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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

DNA Methylation in Repaired DNA Molecules(A) CpG methylation in repaired molecules from ES cells is shown. DNA molecules derived from pooled ES DR-GFP cultures transfected with the I-SceI expression vector or a control plasmid were subjected to bisulfite analysis (Materials and Methods). The number of molecules in each class was as follows: (1) uncut, 40 from cells transfected with control plasmid; (2) HR-H, 25 homologous recombinant molecules from high expressor cells sorted by FACS (23) or picked randomly from mass culture; (3) HR-L, 30 recombinant molecules from low expressor cells sorted by FACS (28) or picked randomly from mass cultures; (4) molecules rearranged at the I-SceI site (NHEJ). The frequency (%) of each class was derived from several independent experiments with mass culture and fluorescent-sorted cells. HR-H 3 ± 0.5; HL 3 ± 1; NHEJ 2 ± 0.3. All CpGs (white circles) flanking the I-SceI site are shown. Gray circles, CpGs methylated in ≥25% of molecules; black circles, CpGs methylated in ≥50% of molecules.(B) CpG methylation in repaired molecules in ES Dnmt1−/− cells is shown. DNA molecules were isolated from ES Dnmt1−/− cells carrying DR-GFP, 16 from control cells and 40 from cells exposed to I-SceI. The frequency of GFP+ cells was 5 ± 1.(C) CpG methylation in repaired molecules from Hela cells is shown. DNA molecules, derived from pooled Hela DR-GFP cultures transfected with the I-SceI expression vector or a control plasmid, were subjected to bisulfite analysis (Materials and Methods). The number of molecules in each class was as follows: (1) 25 molecules from cells transfected with control plasmid; (2) 20 recombinant molecules from low expressor cells sorted by FACS; (3) 15 recombinant molecules from high expressor cells sorted by FACS; (4) six molecules rearranged at the I-SceI site (NHEJ). The frequency (%) of each class was derived from several independent experiments with mass culture and fluorescence sorted cells. HR-H 2 + 0.5; HR-L 2 + 1; NHEJ 2 + 0.4. All CpGs (white circles) flanking the I–SceI site are shown. Gray circles, molecules methylated in ≥20% of molecules; black circles, molecules methylated in ≥40% of molecules.(D) CpG methylation in repaired molecules derived from individual DR-GFP clones is shown. DNA molecules were derived from clones 1, 2, and 3 of Figure 2. DNA was isolated and subjected to bisulfite analysis (Materials and Methods). Shown on the left are nonrecombinant molecules amplified with the 5′-unrec primer (see Figure 1). Shown on the right are recombinant DNA molecules isolated from cells transfected for 4 d with the I-SceI expression vector and selected for GFP expression. The arrows indicate hypermethylated DNA from clones expressing GFP at low levels and hypomethylated DNA from high GFP expressors.(E) Methylation of GFP cassette II is not influenced by recombination. DNA methylation pattern of cassette II in Hela DR-GFP (25 molecules) and ES DR-GFP (30 molecules) cells after transfection with I-SceI or before transfection (seven molecules) is shown. The methylation pattern of cassette II was identical in FACS sorted ES or Hela cells.The molecules analyzed both in ES and Hela cells derived from at least five independent bisulfite reactions and ten independent PCRs for each group: (1) PSVβGal transfected cells; (2) GFP- and (3) GFP+ high, and (4) low expressors from I-SceI transfected cells.
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pgen-0030110-g006: DNA Methylation in Repaired DNA Molecules(A) CpG methylation in repaired molecules from ES cells is shown. DNA molecules derived from pooled ES DR-GFP cultures transfected with the I-SceI expression vector or a control plasmid were subjected to bisulfite analysis (Materials and Methods). The number of molecules in each class was as follows: (1) uncut, 40 from cells transfected with control plasmid; (2) HR-H, 25 homologous recombinant molecules from high expressor cells sorted by FACS (23) or picked randomly from mass culture; (3) HR-L, 30 recombinant molecules from low expressor cells sorted by FACS (28) or picked randomly from mass cultures; (4) molecules rearranged at the I-SceI site (NHEJ). The frequency (%) of each class was derived from several independent experiments with mass culture and fluorescent-sorted cells. HR-H 3 ± 0.5; HL 3 ± 1; NHEJ 2 ± 0.3. All CpGs (white circles) flanking the I-SceI site are shown. Gray circles, CpGs methylated in ≥25% of molecules; black circles, CpGs methylated in ≥50% of molecules.(B) CpG methylation in repaired molecules in ES Dnmt1−/− cells is shown. DNA molecules were isolated from ES Dnmt1−/− cells carrying DR-GFP, 16 from control cells and 40 from cells exposed to I-SceI. The frequency of GFP+ cells was 5 ± 1.(C) CpG methylation in repaired molecules from Hela cells is shown. DNA molecules, derived from pooled Hela DR-GFP cultures transfected with the I-SceI expression vector or a control plasmid, were subjected to bisulfite analysis (Materials and Methods). The number of molecules in each class was as follows: (1) 25 molecules from cells transfected with control plasmid; (2) 20 recombinant molecules from low expressor cells sorted by FACS; (3) 15 recombinant molecules from high expressor cells sorted by FACS; (4) six molecules rearranged at the I-SceI site (NHEJ). The frequency (%) of each class was derived from several independent experiments with mass culture and fluorescence sorted cells. HR-H 2 + 0.5; HR-L 2 + 1; NHEJ 2 + 0.4. All CpGs (white circles) flanking the I–SceI site are shown. Gray circles, molecules methylated in ≥20% of molecules; black circles, molecules methylated in ≥40% of molecules.(D) CpG methylation in repaired molecules derived from individual DR-GFP clones is shown. DNA molecules were derived from clones 1, 2, and 3 of Figure 2. DNA was isolated and subjected to bisulfite analysis (Materials and Methods). Shown on the left are nonrecombinant molecules amplified with the 5′-unrec primer (see Figure 1). Shown on the right are recombinant DNA molecules isolated from cells transfected for 4 d with the I-SceI expression vector and selected for GFP expression. The arrows indicate hypermethylated DNA from clones expressing GFP at low levels and hypomethylated DNA from high GFP expressors.(E) Methylation of GFP cassette II is not influenced by recombination. DNA methylation pattern of cassette II in Hela DR-GFP (25 molecules) and ES DR-GFP (30 molecules) cells after transfection with I-SceI or before transfection (seven molecules) is shown. The methylation pattern of cassette II was identical in FACS sorted ES or Hela cells.The molecules analyzed both in ES and Hela cells derived from at least five independent bisulfite reactions and ten independent PCRs for each group: (1) PSVβGal transfected cells; (2) GFP- and (3) GFP+ high, and (4) low expressors from I-SceI transfected cells.

Mentions: Figure 6A shows the DNA methylation patterns of all classes found in the mass population of ES cells: (1) Before I-SceI cleavage (uncut); (2) recombinant GFP+ molecules (HR) isolated by cell sorting for HR-H or HR-L GFP expression; (3) molecules containing a rearranged I-SceI site generated by NHEJ. The methylation status of the HR molecules corresponded with the GFP expression levels of the sorted cells. Relative to the uncut parent, molecules from HR-L cells were heavily methylated, mostly in a segment of approximately 300 bp downstream to the DSB. Many of these modified CpGs represent de novo methylation sites. In contrast, molecules from HR-H cells were significantly undermethylated, both upstream and downstream to the DSB (Figures 6A and S4A). The ratio of the two classes was 1:1. Note that HR repair in this system is a short-tract strand-conversion event, since cassette II is deleted at both upstream and downstream ends. We suggest that the length of the segment showing an altered methylation pattern in the recombinants is limited by the extent of homology between cassettes I and II (400 bp downstream to the I-SceI/BcgI 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)

DNA Methylation in Repaired DNA Molecules(A) CpG methylation in repaired molecules from ES cells is shown. DNA molecules derived from pooled ES DR-GFP cultures transfected with the I-SceI expression vector or a control plasmid were subjected to bisulfite analysis (Materials and Methods). The number of molecules in each class was as follows: (1) uncut, 40 from cells transfected with control plasmid; (2) HR-H, 25 homologous recombinant molecules from high expressor cells sorted by FACS (23) or picked randomly from mass culture; (3) HR-L, 30 recombinant molecules from low expressor cells sorted by FACS (28) or picked randomly from mass cultures; (4) molecules rearranged at the I-SceI site (NHEJ). The frequency (%) of each class was derived from several independent experiments with mass culture and fluorescent-sorted cells. HR-H 3 ± 0.5; HL 3 ± 1; NHEJ 2 ± 0.3. All CpGs (white circles) flanking the I-SceI site are shown. Gray circles, CpGs methylated in ≥25% of molecules; black circles, CpGs methylated in ≥50% of molecules.(B) CpG methylation in repaired molecules in ES Dnmt1−/− cells is shown. DNA molecules were isolated from ES Dnmt1−/− cells carrying DR-GFP, 16 from control cells and 40 from cells exposed to I-SceI. The frequency of GFP+ cells was 5 ± 1.(C) CpG methylation in repaired molecules from Hela cells is shown. DNA molecules, derived from pooled Hela DR-GFP cultures transfected with the I-SceI expression vector or a control plasmid, were subjected to bisulfite analysis (Materials and Methods). The number of molecules in each class was as follows: (1) 25 molecules from cells transfected with control plasmid; (2) 20 recombinant molecules from low expressor cells sorted by FACS; (3) 15 recombinant molecules from high expressor cells sorted by FACS; (4) six molecules rearranged at the I-SceI site (NHEJ). The frequency (%) of each class was derived from several independent experiments with mass culture and fluorescence sorted cells. HR-H 2 + 0.5; HR-L 2 + 1; NHEJ 2 + 0.4. All CpGs (white circles) flanking the I–SceI site are shown. Gray circles, molecules methylated in ≥20% of molecules; black circles, molecules methylated in ≥40% of molecules.(D) CpG methylation in repaired molecules derived from individual DR-GFP clones is shown. DNA molecules were derived from clones 1, 2, and 3 of Figure 2. DNA was isolated and subjected to bisulfite analysis (Materials and Methods). Shown on the left are nonrecombinant molecules amplified with the 5′-unrec primer (see Figure 1). Shown on the right are recombinant DNA molecules isolated from cells transfected for 4 d with the I-SceI expression vector and selected for GFP expression. The arrows indicate hypermethylated DNA from clones expressing GFP at low levels and hypomethylated DNA from high GFP expressors.(E) Methylation of GFP cassette II is not influenced by recombination. DNA methylation pattern of cassette II in Hela DR-GFP (25 molecules) and ES DR-GFP (30 molecules) cells after transfection with I-SceI or before transfection (seven molecules) is shown. The methylation pattern of cassette II was identical in FACS sorted ES or Hela cells.The molecules analyzed both in ES and Hela cells derived from at least five independent bisulfite reactions and ten independent PCRs for each group: (1) PSVβGal transfected cells; (2) GFP- and (3) GFP+ high, and (4) low expressors from I-SceI transfected cells.
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Show All Figures
getmorefigures.php?uid=PMC1913100&req=5

pgen-0030110-g006: DNA Methylation in Repaired DNA Molecules(A) CpG methylation in repaired molecules from ES cells is shown. DNA molecules derived from pooled ES DR-GFP cultures transfected with the I-SceI expression vector or a control plasmid were subjected to bisulfite analysis (Materials and Methods). The number of molecules in each class was as follows: (1) uncut, 40 from cells transfected with control plasmid; (2) HR-H, 25 homologous recombinant molecules from high expressor cells sorted by FACS (23) or picked randomly from mass culture; (3) HR-L, 30 recombinant molecules from low expressor cells sorted by FACS (28) or picked randomly from mass cultures; (4) molecules rearranged at the I-SceI site (NHEJ). The frequency (%) of each class was derived from several independent experiments with mass culture and fluorescent-sorted cells. HR-H 3 ± 0.5; HL 3 ± 1; NHEJ 2 ± 0.3. All CpGs (white circles) flanking the I-SceI site are shown. Gray circles, CpGs methylated in ≥25% of molecules; black circles, CpGs methylated in ≥50% of molecules.(B) CpG methylation in repaired molecules in ES Dnmt1−/− cells is shown. DNA molecules were isolated from ES Dnmt1−/− cells carrying DR-GFP, 16 from control cells and 40 from cells exposed to I-SceI. The frequency of GFP+ cells was 5 ± 1.(C) CpG methylation in repaired molecules from Hela cells is shown. DNA molecules, derived from pooled Hela DR-GFP cultures transfected with the I-SceI expression vector or a control plasmid, were subjected to bisulfite analysis (Materials and Methods). The number of molecules in each class was as follows: (1) 25 molecules from cells transfected with control plasmid; (2) 20 recombinant molecules from low expressor cells sorted by FACS; (3) 15 recombinant molecules from high expressor cells sorted by FACS; (4) six molecules rearranged at the I-SceI site (NHEJ). The frequency (%) of each class was derived from several independent experiments with mass culture and fluorescence sorted cells. HR-H 2 + 0.5; HR-L 2 + 1; NHEJ 2 + 0.4. All CpGs (white circles) flanking the I–SceI site are shown. Gray circles, molecules methylated in ≥20% of molecules; black circles, molecules methylated in ≥40% of molecules.(D) CpG methylation in repaired molecules derived from individual DR-GFP clones is shown. DNA molecules were derived from clones 1, 2, and 3 of Figure 2. DNA was isolated and subjected to bisulfite analysis (Materials and Methods). Shown on the left are nonrecombinant molecules amplified with the 5′-unrec primer (see Figure 1). Shown on the right are recombinant DNA molecules isolated from cells transfected for 4 d with the I-SceI expression vector and selected for GFP expression. The arrows indicate hypermethylated DNA from clones expressing GFP at low levels and hypomethylated DNA from high GFP expressors.(E) Methylation of GFP cassette II is not influenced by recombination. DNA methylation pattern of cassette II in Hela DR-GFP (25 molecules) and ES DR-GFP (30 molecules) cells after transfection with I-SceI or before transfection (seven molecules) is shown. The methylation pattern of cassette II was identical in FACS sorted ES or Hela cells.The molecules analyzed both in ES and Hela cells derived from at least five independent bisulfite reactions and ten independent PCRs for each group: (1) PSVβGal transfected cells; (2) GFP- and (3) GFP+ high, and (4) low expressors from I-SceI transfected cells.
Mentions: Figure 6A shows the DNA methylation patterns of all classes found in the mass population of ES cells: (1) Before I-SceI cleavage (uncut); (2) recombinant GFP+ molecules (HR) isolated by cell sorting for HR-H or HR-L GFP expression; (3) molecules containing a rearranged I-SceI site generated by NHEJ. The methylation status of the HR molecules corresponded with the GFP expression levels of the sorted cells. Relative to the uncut parent, molecules from HR-L cells were heavily methylated, mostly in a segment of approximately 300 bp downstream to the DSB. Many of these modified CpGs represent de novo methylation sites. In contrast, molecules from HR-H cells were significantly undermethylated, both upstream and downstream to the DSB (Figures 6A and S4A). The ratio of the two classes was 1:1. Note that HR repair in this system is a short-tract strand-conversion event, since cassette II is deleted at both upstream and downstream ends. We suggest that the length of the segment showing an altered methylation pattern in the recombinants is limited by the extent of homology between cassettes I and II (400 bp downstream to the I-SceI/BcgI 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