Widespread recovery of methylation at gametic imprints in hypomethylated mouse stem cells following rescue with DNMT3A2
Bibliographic record
Abstract
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). 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. 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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".