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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

Mapping of GFP Transcription in Recombinant and Nonrecombinant Cells with and without 5-AzadC Treatment(A) A schematic of the DR-GFP transcriptional unit shows the location of the CMV promoter, intron, and GFP coding sequence. Primers used for quantitative RT-PCR and RT-PCR are indicated by arrows. Different sets of primers were derived from the intron (738–757, forward 5′-CGTTACTCCCACAGGTGAGC-3′; 966–948, reverse 5′-CGCCCGTAGCGCTCACAGC-3′), AUG (1,666–1,685, forward 5′-TACAGCTCCTGGGCAACGTG-3′; 1,911–1,892, reverse, 5′-TCCTGCTCCTGGGCTTCTCG-3′), and BcgI/I-SceI (described in Figure 1A) segments of GFP gene.Control (DR-GFP cells transfected with pBluescript), HR-L, and HR-H cells were treated with 40 μM 5-AzadC for 48 h. Total RNA was extracted 24 h later and subjected to quantitative RT-PCR with the primers indicated. The data, derived from three independent cDNAs, are shown as fold induction by 5-AzadC over the basal control, normalized to GADPH and β-actin. The primers used to amplify the control samples were those indicated as I-SceI unrec (Figure 1A).(B) Shown is RT-PCR with the same cDNAs indicated in (A) at 30 cycles.
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pgen-0030110-g010: Mapping of GFP Transcription in Recombinant and Nonrecombinant Cells with and without 5-AzadC Treatment(A) A schematic of the DR-GFP transcriptional unit shows the location of the CMV promoter, intron, and GFP coding sequence. Primers used for quantitative RT-PCR and RT-PCR are indicated by arrows. Different sets of primers were derived from the intron (738–757, forward 5′-CGTTACTCCCACAGGTGAGC-3′; 966–948, reverse 5′-CGCCCGTAGCGCTCACAGC-3′), AUG (1,666–1,685, forward 5′-TACAGCTCCTGGGCAACGTG-3′; 1,911–1,892, reverse, 5′-TCCTGCTCCTGGGCTTCTCG-3′), and BcgI/I-SceI (described in Figure 1A) segments of GFP gene.Control (DR-GFP cells transfected with pBluescript), HR-L, and HR-H cells were treated with 40 μM 5-AzadC for 48 h. Total RNA was extracted 24 h later and subjected to quantitative RT-PCR with the primers indicated. The data, derived from three independent cDNAs, are shown as fold induction by 5-AzadC over the basal control, normalized to GADPH and β-actin. The primers used to amplify the control samples were those indicated as I-SceI unrec (Figure 1A).(B) Shown is RT-PCR with the same cDNAs indicated in (A) at 30 cycles.

Mentions: Our data indicate that methylation of a short segment of DNA flanking the DSB (Figure 7) is sufficient to silence GFP expression in a significant fraction of cells (HR-L) (Figures 2C, 6, and 8). Since the CMV promoter and chicken β-actin enhancer that drive GFP expression are located ~1,000 bp from the BcgI/I-SceI sites and are insulated from surrounding genomic regions (see Materials and Methods), the link between methylation and silencing is not readily evident. To explore this question, we asked if methylation inhibited transcription initiation and/or elongation. We performed RT-PCR analysis of RNA with primers derived from the upstream intron (close to the transcription initiation site), from the beginning of the GFP gene, and from the I-SceI (control cells) or BcgI (HR-L and HR-H cells) sites (Figure 10A). Since PCR reactions performed with different primers cannot be directly compared, we measured amplification of the PCR signal in a particular region of the gene by 5-AzadC. This value indicates how methylation affects transcription near the promoter and at downstream regions.


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)

Mapping of GFP Transcription in Recombinant and Nonrecombinant Cells with and without 5-AzadC Treatment(A) A schematic of the DR-GFP transcriptional unit shows the location of the CMV promoter, intron, and GFP coding sequence. Primers used for quantitative RT-PCR and RT-PCR are indicated by arrows. Different sets of primers were derived from the intron (738–757, forward 5′-CGTTACTCCCACAGGTGAGC-3′; 966–948, reverse 5′-CGCCCGTAGCGCTCACAGC-3′), AUG (1,666–1,685, forward 5′-TACAGCTCCTGGGCAACGTG-3′; 1,911–1,892, reverse, 5′-TCCTGCTCCTGGGCTTCTCG-3′), and BcgI/I-SceI (described in Figure 1A) segments of GFP gene.Control (DR-GFP cells transfected with pBluescript), HR-L, and HR-H cells were treated with 40 μM 5-AzadC for 48 h. Total RNA was extracted 24 h later and subjected to quantitative RT-PCR with the primers indicated. The data, derived from three independent cDNAs, are shown as fold induction by 5-AzadC over the basal control, normalized to GADPH and β-actin. The primers used to amplify the control samples were those indicated as I-SceI unrec (Figure 1A).(B) Shown is RT-PCR with the same cDNAs indicated in (A) at 30 cycles.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-0030110-g010: Mapping of GFP Transcription in Recombinant and Nonrecombinant Cells with and without 5-AzadC Treatment(A) A schematic of the DR-GFP transcriptional unit shows the location of the CMV promoter, intron, and GFP coding sequence. Primers used for quantitative RT-PCR and RT-PCR are indicated by arrows. Different sets of primers were derived from the intron (738–757, forward 5′-CGTTACTCCCACAGGTGAGC-3′; 966–948, reverse 5′-CGCCCGTAGCGCTCACAGC-3′), AUG (1,666–1,685, forward 5′-TACAGCTCCTGGGCAACGTG-3′; 1,911–1,892, reverse, 5′-TCCTGCTCCTGGGCTTCTCG-3′), and BcgI/I-SceI (described in Figure 1A) segments of GFP gene.Control (DR-GFP cells transfected with pBluescript), HR-L, and HR-H cells were treated with 40 μM 5-AzadC for 48 h. Total RNA was extracted 24 h later and subjected to quantitative RT-PCR with the primers indicated. The data, derived from three independent cDNAs, are shown as fold induction by 5-AzadC over the basal control, normalized to GADPH and β-actin. The primers used to amplify the control samples were those indicated as I-SceI unrec (Figure 1A).(B) Shown is RT-PCR with the same cDNAs indicated in (A) at 30 cycles.
Mentions: Our data indicate that methylation of a short segment of DNA flanking the DSB (Figure 7) is sufficient to silence GFP expression in a significant fraction of cells (HR-L) (Figures 2C, 6, and 8). Since the CMV promoter and chicken β-actin enhancer that drive GFP expression are located ~1,000 bp from the BcgI/I-SceI sites and are insulated from surrounding genomic regions (see Materials and Methods), the link between methylation and silencing is not readily evident. To explore this question, we asked if methylation inhibited transcription initiation and/or elongation. We performed RT-PCR analysis of RNA with primers derived from the upstream intron (close to the transcription initiation site), from the beginning of the GFP gene, and from the I-SceI (control cells) or BcgI (HR-L and HR-H cells) sites (Figure 10A). Since PCR reactions performed with different primers cannot be directly compared, we measured amplification of the PCR signal in a particular region of the gene by 5-AzadC. This value indicates how methylation affects transcription near the promoter and at downstream regions.

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