Transcription influences repair-induced DNA methylation

Morano, Annalisa (2008) Transcription influences repair-induced DNA methylation. [Tesi di dottorato] (Inedito)

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Abstract

This work is aimed at the dissection of the molecular mechanism(s) linking DNA damage and gene silencing. To this end, we have developed a genetic system that allows a rapid assessment of homologous-directed repair (HR) of an unique DNA double strand break (DSB). Briefly, we induced a DBS in the genome of HeLa or mouse embryonic stem (ES) cells using the I-SceI restriction endonuclease. Homologous recombination repair by gene conversion, initiated at the site of the double strand break, converts 2 inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP) in an intact functional gene. The efficiency of HR, under our conditions, is approximately 2%–4% and can be easily quantified by analyzing GFP+ cells. Half of these recombinants expressed GFP poorly, because GFP gene was silenced. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since HeLa DR-GFP treatment with 5-aza-2’-deoxycytidine, a DNA demethylating drug, significantly increased the fraction of GFP expressing cells. Methylation did not alter recombination frequency in both cell types. ES cells deficient in DNA methyl-transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Bisulfite analysis of GFP DNA molecules revealed that approximately half of the HR repaired molecules were de novo methylated, principally at the 3’-end of the DSB in a range of ~300bp. The other half GFP molecules were hypomethylated. Uncleaved and non-homologous repaired molecules did not show changes of the methylation profile. DNA methyl-transferase 1 bound specifically to HR GFP DNA, as revealed by chromatin immunoprecipitation and RNA analysis. HR induced novel methylation profiles on top of the old patterns and contributed to the silencing of GFP expression. Inhibition of transcription by 􀀁-amanitin for a very short period (6-24 h during ISceI cleavage) significantly reduced the frequency of recombination. Surprisingly, the 2 classes of recombinants were better separated in terms of GFP expression. Methylation analysis showed that the methylated molecules were hypermethylated, whereas the hypomethylated GFP gene molecules were un-methylated, relative to the untreated samples. Taken together, our data support a mechanistic link between HR, DNA methylation and transcription. We propose that stalled RNA polymerase molecules slow down homologous recombination by interfering possibly with DNA polymerase complex or strand invasion. At the same time, RNA polymerase II transcription complex signals to DNMT1 the coding strand and facilitates strand selective DNA methylation. Overall, these data highlight a new and unexpected opportunity to understand the mechanisms of silencing of damaged and repaired genes.

Tipologia di documento:Tesi di dottorato
Parole chiave:DNA damage,gene silencing, homologous-directed repair
Settori scientifico-disciplinari MIUR:Area 06 Scienze mediche > MED/03 GENETICA MEDICA
Area 06 Scienze mediche > MED/04 PATOLOGIA GENERALE
Coordinatori della Scuola di dottorato:
Coordinatore del Corso di dottoratoe-mail (se nota)
Avvedimento, Vittorio Enricoavvedim@unina.it
Tutor della Scuola di dottorato:
Tutor del Corso di dottoratoe-mail (se nota)
Avvedimento, Vittorio Enricoavvedim@unina.it
Gottesman, Maxwellmeg8@columbia.edu
Stato del full text:Accessibile
Data:01 Dicembre 2008
Numero di pagine:80
Istituzione:Università degli studi di Napoli "Federico II"
Dipartimento o Struttura:Biologia e Patologia Cellulare e Molecolare "L. Califano"
Tipo di tesi:Dottorato
Stato dell'Eprint:Inedito
Scuola di dottorato:Medicina Molecolare
Denominazione del dottorato:Patologia e Fisiopatologia Molecolare
Ciclo di dottorato:XXI
Numero di sistema:3231
Depositato il:11 Novembre 2009 15:22
Ultima modifica:11 Novembre 2009 15:22

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