Novel imprinted single CpG sites found by global DNA methylation analysis in human parthenogenetic induced pluripotent stem cells

Figures & data

Figure 1. Characterization of parthenogenetic HiPSC lines. (A) Morphology of HiPSCs and parthenogenetic (Pg) HiPSC-1. (B) Immunocytochemistry for pluripotency markers (OCT4 and SSEA4) in biparental HiPSCs and parthenogenetic HiPSC-1. Scale bar = 100 μm. (C) RT-PCR analysis of pluripotency-specific gene expression in WtFibs, PgFib-1, HiPSCs, PgHiPSC-1, and H9. (D) Scatter plots comparing the global gene expression patterns between WtFibs and HiPSCs, PgFib-1 and PgHiPSC-1, H9 and HiPSCs, and H9 and PgHiPSC-1 analyzed by oligonucleotide microarrays. (E) Bisulfite sequencing analysis of OCT4 and NANOG promoter regions in WtFibs HiPSCs, PgFib-1, PgHiPSC-1, and H9. (F) Confirmation of a normal 46, XX karyotype of PgFib-1 and PgHiPSC-1.

Figure 2. Analysis of expression and DNA methylation of known imprinted genes. (A) Relative mRNA expression levels of known paternally expressed genes in WtFibs, HiPSCs, PgFib-1, PgHiPSC-1, and H9. The data shown are averages of gene expression. (B) DNA methylation status of known paternally and maternally imprinted genes (H19, GRB10, and MEST) in PgFib-1, PgHiPSC-1, WtFibs, HiPSCs, and H9 was analyzed by bisulfite sequencing. Each line represents a separate clone. Black and white circles represent methylated and unmethylated CpGs, respectively.

Table 1. A survey for candidate imprinted CpG sites

Illumina Probe ID	Gene names	PgFib-1	PgHiPSC-1	WtFibs	HiPSCs	H9
cg00143986	GPR133	0.977	0.957	0.413	0.466	0.458
cg25465065	PMF1	0.946	0.952	0.516	0.502	0.504
cg11438287	COL4A2	0.958	0.952	0.594	0.532	0.604
cg25249362	PFKP	0.964	0.948	0.487	0.605	0.538
cg02380521	FCHO1	0.944	0.947	0.502	0.509	0.554
cg02945674	ABCA1^a	0.934	0.945	0.456	0.459	0.476
cg05730108	FARP1	0.938	0.945	0.423	0.429	0.438
cg17042243	HRNBP3	0.907	0.941	0.559	0.524	0.542
cg00574513	MYLK	0.965	0.932	0.656	0.623	0.647
cg07895329	CNKSR3	0.911	0.931	0.653	0.665	0.644
cg27160141	C4orf23	0.942	0.929	0.463	0.534	0.559
cg12432807	KRT23	0.935	0.926	0.614	0.611	0.594
cg04497611	TBCD	0.937	0.925	0.662	0.638	0.579
cg23947138	RASA3	0.931	0.919	0.503	0.500	0.470
cg14609402	UHRF1	0.905	0.910	0.495	0.479	0.395
cg11106864	RAP1GDS1	0.932	0.903	0.611	0.606	0.623
cg15242686	GSTTP1	0.067	0.095	0.381	0.590	0.599
cg27288829	RAX2	0.079	0.084	0.515	0.484	0.517
cg06352616	CCDC154^a	0.065	0.081	0.447	0.517	0.593
cg11917734	HLA-C	0.058	0.080	0.512	0.517	0.505
cg05161773	SEPT9	0.082	0.075	0.425	0.411	0.379
cg23620282	IRX4	0.065	0.069	0.363	0.395	0.412
cg25831233	POLR2J	0.095	0.063	0.315	0.380	0.311
cg09819502	C5orf33	0.050	0.058	0.505	0.574	0.550
cg02665463	ARL17A	0.056	0.055	0.378	0.353	0.344
cg15988569	PNLIPRP2	0.047	0.051	0.459	0.495	0.527
cg22337626	MAST2	0.082	0.051	0.401	0.648	0.364
cg11008123	LOC283267	0.046	0.046	0.447	0.466	0.423
cg26995506	ZEB2	0.046	0.044	0.556	0.505	0.516
cg03493520	PRCP	0.043	0.044	0.436	0.551	0.565
cg12648931	C5orf33	0.054	0.042	0.430	0.415	0.454
cg20370184	SLC44A4	0.045	0.038	0.437	0.420	0.443
cg17770035	NCR2	0.031	0.033	0.326	0.416	0.473

Methylation range (β≤0.1 or 0.9≤β in PgFib-1 and PgHiPSC-1, and 0.3≤β≤0.7 in WtFibs, HiPSCs, and H9)

a The candidate imprinted CpG sites are significantly correlated with sperm sample from the Gene Expression Omnibus (GEO) database (GSE72444)

Figure 3. Confirmation of DNA methylation status at novel imprinted CpG sites. Bisulfite sequencing showed imprinted DNA methylation patterns of ABCA1 and CCDC154. Each line represents a separate clone. Black and white circles represent methylated and unmethylated CpGs, respectively.