Depletion of DNMT1 in differentiated human cells highlights key classes of sensitive genes and an interplay with polycomb repression

Additional file 3: Figure S2. Changes in methylation levels by genomic element. (A) Protein levels in knockdown lines by western blotting. As a control HCT116 colon cancer cells which are WT or have a homozygous mutation in DNMT1 (KO) are shown: the DNMT1-specific top band is indicated by the arrowhead at right. (B) Median levels of methylation are shown for each genomic element (listed at top). The positions of medians are also indicated at right (arrowheads). The differences between WT and KD medians were used to plot Fig. 1d. (C) Density distribution of methylation at the three main elements involved in gene regulation, shown by cell line. Demethylation seems most marked at gene bodies (Genes), indicated by increased density of probes at low methylation (β) values

Widespread recovery of methylation at gametic imprints in hypomethylated mouse stem cells following rescue with DNMT3A2

BACKGROUND: Imprinted loci are paradigms of epigenetic regulation and are associated with a number of genetic disorders in human. A key characteristic of imprints is the presence of a gametic differentially methylated region (gDMR). Previous studies have indicated that DNA methylation lost from gDMRs could not be restored by DNMT1, or the de novo enzymes DNMT3A or 3B in stem cells, indicating that imprinted regions must instead undergo passage through the germline for reprogramming. However, previous studies were non-quantitative, were unclear on the requirement for DNMT3A/B and showed some inconsistencies. In addition, new putative gDMR has recently been described, along with an improved delineation of the existing gDMR locations. We therefore aimed to re-examine the dependence of methylation at gDMRs on the activities of the methyltransferases in mouse embryonic stem cells (ESCs). RESULTS: We examined the most complete current set of imprinted gDMRs that could be assessed using quantitative pyrosequencing assays in two types of ESCs: those lacking DNMT1 (1KO) and cells lacking a combination of DNMT3A and DNMT3B (3abKO). We further verified results using clonal analysis and combined bisulfite and restriction analysis. Our results showed that loss of methylation was approximately equivalent in both cell types. 1KO cells rescued with a cDNA-expressing DNMT1 could not restore methylation at the imprinted gDMRs, confirming some previous observations. However, nearly all gDMRs were remethylated in 3abKO cells rescued with a DNMT3A2 expression construct (3abKO + 3a2). Transcriptional activity at the H19/Igf2 locus also tracked with the methylation pattern, confirming functional reprogramming in the latter. CONCLUSIONS: These results suggested (1) a vital role for DNMT3A/B in methylation maintenance at imprints, (2) that loss of DNMT1 and DNMT3A/B had equivalent effects, (3) that rescue with DNMT3A2 can restore imprints in these cells. This may provide a useful system in which to explore factors influencing imprint reprogramming. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13072-016-0104-2) contains supplementary material, which is available to authorized users

MOESM1 of Widespread recovery of methylation at gametic imprints in hypomethylated mouse stem cells following rescue with DNMT3A2

Additional file 1: Table S1. Details of the primers used for RT-qPCR during the study

MOESM2 of Widespread recovery of methylation at gametic imprints in hypomethylated mouse stem cells following rescue with DNMT3A2

Additional file 2: Table S2. Details of the primers used for pyrosequencing during the study. The gametic differentially methylated region (gDMR) is given at left. Some primer sets are commercially available from Qiagen, as indicated. The sequence of the unconverted DNA, as well as the bisulfite-converted sequence, is given for ease of identification. Which primer carried the biotin modification is also indicated

MOESM3 of Widespread recovery of methylation at gametic imprints in hypomethylated mouse stem cells following rescue with DNMT3A2

Additional file 3: Figure S1. Controls to confirm the methyltransferase activities in the ESC used during the study. (A) Westerns showing the presence or absence of DNMT1 (top) or of DNMT3A2 (bottom) in the various cell lines indicated. ACTB and GAPDH are loading controls. The size of the expected protein is shown at left. A number of DNMT3B antibodies tried proved unreliable. (B) RT-PCR showing the presence or absence of transcripts for the various enzymes in the ESC used. The expected sizes are shown at left. Hprt was a loading control