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

Irreversible inactivation or silencing of tumor suppressor genes occurs fre quently in the development of cancer. A similar process of silencing can oc cur after the integration of transfected or microinjected genes into the ge nomes of recipient cells. The inactivation of transfected genes seems parti cularly efficient in cells with stem cell characteristics. We have been stu dying the inactivation of genes transfected into cultured P19 embryonal car cinoma cells and found that the CpG-rich sequence comprising the coding reg ion of the lacZ reporter gene becomes extensively methylated after integrat ion into the genome. 5-Aza-2'-deoxycytidine (5AdC), an inhibitor of DNA met hylation, induced the reexpression of silent transgenes in one clone of P19 cells studied in detail. However, the reexpressed genes remained heavily m ethylated over the lacZ coding sequence. We used pulsed-field gel electroph oresis to analyze the structure of the transgenic locus in the parental and in 5AdC-treated cells and found that, in each of the cells reexpressing th e transgene, the cluster of transgenes had been rearranged. Each clone had undergone a different rearrangement that appeared to involve recombination within the tandemly repeated copies of the transgene. Our data seem consist ent with the idea that 5AdC induces efficient DNA recombination between tan demly repeated genes and that the reexpression of silenced genes induced by 5AdC might be triggered by the chromatin reorganization at the site of DNA recombination. (C) 2001 Wiley-Liss, Inc.