DNA Methylation Supports Intrinsic Epigenetic Memory in Mammalian Cells

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Abstract
We have investigated the role of DNA methylation in the initiation and maintenance of silenced chromatin in somatic mammalian cells. We found that a mutated transgene, in which all the CpG dinucleotides have been eliminated, underwent transcriptional silencing to the same extent as the unmodified transgene. These observations demonstrate that DNA methylation is not required for silencing. The silenced CpG-free transgene exhibited all the features of heterochromatin, including silencing of transcriptional activity, delayed DNA replication, lack of histone H3 and H4 acetylation, lack of H3-K4 methylation, and enrichment in tri-methyl-H3-K9. In contrast, when we tested for transgene reactivation using a Cre recombinase-mediated inversion assay, we observed a marked difference between a CpG-free and an unmodified transgene: the CpG-free transgene resumed transcription and did not exhibit markers of heterochromatin whereas the unmodified transgene remained silenced. These data indicate that methylation of CpG residues conferred epigenetic memory in this system. These results also suggest that replication delay, lack of histone H3 and H4 acetylation, H3-K4 methylation, and enrichment in tri-methyl-H3-K9 are not sufficient to confer epigenetic memory. We propose that DNA methylation within transgenes serves as an intrinsic epigenetic memory to permanently silence transgenes and prevent their reactivation. Regulation of gene expression during development and differentiation occurs at many levels including transcriptional gene silencing and activation. Understanding the mechanism of gene silencing has practical importance because genes inserted in the genome for gene therapy are often silenced, decreasing the efficiency and increasing the risk associated with these procedures. In this report, the authors created a gene that cannot be methylated by mutating all of its CpG dinucleotides and examined in detail the role of DNA methylation and other epigenetic modifications in the establishment and maintenance of gene silencing. The authors found that DNA methylation is not required for the establishment or the maintenance of the silent chromatin state, but that this covalent DNA modification was unique among the epigenetic marks tested because it conferred to the chromatin structure a long-term intrinsic epigenetic memory that prevents gene reactivation.