Differential responses of clinically important gene classes to transient loss of DNA methylation in human differentiated cells.

Sarah-Jayne Mackin, Karla O'Neill, Colum P Walsh

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Abstract

Background: Methylation of DNA sequences at promoters, CpGislands and other elements plays a vital role in regulating gene activity. In human, loss of methylation is known to play a causativ erole in imprinting disorders and in inappropriate germline gene expression in cancers. While in mouse, loss of function mutants have given great insight into the targets of methylation, functional studies in human have been largely limited to cancer cells and more recently stem cells, not normal adult cells. Methods: Stable knockdown sof the maintenance methyltransferase DNMT1 were generated innormosomic hTERT-immortalised adult fibroblasts. Genome-wide methylation levels were assayed using the Illumina 450K bead array. Results were analysed using RnBeads and Galaxy. Locus specific methylation was verified using pyrosequencing and clonal analysis. Validation was achieved using transient siRNA. Results:Loss of function was poorly tolerated and all clonally-expanded cell lines had spontaneously restored DNMT1 levels by silencing of the shRNA. Evidence for a genome-wide methylation erasure event followed by a wave of remethylation could be clearly traced.Gene bodies and the shores of CpG islands showed the clearest loss of methylation overall. While most CpG islands are normally unmethylated and so unaffected, both imprints and germ line genes fall into the rarer category of normally methylated islands: ofthese two, lasting loss of methylation was much more common among imprints than germ line genes. Conclusions: 1: transient loss of methylation is poorly tolerated; 2: a robust mechanism for remethylation exists even in adult cells; 3: aberrant remethylationis frequent on recovery and 4: Imprints are particularly sensitive.
LanguageEnglish
Title of host publicationUnknown Host Publication
Pages212-212
Number of pages1
Volume84
Publication statusAccepted/In press - 3 Sep 2015
Event18th Meeting of the Irish Society of Human Genetics - Dublin City University
Duration: 3 Sep 2015 → …

Conference

Conference18th Meeting of the Irish Society of Human Genetics
Period3/09/15 → …

Fingerprint

DNA Methylation
Methylation
Genes
CpG Islands
Germ Cells
Small Interfering RNA
Galaxies
Genome
Methyltransferases
Islands
Neoplasms
Stem Cells
Fibroblasts
Maintenance
Gene Expression
Cell Line

Keywords

  • Methylation
  • DNMT1
  • 450k
  • Imprints.

Cite this

Mackin, S-J., O'Neill, K., & Walsh, C. P. (Accepted/In press). Differential responses of clinically important gene classes to transient loss of DNA methylation in human differentiated cells. In Unknown Host Publication (Vol. 84, pp. 212-212)
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abstract = "Background: Methylation of DNA sequences at promoters, CpGislands and other elements plays a vital role in regulating gene activity. In human, loss of methylation is known to play a causativ erole in imprinting disorders and in inappropriate germline gene expression in cancers. While in mouse, loss of function mutants have given great insight into the targets of methylation, functional studies in human have been largely limited to cancer cells and more recently stem cells, not normal adult cells. Methods: Stable knockdown sof the maintenance methyltransferase DNMT1 were generated innormosomic hTERT-immortalised adult fibroblasts. Genome-wide methylation levels were assayed using the Illumina 450K bead array. Results were analysed using RnBeads and Galaxy. Locus specific methylation was verified using pyrosequencing and clonal analysis. Validation was achieved using transient siRNA. Results:Loss of function was poorly tolerated and all clonally-expanded cell lines had spontaneously restored DNMT1 levels by silencing of the shRNA. Evidence for a genome-wide methylation erasure event followed by a wave of remethylation could be clearly traced.Gene bodies and the shores of CpG islands showed the clearest loss of methylation overall. While most CpG islands are normally unmethylated and so unaffected, both imprints and germ line genes fall into the rarer category of normally methylated islands: ofthese two, lasting loss of methylation was much more common among imprints than germ line genes. Conclusions: 1: transient loss of methylation is poorly tolerated; 2: a robust mechanism for remethylation exists even in adult cells; 3: aberrant remethylationis frequent on recovery and 4: Imprints are particularly sensitive.",
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Mackin, S-J, O'Neill, K & Walsh, CP 2015, Differential responses of clinically important gene classes to transient loss of DNA methylation in human differentiated cells. in Unknown Host Publication. vol. 84, pp. 212-212, 18th Meeting of the Irish Society of Human Genetics, 3/09/15.

Differential responses of clinically important gene classes to transient loss of DNA methylation in human differentiated cells. / Mackin, Sarah-Jayne; O'Neill, Karla; Walsh, Colum P.

Unknown Host Publication. Vol. 84 2015. p. 212-212.

Research output: Chapter in Book/Report/Conference proceedingConference contribution

TY - GEN

T1 - Differential responses of clinically important gene classes to transient loss of DNA methylation in human differentiated cells.

AU - Mackin, Sarah-Jayne

AU - O'Neill, Karla

AU - Walsh, Colum P

PY - 2015/9/3

Y1 - 2015/9/3

N2 - Background: Methylation of DNA sequences at promoters, CpGislands and other elements plays a vital role in regulating gene activity. In human, loss of methylation is known to play a causativ erole in imprinting disorders and in inappropriate germline gene expression in cancers. While in mouse, loss of function mutants have given great insight into the targets of methylation, functional studies in human have been largely limited to cancer cells and more recently stem cells, not normal adult cells. Methods: Stable knockdown sof the maintenance methyltransferase DNMT1 were generated innormosomic hTERT-immortalised adult fibroblasts. Genome-wide methylation levels were assayed using the Illumina 450K bead array. Results were analysed using RnBeads and Galaxy. Locus specific methylation was verified using pyrosequencing and clonal analysis. Validation was achieved using transient siRNA. Results:Loss of function was poorly tolerated and all clonally-expanded cell lines had spontaneously restored DNMT1 levels by silencing of the shRNA. Evidence for a genome-wide methylation erasure event followed by a wave of remethylation could be clearly traced.Gene bodies and the shores of CpG islands showed the clearest loss of methylation overall. While most CpG islands are normally unmethylated and so unaffected, both imprints and germ line genes fall into the rarer category of normally methylated islands: ofthese two, lasting loss of methylation was much more common among imprints than germ line genes. Conclusions: 1: transient loss of methylation is poorly tolerated; 2: a robust mechanism for remethylation exists even in adult cells; 3: aberrant remethylationis frequent on recovery and 4: Imprints are particularly sensitive.

AB - Background: Methylation of DNA sequences at promoters, CpGislands and other elements plays a vital role in regulating gene activity. In human, loss of methylation is known to play a causativ erole in imprinting disorders and in inappropriate germline gene expression in cancers. While in mouse, loss of function mutants have given great insight into the targets of methylation, functional studies in human have been largely limited to cancer cells and more recently stem cells, not normal adult cells. Methods: Stable knockdown sof the maintenance methyltransferase DNMT1 were generated innormosomic hTERT-immortalised adult fibroblasts. Genome-wide methylation levels were assayed using the Illumina 450K bead array. Results were analysed using RnBeads and Galaxy. Locus specific methylation was verified using pyrosequencing and clonal analysis. Validation was achieved using transient siRNA. Results:Loss of function was poorly tolerated and all clonally-expanded cell lines had spontaneously restored DNMT1 levels by silencing of the shRNA. Evidence for a genome-wide methylation erasure event followed by a wave of remethylation could be clearly traced.Gene bodies and the shores of CpG islands showed the clearest loss of methylation overall. While most CpG islands are normally unmethylated and so unaffected, both imprints and germ line genes fall into the rarer category of normally methylated islands: ofthese two, lasting loss of methylation was much more common among imprints than germ line genes. Conclusions: 1: transient loss of methylation is poorly tolerated; 2: a robust mechanism for remethylation exists even in adult cells; 3: aberrant remethylationis frequent on recovery and 4: Imprints are particularly sensitive.

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KW - DNMT1

KW - 450k

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BT - Unknown Host Publication

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