DNA Methylation Supports Intrinsic Epigenetic Memory in Mammalian Cells

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.

An Unmethylated 3' Promoter-proximal Region is Required for Efficient Transcription Initiation

“…Surprisingly, analysis of the TATA and GFP regions of the transgene revealed significant enrichment of H3 acetylation exclusively in the unmethylated clone, despite the fact that the promoter/TATA box region of the patchmethylated cassette is also unmethylated. Given the absence of H3K9/K14ac throughout the patchmethylated cassette, and the reported association of DNA methylation and H3K9 methylation [36][37][38], we next tested whether the patch-methylated cassette is marked by H3K9me3, using the endogenous Gnas gene, previously shown to be marked by H3K9me3 in MEL cells [39], as a positive control ( Figure 5B). Analysis of the Gnas gene revealed similar levels of enrichment in the unmethylated and patchmethylated clones (;26-and 23-fold, respectively, relative to the IgG control), indicating that the H3K9me3 ChIP worked with similar efficiency in both chromatin preparations.…”

Replication Fork Velocities at Adjacent Replication Origins Are Coordinately Modified during DNA Replication in Human Cells

“…Efficient duplication of large genomes requires the coordinated activities of several proteins, including not only replication factors, but also cell cycle regulators (Cardoso et al, 1993), base excision repair enzymes (Otterlei et al, 1999), and DNA methyltransferases (Leonhardt et al, 1992;Feng et al, 2006). Chromatin modifications, namely methylation and acetylation, are coordinated with DNA replication (Feng et al, 2006;Ozdemir et al, 2006).…”

“…Chromatin modifications, namely methylation and acetylation, are coordinated with DNA replication (Feng et al, 2006;Ozdemir et al, 2006). In the cell nucleus, proteins bound to the DNA are spatially organized into microscop- ically visible functional structures, which are referred to as replication factories, or replication foci (Leonhardt and Cardoso, 1995;Newport and Yan, 1996;Cook, 1999).…”

“…Initiation of DNA replication from a reduced number of origins is associated with gross chromosomal abnormalities in Saccharomyces cerevisiae Sic1 mutants (Lengronne and Schwob, 2002). The number of origins and their initiation timing are also checkpointrestricted in unstressed Xenopus extracts, perhaps in order to limit fork density and fork arrest (Marheineke and Hyrien, 2004;Shechter et al, 2004), which are potential sources of genomic instability (Rothstein et al, 2000;McGlynn and Lloyd, 2002;Nyberg et al, 2002;Cleary and Pearson, 2005).Efficient duplication of large genomes requires the coordinated activities of several proteins, including not only replication factors, but also cell cycle regulators (Cardoso et al, 1993), base excision repair enzymes (Otterlei et al, 1999), and DNA methyltransferases (Leonhardt et al, 1992;Feng et al, 2006). Chromatin modifications, namely methylation and acetylation, are coordinated with DNA replication (Feng et al, 2006;Ozdemir et al, 2006).…”

Identification of transgenic cloned dairy goats harboring human lactoferrin and methylation status of the imprinted gene IGF2R in their lungs

“…DNA methylation is a principal epigenetic modification of the genome and plays a critical role in nuclear reprogramming (Feng et al, 2006). It mainly occurs at the 5'cytosine in CpG dinucleotides, which are usually found in clusters, termed CpG islands (Gardiner-Garden and Frommer, 1987).…”