The autosomal dominant genetic disease Facioscapulohumeral Dystrophy (FSHD) represents the most common myopathy found in adults. The genetic defect of FSHD does not reside in any protein-coding gene, but instead is linked to the contraction below 11 copies of the 3.3kb D4Z4 tandem repeat macrosatellite located on chromosome 4q35 (FSHD locus). Several mechanisms underlying FSHD have been described, however they don’t explain completely the variability of this complex epigenetic disease. With our work we aimed to elucidate the contribution of D4Z4 repeat in mediating the 3D genomic architecture during normal and FSHD-associated myogenesis. We have used a combination of genome-wide approaches including circular chromosome conformation capture (4C)-seq specific for the 4q chromosome, ChIP-seq with chromatin state dynamics analysis as well as RNA-seq datasets to investigate the dynamics of chromatin epigenetic landscapes and folding in FSHD. From these analysis we retrived that genome topology of FSHD cells was altered as well as the epigenetic landscape. Moreover in FSHD we retrived number of genes not regulated by DUX4 that instead present a deregulated interaction with 4q (lost and gained interactions) and showing active enhancers that are involved in the atrophic pathway. Among others, we further investigated a trans interaction with the muscle atrophy marker Atrogin1 (8q24) and showed that contact frequency between D4Z4 and Atrogin1, genomic locus topology, revealed by 4C-seq, chromosome conformation capture (3C) and 3D-FISH assays as well as Atrogin1 transcription, as well as epigenetic marks on the promoter, were deregulated during FSHD muscle cell differentiation. We have revealed through 4C assay that Atrogin1 3D-interactome comprise also important players in atrophic pathway, differentially regulated in FSHD cells, and therefore contributing to the FSHD patients phenotype. Finally we suggest that is the impairment in the epigentic environment of the FSHD locus, that occurs in patients, responsible for the deregulation of 3D contacts, at least for Atrogin1. Indeed when we restore another 4q D4Z4 array we are able to recover Atrogin1 expression. Altogether our data highlight the contribution of the macrosatellite D4Z4 in regulating the 3D genome architecture during normal myogenic differentiation. We propose that the profound coordinated alterations of chromatin folding and functional states observed in FSHD may be a key trait for the disease.

La Distrofia Facio-scapolo-omerale (FSHD)è una malattia genetica autosomica dominante che rappresenta la più comune miopatia degli adulti. Il difetto genetico della FSHD non risiede in alcun gene codificante per proteina, ma è invece associato alla contrazione al di sotto delle 11 copie dell’array di D4Z4, macrosatellite di 3.3. Kb ripetuto in tandem a livello del cromosoma 4q35 (FSHD locus). Diversi meccanismi alla base della FSHD sono stati descritti, comunque essi non spiegano completamente la variabilità di questa complessa malattia epigenetica. Col nostro lavoro cerchiamo di elucidare il contributo della sequenza ripetuta D4Z4 nel mediare un’architettura genomica in 3D durante la miogenesi di persone sane e affette da FSHD. Abbiamo utilizzato una combinazione di approcci genome-wide che includono circular chromosome conformation capture (4C)-seq specifica per il cromosoma 4q, ChIP-seq con chromatin state dynamics analysis e RNA-seq per investigare la dinamica degli scenari epigenetici cromatinici e strutturali nella FSHD. Da queste analisi ricaviamo che la topologia genomica delle cellule FSHD è alterata così come lo scenario epigenetico. Inoltre, ricaviamo numerosi geni non regolati da DUX4 che invece presentano una deregolata interazione con 4q (interazioni perse o acquisite) e enhancers attivi, che sono coinvolti nel pathway atrofico. Tra questi, abbiamo investigato approfonditamente un’interazione in trans con il marcatore di atrofia muscolare Atrogin1 (8q24) e mostrato che la frequenza di contatto tra D4Z4 e Atrogin1 rivelata tramite 4C-seq, chromosome conformation capture (3C) e 3D-FISH, così come la trascrizione di Atrogin1 e i marcatori epigentici a livello del promotore sono deregolati durante il differenziamento delle cellule muscolari in FSHD. Abbiamo rivelato tramite esperimenti di 4C che l’interattoma mediato da Atrogin1 comprende anche partners importanti nel pathway atrofico, differenzialmente regolati in cellule FSHD, e che quindi contribuiscono al fenotipo dei pazienti FSHD. In conclusione suggeriamo che è questa deregolazione nell’ambiente epigenetico del locus FSHD, che avviene nei pazienti, responsabile della deregolazione dei contatti 3D, almeno per Atrogin1. Quando reintroduciamo un altro 4q D4Z4 array siamo in grado di recuperare l’espressione di Atrogin1. Nel complesso i nostri dati sottolineano il contributo del macrosatellite D4Z4 nella regolazione dell’architettura genomica in 3D durante il normale differenziamento miogenico. Proponiamo infine che le alterazioni profonde e coordinate dell’impacchettamento cromatico e degli stati funzionali osservati nella FSHD possano essere un tratto chiave per la malattia.

(2017). Chromatin landscape of D4Z4 repeat interactome reveals a muscle atrophy signature in Facioscapulohumeral Dystrophy. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2017).

Chromatin landscape of D4Z4 repeat interactome reveals a muscle atrophy signature in Facioscapulohumeral Dystrophy

CORTESI, ALICE
2017

Abstract

The autosomal dominant genetic disease Facioscapulohumeral Dystrophy (FSHD) represents the most common myopathy found in adults. The genetic defect of FSHD does not reside in any protein-coding gene, but instead is linked to the contraction below 11 copies of the 3.3kb D4Z4 tandem repeat macrosatellite located on chromosome 4q35 (FSHD locus). Several mechanisms underlying FSHD have been described, however they don’t explain completely the variability of this complex epigenetic disease. With our work we aimed to elucidate the contribution of D4Z4 repeat in mediating the 3D genomic architecture during normal and FSHD-associated myogenesis. We have used a combination of genome-wide approaches including circular chromosome conformation capture (4C)-seq specific for the 4q chromosome, ChIP-seq with chromatin state dynamics analysis as well as RNA-seq datasets to investigate the dynamics of chromatin epigenetic landscapes and folding in FSHD. From these analysis we retrived that genome topology of FSHD cells was altered as well as the epigenetic landscape. Moreover in FSHD we retrived number of genes not regulated by DUX4 that instead present a deregulated interaction with 4q (lost and gained interactions) and showing active enhancers that are involved in the atrophic pathway. Among others, we further investigated a trans interaction with the muscle atrophy marker Atrogin1 (8q24) and showed that contact frequency between D4Z4 and Atrogin1, genomic locus topology, revealed by 4C-seq, chromosome conformation capture (3C) and 3D-FISH assays as well as Atrogin1 transcription, as well as epigenetic marks on the promoter, were deregulated during FSHD muscle cell differentiation. We have revealed through 4C assay that Atrogin1 3D-interactome comprise also important players in atrophic pathway, differentially regulated in FSHD cells, and therefore contributing to the FSHD patients phenotype. Finally we suggest that is the impairment in the epigentic environment of the FSHD locus, that occurs in patients, responsible for the deregulation of 3D contacts, at least for Atrogin1. Indeed when we restore another 4q D4Z4 array we are able to recover Atrogin1 expression. Altogether our data highlight the contribution of the macrosatellite D4Z4 in regulating the 3D genome architecture during normal myogenic differentiation. We propose that the profound coordinated alterations of chromatin folding and functional states observed in FSHD may be a key trait for the disease.
Bodega, Beatrice
FSHD; D4Z4; 3D structure; Epigenetics
MED/04 - PATOLOGIA GENERALE
English
20-mar-2017
MEDICINA TRASLAZIONALE E MOLECOLARE - DIMET - 76R
29
2015/2016
open
(2017). Chromatin landscape of D4Z4 repeat interactome reveals a muscle atrophy signature in Facioscapulohumeral Dystrophy. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2017).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/154433
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