Normal DNA methylation dynamics in DICER1-deficient mouse embryonic stem cells

Abstract

Reduced DNA methylation has been reported in DICER1-deficient mouse ES cells. Reductions seen at pericentric satellite repeats have suggested that siRNAs are required for the proper assembly of heterochromatin. More recent studies have postulated that the reduced methylation is an indirect effect: the loss of Mir290 cluster miRNAs leads to upregulation of the transcriptional repressor RBL2 that targets the downregulation of DNA methyltransferase (Dnmt) genes. However, the observations have been inconsistent. We surmised that the inconsistency could be related to cell line "age," given that DNA methylation is lost progressively with passage in DNMT-deficient ES cells. We therefore subjected Dicer1(-/-) ES cells to two experimental regimes to rigorously test the level of functional DNMT activity. First, we cultured them for a prolonged period. If DNMT activity was reduced, further losses of methylation would occur. Second, we measured their DNMT activity in a rebound DNA methylation assay: DNA methylation was stripped from Cre/loxP conditionally mutant Dicer1 ES cells using a shRNA targeting Dnmt1 mRNA. Cre expression then converted these cells to Dicer1(-/-), allowing for DNMT1 recovery and forcing the cells to remethylate in the absence of RNAi. In both cases, we found functional DNMT activity to be normal. Finally, we also show that the level of RBL2 protein is not at excess levels in Dicer1(-/-) ES cells as has been assumed. These studies reveal that reduced functional DNMT activity is not a salient feature of DICER1-deficient ES cells. We suggest that the reduced DNA methylation sometimes observed in these cells could be due to stochastic alterations in DNA methylation patterns that could offer growth or survival advantages in culture, or to the dysregulation of pathways acting in opposition to the DNMT pathway.

Figure 1. qPCR assays.

(A) Values obtained for the parental Dicer1 ^c/− line were used to calibrate all other values, and are set at 1.0. The y axis is a linear scale. Bars are mean ± s.d. Probabilities obtained from Student's t-test, 2-tail, type 2: P<0.05 (*), P<0.01 (**), not significantly different (//). Bar 1; Dicer1 ^+/+ cell line 2A at early passage (EP). Bar 2; parental Dicer1 ^c/− EP line. Bars 3 and 4; Two independent Dicer1 ^−/− EP lines derived from Cre transfection of the parental Dicer1 ^c/− EP line. Bars 5 and 6; Two independent sublines (MC1 and MC2) derived from concatemeric targeting at Hprt with the Dnmt1 shRNA. Bars 7–9 and 10–12; three sublines derived from exposure of MC1 (two sublines) and MC2 (one subline) to Cre (MC1/2) that did not excise (c/−) and did excise (−/−) the floxed Dicer1 sequence, respectively. (B) Measurements were of the parental Dicer1 ^c/− line and one Dicer1 ^−/− subline. The y axis is a linear scale. Values are normalized to those obtained for Rps7, which are set at 1.0. Other details are contained in the Materials and Methods section.

Figure 2. Immunoblots.

Values below each band represent the densitometry reading taken for that band. These were normalized to the reading taken for lane 1, which was for the WT Dicer1 ^+/+ EP cell line (+/+, EP), or the parental Dicer1 ^c/− EP cell line (c/−, EP). The loading was consistent throughout as shown by the detection of Tubulin β (TUBβ).

Figure 3. Stability of DNA methylation in DICER1-deficient ES cells.

Methylation-sensitive Southern blots. Details are provided in the legend to Figure 1, except that later passage (LP) cells were also used. Passage (p) numbers were: +/+, EP (p8), c/−, EP (p7), c/−, L (p24), −/− 1, EP and −/− 2, EP (p5), −/− 1, LP and −/− 2, LP (p20). Generally, a single passage for +/+ and c/− lines lasted 3 days, while for −/− cells a single passage lasted 5 days. The restriction enzyme and its recognition sequence are at the bottom right of each plate. At CCGG sites, HpaII cuts only when the CpG dyad is unmethylated. The isoschizomer MspI can cut when the CpG dyad is fully, hemi- or unmethylated.

Figure 4. DNA methylation analysis of single copy sequences.

(A) Heat-map showing the degree of methylation at CpG units as assayed by EpiTYPER. For example, a yellow rectangle indicates a small fraction of the CpG unit was methylated. Actual values obtained are provided (Dataset S1). The Xist_1 and _2 assays correspond to CpG-rich region 2 within exon 1, while the Xist_4 assay corresponds to CpG-rich region 1 directly adjacent to the 5′ end of exon 1. These regions correspond to the Xist promoter . The Gja8 and Trpc1 assays correspond to 5′ upstream regions of these genes. The H19_1 assay corresponds to the H19 promoter region, while the H19_4 and _5 assays correspond to the insulin-like growth factor 2 (Igf2)/H19 imprinting control region located 5′ upstream of H19. In EpiTYPER assays, CpGs are analysed as ‘units’, that is, the methylated fraction measured may correspond to one CpG, e.g. (Xist_2, 8), or be the average of more than one CpG, for example (Xist_2, 20.21.22). SNL, mitomycin C-inactivated SNL feeder cells. (B) Bisulphite sequencing assays. Each row represents a sequenced clone. Closed and open circles represent a methylated and non-methylated CpG, respectively.

Figure 5. Experimental strategy to test rebound DNA methylation after Dicer1 ablation.

(A) (a) The parental Dicer1 ^c/− XY ES cell line is normally methylated. (b) A construct containing an shRNA targeting Dnmt1 mRNA (shRNA-Dnmt1) is homologously recombined into the X-linked Hprt locus. (c) The integrated shRNA effects substantial reduction in genome-wide DNA methylation content, although a background of DNA methylation is retained due to ongoing de novo DNMT activity. (d) Recombinant clones are transiently transfected with Cre to ablate the Dicer1 ^c allele. After ablation, clearing of residual DICER1 and si- and miRNAs, as well as restoration of functional DNMT1 activity, is expected to occur within a few cell divisions. (e) DNA methylation would potentially be re-established over ∼5 passages, corresponding to ∼25 cell divisions through the combined action of the DNMT system as a whole. The extent of rebound DNA methylation serves as an indicator of the dependency of this process on DICER1 activity and the presence of si- and miRNAs. (B) The targeting vector consisted of a 6.8 kb BamHI (B) genomic fragment of Hprt spanning exons 2 and 3 (ex2 and ex3). The mU6 promoter (pr) driving the shRNA and a sneo selection cassette were inserted into ex3. The sequence of the shRNA cassette is provided (Text S1). All clones surviving G418 and 6TG dual selection are expected to be targeted. pb, probe used in Southern blot, hybridizing to EcoRI (E) bands as indicated by horizontal bars. (C) Southern blot to detect gene targeting. WT bands are seen in the parental Dicer1 ^c/− line, and in a Dicer1 ^+/+ line. An increase in band size is occurs when the single Hprt sequence is replaced by a single copy (SC) or multiple copy concatemer (MC) of the targeting vector. In addition to the mutant band, clone MC2 clone shows a WT band of normal intensity (*). This results from recombination occurring in the 5′ arm, rather than in the 3′ arm, of the terminal vector in the concatemer. (D) qPCR of Dnmt1 mRNA. All values were calibrated to that obtained for the Dicer1 ^c/− line, adjusted to 1.0. SNL, mitomycin C-inactivated feeder cells. Other details as above. (E) Southern blot to detect methylation levels at MinS repeats. Details as above.

Figure 6. Rebound DNA methylation at repetitive sequences after Dicer1 ablation.

Southern blots. MC1^Cre and MC2^Cre are sublines derived from Cre transfection of line MC1 and MC2 respectively. Other details are as provided in the legends to previous figures.