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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 and Recombination(A) In the recombination assay primers for PCR and RT-PCR amplification were: (1) 5′-unrec (unrecombinant) centered on the I-SceI site, present only in cassette I and (2) 5′-rec (recombinant) centered in the BcgI site present only in the converted GFP or in cassette II. The 3′ end primer (3′) is located in cassette I in a sequence deleted in cassette II (X). The 5′-rec primer can pair with the 3′ primer only after its reconstitution in the 5′ cassette by gene conversion. The sequence of the 5′ primers is indicated with the distinguishing bases in capital letters.Stable Hela cells containing the DR-GFP plasmid were transiently transfected with I-SceI and pSVβGal (Promega) expression vectors as described in Materials and Methods. After 3 d, GFP cells were scored by FACS. The histogram shows data from three experiments. Transfection efficiency in these three experiments was 70% ± 5%.(B) Inhibition of methylation reveals silenced recombinants. Hela cells carrying the DR-GFP plasmid were transfected with I-SceI and PSVßGal expression vectors and treated with 5 μM 5-AzadC 48 h posttransfection, as described in Materials and Methods. (a) GFP+ cells were analyzed by FACS. The ordinate shows the fraction of GFP+ cells over total cells transfected. The efficiency of transfection was 70% +/− 5%. To determine the effect of 5-AzadC on GFP fluorescence, we compared the percentage of GFP+ cells before and after 5-AzadC treatment in six experiments (three in duplicate) with a nonparametric matched pairs test, such as the Wilcoxon sign-rank (p < 0.016). (b) For the DNA analysis GFP clones were analyzed by PCR with the primers indicated in Figure 1A. The results shown were derived from DNA of I-SceI treated cells (see Materials and Methods). (c) The cells were treated with 5-AzadC 48 h after transfection as described in Materials and Methods and analyzed by FACS. Treatment with 5-AzadC after I-SceI transfection significantly increased the number and the intensity of fluorescence of GFP+ cells, while 5-AzadC alone was ineffective. The area indicated as “GFP+” includes 95% of GFP-expressing cells after I-SceI transfection.
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pgen-0030110-g001: DNA Methylation and Recombination(A) In the recombination assay primers for PCR and RT-PCR amplification were: (1) 5′-unrec (unrecombinant) centered on the I-SceI site, present only in cassette I and (2) 5′-rec (recombinant) centered in the BcgI site present only in the converted GFP or in cassette II. The 3′ end primer (3′) is located in cassette I in a sequence deleted in cassette II (X). The 5′-rec primer can pair with the 3′ primer only after its reconstitution in the 5′ cassette by gene conversion. The sequence of the 5′ primers is indicated with the distinguishing bases in capital letters.Stable Hela cells containing the DR-GFP plasmid were transiently transfected with I-SceI and pSVβGal (Promega) expression vectors as described in Materials and Methods. After 3 d, GFP cells were scored by FACS. The histogram shows data from three experiments. Transfection efficiency in these three experiments was 70% ± 5%.(B) Inhibition of methylation reveals silenced recombinants. Hela cells carrying the DR-GFP plasmid were transfected with I-SceI and PSVßGal expression vectors and treated with 5 μM 5-AzadC 48 h posttransfection, as described in Materials and Methods. (a) GFP+ cells were analyzed by FACS. The ordinate shows the fraction of GFP+ cells over total cells transfected. The efficiency of transfection was 70% +/− 5%. To determine the effect of 5-AzadC on GFP fluorescence, we compared the percentage of GFP+ cells before and after 5-AzadC treatment in six experiments (three in duplicate) with a nonparametric matched pairs test, such as the Wilcoxon sign-rank (p < 0.016). (b) For the DNA analysis GFP clones were analyzed by PCR with the primers indicated in Figure 1A. The results shown were derived from DNA of I-SceI treated cells (see Materials and Methods). (c) The cells were treated with 5-AzadC 48 h after transfection as described in Materials and Methods and analyzed by FACS. Treatment with 5-AzadC after I-SceI transfection significantly increased the number and the intensity of fluorescence of GFP+ cells, while 5-AzadC alone was ineffective. The area indicated as “GFP+” includes 95% of GFP-expressing cells after I-SceI transfection.

Mentions: Our recombination assay relies on the two inactivated tandem repeated (DR)-GFP plasmid developed by M. Jasin [16], which contains two mutated GFP genes oriented as direct repeats and separated by a drug selection marker, the puromycin N-acetyltransferase gene (Figure 1A). An upstream cytomegalovirus (CMV) enhancer fused to the chicken β-actin promoter provided a strong and insulated transcriptional unit (see Materials and Methods for the structure of DR-GFP). The upstream (5′) GFP gene (cassette I) carries a recognition site for I-SceI, a rare-cutting endonuclease that does not cleave several eukaryotic genomes tested [17]. The I-SceI recognition sequence was incorporated into a naturally occurring BcgI restriction site by substituting 11 bp of the wild-type gene. These substituted base pairs supply two in-frame stop codons that terminate translation, thereby inactivating cassette I. The downstream (3′) GFP (cassette II) is inactivated by upstream and downstream truncations, leaving only ~502 bp of GFP (see Materials and Methods for the structure of DR-GFP). Hela cells were stably transfected with DR-GFP and selected in the presence of puromycin. Puromycin-resistant pools of cells carrying DR-GFP at various loci were then transiently transfected with a vector expressing I-SceI. The resultant DSB induced homologous recombination. Fluorescence-activated cell sorter (FACS) analysis was used to reveal the percentage of cells expressing GFP (Figure 1A, upper panel). We maintained the cells after transfection in the presence of puromycin to eliminate recombinant clones generated by homology-mediated deletion [16].


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 and Recombination(A) In the recombination assay primers for PCR and RT-PCR amplification were: (1) 5′-unrec (unrecombinant) centered on the I-SceI site, present only in cassette I and (2) 5′-rec (recombinant) centered in the BcgI site present only in the converted GFP or in cassette II. The 3′ end primer (3′) is located in cassette I in a sequence deleted in cassette II (X). The 5′-rec primer can pair with the 3′ primer only after its reconstitution in the 5′ cassette by gene conversion. The sequence of the 5′ primers is indicated with the distinguishing bases in capital letters.Stable Hela cells containing the DR-GFP plasmid were transiently transfected with I-SceI and pSVβGal (Promega) expression vectors as described in Materials and Methods. After 3 d, GFP cells were scored by FACS. The histogram shows data from three experiments. Transfection efficiency in these three experiments was 70% ± 5%.(B) Inhibition of methylation reveals silenced recombinants. Hela cells carrying the DR-GFP plasmid were transfected with I-SceI and PSVßGal expression vectors and treated with 5 μM 5-AzadC 48 h posttransfection, as described in Materials and Methods. (a) GFP+ cells were analyzed by FACS. The ordinate shows the fraction of GFP+ cells over total cells transfected. The efficiency of transfection was 70% +/− 5%. To determine the effect of 5-AzadC on GFP fluorescence, we compared the percentage of GFP+ cells before and after 5-AzadC treatment in six experiments (three in duplicate) with a nonparametric matched pairs test, such as the Wilcoxon sign-rank (p < 0.016). (b) For the DNA analysis GFP clones were analyzed by PCR with the primers indicated in Figure 1A. The results shown were derived from DNA of I-SceI treated cells (see Materials and Methods). (c) The cells were treated with 5-AzadC 48 h after transfection as described in Materials and Methods and analyzed by FACS. Treatment with 5-AzadC after I-SceI transfection significantly increased the number and the intensity of fluorescence of GFP+ cells, while 5-AzadC alone was ineffective. The area indicated as “GFP+” includes 95% of GFP-expressing cells after I-SceI transfection.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-0030110-g001: DNA Methylation and Recombination(A) In the recombination assay primers for PCR and RT-PCR amplification were: (1) 5′-unrec (unrecombinant) centered on the I-SceI site, present only in cassette I and (2) 5′-rec (recombinant) centered in the BcgI site present only in the converted GFP or in cassette II. The 3′ end primer (3′) is located in cassette I in a sequence deleted in cassette II (X). The 5′-rec primer can pair with the 3′ primer only after its reconstitution in the 5′ cassette by gene conversion. The sequence of the 5′ primers is indicated with the distinguishing bases in capital letters.Stable Hela cells containing the DR-GFP plasmid were transiently transfected with I-SceI and pSVβGal (Promega) expression vectors as described in Materials and Methods. After 3 d, GFP cells were scored by FACS. The histogram shows data from three experiments. Transfection efficiency in these three experiments was 70% ± 5%.(B) Inhibition of methylation reveals silenced recombinants. Hela cells carrying the DR-GFP plasmid were transfected with I-SceI and PSVßGal expression vectors and treated with 5 μM 5-AzadC 48 h posttransfection, as described in Materials and Methods. (a) GFP+ cells were analyzed by FACS. The ordinate shows the fraction of GFP+ cells over total cells transfected. The efficiency of transfection was 70% +/− 5%. To determine the effect of 5-AzadC on GFP fluorescence, we compared the percentage of GFP+ cells before and after 5-AzadC treatment in six experiments (three in duplicate) with a nonparametric matched pairs test, such as the Wilcoxon sign-rank (p < 0.016). (b) For the DNA analysis GFP clones were analyzed by PCR with the primers indicated in Figure 1A. The results shown were derived from DNA of I-SceI treated cells (see Materials and Methods). (c) The cells were treated with 5-AzadC 48 h after transfection as described in Materials and Methods and analyzed by FACS. Treatment with 5-AzadC after I-SceI transfection significantly increased the number and the intensity of fluorescence of GFP+ cells, while 5-AzadC alone was ineffective. The area indicated as “GFP+” includes 95% of GFP-expressing cells after I-SceI transfection.
Mentions: Our recombination assay relies on the two inactivated tandem repeated (DR)-GFP plasmid developed by M. Jasin [16], which contains two mutated GFP genes oriented as direct repeats and separated by a drug selection marker, the puromycin N-acetyltransferase gene (Figure 1A). An upstream cytomegalovirus (CMV) enhancer fused to the chicken β-actin promoter provided a strong and insulated transcriptional unit (see Materials and Methods for the structure of DR-GFP). The upstream (5′) GFP gene (cassette I) carries a recognition site for I-SceI, a rare-cutting endonuclease that does not cleave several eukaryotic genomes tested [17]. The I-SceI recognition sequence was incorporated into a naturally occurring BcgI restriction site by substituting 11 bp of the wild-type gene. These substituted base pairs supply two in-frame stop codons that terminate translation, thereby inactivating cassette I. The downstream (3′) GFP (cassette II) is inactivated by upstream and downstream truncations, leaving only ~502 bp of GFP (see Materials and Methods for the structure of DR-GFP). Hela cells were stably transfected with DR-GFP and selected in the presence of puromycin. Puromycin-resistant pools of cells carrying DR-GFP at various loci were then transiently transfected with a vector expressing I-SceI. The resultant DSB induced homologous recombination. Fluorescence-activated cell sorter (FACS) analysis was used to reveal the percentage of cells expressing GFP (Figure 1A, upper panel). We maintained the cells after transfection in the presence of puromycin to eliminate recombinant clones generated by homology-mediated deletion [16].

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