E2f6-mediated repression of the meiotic Stag3 and Smc1β genes during early embryonic development requires Ezh2 and not the de novo methyltransferase Dnmt3b

Figures & data

Table 1. Data from microarray performed on E2f6 wild-type and E2f6 knockout MEFs

GENE	E2f6 wt	E2f6 KO	E2f6 KO/wt	Description
Stag3	0.08	1.43	17.39	Stromal antigen 3
Smc1β	0.03	2.28	74.14	Structural maintenance of chromosomes 1B
Tuba3a	0.20	67.57	332.22	Tubulin α, 3A
Slc25a31	0.02	0.67	43.00	Solute carrier family 25, member 31
E2f6	2.37	0.64	0.27	E2F transcription factor 6
Dp1	5.64	12.24	2.17	Transcription factor Dp1
Dp2	0.15	0.16	1.12	Transcription factor Dp2

Table 2. CGI class definition of Stag3, Smc1β, Tuba3a, and Slc25a31 according to the more stringent criteria proposed by Takai and Jones¹⁷

Gene	Region*	G+C%	No. CpGs	Obs./Exp.	CGI class
Stag3	−389/+111	61.6	44	0.94	strong
Smc1β	−204/+416	62.3	48	0.81	strong
Tuba3a	−356/+249	64.5	33	0.53	weak
Slc25a31	−128/+372	61.4	41	0.87	strong

* Flanking the region amplified with the primers for bisulfite sequencing used in this study (seeTable S1).

Figure 1. Deletion of E2f6 disrupts somatic cell CGI methylation at E2f6 dependent germline specific gene promoters. (A, C, E, and G) Bisulfite DNA sequencing analysis for CGI methylation of the Stag3, Smc1β, Tuba3a, and Slc25a31 promoters, respectively, in wild-type and E2f6^−/− MEFs. Each gene representation includes the first exon in black and the position of the CGI in gray. Short lines with numbers depict the first nucleotide from the forward and reverse primers used for bisulfite sequencing. Percent methylation for the region amplified by the primers is given in parenthesis. (B, D, F, and H) Quantification of bisulfite sequencing data demonstrating the dynamics of methylation at each individual CpG dinucleotide in Stag3, Smc1β, Tuba3a, and Slc25a31 promoters, respectively. A value of one corresponds to 100% methylation. *Designates the position of E2f6 binding; +1 designates the TSS.

Table 3. Data from microarray performed on ESCs, EpiSCs, and MEFs

GENE	ESCs	EpiSCs	MEFs	Description
Stag3	2.72	0.07	0.12	Stromal antigen 3
Smc1β	1.37	0.05	0.02	Structural maintenance of chromosomes 1B
Tuba3a	9.68	0.06	0.01	Tubulin α, 3A
Slc25a31	0.33	0.01	0.01	Solute carrier family 25, member 31
E2f6	1.92	5.03	5.00	E2F transcription factor 6
Dp1	12.21	11.82	7.67	Transcription factor Dp1
Dp2	1.45	1.01	0.61	Transcription factor Dp2
Ezh1	0.27	0.15	0.19	Enhancer of Zeste homolog 1
Ezh2	3.77	3.79	0.97	Enhancer of Zeste homolog 2
Suz12	10.64	4.53	1.89	Suppressor of Zeste 12 homolog
Eed	4.29	3.13	1.52	Embryonic ectoderm development
Dnmt1	7.78	8.03	3.95	DNA methyltransferase 1
Dnmt3a	3.15	5.87	0.13	DNA methyltransferase 3A
Dnmt3b	7.25	14.83	0.15	DNA methyltransferase 3B
Dnmt3L	45.07	5.48	0.04	DNA methyltransferase 3-like
Pou5f1	26.38	59.76	0.01	POU domain class 5, transcription factor 1
Nanog	9.47	6.62	0.03	Nanog homeobox
Sox2	6.52	4.11	0.04	SRY-box containing gene 2
Klf4	32.37	2.21	13.68	Kruppel-like factor 4
Zfp42	50.81	0.89	0.01	Zinc finger protein 42
Fgf5	0.04	3.33	0.04	Fibroblast growth factor 5
Col11a	0.09	0.07	0.32	Pro-collagen, type XI, α

The values of expression are given normalized to the median value of the array. Genes are grouped together according to their functions.

Figure 2. Germline-specific genes regulated by E2f6 are first silenced in primed pluripotent stem cells. (A) RNA expression levels of Stag3, Smc1β, Tuba3a, and Slc25a31 in ESCs, EpiSCs, and MEFs shown by qPCR. The expression of each gene in embryonic stem cells was set to a value of one. (B) ChIP-qPCR analysis for H3K4me3 enrichment spanning 2.5 kb downstream and 3 kb upstream (relative to the TSS) of the Stag3 promoter in ESCs, EpiSCs, and MEFs. Enrichment is shown normalized to the total H3 content at each specific primer pair position. (C) ChIP-qPCR analysis for enrichment of E2f6 at the Stag3 promoter in ESCs, EpiSCs, and MEFs. Enrichment is shown as the percent of Input DNA.

Figure 3. Differential DNA methylation of the Stag3 and Smc1β promoters in ESCs, EpiSCs, and MEFs. (A) CGI methylation of the Stag3 promoter in ESCs, EpiSCs, and MEFs. (B) CGI methylation of the Smc1β promoter in ESCs, EpiSCs, and MEFs. *Designates the position of E2f6 binding site; +1 designates the TSS.

Figure 4. The effects of E2f6 and Dnmt3b overexpression on RNA expression and DNA methylation of meiotic genes in ESCs. (A) qPCR analysis of Stag3, Smc1β, and E2f6 RNA expression in ESCs with or without stable overexpression of E2f6. (B) Bisulfite DNA sequencing analysis of the Stag3 promoter in ESCs with or without stable overexpression of E2f6. (C) ChIP-qPCR analysis for E2f6 occupancy at the Stag3 promoter in ESCs ectopically overexpressing FLAG-tagged E2f6. (D) RNA expression levels of Stag3, Smc1β, and Dnmt3b in ESCs with or without stable overexpression of Dnmt3b. (E) Bisulfite DNA sequencing analysis for methylation of the Stag3 promoter in ESCs with or without stable overexpression of Dnmt3b.

Figure 5. Downregulation of Stag3 and Smc1β gene expression during embryoid body (EB) differentiation of wild-type, Dnmt3b knockout (KO), and Dnmt3a/3b double knockout (DKO) J1 ESCs. (A) qPCR analysis of Stag3 and Smc1β RNA expression during EB differentiation of wild type, 3b KO, and 3a/3b DKO J1 ESCs at Day0 and Day 10. (B) CGI methylation of the downstream region of the Smc1β promoter measured using bisulfite sequencing from the sample of Day 0 and Day 10 following EB differentiation of wild-type J1 ESCs, Dnmt3b KO (C), and Dnmt3a/3b DKO (D).

Figure 6. Downregulation of Stag3 and Smc1β gene expression during embryoid body (EB) differentiation of wild-type and Ezh2-deficient pluripotent stem cells. (A) Confirmation of Ezh2^fl/fl and Ezh2^∆/∆ genotype using PCR and RT-PCR, upper and lower panel, respectively. The amplicons for the flox and the Δ allele are 450 bp and 370 bp (for genomic PCR) and 587 bp and 254 bp (for cDNA PCR), respectively. (B) ChIP-qPCR analysis for H3K4me3 enrichment of the Stag3 promoter in Ezh2 ^fl/fl iPSCs at 3 differentiation time points (Day 0, Day 6, and Day 10). Enrichment is shown normalized to the total H3 content at each specific primer pair position. (C) ChIP-qPCR analysis for Ezh2 occupancy at the Stag3 promoter in Ezh2 ^fl/fl iPSCs at 3 differentiation time points (Day 0, Day 6, and Day 10). Enrichment is shown normalized to the background from a ChIP with mouse IgG. (D) qPCR analysis of Stag3 and Smc1β RNA expression during EB differentiation of Ezh2^fl/fl iPSCs and Ezh2^∆/∆ iPSCs at Day 0 and Day 10. The expression of each gene at Day 0 was set to a value of one. (E and F) CGI methylation of the downstream region of the Smc1β promoter measured using bisulfite sequencing from the sample of Day 0 and Day 10 following EB differentiation of Ezh2^fl/fl iPSCs (E) and Ezh2^∆/∆ iPSCs (F).

Takai D, Jones PA. Comprehensive analysis of CpG islands in human chromosomes 21 and 22. Proc Natl Acad Sci U S A 2002; 99:3740 - 5; http://dx.doi.org/10.1073/pnas.052410099; PMID: 11891299

 PubMed Web of Science ®Google Scholar