DNA damage, homology-directed repair, and DNA methylation.
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.
Cuozzo
Concetta
C
Dipartimento di Biologia e Patologia Molecolare e Cellulare, Istituto di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Università Federico II, Naples, Italy.
Porcellini
Antonio
A
Angrisano
Tiziana
T
Morano
Annalisa
A
Lee
Bongyong
B
Di Pardo
Alba
A
Messina
Samantha
S
Iuliano
Rodolfo
R
Fusco
Alfredo
A
Santillo
Maria R
MR
Muller
Mark T
MT
Chiariotti
Lorenzo
L
Gottesman
Max E
ME
Avvedimento
Enrico V
EV
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D018899:CpG Islands; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D019175:DNA Methylation; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair; D015870:Gene Expression; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D019656:Loss of Heterozygosity; D051379:Mice; D008957:Models, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D011995:Recombination, Genetic; D013964:Thyroid Neoplasms / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D002843:Chromatin; D017931:DNA Primers; D012333:RNA, Messenger; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases