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DNA damage, homology-directed repair, and DNA methylation.

Cuozzo C, Porcellini A, Angrisano T, Morano A, Lee B, Di Pardo A, Messina S, Iuliano R, Fusco A, Santillo MR, Muller MT, Chiariotti L, Gottesman ME, Avvedimento EV - PLoS Genet. (2007)

Bottom Line: Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2'-deoxycytidine.Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression.Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biologia e Patologia Molecolare e Cellulare, Istituto di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Università Federico II, Naples, Italy.

ABSTRACT
To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%-4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, approximately 50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2'-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.

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Related in: MedlinePlus

Methylation Patterns of Individual GFP DNA Molecules before or after HRClustalW analysis of individual GFP molecules (−50 to +420 bp relative to the DSB) derived from (A) ES, (B) Dnmt1−/−, and (C) Hela cell lines. Recombinant molecules were derived from cells sorted by GFP immunofluorescence as HR-L and HR-H in Figure 2B and 2C. To compare only methylation patterns, the same unrecombinant molecules shown in Figure 6A–6C were artificially transformed to recombinant molecules by substituting the I-SceI with the Bcg1 site. The percent of sequence variation is shown on the ordinate of the phylogenetic tree (dendrogram). The three main groups indicated in the dendrogram correspond to nonrecombinant (red) and recombinant sequences (black, HR-L and blue, HR-L clones). The variation in each group depends solely on the methylation pattern. The analysis has been performed on molecules derived from ~60 independent bisulfite reactions. The sequences that showed an identical pattern of methylation from the same bisulfite analysis were eliminated. For each sequence the percent of methylation is indicated at the bottom. The sequence analysis was performed using the MegAlign 7.0.1 a module of the Lasergene-DNASTAR software.
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pgen-0030110-g008: Methylation Patterns of Individual GFP DNA Molecules before or after HRClustalW analysis of individual GFP molecules (−50 to +420 bp relative to the DSB) derived from (A) ES, (B) Dnmt1−/−, and (C) Hela cell lines. Recombinant molecules were derived from cells sorted by GFP immunofluorescence as HR-L and HR-H in Figure 2B and 2C. To compare only methylation patterns, the same unrecombinant molecules shown in Figure 6A–6C were artificially transformed to recombinant molecules by substituting the I-SceI with the Bcg1 site. The percent of sequence variation is shown on the ordinate of the phylogenetic tree (dendrogram). The three main groups indicated in the dendrogram correspond to nonrecombinant (red) and recombinant sequences (black, HR-L and blue, HR-L clones). The variation in each group depends solely on the methylation pattern. The analysis has been performed on molecules derived from ~60 independent bisulfite reactions. The sequences that showed an identical pattern of methylation from the same bisulfite analysis were eliminated. For each sequence the percent of methylation is indicated at the bottom. The sequence analysis was performed using the MegAlign 7.0.1 a module of the Lasergene-DNASTAR software.

Mentions: The data shown in Figures 6 and 7 summarize the statistical analysis of GFP DNA methylation before and after recombination. However, these data do not reveal the impact of recombination on the methylation pattern of individual GFP molecules. To visualize changes in individual molecules, we performed ClustalW analysis on the complete collection of GFP molecules. The difference in DNA sequence between recombinant and nonrecombinant molecules may obscure changes due to methylation. To eliminate this problem and to better assess the impact of recombination on de novo methylation, we converted the I-SceI site into a BcgI restriction site in all nonrecombinant sequences and repeated the ClustalW analysis on the total pool of sequences. The molecules now are identical in sequence and differ only in methylated CpGs. ClustalW analysis of these molecules shows the methylation profiles and the degree of similarity among different molecules. Sequences containing the same methylated CpG are clustered in branches of the dendrogram (Figure 8). Figure 8A shows that nonrecombinant sequences (uncut, red) can be distinguished from recombinant HR-H (black) or recombinant HR-L (blue).


DNA damage, homology-directed repair, and DNA methylation.

Cuozzo C, Porcellini A, Angrisano T, Morano A, Lee B, Di Pardo A, Messina S, Iuliano R, Fusco A, Santillo MR, Muller MT, Chiariotti L, Gottesman ME, Avvedimento EV - PLoS Genet. (2007)

Methylation Patterns of Individual GFP DNA Molecules before or after HRClustalW analysis of individual GFP molecules (−50 to +420 bp relative to the DSB) derived from (A) ES, (B) Dnmt1−/−, and (C) Hela cell lines. Recombinant molecules were derived from cells sorted by GFP immunofluorescence as HR-L and HR-H in Figure 2B and 2C. To compare only methylation patterns, the same unrecombinant molecules shown in Figure 6A–6C were artificially transformed to recombinant molecules by substituting the I-SceI with the Bcg1 site. The percent of sequence variation is shown on the ordinate of the phylogenetic tree (dendrogram). The three main groups indicated in the dendrogram correspond to nonrecombinant (red) and recombinant sequences (black, HR-L and blue, HR-L clones). The variation in each group depends solely on the methylation pattern. The analysis has been performed on molecules derived from ~60 independent bisulfite reactions. The sequences that showed an identical pattern of methylation from the same bisulfite analysis were eliminated. For each sequence the percent of methylation is indicated at the bottom. The sequence analysis was performed using the MegAlign 7.0.1 a module of the Lasergene-DNASTAR software.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1913100&req=5

pgen-0030110-g008: Methylation Patterns of Individual GFP DNA Molecules before or after HRClustalW analysis of individual GFP molecules (−50 to +420 bp relative to the DSB) derived from (A) ES, (B) Dnmt1−/−, and (C) Hela cell lines. Recombinant molecules were derived from cells sorted by GFP immunofluorescence as HR-L and HR-H in Figure 2B and 2C. To compare only methylation patterns, the same unrecombinant molecules shown in Figure 6A–6C were artificially transformed to recombinant molecules by substituting the I-SceI with the Bcg1 site. The percent of sequence variation is shown on the ordinate of the phylogenetic tree (dendrogram). The three main groups indicated in the dendrogram correspond to nonrecombinant (red) and recombinant sequences (black, HR-L and blue, HR-L clones). The variation in each group depends solely on the methylation pattern. The analysis has been performed on molecules derived from ~60 independent bisulfite reactions. The sequences that showed an identical pattern of methylation from the same bisulfite analysis were eliminated. For each sequence the percent of methylation is indicated at the bottom. The sequence analysis was performed using the MegAlign 7.0.1 a module of the Lasergene-DNASTAR software.
Mentions: The data shown in Figures 6 and 7 summarize the statistical analysis of GFP DNA methylation before and after recombination. However, these data do not reveal the impact of recombination on the methylation pattern of individual GFP molecules. To visualize changes in individual molecules, we performed ClustalW analysis on the complete collection of GFP molecules. The difference in DNA sequence between recombinant and nonrecombinant molecules may obscure changes due to methylation. To eliminate this problem and to better assess the impact of recombination on de novo methylation, we converted the I-SceI site into a BcgI restriction site in all nonrecombinant sequences and repeated the ClustalW analysis on the total pool of sequences. The molecules now are identical in sequence and differ only in methylated CpGs. ClustalW analysis of these molecules shows the methylation profiles and the degree of similarity among different molecules. Sequences containing the same methylated CpG are clustered in branches of the dendrogram (Figure 8). Figure 8A shows that nonrecombinant sequences (uncut, red) can be distinguished from recombinant HR-H (black) or recombinant HR-L (blue).

Bottom Line: Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2'-deoxycytidine.Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression.Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biologia e Patologia Molecolare e Cellulare, Istituto di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Università Federico II, Naples, Italy.

ABSTRACT
To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%-4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, approximately 50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2'-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.

Show MeSH
Related in: MedlinePlus