Hemi-methylated CpG sites connect Dnmt1-knockdown-induced and Tet1-induced DNA demethylation during somatic cell reprogramming

The relationship between active DNA demethylation induced by overexpressing Tet1 and passive DNA demethylation induced by suppressing Dnmt1 remains unclear. Here, we found that DNMT1 preferentially methylated, but TET1 preferentially demethylated, hemi-methylated CpG sites. These phenomena resulted in a significant overlap in the targets of these two types of DNA demethylation and the counteractions of Dnmt1 and Tet1 during somatic cell reprogramming. Since the hemi-methylated CpG sites generated during cell proliferation were enriched at core pluripotency loci, DNA demethylation induced by Tet1 or sh-RNA against Dnmt1 (sh-Dnmt1) was enriched in these loci, which, in combination with Yamanaka factors, led to the up-regulation of these genes and promoted somatic cell reprogramming. In addition, since sh-Dnmt1 induces DNA demethylation by impairing the further methylation of hemi-methylated CpG sites generated during cell proliferation, while Tet1 induced DNA demethylation by demethylating these hemi-methylated CpG sites, Tet1-induced DNA demethylation, compared with sh-Dnmt1-induced DNA demethylation, exhibited a higher ability to open the chromatin structure and up-regulate gene expression. Thus, Tet1-induced but not sh-Dnmt1-induced DNA demethylation led to the up-regulation of an additional set of genes that can promote the epithelial-mesenchymal transition and impair reprogramming. When vitamin C was used to further increase the demethylation ability of TET1 during reprogramming, Tet1 induced a larger up-regulation of these genes and significantly impaired reprogramming. Therefore, the current studies provide additional information regarding DNA demethylation during somatic cell reprogramming.


Supplementary Figure S1
Two kinds of DNA demethylation share targets (related to Fig. 1) (a) The influences of sh-Dnmt1 and Tet1 on methylation levels of all CpG sites detected in current RRBS were summarized.
(b-d) CpG sites with larger demethylation than average were further selected. The overlapped targets of the two kinds of demethylation were summarized in (b). In addition, the correlation between demethylation induced by sh-Dnmt1 and Tet1 were listed in (c). Constantly demethylated CpG sites (about 165 thousand CpG sites, d) were summarized by overlapping the results in (b).
(e) Dnmt1 and sh-Dnmt1 were over-expressed with Tet1. Methylation levels of all CpG sites in genome were summarized.

Supplementary Figure S2
DNMT1 counteracts with TET1 (related to Fig. 2) (a) Schematic illustration of the generation of hemi-methylated CpG sites, and the four methylation reads of one particular CpG site obtained by performing WGBS.
(b-c) The methylation differences between indicated reads were averaged. Absolute methylation differences (AMDs) were calculated by averaging the absolute values of differences in methylation between positive and negative strands of all CpG sites (ave. of abs.[+vs-]). The theoretical expectation was calculated as described in Materials and Methods. Significant higher AMDs (both in G1 and G2 phase, and both in CpG and gene levels) than theoretical expectation confirmed the existence of hemimethylated CpG sites.
(d-e) CpG sites and genes were sorted according to the enrichment of hemimethylation (AMDs between two strands) and grouped into 14 and 20 groups, respectively. The average methylation levels of different groups were plotted against their AMDs.
(f-k) 20% CpG sites (around TSS, f, h, j) and genes (g, i, k) with higher enrichment of hemi-methylation (AMDs) were analyzed separately from the remaining 80%. The abilities of sh-Dnmt1 and Tet1 to induce demethylation were listed in (f-g). The abilities of Tet1 to promote sh-Dnmt1-induced demethylation were summarized in (hi). The counteraction between Dnmt1 and Tet1 was listed in (j-k).

Supplementary Figure S3
Enrichments of hemi-methylation in specific genes (related to Fig. 5) (a) CpG sites were divided into different groups basing on their methylation levels and distances from surrounding CpG sites. The enrichment of hemi-methylation (AMDs between two strands) was summarized. CpG sites with particular methylation levels in MEFs (20~85%) and distances from surrounding CpG sites (45~90 bp) were enriched with hemi-methylated CpG sites.
(b) Genes were divided into 10 groups based on their methylation levels. The enrichment of CpG sites with different levels of methylation was plotted for these ten groups of genes. CpG sites with particular methylation levels in MEFs (20~85%) were enriched in genes with methylation levels between 20% and 60%.
(c) Correlation between gene expression and methylation in MEFs were plotted. Particular levels of demethylation were used to simulate the influences of demethylation on gene expression. With similar demethylation, the closer to 60% of the methylation levels of genes the larger up-regulation was proposed.

Supplementary Figure S4
Vc increases the ability of Tet1 to induce DNA demethylation (related to Fig. 6) (a) HPLC was used to determine overall DNA methylation levels 3 days after overexpressing Tet1 or sh-Dnmt1.
(b) The abilities of purified TET1 protein to demethylate hemi-and full-methylated CpG sites in the presence or in the absence of Vc.
(c-e) To confirm this, the demethylation on hemi-methylated CpG sites was determined by analyzing CpG sites with methylation levels between 0~10% and large AMDs between the two strands (top 10%). The demethylation on full-methylated CpG sites was determined by analyzing CpG sites with methylation levels between 60~90% and small AMDs between the two strands (top 10%). To incorporate the different expression of Tet1 and Dnmt1 in different experimental systems (c-d), the demethylation activities of Tet1 or sh-Dnmt1 were assumed at the same levels in MEFs and during reprogramming without Vc. As summarized in (e), the abilities of sh-Dnmt1 to demethylate full-and hemi-methylated CpG sites were constant in all three experimental systems. The abilities of Tet1 to demethylate hemi-methylated CpG sites increased by about 20% during reprogramming with Vc when compared with the other two systems. The abilities of Tet1 to demethylate hemi-methylated CpG sites increased by about 100% in the presence of Vc. Tables   Supplementary Table S1 1680 genes that were constantly demethylated by both sh-Dnmt1 and Tet1 (related to Fig. 1) As title.

Supplementary Table S2
Gene methylation and chromatin accessibility (related to Fig. 4) Gene methylation was calculated by averaging the methylation levels of detected CpG sites around TSS (-1.5~+2.0kb). Chromatin accessibility was calculated by determining the average reading of ATAC-seq around TSS (-1.5~+0.2kb). Table S3 698 Genes with particular methylation levels and CpG densities (related to Fig.

5)
As title. Table S4 606 Genes whose expression were reversed by Tet1 and Vc (related to Fig. 6) As title. Table S5