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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

Dnmt1 Inhibits the Expression of Recombinant GFP GenesWild-type or Dnmt1−/− ES cells carrying DR-GFP were transfected with the I-SceI expression vector and PSVbGal, grown 4 d, and analyzed for GFP recombination and expression.(A) Genomic DNA from the two cell lines was PCR-amplified with nonrecombinant (5′-unrec) and recombinant (5′-rec) primers. The specificity of the products and the linearity of the reactions were controlled as described in Materials and Methods. qPCR of the same samples was carried out as described in Materials and Methods.(B) FACS analysis of cells transfected with I-SceI is shown. The gating of GFP+ cells was created to exclude up the 99.5% of wild-type untransfected ES cells. The same gating applied to Dnmt1−/− cells shows a significant increase in the population expressing GFP. Following I-SceI transfection, Dnmt1−/− cells were treated with 5-AzadC as described in Materials and Methods. Treatment with 5-AzadC increased the fraction of cells expressing GFP in wild-type ES but did not enhance the expression of GFP in the Dnmt1−/− cells(C) The histogram showing the fraction of GFP+ cells derived from three experiments is shown. To obtain reliable values of differential GFP fluorescence in ES and Dnmt1−/− cells, we compared the percentage of GFP+ cells, normalized for the transfection efficiency in six experiments (three in duplicate), with the Wilcoxon Kruskal-Wallis Test, *, p < 0.012 versus wild type.
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pgen-0030110-g005: Dnmt1 Inhibits the Expression of Recombinant GFP GenesWild-type or Dnmt1−/− ES cells carrying DR-GFP were transfected with the I-SceI expression vector and PSVbGal, grown 4 d, and analyzed for GFP recombination and expression.(A) Genomic DNA from the two cell lines was PCR-amplified with nonrecombinant (5′-unrec) and recombinant (5′-rec) primers. The specificity of the products and the linearity of the reactions were controlled as described in Materials and Methods. qPCR of the same samples was carried out as described in Materials and Methods.(B) FACS analysis of cells transfected with I-SceI is shown. The gating of GFP+ cells was created to exclude up the 99.5% of wild-type untransfected ES cells. The same gating applied to Dnmt1−/− cells shows a significant increase in the population expressing GFP. Following I-SceI transfection, Dnmt1−/− cells were treated with 5-AzadC as described in Materials and Methods. Treatment with 5-AzadC increased the fraction of cells expressing GFP in wild-type ES but did not enhance the expression of GFP in the Dnmt1−/− cells(C) The histogram showing the fraction of GFP+ cells derived from three experiments is shown. To obtain reliable values of differential GFP fluorescence in ES and Dnmt1−/− cells, we compared the percentage of GFP+ cells, normalized for the transfection efficiency in six experiments (three in duplicate), with the Wilcoxon Kruskal-Wallis Test, *, p < 0.012 versus wild type.

Mentions: Stimulation of recombinant GFP gene expression by 5-AzadC suggested that a significant fraction of recombinant genes was silenced by methylation. We confirmed this conclusion in another system in which global methylation was impaired by inactivation of Dnmt1. Dnmt1 is responsible for methylation maintenance in the mouse genome [5]. We transfected a Dnmt1−/− ES cell line [5] with DR-GFP. The pool of puromycin-resistant clones was then transfected with I-SceI and analyzed as described above for Hela cells. Our results indicate that the frequency of HR was the same in wild-type and Dnmt1−/− ES cells, as shown by PCR and quantitative (q)PCR (Figure 5A). FACS analysis indicates that the percentage of Dnmt1−/− cells that expressed GFP at elevated levels was higher than wild-type cells (Figure 5B and 5C). Finally, treatment with 5-AzadC increased the fraction of wild-type ES high expressors but did not amplify the expression of GFP in Dnmt1−/− cells (Figure 5B and 5C). These data suggest that Dnmt1-dependent methylation silences GFP expression in recombinant clones.


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)

Dnmt1 Inhibits the Expression of Recombinant GFP GenesWild-type or Dnmt1−/− ES cells carrying DR-GFP were transfected with the I-SceI expression vector and PSVbGal, grown 4 d, and analyzed for GFP recombination and expression.(A) Genomic DNA from the two cell lines was PCR-amplified with nonrecombinant (5′-unrec) and recombinant (5′-rec) primers. The specificity of the products and the linearity of the reactions were controlled as described in Materials and Methods. qPCR of the same samples was carried out as described in Materials and Methods.(B) FACS analysis of cells transfected with I-SceI is shown. The gating of GFP+ cells was created to exclude up the 99.5% of wild-type untransfected ES cells. The same gating applied to Dnmt1−/− cells shows a significant increase in the population expressing GFP. Following I-SceI transfection, Dnmt1−/− cells were treated with 5-AzadC as described in Materials and Methods. Treatment with 5-AzadC increased the fraction of cells expressing GFP in wild-type ES but did not enhance the expression of GFP in the Dnmt1−/− cells(C) The histogram showing the fraction of GFP+ cells derived from three experiments is shown. To obtain reliable values of differential GFP fluorescence in ES and Dnmt1−/− cells, we compared the percentage of GFP+ cells, normalized for the transfection efficiency in six experiments (three in duplicate), with the Wilcoxon Kruskal-Wallis Test, *, p < 0.012 versus wild type.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-0030110-g005: Dnmt1 Inhibits the Expression of Recombinant GFP GenesWild-type or Dnmt1−/− ES cells carrying DR-GFP were transfected with the I-SceI expression vector and PSVbGal, grown 4 d, and analyzed for GFP recombination and expression.(A) Genomic DNA from the two cell lines was PCR-amplified with nonrecombinant (5′-unrec) and recombinant (5′-rec) primers. The specificity of the products and the linearity of the reactions were controlled as described in Materials and Methods. qPCR of the same samples was carried out as described in Materials and Methods.(B) FACS analysis of cells transfected with I-SceI is shown. The gating of GFP+ cells was created to exclude up the 99.5% of wild-type untransfected ES cells. The same gating applied to Dnmt1−/− cells shows a significant increase in the population expressing GFP. Following I-SceI transfection, Dnmt1−/− cells were treated with 5-AzadC as described in Materials and Methods. Treatment with 5-AzadC increased the fraction of cells expressing GFP in wild-type ES but did not enhance the expression of GFP in the Dnmt1−/− cells(C) The histogram showing the fraction of GFP+ cells derived from three experiments is shown. To obtain reliable values of differential GFP fluorescence in ES and Dnmt1−/− cells, we compared the percentage of GFP+ cells, normalized for the transfection efficiency in six experiments (three in duplicate), with the Wilcoxon Kruskal-Wallis Test, *, p < 0.012 versus wild type.
Mentions: Stimulation of recombinant GFP gene expression by 5-AzadC suggested that a significant fraction of recombinant genes was silenced by methylation. We confirmed this conclusion in another system in which global methylation was impaired by inactivation of Dnmt1. Dnmt1 is responsible for methylation maintenance in the mouse genome [5]. We transfected a Dnmt1−/− ES cell line [5] with DR-GFP. The pool of puromycin-resistant clones was then transfected with I-SceI and analyzed as described above for Hela cells. Our results indicate that the frequency of HR was the same in wild-type and Dnmt1−/− ES cells, as shown by PCR and quantitative (q)PCR (Figure 5A). FACS analysis indicates that the percentage of Dnmt1−/− cells that expressed GFP at elevated levels was higher than wild-type cells (Figure 5B and 5C). Finally, treatment with 5-AzadC increased the fraction of wild-type ES high expressors but did not amplify the expression of GFP in Dnmt1−/− cells (Figure 5B and 5C). These data suggest that Dnmt1-dependent methylation silences GFP expression in recombinant clones.

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