WIDESPREAD RECOVERY OF METHYLATION AT GAMETIC IMPRINTS IN HYPOMETHYLATED MOUSE STEM CELLS FOLLOWING RESCUE WITH DNMT3A2

Avinash Thakur, Sarah-Jayne Mackin, Rachelle E Irwin, Karla O'Neill, Gareth Pollin, CP Walsh

Research output: Contribution to conferenceAbstract

Abstract

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 new putative gDMR have 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).

Method:
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).

Results:
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 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:
DNMT3A/B plays a vital role in methylation maintenance at imprints as the rescue with DNMT3A2 can restore imprints in these cells. This provides a useful system to explore factors influencing imprint reprogramming.

Conference

Conference20th Meeting of the Irish Society of Human Genetics
CountryIreland
CityDublin
Period15/09/1715/09/18
Internet address

Fingerprint

Methylation
Stem Cells
Inborn Genetic Diseases
Methyltransferases
DNA Methylation
Embryonic Stem Cells
Epigenomics
Complementary DNA
Maintenance
Enzymes

Cite this

Thakur, A., Mackin, S-J., Irwin, R. E., O'Neill, K., Pollin, G., & Walsh, CP. (2018). WIDESPREAD RECOVERY OF METHYLATION AT GAMETIC IMPRINTS IN HYPOMETHYLATED MOUSE STEM CELLS FOLLOWING RESCUE WITH DNMT3A2. 54-64. Abstract from 20th Meeting of the Irish Society of Human Genetics, Dublin, Ireland.
Thakur, Avinash ; Mackin, Sarah-Jayne ; Irwin, Rachelle E ; O'Neill, Karla ; Pollin, Gareth ; Walsh, CP. / WIDESPREAD RECOVERY OF METHYLATION AT GAMETIC IMPRINTS IN HYPOMETHYLATED MOUSE STEM CELLS FOLLOWING RESCUE WITH DNMT3A2. Abstract from 20th Meeting of the Irish Society of Human Genetics, Dublin, Ireland.
@conference{3f6bb30b1c994e5f813e4fa0d2f59b04,
title = "WIDESPREAD RECOVERY OF METHYLATION AT GAMETIC IMPRINTS IN HYPOMETHYLATED MOUSE STEM CELLS FOLLOWING RESCUE WITH DNMT3A2",
abstract = "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 new putative gDMR have 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).Method: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).Results: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 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:DNMT3A/B plays a vital role in methylation maintenance at imprints as the rescue with DNMT3A2 can restore imprints in these cells. This provides a useful system to explore factors influencing imprint reprogramming.",
author = "Avinash Thakur and Sarah-Jayne Mackin and Irwin, {Rachelle E} and Karla O'Neill and Gareth Pollin and CP Walsh",
year = "2018",
language = "English",
pages = "54--64",
note = "20th Meeting of the Irish Society of Human Genetics ; Conference date: 15-09-2017 Through 15-09-2018",
url = "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5849961/",

}

Thakur, A, Mackin, S-J, Irwin, RE, O'Neill, K, Pollin, G & Walsh, CP 2018, 'WIDESPREAD RECOVERY OF METHYLATION AT GAMETIC IMPRINTS IN HYPOMETHYLATED MOUSE STEM CELLS FOLLOWING RESCUE WITH DNMT3A2' 20th Meeting of the Irish Society of Human Genetics, Dublin, Ireland, 15/09/17 - 15/09/18, pp. 54-64.

WIDESPREAD RECOVERY OF METHYLATION AT GAMETIC IMPRINTS IN HYPOMETHYLATED MOUSE STEM CELLS FOLLOWING RESCUE WITH DNMT3A2. / Thakur, Avinash; Mackin, Sarah-Jayne; Irwin, Rachelle E; O'Neill, Karla; Pollin, Gareth; Walsh, CP.

2018. 54-64 Abstract from 20th Meeting of the Irish Society of Human Genetics, Dublin, Ireland.

Research output: Contribution to conferenceAbstract

TY - CONF

T1 - WIDESPREAD RECOVERY OF METHYLATION AT GAMETIC IMPRINTS IN HYPOMETHYLATED MOUSE STEM CELLS FOLLOWING RESCUE WITH DNMT3A2

AU - Thakur, Avinash

AU - Mackin, Sarah-Jayne

AU - Irwin, Rachelle E

AU - O'Neill, Karla

AU - Pollin, Gareth

AU - Walsh, CP

PY - 2018

Y1 - 2018

N2 - 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 new putative gDMR have 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).Method: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).Results: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 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:DNMT3A/B plays a vital role in methylation maintenance at imprints as the rescue with DNMT3A2 can restore imprints in these cells. This provides a useful system to explore factors influencing imprint reprogramming.

AB - 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 new putative gDMR have 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).Method: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).Results: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 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:DNMT3A/B plays a vital role in methylation maintenance at imprints as the rescue with DNMT3A2 can restore imprints in these cells. This provides a useful system to explore factors influencing imprint reprogramming.

M3 - Abstract

SP - 54

EP - 64

ER -

Thakur A, Mackin S-J, Irwin RE, O'Neill K, Pollin G, Walsh CP. WIDESPREAD RECOVERY OF METHYLATION AT GAMETIC IMPRINTS IN HYPOMETHYLATED MOUSE STEM CELLS FOLLOWING RESCUE WITH DNMT3A2. 2018. Abstract from 20th Meeting of the Irish Society of Human Genetics, Dublin, Ireland.