	6.17	3.33	3.50	2.50	2.67	1.76	6.17	2.25	2.83	1.44	27.50	0.87
	NC	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	1.76
	BeSO4	90.00	1.73	90.33	1.04	95.17	0.76	97.67	0.29	96.33	1.76	93.33	2.02
	CoCl2	91.67	1.04	93.33	1.44	96.50	0.50	98.17	0.29	97.17	0.29	88.17	11.03
	Ni(II)Cl2	90.67	3.21	89.83	2.93	95.50	1.50	97.50	0.50	96.33	1.76	90.00	12.62
	Al2(SO4)^3	92.83	1.89	91.67	2.84	94.00	0.87	98.17	0.29	94.83	1.61	87.33	13.29
	NC	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	3.51
	BeSO4	92.33	1.04	90.00	4.09	91.00	4.50	98.17	0.58	96.00	1.32	86.17	3.33
	CoCl2	92.17	1.89	90.83	2.02	92.00	1.80	97.83	0.76	95.33	1.44	88.00	3.12
	Ni(II)Cl2	90.50	1.00	87.00	3.04	92.33	3.06	96.67	1.89	92.33	3.62	83.17	6.01
	Al2(SO4)^3	88.33	2.89	85.83	3.21	90.17	1.53	97.83	0.58	95.33	1.26	86.50	0.50
	NC	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	15.89
	BeSO4	90.17	1.26	91.67	3.33	95.83	1.44	97.83	1.04	97.00	1.00	89.33	11.56
	CoCl2	92.33	0.76	94.00	2.60	96.00	1.73	96.50	2.18	96.33	2.02	93.33	2.52
	Ni(II)Cl2	90.67	0.58	93.00	1.00	96.67	0.29	97.33	0.76	95.67	1.04	92.67	4.48
	Al2(SO4)^3	89.50	3.91	91.67	3.18	96.33	0.76	97.83	0.76	96.67	1.15	93.50	4.36
	NC	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 TNFα and IFNγ promoter regions. TNFα CpG sites TNF1-1: 35 337 466, TNF1-2: 35 337 464, TNF1-3: 35 337 458, TNF1-4: 35 337 443, TNF2-1: 35 337 403 and TNF4-1: 35 337 274 of NCBI Accessions Number NC_8000083.5. IFNγ 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.

Figure 3. H36.12J percentage TNFα promoter CpG methylation (mean ± standard deviation) of cells treated with 100 μM BeSO₄, 100 μM CoCl₂, 100 μM Ni(II)Cl₂, 100 μM Al₂(SO₄)₃, 100 μl phosphate-buffered saline (PBS, pH 7.4) or a non-addition control (NC); n = 3 experiments. Percentage methylation observed with each treatment at each of the six TNFα promoter CpG sites investigated showed no significant difference between treatments.

Figure 4. Percentage TNFα promoter CpG methylation (mean ± standard deviation) of cells treated with 100 μM BeSO₄, 100 μM CoCl₂, 100 μM Ni(II)Cl₂, 100 μM Al₂(SO₄)₃, 100 μl phosphate-buffered saline (PBS, pH 7.4) or a non-addition control (NC); n = 3 experiments. Comparing the levels of CpG promoter methylation between the three mouse cell lines cultured with 100 μM BeSO₄ overnight revealed significantly less (p < 0.005) methylation at CpG site TNF 4-1 in H36.12J cells when compared to in P388D.1 cells, the parental cell line.

Figure 5. Comparisons of TNFα and IFNγ CpG percentage methylation (mean ± standard deviation) of cells treated with 100 μM BeSO₄, 100 μM CoCl₂, 100 μM Ni(II)Cl₂, 100 μM Al₂(SO₄)₃, 100 μl phosphate-buffered saline (PBS, pH 7.4) or non-addition control (NC); n = 3 experiments. Comparison of overall percentage CpG methylation for each promoter region separated by cell type, regardless of cell treatment, shows percentage CpG methylation of the TNFα promoter was much lower than that of the IFNγ promoter within each cell type.