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. 2016 Apr 28;7:11453.
doi: 10.1038/ncomms11453.

DNMT3B Isoforms Without Catalytic Activity Stimulate Gene Body Methylation as Accessory Proteins in Somatic Cells

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Free PMC article

DNMT3B Isoforms Without Catalytic Activity Stimulate Gene Body Methylation as Accessory Proteins in Somatic Cells

Christopher E Duymich et al. Nat Commun. .
Free PMC article

Abstract

Promoter DNA methylation is a key epigenetic mechanism for stable gene silencing, but is correlated with expression when located in gene bodies. Maintenance and de novo DNA methylation by catalytically active DNA methyltransferases (DNMT1 and DNMT3A/B) require accessory proteins such as UHRF1 and DNMT3L. DNMT3B isoforms are widely expressed, although some do not have active catalytic domains and their expression can be altered during cell development and tumourigenesis, questioning their biological roles. Here, we show that DNMT3B isoforms stimulate gene body methylation and re-methylation after methylation-inhibitor treatment. This occurs independently of the isoforms' catalytic activity, demonstrating a similar functional role to the accessory protein DNMT3L, which is only expressed in undifferentiated cells and recruits DNMT3A to initiate DNA methylation. This unexpected role for DNMT3B suggests that it might substitute for the absent accessory protein DNMT3L to recruit DNMT3A in somatic cells.

Figures

Figure 1
Figure 1. Stable reintroductions of DNMT3B isoforms and DNMT3L in DKO8 and 3BKO cell lines.
(a) Schematic diagram of DNMT3A (3A), DNMT3B isoforms (3B1, 3B1-M, 3B3, 3B4, Δ3B2, Δ3B2-M and Δ3B4) and DNMT3L (3L) showing conserved PWWP (purple), PHD-like domain (green) and DNMT catalytic motifs (black). There are five catalytic domains (I, IV, VI, IX and X) in DNMT3A and DNMT3B, all of which are absent in DNMT3L. A red-coloured VI domain indicates inactivating mutations (Cys to Ser) of amino acids 651 and 452 in 3B1-M and Δ3B2-M, respectively. 3B4 has a frameshift with a unique protein sequence shown in orange. ^indicates alternative splicing. (b,c) mRNA expression level of endogenous DNMT3B in HCT116 cells, exogenous DNMT3B isoforms and DNMT3L assessed by qRT-PCR and normalized to the expression of the TATA Box Binding Protein (TBP) in DKO8 cells, 56 days post transfection and 3BKO cells, 14 days post transfection, respectively. Error bars indicate standard deviation from the mean of three biological replicates. The empty vector (EV) cell line is the transfection control. (d,e) Protein-expression levels of exogenous MYC-tagged DNMT isoforms by western blot analysis in DKO8 cells, 56 days post transfection and 3BKO cells, 14 days post transfection, respectively. β-ACTIN was used as a loading control.
Figure 2
Figure 2. DNMT3B isoforms and DNMT3L restore DNA methylation at specific CpG sites.
(a) Heatmap showing 54,911 CpG sites in DKO8 cells, expressing the indicated DNMT isoforms. CpGs were selected because they were targeted by at least one construct. CpG sites in genomic locations targeted by DNMT3Bs and 3L with respect to promoter (maroon), gene body (orange) and other regions, excluding promoters and gene bodies (grey), are shown in the left panel. CpG sites in CpG islands are represented in green in the left panel. Endogenous methylation levels in HCT116 and DKO8 cells are represented by a cold to warm colour scale (β-value 0–1, 0–100% methylated), where every row represents one CpG. DNA-methylation levels of DKO8 cells expressing a DNMT3B isoforms or DNMT3L are shown in the right panel. (b) Boxplots showing the distribution of DNA-methylation levels of 54,911 CpG sites for each indicated cell line. HCT116 cells' methylation level is included for comparison with the derivative cell line DKO8 EV. A dashed red line indicates the median of DKO8 EV, outliers outside the 5th to 95th percentiles are not shown. (c) Distribution of genomic locations targeted by individual DNMT3Bs and 3L with respect to promoter (maroon), gene body (orange) and other regions excluding promoters and gene bodies (grey), from the 450 K array. (d) Distribution of genomic locations targeted by DNMT3Bs and 33LL in respect to CpG island (green) or non-CpG island (grey) from the 450 K array. Background distribution is the representative population of CpG sites that could be targeted by the individual DNMTs. Statistical enrichment was performed using a z-test comparing the targeted CpG sites for each DNMT isoform with the overall distribution of probes on the 450 K array, ***P<0.001.
Figure 3
Figure 3. DNMT3B isoforms restore DNA methylation in a DNMT3B knock-out cell line.
Heatmaps and Boxplots showing previously (Yang et al.23) defined H3K36me3-enriched and fast-rebounding Group I CpG sites in cell lines expressing different DNMTs before 24 h 5-Aza-CdR treatment and at Day 5 and Day 42 post treatment for 3BKO (a) and 3ABDKO (b) cells. Individual CpG sites falling in genomic locations targeted by DNMT3Bs and 3L with respect to promoter (maroon), gene body (orange) and other regions, excluding promoters and gene bodies (grey) are shown in the left panel. CpG sites in CpG islands are represented in green in the left panel. Endogenous methylation levels in cell lines are represented by a cold to warm colour scale (β-value 0–1, 0–100% methylation) where every row represents one CpG site in the three right panels.
Figure 4
Figure 4. Differential expression levels of DNMT3A and 3B between normal and various tumour tissues.
Normalized read counts from RNA-Seq data were calculated for DNMT3A, DNMT3B and isoforms from matched normal and tumour tissue samples by expectation-maximization (RSEM). Expression fold change is shown as log2 (tumour/normal) for each cancer type. N indicates the number of sample pairs for each tumour type. Mann–Whitney's unpaired statistical test was used to assess expression differences on log2 (RSEM) values between tumour and normal tissue. *P<0.05, **P<0.01 or ***P<0.001.
Figure 5
Figure 5. The changing landscape of de novo DNMTs during development.
In ES cells the catalytically active CpG methyltransferases DNMT3A1 (3A1), DNMT3A2 (3A2) and DNMT3B1 (3B1) are expressed, as well as the accessory protein DNMT3L (3L) (refs 40, 44, 53, 54). Our work suggests that catalytically inactive DNMT3B3 (3B3) may also participate as an accessory protein in the establishment of DNA-methylation patterns. Following differentiation 3A2 and 3L are no longer expressed, while expression of 3B1 is decreased markedly, leaving 3A1 and the accessory protein 3B3 as potential mediators of methylation. Our work also suggests that 3B3 preferentially targets 3A1 to gene bodies. Active methyltransferase enzymes and accessory proteins are shown in red and blue boxes, respectively.

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