Abstract
We report that homology-directed repair of a DNA double-strand break within a single copy Green Fluorescent Protein (GFP) gene in HeLa cells alters the methylation pattern at the site of recombination. DNA methyl transferase (DNMT)1, DNMT3a and two proteins that regulate methylation, Np95 and GADD45A, are recruited to the site of repair and are responsible for selective methylation of the promoter-distal segment of the repaired DNA. The initial methylation pattern of the locus is modified in a transcription-dependent fashion during the 15–20 days following repair, at which time no further changes in the methylation pattern occur. The variation in DNA modification generates stable clones with wide ranges of GFP expression. Collectively, our data indicate that somatic DNA methylation follows homologous repair and is subjected to remodeling by local transcription in a discrete time window during and after the damage. We propose that DNA methylation of repaired genes represents a DNA damage code and is source of variation of gene expression.
Highlights
DNA methylation is a feature of higher eukaryote genomes
We report here that methylation induced by homology-directed repair (HDR) is influenced by recruitment of Np95 and GADD45a to the DSB and that DNMT3a is active at the DSB
The integrated DRGFP undergoes several cycles of cutting and resealing until the I-SceI site is lost by nonhomologous end joining (NHEJ) or homology-dependent repair (HDR)
Summary
DNA methylation is a feature of higher eukaryote genomes. DNA methylation is critical during development in plants and mammals. Patterns of methylated CpGs are transmitted to daughter cells with high fidelity [2,3]. Aberrant methylation, both hyper- and hypo-methylation, has been found in cancer cells [4]. There are two patterns of DNA methylation: (i) Stable methylation, which is the basis of imprinting, is inherited in a sex-specific fashion and is invariant among individuals and cell types. Loss or modification of stable methylation results in significant phenotypic and genetic alterations. (ii) Unstable or metastable methylation, which is variable among individuals and cell types Loss or modification of stable methylation results in significant phenotypic and genetic alterations. (ii) Unstable or metastable methylation, which is variable among individuals and cell types
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