Spinal Muscular Atrophy (SMA) and Familial Dysautonomia (FD) represent two paradigmatic diseases with a genetic origin associated to exon skipping defects. In both cases, the degree of exon skipping is directly related to the tissue- and phenotypic- severity and rescue of the underlying aberrant splicing events is therapeutic. In common with normal alternative splicing, exon skipping defects might be regulated co-transcriptionally by RNA Polymerase II (Pol II) elongation rate and/or chromatin histone marks. In addition, age-dependent genetic programs that include also co-transcriptional regulation can not only modify splicing globally, but potentially affect disease-causing splicing mutations. In this thesis, I have evaluated temporal changes in aberrant splicing patterns for two exon skipping events in SMA (Smn2 exon 7) and FD (Elp1 exon 20) asymptomatic mice and studied in detail the relationships between chromatin marks and splicing changes in the FD animal and in cellular models. Comparison of patterns at PostNatal Days (PND or P) 10, 90 and 365 showed tissue- and gene-specific splicing changes of the exon skipping events. In muscle, liver, lung and kidney, the Smn2 exon 7 inclusion showed an age-dependent increase, mainly during post-natal development. In central nervous system (CNS), exon 7 inclusion decreased within 3 months of age to then returned to its baseline level at P365 while no changes were observed in heart. In the FD mouse, the inclusion levels of Elp1 exon 20 are reduced in the CNS, liver, muscle and lung between P10 and P90, remaining low at one year of life: no changes were observed for the other organs. To explore the potential co-transcriptional regulation of the exon skipping events in vivo, I focused on the FD model and I performed on liver and brain tissues chromatin immunoprecipitation-qPCR experiments (ChIP-qPCR) of Pol II and chromatin marks (H3K4me3, H3K9me3, H3K27me3 and H3K36me3). The analysis was performed on three genomic Elp1 regions corresponding to constitutive exons (exons 10 and 29) and the alternative exon 20. The results showed in both tissues and for all genomic regions an age-dependent decrease in Pol II density profile and a robust increase (approximately 3-fold in liver and 20-fold in brain) of the repressive H3K27me3 mark. Interestingly, the H3K27me3 increased also globally in these tissues. To clarify the role of Pol II and histone modifications on Elp1 splicing regulation, I performed minigene experiments evaluating Pol II mutants with different elongation rate and chemicals that modify the chromatin structure (EED-226 and Valproic Acid) or affect the Pol II elongation (Camptothecin and DRB). The result I have obtained indicate that a more relaxed chromatin results in exon 20 inclusion while a more compact chromatin or a slow Pol II induce exon 20 skipping. These treatments specifically affect the defective exon 20 with no changes on splicing of minigene or endogenous constitutive exons. Interestingly, EED-226, a drug the specifically targets the repressive H3K27me3 mark induced exon 20 inclusion. These data suggest a co-transcriptional model of Elp1 exon 20 regulation in which an age-dependent increase in the repressive mark H3K27me3 will negatively affect the rate of Pol II elongation along the entire Elp1 gene. This will have a specific effect on the processing of defective exon 20 by inducing its skipping but will spare the other constitutive exons.

L’Atrofia Muscolare Spinale (SMA) e la Disautonomia Familiare (FD) sono due malattie paradigmatiche di origine genetica associate a un difetto di inclusione dell’esone. In entrambi i casi, la severità del salto dell’esone (exon skipping) è direttamente in relazione con la severità della malattia a livello tessutale e fenotipico ed il trattamento del difetto di splicing è terapeutico. In comune con lo splicing fisiologico, i difetti di exon skipping potrebbero essere regolati co-trascrizionalmente dal tasso di elongazione della RNA Polimerasi II (Pol II) e/o dalle modificazioni istoniche della cromatina. In aggiunta, programmi genetici dipendenti dall’età che includono anche una regolazione co-trascrizionale non solo possono modificare lo splicing a livello globale, ma potenzialmente interessare lo splicing aberrante dovuto a mutazioni che causano malattia. In questa tesi, ho valutato i cambi nel tempo di due eventi di splicing difettivi in topi asintomatici SMA (Smn2 esone 7) e FD (Elp1 esone 20) e studiato nel dettaglio la relazione tra modificazioni della cromatina e cambi di splicing nel topo FD e in modelli cellulari. La comparazione dei pattern di splicing a 10, 90 e 365 giorni post-nascita (PND or P) ha mostrato cambi di splicing specifici nello skipping dell’esone per ciascun gene. In muscolo, fegato, polmone e rene, l’inclusione dell’esone 7 del gene Smn2 ha mostrato un incremento dipendente dall’età, soprattutto durante lo sviluppo post-natale. Nel sistema nervoso centrale (CNS), l’inclusione dell’esone 7 è diminuita entro i 3 mesi di età per poi ritornare al livello basale a P365 mentre nessun cambio è stato osservato nel cuore. Nel topo FD, l’inclusione dell’esone 20 del gene Elp1 è ridotta nel CNS, fegato, muscolo e polmone tra P10 e P90, rimanendo bassa all’anno di vita: nessun cambio è stato visto in altri organi. Per esplorare la potenziale regolazione co-trascrizionale degli eventi di exon skipping in vivo, mi sono focalizzato sul modello FD e ho effettuato su fegato e cervello l’immunoprecipitazione della cromatina seguita da PCR quantitativa (ChIP-qPCR) verso la Pol II e alcune modificazioni della cromatina (H3K4me3, H3K9me3, H3K27me3 and H3K36me3). Questa analisi è stata fatta su tre regioni genomiche del gene Elp1 corrispondenti a esoni costitutivi (esone 10 e 29) e l’esone alternativo 20. I risultati mostrano che in entrambi i tessuti e su tutte le regioni genomiche avviene un calo nel profilo di densità della Pol II dipendente dall’età ed un robusto incremento (circa 3 volte nel fegato e 20 volte nel cervello) della modificazione repressiva H3K27me3. Interessante notare il fatto che il livello di H3K27me3 aumenta globalmente in entrambi i tessuti. Per chiarire il ruolo della Pol II e delle modificazioni istoniche nella regolazione dello splicing di Elp1, ho effettuato esperimenti sul minigene valutando l’effetto di mutanti di Pol II con diversi tassi di elongazione della Pol II e composti che modificano la struttura della cromatina (EED-226 e Acido Valproico) o che interessano l’elongazione della Pol II (Camptotecina e DRB). I risultati da me ottenuti indicano che uno stato più rilassato della cromatina risulta nell’inclusione dell’esone 20 mentre una struttura della cromatina più compatta o una Pol II lenta inducono skipping dell’esone 20. Questi trattamenti interessano in maniera specifica questo esone e non indicano cambi nello splicing di esoni costitutivi né sul minigene né su geni endogeni. Interessante il fatto che EED-226, un composto che specificatamente ha come target la modificazione repressiva H3K27me3, induce inclusione dell’esone 20. Questi dati suggeriscono un modello co-trascrizionale di regolazione dell’esone 20 del gene Elp1 in cui un incremento della modificazione repressiva H3K27me3 dovuto al tempo ha un impatto negativo sul tasso di elongazione della Pol II lungo l’intero gene.

Age-dependent regulation of co-transcriptional Elp1 exon 20 splicing in Familial Dysautonomia

-
2022

Abstract

Spinal Muscular Atrophy (SMA) and Familial Dysautonomia (FD) represent two paradigmatic diseases with a genetic origin associated to exon skipping defects. In both cases, the degree of exon skipping is directly related to the tissue- and phenotypic- severity and rescue of the underlying aberrant splicing events is therapeutic. In common with normal alternative splicing, exon skipping defects might be regulated co-transcriptionally by RNA Polymerase II (Pol II) elongation rate and/or chromatin histone marks. In addition, age-dependent genetic programs that include also co-transcriptional regulation can not only modify splicing globally, but potentially affect disease-causing splicing mutations. In this thesis, I have evaluated temporal changes in aberrant splicing patterns for two exon skipping events in SMA (Smn2 exon 7) and FD (Elp1 exon 20) asymptomatic mice and studied in detail the relationships between chromatin marks and splicing changes in the FD animal and in cellular models. Comparison of patterns at PostNatal Days (PND or P) 10, 90 and 365 showed tissue- and gene-specific splicing changes of the exon skipping events. In muscle, liver, lung and kidney, the Smn2 exon 7 inclusion showed an age-dependent increase, mainly during post-natal development. In central nervous system (CNS), exon 7 inclusion decreased within 3 months of age to then returned to its baseline level at P365 while no changes were observed in heart. In the FD mouse, the inclusion levels of Elp1 exon 20 are reduced in the CNS, liver, muscle and lung between P10 and P90, remaining low at one year of life: no changes were observed for the other organs. To explore the potential co-transcriptional regulation of the exon skipping events in vivo, I focused on the FD model and I performed on liver and brain tissues chromatin immunoprecipitation-qPCR experiments (ChIP-qPCR) of Pol II and chromatin marks (H3K4me3, H3K9me3, H3K27me3 and H3K36me3). The analysis was performed on three genomic Elp1 regions corresponding to constitutive exons (exons 10 and 29) and the alternative exon 20. The results showed in both tissues and for all genomic regions an age-dependent decrease in Pol II density profile and a robust increase (approximately 3-fold in liver and 20-fold in brain) of the repressive H3K27me3 mark. Interestingly, the H3K27me3 increased also globally in these tissues. To clarify the role of Pol II and histone modifications on Elp1 splicing regulation, I performed minigene experiments evaluating Pol II mutants with different elongation rate and chemicals that modify the chromatin structure (EED-226 and Valproic Acid) or affect the Pol II elongation (Camptothecin and DRB). The result I have obtained indicate that a more relaxed chromatin results in exon 20 inclusion while a more compact chromatin or a slow Pol II induce exon 20 skipping. These treatments specifically affect the defective exon 20 with no changes on splicing of minigene or endogenous constitutive exons. Interestingly, EED-226, a drug the specifically targets the repressive H3K27me3 mark induced exon 20 inclusion. These data suggest a co-transcriptional model of Elp1 exon 20 regulation in which an age-dependent increase in the repressive mark H3K27me3 will negatively affect the rate of Pol II elongation along the entire Elp1 gene. This will have a specific effect on the processing of defective exon 20 by inducing its skipping but will spare the other constitutive exons.
RICCARDI, FEDERICO
PINTON, Paolo
PINTON, Paolo
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2481328
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