Genome-Wide Methylation Analysis of DNMT3B Gene Isoforms Revealed Specific Methylation Profiles in Breast Cell Lines

Figures & data

Figure 1.  Expression analysis of DNMT3B2 and DNMT3B3 isoforms in human breast cancer and noncancerous cell lines.

Relative mRNA expression of human DNMT3B2 (gray bar) and its alternative spliced isoform DNMT3B3 (black bar). Results are expressed in mRNA copy number corrected with the reference gene HPRT1 as mean ± SEM; n = 4.

Figure 2.  Unsupervised hierarchical clustering of breast cell lines based on their methylation profile.

Hierarchical clustering of parental (untransfected) cell lines based on genome-wide methylation data of each cell line. This analysis was performed with GenomeStudio calculating dissimilarities based on absolute correlation using a 1-r distance measure. Rows represent each parental (untransfected) cell line.

Table 1.  DNMT3B3 and DNMT3B2 expression levels in untransfected and transfected/transduced breast cell lines.

	Untransfected cells			DNMT3B2-overexpressing cells^†			DNMT3B3-overexpressing cells^‡
Cell line	DNMT3B2^§	DNMT3B3^§	Ratio^¶	DNMT3B2^§	DNMT3B3^§	Ratio^¶	DNMT3B2^§	DNMT3B3^§	Ratio^¶	Fold^#
MCF7	31,748 ± 3528	8170 ± 770	0.26	23,260 ± 1526	6049 ± 1051	0.26	8873 ± 814	4,373,123 ± 891,428	492	1895
T-47D	47,923 ± 2904	3372 ± 497	0.07	31,887 ± 2599	2964 ± 198	0.09	13,034 ± 1395	1,034,702 ± 129,142	79.38	854
ZR-75-1	26,189 ± 1701	1487 ± 148	0.06	39,935 ± 1592	1285 ± 196	0.03	17,843 ± 1276	50,873 ± 3192	2.85	82
CAMA-1	97,931 ± 15,966	4308 ± 529	0.04	95,814 ± 6028	6099 ± 502	0.06	36,613 ± 2862	2,112,420 ± 157,597	57.70	906
MDA-231	171 ± 71	5419 ± 869	31.75	45,429,566 ± 4,304,468	10,222 ± 690	0.0002	263 ± 94	4453 ± 616	16.91	75,153
BT-549	58 ± 19	894 ± 97	15.54	1,330,021 ± 131,616	5457 ± 730	0.004	634 ± 202	7132 ± 704	11.25	2742
MCF10A	241 ± 67	3378 ± 414	14.01	12,911,142 ± 3,577,933	2685 ± 803	0.0002	430 ± 166	4178 ± 647	9.71	46,694

^†Treatments: MCF7, T-47D, ZR-75-1 and CAMA-1 cells were stably transduced with nonspecific shRNA and transfected with empty pcDNA3 vector, while MDA-231, BT-549 and MCF10A cells were transfected with pcDNA3–DNMT3B2 expressing vector.

^‡Treatments: MCF7, T-47D, ZR-75-1 and CAMA-1 cells were stably transduced with shRNA targeting specifically DNMT3B2 and transfected with pcDNA3–DNMT3B3 expressing vector, while MDA-231, BT-549 and MCF10A cells were transfected with empty pcDNA3 vector.

^§ DNMT3B2 and DNMT3B3 mRNA levels expressed as copy numbers/μg RNA.

^¶ DNMT3B3/DNMT3B2 expression ratio is expressed as: mRNA copy number of DNMT3B3/mRNA copy number of DNMT3B2.

^#Fold difference is expressed as the expression ratio found in DNMT3B3-overexpressing cells/expression ratio found in DNMT3B2-overexpressing cells.

Table 2.  Number of CpG sites and associated genes identified as differentially methylated in ER+/PR+ breast cell lines.

Cell lines	Whole genome				Promoter region only^†
	CpG sites		Genes		CpG sites		Genes
	Undermet.	Overmet.	Undermet.	Overmet.	Undermet.	Overmet.	Undermet.	Overmet.
MCF7	33,453	3403	8815	1715	10,692	1101	6147	893
T-47D	2222	3791	1254	1898	695	1 278	617	1069
ZR-75-1	115	331	83	224	20	158	20	141
CAMA-1	76	6	49	4	17	1	16	1
MCF7 + T-47D	10,795	3824	4527	1927	3234	1276	2431	1033
Number of CpG sites & associated genes identified as differentially methylated in ER-/PR- breast cell lines
MDA-231	1521	6	1089	5	1033	2	856	2
BT-549	47	3	37	2	16	0	16	0
MCF10A	727	12	487	9	243	5	207	5
MDA-231 + BT-549	1453	8	1054	5	994	2	845	2

The differential analysis was performed between DNMT3B2-overexpressing and DNMT3B3-overexpressing cells. Only CpG sites with DiffScore ≥ |22| (p-value ≤ 0.01) were displayed.

^†Promoter region includes 1500 bp of the Transcription Start Site (TSS1500), 200 bp of the Transcription Start Site (TSS200), 5′UTR and first exon regions.

Table 3.  Number of differentially methylated CpG sites according to their gene location.

Genomic location	ER+/PR+ (MCF7 and T-47D) cell lines		ER-/PR- (MDA-231 and BT-549) cell lines
	Undermethylated	Overmethylated	Undermethylated	Overmethylated
Total^†	10,795	3824	1453	8
TSS1500^‡	1641	573	176	1
TSS200^§	593	249	395	0
5′UTR	760	301	194	0
1st exon	240	153	229	1
Body	3551	1252	272	2
3′UTR	452	99	15	1
Intergenic^¶	3558	1197	172	3

^†Only CpG sites with significant differential methylation level are displayed.

^‡TSS1500 = 1500 bp of the Transcription Start Site.

^§TSS200 = 200 bp of the Transcription Start Site.

^¶Intergenic: CpG not associated with a gene.

Table 4.  Over-represented pathways, functions and diseases triggered by the overexpression of the DNMT3B3 isoform in ER+/PR+ cell lines.

Undermethylated genes^†		Overmethylated genes^‡		All genes combined^§
Pathways	p-value	Pathways	p-value	Pathways	p-value
G-protein coupled receptor signaling (95)	1.03E-10	Transcriptional regulatory network in embryonic stem cells (13)	2.07E-5	G-Protein coupled receptor signaling (107)	6.42E-9
cAMP-mediated signaling (77)	8.51E-8	Molecular mechanisms of cancer (54)	8.67E-5	Axonal guidance signaling (162)	2.08E-8
Cardiac-adrenergic signaling (53)	9.19E-8	Factors promoting cardiogenesis in vertebrates (20)	1.07E-4	Glutamate receptor signaling (32)	7.71E-7
Axonal guidance signaling (132)	9.22E-8	Regulation of the epithelial-mesenchymal transition pathway (32)	1.25E-4	Cardiac-adrenergic signaling (59)	1.53E-6
Glutamate receptor signaling (29)	1.72E-7	GABA receptor signaling (16)	1.62E-4	cAMP-mediated signaling (87)	2.12E-6
Functions	Top p-value^¶	Functions	Top p-value^¶	Functions	Top p-value^¶
Cellular movement (867)	3.47E-23	Cellular development (601)	6.76E-17	Cell-to-cell signaling and interaction (808)	3.33E-26
Cell-to-cell signaling and interaction (659)	1.32E-21	Cell death and survival (586)	2.38E-16	Cellular movement (1078)	1.05E-23
Molecular transport (926)	2.29E-19	Cell morphology (460)	2.73E-14	Cellular function and maintenance (1357)	1.42E-23
Cellular function and maintenance (1026)	1.80E-18	Cell-to-cell signaling and interaction (167)	1.11E-13	Molecular transport (1124)	5.40E-23
Cellular growth and proliferation (1360)	1.09E-17	Cellular assembly and organization (311)	2.11E-13	Cellular development (1492)	6.55E-21
Diseases	Top p-value^¶	Diseases	Top p-value^¶	Diseases	Top p-value^¶
Cancer (3743)	2.24E-120	Cancer (1656)	1.11E-53	Cancer (4772)	1.18E-156
Organismal injury and abnormalities (3772)	2.24E-120	Organismal injury and abnormalities (1660)	1.11E-53	Organismal injury and abnormalities (4792)	1.18E-156

The number of genes involved in the process is given in parentheses.

^†Analyses were performed using genes associated with significant CpG sites differentially undermethylated.

^‡Analyses were performed using genes associated with significant CpG sites differentially overmethylated.

^§Analyses were performed using genes associated with significant CpG sites differentially undermethylated or overmethylated.

^¶Top p-value of the p-value range.

Table 5.  Overrepresented pathways, functions and diseases triggered by the overexpression of the DNMT3B3 isoform in ER-/PR- cell lines.

Undermethylated genes^†		Overmethylated genes^‡		All genes combined^§
Pathways	p-value	Pathways^¶	p-value	Pathways	p-value
Signaling by Rho family GTPases (25)	1.05E-4	N/A	N/A	Signaling by Rho Family GTPases (25)	1.14E-4
RhoGDI signaling (18)	1.26E-3	N/A	N/A	RhoGDI signaling (18)	1.34E-3
Breast cancer regulation by Stathmin1 (19)	1.60E-3	N/A	N/A	Breast cancer regulation by Stathmin1 (19)	1.69E-3
Phospholipase C signaling (22)	1.76E-3	N/A	N/A	Phospholipase C signaling (22)	1.87E-3
AMPK signaling (18)	1.85E-3	N/A	N/A	IGF-1 signaling (12)	1.94E-3
Functions	Top p-value^#	Functions	Top p-value^#	Functions	Top p-value^#
Cellular growth and proliferation (346)	1.61E-8	Cellular assembly and organization (1)	3.65E-3	Cellular growth and proliferation (346)	2.90E-8
Cell death and survival (314)	5.10E-7	RNA post-transcriptional modification (1)	3.65E-3	Cell death and survival (314)	8.29E-7
Cellular assembly and organization (203)	1.07E-6	Molecular transport (2)	9.96E-3	Cellular assembly and organization (203)	1.41E-6
Cellular function and maintenance (277)	1.07E-6	RNA trafficking (1)	9.96E-3	Cellular function and maintenance (277)	1.41E-6
Cellular development (282)	2.91E-6	Cell cycle (1)	2.63E-2	Cellular development (282)	3.35E-6
Diseases	Top p-value^#	Diseases	Top p-value^#	Diseases	Top p-value^#
Cancer (820)	1.95E-9	Endocrine system disorders (2)	3.17E-3	Cancer (825)	2.85E-9
Endocrine system disorders (205)	1.95E-9	Organismal injury and abnormalities (2)	3.17E-3	Endocrine system disorders (205)	2.85E-9

The number of genes involved in the process is given in parentheses.

^†Analyses were performed using genes associated with significant CpG sites differentially undermethylated.

^‡Analyses were performed using genes associated with significant CpG sites differentially overmethylated.

^§Analyses were performed using genes associated with significant CpG sites differentially undermethylated or overmethylated.

^¶Given the limited number of genes which have been submitted for IPA analysis, no significant pathway has been identified.

^#Top p-value of the p-value range.

Table 6.  Over-represented pathways, functions and diseases triggered by the overexpression of the DNMT3B3 isoform using affected genes commonly identified in ER+/PR+ and ER-/PR- cell lines.

Pathways	p-value
Axonal guidance signaling (14)	5.35E-4
Breast cancer regulation by Stathmin1 (8)	1.71E-3
nNOS signaling in neurons (4)	2.07E-3
GNRH signaling (6)	3.83E-3
PPAR/RXR activation (7)	4.78E-3
Functions	Top p-value^†
Cellular assembly and organization (60)	2.04E-6
Cellular function and maintenance (80)	2.48E-5
Cellular growth and proliferation (88)	3.54E-5
Cellular response to therapeutics (3)	3.93E-4
Drug metabolism (3)	3.93E-4
Diseases	Top p-value^†
Cancer (210)	1.27E-5
Organismal injury and abnormalities (211)	1.27E-5

The number of genes involved in the process is given in parentheses.

^†Top p-value of the p-value range.

Table 7.  Predicted effect of DNMT3B isoform expression on relevant biological processes in ER+/PR+ cell lines.

Category	Disease/function annotation	p-value	Predicted activation state upon DNMT3B3 expression	Predicted activation state upon DNMT3B2 expression
Organism survival	Morbidity or mortality	1.83E-11	↑	↓
Organism survival	Organismal death	2.22E-11	↑	↓
Organism survival	Neonatal death	2.25E-10	↑	↓
Developmental disorder	Dysgenesis	2.22E-07	↑	↓
Neurological disease	Seizures	2.30E-07	↑	↓
Developmental disorder	Hypoplasia of organ	1.29E-06	↑	↓
Inflammatory response	Inflammation of organ	2.32E-06	↑	↓
Metabolic disease	Glucose metabolism disorder	4.80E-06	↑	↓
Cell death and survival	Cell death of brain	4.39E-05	↑	↓
Inflammatory response	Inflammation of body region	1.30E-04	↑	↓
Cellular movement	Cell movement	5.03E-12	↓	↑
Cellular movement	Migration of cells	9.33E-12	↓	↑
Cellular growth and proliferation	Proliferation of cells	1.73E-11	↓	↑
Cellular development	Differentiation of cells	2.88E-11	↓	↑
Molecular transport	Transport of molecule	5.22E-11	↓	↑
Cellular movement	Chemotaxis	3.50E-09	↓	↑
Cellular assembly and organization	Organization of cytoskeleton	6.84E-09	↓	↑
Cellular growth and proliferation, tissue development	Proliferation of epithelial cells	8.09E-08	↓	↑
Cellular development, cellular growth and proliferation	Cell proliferation of tumor cell lines	8.40E-08	↓	↑
Cardiovascular system development	Angiogenesis	9.77E-08	↓	↑
Predicted effect of DNMT3B isoform expression on relevant biological processes in ER-/PR- cell lines
Organism survival	Morbidity or mortality	4.65E-04	↑	↓
Organism survival	Organismal death	8.97E-04	↑	↓
Cancer, dermatological diseases and conditions	Hyperplasia of skin	3.44E-03	↑	↓
Organismal injury and abnormalities	Bleeding of tissue	4.66E-03	↑	↓
Cancer, dermatological diseases and conditions	Hyperplasia of epidermis	5.38E-03	↑	↓
Cancer, organismal injury and abnormalities	Incidence of benign tumor	1.16E-02	↑	↓
Organismal injury and abnormalities, reproductive system disease	Atresia of reproductive tract	1.82E-02	↑	↓
Endocrine system disorders	Hypoglycemia	2.01E-02	↑	↓
Dermatological diseases and conditions	Loss of hair	2.44E-02	↑	↓
Neurological disease	Movement disorders	2.48E-02	↑	↓
Cellular growth and proliferation	Proliferation of cells	3.71E-05	↓	↑
Cellular assembly and organization	Organization of cytoskeleton	1.03E-04	↓	↑
Cellular assembly and organization	Organization of cytoplasm	3.25E-04	↓	↑
Carbohydrate metabolism	Metabolism of carbohydrate	3.49E-04	↓	↑
Cellular movement, embryonic development	Invasion of embryonic cell lines	4.47E-04	↓	↑
Cellular movement	Cell movement of tumor cell lines	4.59E-04	↓	↑
Cell death and survival, connective tissue disorders	Apoptosis of fibroblast cell lines	7.66E-04	↓	↑
Cellular development, cellular growth and proliferation	Cell proliferation of tumor cell lines	8.09E-04	↓	↑
Protein synthesis	Metabolism of protein	1.11E-03	↓	↑
Cellular movement	Migration of tumor cell lines	1.65E-03	↓	↑

↑ Activation of the biological process.

↓ Repression of the biological process.