La metilazione del DNA in posizione 5 della citosina (5mC) è una modificazione epigenetica con un ruolo importante nella regolazione della trascrizione, nel differenziamento cellulare, nel mantenimento della stabilità della cromatina e nella funzionalità e la sopravvivenza dei tessuti adulti. Nei mammiferi, la DNA metiltrasferasi 1 (DNMT1) assicura il mantenimento dei profili di metilazione del DNA, instaurati dalle DNMT3A e DNMT3B, durante la replicazione e la riparazione. Di recente sono state identificate mutazioni nel gene DNMT1 umano responsabili di due distinti disordini neurodegenerativi: la neuropatia sensitiva ereditaria con demenza e perdita dell'udito (HSAN1E) e la atassia cerebellare dominante autonomica con sordità e narcolessia (ADCA-DN). Tali patologie appartengono allo stesso spettro clinico e sono caratterizzate da un coinvolgimento del sistema nervoso centrale e periferico. Tutte le mutazioni associate ad ADCA-DN e HSAN1E sono localizzate a livello del motivo RFTS (Replication Foci Targeting Sequence), che indirizza la DNMT1 sui siti emi-metilati durante la fase S e ne regola l'associazione stabile all'eterocromatina durante la tarda fase S e la fase G2. La DNMT1 mutata potrebbe essere implicata nella patogenesi delle due sindromi attraverso un meccanismo di loss o di gain-of-funtion che potrebbe condurre ad una deregolazione dell'espressione genica dovuta all'alterazione dei profili di metilazione del DNA. Per studiare le alterazioni epigenetiche e gli effetti patogenetici correlati alle mutazioni della DNMT1 abbiamo generato un modello in vitro di ADCA-DN a partire da cellule staminali embrionali murine (mESC). Inizialmente abbiamo ottenuto, tramite integrazione casuale, cloni che esprimono la biotina ligasi di E. coli BirA: questo enzima lega covalentemente la biotina a specifici peptidi accettori (ad es. AviTag). Successivamente, attraverso la tecnologia CRISPR/Cas9, che facilita la ricombinazione omologa, abbiamo generato un clone che esprime il peptide AviTag fuso in frame all'estremità amino-terminale della Dnmt1 endogena. Il clone AviTag-Dnmt1 verrà utilizzato per generare un clone con una delle mutazioni missenso (DNMT1A570V) riscontrate nella ADCA-DN e potrà essere altresì utilizzato per la generazione e lo studio delle altre mutazioni puntiformi riscontrate in entrambe le patologie. Sfruttando l'elevata affinità della streptavidina per la biotina potremo effettuare delle immunoprecipitazioni ad alta stringenza delle proteine wild type e mutante AviTag-Dnmt1 e analizzare il legame della proteina al DNA in esperimenti di immunoprecipitazione della cromatina seguiti da sequenziamento massivo (ChIP-Seq). Il clone Avitag-Dnmt1 che abbiamo generato sarà fondamentale per studiare l'espressione e l'attività della Dnmt1 wild type e mutante, la metilazione del DNA e i profili di espressione genica sia in cellule staminali sia in cellule differenziate nella linea neurale. I cloni mutanti potranno inoltre essere utilizzati per la generazione di topi knock-in, consentendo così lo sviluppo di un modello murino che permetta di analizzare in vivo i meccanismi patogenetici di ADCA-DN e HSAN1E. In conclusione, la generazione di un modello mESC in vitro consentirà di comprendere meglio i meccanismi patogenetici delle malattie legate alla DNMT1, ponendo così le basi per un futuro sviluppo di eventuali approcci terapeutici.
DNA methylation on position 5 of cytosine (5mC) is an epigenetic modification that plays a major role in transcriptional regulation, cell differentiation, chromatin stability, and adult tissues' function and survival. In mammals, DNA methyltransferase1 (DNMT1) ensures the maintenance of DNA methylation patterns established by DNMT3a and DNMT3b during replication and repair. Mutations in the human DNMT1 gene have been recently identified as causative for two monogenic neurodegenerative disorders, with adult-onset and age-dependent progression: hereditary sensory autonomic neuropathy with dementia and hearing loss (HSAN1E) and autosomal dominant cerebellar ataxia-deafness and narcolepsy (ADCA-DN). Both phenotypes belong to the same clinical spectrum with involvement of central and peripheral nervous system. Interestingly, all mutations associated with ADCA-DN and HSAN1E lie within the replication foci targeting sequence (RFTS) motif, which directs DNMT1 to hemimethylated sites during S phase and regulates its association to heterochromatin during the late S and G2 phases. Mutated DNMT1 could exert its pathogenic effect through a loss of function or through a toxic gain of function mechanism which may lead to deregulation of gene expression profiles due to aberrant DNA methylation patterns. To gain insight into epigenetic alterations and pathogenic effects of DNMT1 mutations, we generated an in vitro model of the ADCA-DN and HSAN1E diseases. Taking advantage of the recently developed CRISPR/Cas9 technology which greatly facilitates homologous recombination, we generated a mouse embryonic stem cell (mESC) clone expressing a short biotin acceptor domain (AviTag) fused in frame to the N-terminus of the endogenous Dnmt1 protein. The AviTag domain gets biotinylated by the E. coli biotin ligase BirA that we previously inserted into the mESC genome by random integration. The AviTag-Dnmt1 clone will be used to generate one of the heterozygous missense mutations of the ADCA-DN disease (DNMT1A570V) and could be used to generate and study the other point mutations found in the abovementioned disorders. The biotinylated AviTag permits performing high stringent streptavidin-based immunoprecipitation of the fused protein. Thus, it allows to precisely investigate the binding of the mutated Dnmt1 protein with respect to the wild type protein in Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) experiments. The AviTag-Dnmt1 clone we generated will be instrumental in studying Dnmt1 expression and activity as well as DNA methylation and gene expression profiles in wild type and mutant stem cells and in cells differentiated towards the neural lineage. Moreover, mutant clones could be used to generate knock-in mice, thus obtaining an in vivo model that would better recapitulate the pathogenic mechanisms of the ADCA-DN disease. Summarizing, the generation of an in vitro mESC model of DNMT1-related diseases will provide comprehensive insights on the pathogenic mechanisms of DNMT1-related diseases in order to lay the basis for future development of therapeutic approaches.
Generating an in vitro model for the study of DNMT1-related diseases
LUPOI, ROSA ILARIA
2016/2017
Abstract
DNA methylation on position 5 of cytosine (5mC) is an epigenetic modification that plays a major role in transcriptional regulation, cell differentiation, chromatin stability, and adult tissues' function and survival. In mammals, DNA methyltransferase1 (DNMT1) ensures the maintenance of DNA methylation patterns established by DNMT3a and DNMT3b during replication and repair. Mutations in the human DNMT1 gene have been recently identified as causative for two monogenic neurodegenerative disorders, with adult-onset and age-dependent progression: hereditary sensory autonomic neuropathy with dementia and hearing loss (HSAN1E) and autosomal dominant cerebellar ataxia-deafness and narcolepsy (ADCA-DN). Both phenotypes belong to the same clinical spectrum with involvement of central and peripheral nervous system. Interestingly, all mutations associated with ADCA-DN and HSAN1E lie within the replication foci targeting sequence (RFTS) motif, which directs DNMT1 to hemimethylated sites during S phase and regulates its association to heterochromatin during the late S and G2 phases. Mutated DNMT1 could exert its pathogenic effect through a loss of function or through a toxic gain of function mechanism which may lead to deregulation of gene expression profiles due to aberrant DNA methylation patterns. To gain insight into epigenetic alterations and pathogenic effects of DNMT1 mutations, we generated an in vitro model of the ADCA-DN and HSAN1E diseases. Taking advantage of the recently developed CRISPR/Cas9 technology which greatly facilitates homologous recombination, we generated a mouse embryonic stem cell (mESC) clone expressing a short biotin acceptor domain (AviTag) fused in frame to the N-terminus of the endogenous Dnmt1 protein. The AviTag domain gets biotinylated by the E. coli biotin ligase BirA that we previously inserted into the mESC genome by random integration. The AviTag-Dnmt1 clone will be used to generate one of the heterozygous missense mutations of the ADCA-DN disease (DNMT1A570V) and could be used to generate and study the other point mutations found in the abovementioned disorders. The biotinylated AviTag permits performing high stringent streptavidin-based immunoprecipitation of the fused protein. Thus, it allows to precisely investigate the binding of the mutated Dnmt1 protein with respect to the wild type protein in Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) experiments. The AviTag-Dnmt1 clone we generated will be instrumental in studying Dnmt1 expression and activity as well as DNA methylation and gene expression profiles in wild type and mutant stem cells and in cells differentiated towards the neural lineage. Moreover, mutant clones could be used to generate knock-in mice, thus obtaining an in vivo model that would better recapitulate the pathogenic mechanisms of the ADCA-DN disease. Summarizing, the generation of an in vitro mESC model of DNMT1-related diseases will provide comprehensive insights on the pathogenic mechanisms of DNMT1-related diseases in order to lay the basis for future development of therapeutic approaches.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14240/52153