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Reexpression of a cluster of silenced transgenes is associated with their rearrangement

M W McBurney1, S Lau, K Jardine

  • 1Ottawa Regional Cancer Center and Department of Medicine, University of Ottawa, Ottawa, Ontario, Canada. michael.mcburney@orcc.on.ca

Genes, Chromosomes & Cancer
|December 18, 2001
PubMed

Insights

DNA methylation silences genes in cancer cells and after gene transfer. A drug inhibiting DNA methylation, 5-Aza-2'-deoxycytidine (5AdC), reactivated silenced genes by causing DNA recombination and chromatin changes.

Area of Science:

  • Molecular Biology
  • Epigenetics
  • Cancer Research

Background:

  • Gene silencing is crucial in cancer development and can occur after gene integration into host genomes.
  • Stem cells exhibit efficient inactivation of transfected genes, a process linked to DNA methylation.
  • P19 embryonal carcinoma cells were used to study transgene inactivation and its mechanisms.

Purpose of the Study:

  • To investigate the mechanisms of gene silencing in P19 embryonal carcinoma cells after gene transfection.
  • To explore the role of DNA methylation in transgene inactivation.
  • To determine the effect of 5-Aza-2'-deoxycytidine (5AdC) on silenced transgenes and associated genomic structures.

Main Methods:

  • Transfection of the lacZ reporter gene into P19 embryonal carcinoma cells.
  • Analysis of DNA methylation patterns within the lacZ coding sequence.
  • Treatment with 5-Aza-2'-deoxycytidine (5AdC), a DNA methylation inhibitor.
  • Pulsed-field gel electrophoresis to analyze the structure of the transgenic locus.

Main Results:

  • The CpG-rich coding region of the lacZ transgene became extensively methylated after integration into the P19 cell genome.
  • 5AdC treatment induced reexpression of the silenced transgene in a specific P19 cell clone.
  • Reexpression of the transgene was associated with DNA recombination and rearrangement within the tandemly repeated transgene cluster.
  • The rearrangements were unique to each clone that reexpressed the transgene.

Conclusions:

  • DNA methylation plays a significant role in silencing integrated transgenes in P19 embryonal carcinoma cells.
  • 5AdC can induce reexpression of silenced transgenes, potentially by triggering DNA recombination.
  • Chromatin reorganization at the site of DNA recombination may be a key event in reactivating silenced genes.

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