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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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Recombination in Individual DR-GFP ClonesSingle clones were isolated from pooled cultures of Hela carrying inactive copies of DR-GFP. The three clones analyzed indicated as 1, 2, and 3 contained a single insertion with one or four copies of DR-GFP by Southern blot and qPCR (Figure S2). These clones were transfected in several independent experiments with I-SceI and pSVβGal vectors and scored for GFP+ cells.(A) Shown is FACS analysis of three clones 72 h after I-SceI transfection. The ordinate shows the number of cells and the abscissa the intensity of the fluorescence, respectively. The inset shows the dot plot of the bivariate analysis.(B) The experiment illustrated in (A) was repeated several times, and the mean of intensity of fluorescence of GFP+ cells is shown in the histogram. The roman numerals indicate an individual experiment.
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pgen-0030110-g003: Recombination in Individual DR-GFP ClonesSingle clones were isolated from pooled cultures of Hela carrying inactive copies of DR-GFP. The three clones analyzed indicated as 1, 2, and 3 contained a single insertion with one or four copies of DR-GFP by Southern blot and qPCR (Figure S2). These clones were transfected in several independent experiments with I-SceI and pSVβGal vectors and scored for GFP+ cells.(A) Shown is FACS analysis of three clones 72 h after I-SceI transfection. The ordinate shows the number of cells and the abscissa the intensity of the fluorescence, respectively. The inset shows the dot plot of the bivariate analysis.(B) The experiment illustrated in (A) was repeated several times, and the mean of intensity of fluorescence of GFP+ cells is shown in the histogram. The roman numerals indicate an individual experiment.

Mentions: The data shown above suggest that recombination products induced by I-SceI cleavage are silenced by methylation. These results were obtained from pools of cells carrying DR-GFP integrated randomly in the genome and did not distinguish among individual clones. We therefore asked if the integration locus influenced the expression of GFP recombination products, and by inference, their methylation status. We isolated several HeLa DR-GFP clones with single insertions (Figure S3) and transfected them with I-SceI. Figure S3A (inserts) shows the fluorescent mean intensity as dot plots in red and the fraction of GFP-expressing cells in three individual clones. Both the frequency (ordinate) and the fluorescence intensity (abscissa) segregated in discrete peaks. We repeatedly transfected the individual clones with I-SceI and determined GFP fluorescence intensity after normalization for transfection efficiency. The results, shown in Figure 3B, indicate differences in GFP expression from experiment to experiment. In all cases, I-SceI induced GFP expression, and each clone displayed a particular range of GFP levels, probably due to the specific integration site.


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)

Recombination in Individual DR-GFP ClonesSingle clones were isolated from pooled cultures of Hela carrying inactive copies of DR-GFP. The three clones analyzed indicated as 1, 2, and 3 contained a single insertion with one or four copies of DR-GFP by Southern blot and qPCR (Figure S2). These clones were transfected in several independent experiments with I-SceI and pSVβGal vectors and scored for GFP+ cells.(A) Shown is FACS analysis of three clones 72 h after I-SceI transfection. The ordinate shows the number of cells and the abscissa the intensity of the fluorescence, respectively. The inset shows the dot plot of the bivariate analysis.(B) The experiment illustrated in (A) was repeated several times, and the mean of intensity of fluorescence of GFP+ cells is shown in the histogram. The roman numerals indicate an individual experiment.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-0030110-g003: Recombination in Individual DR-GFP ClonesSingle clones were isolated from pooled cultures of Hela carrying inactive copies of DR-GFP. The three clones analyzed indicated as 1, 2, and 3 contained a single insertion with one or four copies of DR-GFP by Southern blot and qPCR (Figure S2). These clones were transfected in several independent experiments with I-SceI and pSVβGal vectors and scored for GFP+ cells.(A) Shown is FACS analysis of three clones 72 h after I-SceI transfection. The ordinate shows the number of cells and the abscissa the intensity of the fluorescence, respectively. The inset shows the dot plot of the bivariate analysis.(B) The experiment illustrated in (A) was repeated several times, and the mean of intensity of fluorescence of GFP+ cells is shown in the histogram. The roman numerals indicate an individual experiment.
Mentions: The data shown above suggest that recombination products induced by I-SceI cleavage are silenced by methylation. These results were obtained from pools of cells carrying DR-GFP integrated randomly in the genome and did not distinguish among individual clones. We therefore asked if the integration locus influenced the expression of GFP recombination products, and by inference, their methylation status. We isolated several HeLa DR-GFP clones with single insertions (Figure S3) and transfected them with I-SceI. Figure S3A (inserts) shows the fluorescent mean intensity as dot plots in red and the fraction of GFP-expressing cells in three individual clones. Both the frequency (ordinate) and the fluorescence intensity (abscissa) segregated in discrete peaks. We repeatedly transfected the individual clones with I-SceI and determined GFP fluorescence intensity after normalization for transfection efficiency. The results, shown in Figure 3B, indicate differences in GFP expression from experiment to experiment. In all cases, I-SceI induced GFP expression, and each clone displayed a particular range of GFP levels, probably due to the specific integration site.

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