Determinants of epigenetic resistance to HDAC inhibitors in dystrophic fibro‐adipogenic progenitors
2022; Springer Nature; Volume: 23; Issue: 6 Linguagem: Inglês
10.15252/embr.202254721
ISSN1469-3178
AutoresSilvia Consalvi, Luca Tucciarone, Elisa Macrì, Marco De Bardi, Mario Picozza, Illari Salvatori, Alessandra Renzini, Sérgio Valente, Antonello Mai, Viviana Moresi, Prem Puri,
Tópico(s)GDF15 and Related Biomarkers
ResumoArticle4 April 2022Open Access Transparent process Determinants of epigenetic resistance to HDAC inhibitors in dystrophic fibro-adipogenic progenitors Silvia Consalvi Corresponding Author Silvia Consalvi [email protected] orcid.org/0000-0002-8640-8212 Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Fondazione Santa Lucia, Rome, Italy UniCamillus - Saint Camillus International University of Health Sciences, Rome, Italy Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Project administration Search for more papers by this author Luca Tucciarone Luca Tucciarone Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Fondazione Santa Lucia, Rome, Italy Contribution: Data curation, Formal analysis, Investigation Search for more papers by this author Elisa Macrì Elisa Macrì orcid.org/0000-0001-9232-0578 Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Fondazione Santa Lucia, Rome, Italy Search for more papers by this author Marco De Bardi Marco De Bardi orcid.org/0000-0001-8217-970X Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Fondazione Santa Lucia, Rome, Italy Contribution: Formal analysis Search for more papers by this author Mario Picozza Mario Picozza orcid.org/0000-0002-2529-6456 Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Fondazione Santa Lucia, Rome, Italy Contribution: Formal analysis Search for more papers by this author Illari Salvatori Illari Salvatori Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Fondazione Santa Lucia, Rome, Italy Department of Experimental Medicine, University of Rome "La Sapienza", Rome, Italy Contribution: Formal analysis Search for more papers by this author Alessandra Renzini Alessandra Renzini orcid.org/0000-0002-4265-7188 Unit of Histology and Medical Embryology, DAHFMO, University of Rome "La Sapienza", Rome, Italy Contribution: Investigation Search for more papers by this author Sergio Valente Sergio Valente orcid.org/0000-0002-2241-607X Department of Drug Chemistry and Technologies, University of Rome "La Sapienza", Rome, Italy Contribution: Investigation Search for more papers by this author Antonello Mai Antonello Mai orcid.org/0000-0001-9176-2382 Department of Drug Chemistry and Technologies, University of Rome "La Sapienza", Rome, Italy Contribution: Investigation Search for more papers by this author Viviana Moresi Viviana Moresi orcid.org/0000-0003-1912-0339 Unit of Histology and Medical Embryology, DAHFMO, University of Rome "La Sapienza", Rome, Italy Institute of Nanotechnology (Nanotec), National Research Council (CNR), Rome Unit, Rome, Italy Contribution: Data curation, Formal analysis, Investigation Search for more papers by this author Pier Lorenzo Puri Corresponding Author Pier Lorenzo Puri [email protected] orcid.org/0000-0003-4964-0095 Development, Aging and Regeneration Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA Contribution: Conceptualization, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Silvia Consalvi Corresponding Author Silvia Consalvi [email protected] orcid.org/0000-0002-8640-8212 Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Fondazione Santa Lucia, Rome, Italy UniCamillus - Saint Camillus International University of Health Sciences, Rome, Italy Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Project administration Search for more papers by this author Luca Tucciarone Luca Tucciarone Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Fondazione Santa Lucia, Rome, Italy Contribution: Data curation, Formal analysis, Investigation Search for more papers by this author Elisa Macrì Elisa Macrì orcid.org/0000-0001-9232-0578 Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Fondazione Santa Lucia, Rome, Italy Search for more papers by this author Marco De Bardi Marco De Bardi orcid.org/0000-0001-8217-970X Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Fondazione Santa Lucia, Rome, Italy Contribution: Formal analysis Search for more papers by this author Mario Picozza Mario Picozza orcid.org/0000-0002-2529-6456 Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Fondazione Santa Lucia, Rome, Italy Contribution: Formal analysis Search for more papers by this author Illari Salvatori Illari Salvatori Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Fondazione Santa Lucia, Rome, Italy Department of Experimental Medicine, University of Rome "La Sapienza", Rome, Italy Contribution: Formal analysis Search for more papers by this author Alessandra Renzini Alessandra Renzini orcid.org/0000-0002-4265-7188 Unit of Histology and Medical Embryology, DAHFMO, University of Rome "La Sapienza", Rome, Italy Contribution: Investigation Search for more papers by this author Sergio Valente Sergio Valente orcid.org/0000-0002-2241-607X Department of Drug Chemistry and Technologies, University of Rome "La Sapienza", Rome, Italy Contribution: Investigation Search for more papers by this author Antonello Mai Antonello Mai orcid.org/0000-0001-9176-2382 Department of Drug Chemistry and Technologies, University of Rome "La Sapienza", Rome, Italy Contribution: Investigation Search for more papers by this author Viviana Moresi Viviana Moresi orcid.org/0000-0003-1912-0339 Unit of Histology and Medical Embryology, DAHFMO, University of Rome "La Sapienza", Rome, Italy Institute of Nanotechnology (Nanotec), National Research Council (CNR), Rome Unit, Rome, Italy Contribution: Data curation, Formal analysis, Investigation Search for more papers by this author Pier Lorenzo Puri Corresponding Author Pier Lorenzo Puri [email protected] orcid.org/0000-0003-4964-0095 Development, Aging and Regeneration Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA Contribution: Conceptualization, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Silvia Consalvi *,1,2,†, Luca Tucciarone1,†, Elisa Macrì1, Marco De Bardi1, Mario Picozza1, Illari Salvatori1,3, Alessandra Renzini4, Sergio Valente5, Antonello Mai5, Viviana Moresi4,6 and Pier Lorenzo Puri *,7 1Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Fondazione Santa Lucia, Rome, Italy 2UniCamillus - Saint Camillus International University of Health Sciences, Rome, Italy 3Department of Experimental Medicine, University of Rome "La Sapienza", Rome, Italy 4Unit of Histology and Medical Embryology, DAHFMO, University of Rome "La Sapienza", Rome, Italy 5Department of Drug Chemistry and Technologies, University of Rome "La Sapienza", Rome, Italy 6Institute of Nanotechnology (Nanotec), National Research Council (CNR), Rome Unit, Rome, Italy 7Development, Aging and Regeneration Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA † These authors contributed equally to this work *Corresponding author. Tel: +39 06 501703266; E-mail: [email protected] ***Corresponding author. Tel: +1 858 6463100; E-mail: [email protected] EMBO Reports (2022)23:e54721https://doi.org/10.15252/embr.202254721 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Pharmacological treatment of Duchenne muscular dystrophy (DMD) with histone deacetylase inhibitors (HDACi) is currently being tested in clinical trials; however, pre-clinical studies indicated that the beneficial effects of HDACi are restricted to early stages of disease. We show that FAPs from late-stage mdx mice exhibit aberrant HDAC activity and genome-wide alterations of histone acetylation that are not fully reversed by HDACi. In particular, combinatorial H3K27 and/or H3K9/14 hypo-acetylation at promoters of genes required for cell cycle activation and progression, as well as glycolysis, are associated with their downregulation in late-stage mdx FAPs. These alterations could not be reversed by HDACi, due to a general resistance to HDACi-induced H3K9/14 hyperacetylation. Conversely, H3K9/14 hyper-acetylation at promoters of Senescence Associated Secretory Phenotype (SASP) genes is associated with their upregulation in late-stage mdx FAPs; however, HDACi could reduce promoter acetylation and blunt SASP gene activation. These data reveal that during DMD progression FAPs develop disease-associated features reminiscent of cellular senescence, through epigenetically distinct and pharmacologically dissociable events. They also indicate that HDACi might retain anti-fibrotic effects at late stages of DMD. SYNOPSIS Progressive increase of HDAC activity in Fibro-adipogenic progenitors from Duchenne Muscular Dystrophy model mice leads to altered histone acetylation profiles associated with senescence at late disease stages that are not fully reversed by HDAC inhibitors. HDAC activity increases in FAPs of muscles from mdx mice at late stages of disease progression. FAPs of late stage mdx muscles exhibit altered profiles of histone acetylation and develop features of senescence. HDAC inhibitors fail to reverse cell cycle arrest and glycolysis In FAPs of late stage mdx muscles. HDAC inhibitors inhibit activation of SASP genes in FAPs of late stage mdx muscles. Introduction Duchenne muscular dystrophy (DMD) is a fatal genetic disease caused by lack of dystrophin (dys) expression (Hoffman et al, 1987; Muntoni et al, 2003). Genetic correction by restoration of dys expression with gene therapy approaches (Chamberlain & Chamberlain, 2017; Min et al, 2019; Verhaart & Aartsma-Rus, 2019) is predicted to recover the biochemical and functional integrity of the dystrophin-associated protein complex (DAPC) (Ervasti, 2006) and thereby protect myofiber sarcolemma stability post-contraction (Lapidos et al, 2004). However, a variety of “secondary” pathogenic events caused by dys deficiency can contribute to DMD progression (Constantin, 2014; Garbincius & Michele, 2015; Bhat et al, 2017; Morikawa et al, 2017; Hardee et al, 2021; Rugowska et al, 2021) and might persist even after gene therapy. Targeting these DMD-associated “secondary” events might therefore be necessary to achieve complete and long-lasting therapeutic recovery in DMD patients. Among the “secondary” events caused by dys deficiency, pathogenic activation of specific sub-populations of muscle resident cells is emerging as key event in DMD progression (Serrano & Muñoz-Cánoves, 2017; Cappellari et al, 2020). Recent works have lent further support to the activation of “secondary” pathogenic responses in cell types that do not express dys, by showing alterations of the transcriptional profiles in various muscle-resident cell types from mdx muscles, in addition to myonuclei and MuSCs (Juban et al, 2018; Malecova et al, 2018; Tidball et al, 2018; Chemello et al, 2020; Kim et al, 2020). Pharmacological strategies that target the pathogenic activation of muscle-resident cell types in DMD include the current standard treatment with steroids (Quattrocelli et al, 2021), as well as novel interventions with epigenetic drugs, such as histone deacetylate inhibitors (HDACi) (Consalvi et al, 2011). The therapeutic potential of HDACi for DMD has been shown by multiple lines of evidence, including preclinical (Minetti et al, 2006; Consalvi et al, 2013) and early clinical studies (Bettica et al, 2016), and is currently under evaluation in clinical trials with DMD boys (https://clinicaltrials.gov/ct2/show/NCT03373968). Studies in mdx mice—the DMD murine model—have shown that the beneficial effects of HDACi are restricted to the early stages of disease progression (Mozzetta et al, 2013; Saccone et al, 2014). This loss of beneficial effects observed in DMD mouse models at late stages of disease suggests that development of a disease-associated resistance might limit the efficacy of HDACi in late-stage DMD patients. Among muscle-resident cells, fibro-adipogenic progenitors (FAPs) have been implicated as central cellular effectors of DMD progression and key targets of the beneficial effects of HDACi in mdx mice (Mozzetta et al, 2013; Saccone et al, 2014). FAPs support muscle-stem cell-mediated repair in acutely injured muscles, but turn into cellular effectors of fibrotic and adipogenic degeneration of muscles exposed to conditions of chronic damage, such as DMD and other neuromuscular disorders (Joe et al, 2010; Uezumi et al, 2010, 2011; Mozzetta et al, 2013; Saccone et al, 2014; Lemos et al, 2015; Kopinke et al, 2017; Madaro et al, 2018; Malecova et al, 2018; Mázala et al, 2020). We have previously shown that in mdx mice at early stages of disease FAPs can promote muscle stem cell (MuSC)-mediated compensatory regeneration and are susceptible to both HDACi-mediated enhancement of their pro-regenerative activity and inhibition of their fibro-adipogenic potential (Mozzetta et al, 2013; Saccone et al, 2014). Moreover, recent studies have revealed that exposure to HDACi promotes the formation and release of pro-regenerative and anti-fibrotic extra-cellular vesicles (EVs) from FAPs of DMD muscles at early stages of disease (Sandonà et al, 2020). The progressive loss of pro-regenerative potential and response to HDACi in FAPs from late-stage mdx mice (Mozzetta et al, 2013) suggests that proportional changes in HDAC activity and related histone modifications occur in these cells during DMD progression. However, it remains currently unknown whether HDAC activity is altered in FAPs of dystrophic muscles and can generate aberrant profiles of histone acetylation and gene expression; likewise, it is unknown whether these alterations could be effectively restored by HDACi at progressive stages of disease progression and what is their impact on FAP biology. Results FAPs of late-stage mdx mice exhibit increased HDAC activity, altered patterns of histone acetylation and partial response to HDACi We measured class I and class II HDAC activity from lysates of FAPs isolated by FACS (Appendix Fig S1A and B) from hind limb muscles of mdx mice (or control wild-type (wt) mice) at either early (1.5-month-old) or late (12-month-old) stages of disease—hereinafter also referred to as “young mdx FAPs” or “old mdx FAPs”, respectively. Fig 1A illustrates a general and consensual increase in the activity of all HDAC classes—HDAC I, IIa and I/IIb—observed in FAPs isolated from mdx mice, as compared to their wt counterpart. Moreover, while the activity of all classes of HDACs did not change in FAPs isolated from muscles of wt mice at 1.5 or 12 months of age, the enzymatic activity of all HDAC classes was increased about two folds in mdx old FAPs, as compared to mdx young FAPs (Fig 1A). The progressive increase in enzymatic activity observed in in FAPs of mdx, but not wt, mice during aging suggests that deregulation of HDAC activity does not occur as a consequence of chronological aging, but is a disease-associated event. A 15 day treatment with the pan HDACi Trichostatin A (TSA), which we have previously reported to exert histological and functional beneficial effects in young mdx mice (Minetti et al, 2006), could reduce class I HDAC enzymatic activity with a comparable efficacy in mdx FAPs from either stage (Fig 1A, left panel). By contrast, class IIa HDAC activity was drastically inhibited (about 10 fold reduction) by TSA in young mdx FAPs, whereas it was only reduced by half in old mdx mice (Fig 1A, middle panel). Finally, class I/IIb HDAC activity was moderately inhibited by TSA in young mdx FAPs and minimally affected in old mdx FAPs (Fig 1A, right panel). These results show that HDAC activity increases in FAPs of mdx muscles along with disease progression, and that exposure to TSA could reduce the enzymatic activity of class I and II HDAC at both stages, albeit with a progressive reduction in efficacy at late stages that was proportionate to the increased HDAC activity observed in old mdx FAPs. Figure 1. Differential patterns of HDAC activity and gene expression in FAPs during DMD progression and treatment with HDACi WY: young wild type; WO: old wild type; YC: young mdx control; YT: young mdx in vivo treated with TSA for 15 days; OC: old mdx control; OT: old mdx in vivo treated with TSA for 15 days. Graphs showing the enzymatic activity of class I (left panel), class IIa (middle panel), and class I/IIb (right panel) HDACs performed in FAPs isolated from WY, WO, YC, YT, OC and OT mice. Cartoon illustrating the experimental strategy. Mdx mice at 1.5 months (young) and 12 months (old) of age were treated with TSA or its vehicle of control for 15 days. At the end of the treatment, FAPs were isolated by FACS from hindlimb muscles to perform H3K9/14ac and H3K27ac ChIP-seq and RNA-seq. Heatmap for H3K9/14ac ChIP-seq signal in the experimental conditions described in B). NGS plot showing H3K9/14ac comparative patterns in the experimental conditions described in B). Heatmap for H3K27ac ChIP-seq signal in the experimental conditions described in B). NGS plot showing H3K27ac comparative patterns in the experimental conditions described in B). Upset graph showing the intersection size (in black) between the differentially acetylated loci for H3K9/14ac (in blue) in the experimental conditions described in B). The top 10 intersections are shown. Upset graph showing the intersection size (in black) between the differentially acetylated loci for H3K27ac (in blue) in the experimental conditions described in B). The top 10 intersections are shown. Heatmap showing 2 clusters of DE genes identified across all the experimental conditions described in B). Gene expression is represented as z-score calculated across the rows. Gene Ontology performed on cluster 1 (left panel) and cluster 2 (right panel) genes. Heatmap showing the differential expression levels by log2 Fold Change for representative genes of cluster 1 and cluster 2 in the RNA-seq comparisons of YT vs. YC, OC vs. YC and OT vs. OC FAPs. Data information: In (A) data are presented as average ± SEM (n = 4, biological replicates); (°) indicates statistical analysis by Student’s t-test in the comparison against WY, °°°°P ≤ 0.0001, ns = not significant; (*) indicates statistical analysis by Student’s t-test in the comparison against YC, **P ≤ 0.01, ***P ≤ 0.001 ****P ≤ 0.0001; (†) indicates statistical analysis by Student’s t-test in the comparison against OC, †P ≤ 0.05, ††††P ≤ 0.0001; (#) indicates statistical analysis by one-way ANOVA, ####P ≤ 0.0001. Download figure Download PowerPoint As HDAC activity controls histone acetylation, we next sought to determine whether the different levels of HDAC activity detected in FAPs from muscles of mdx mice at the two stages of disease (namely, 1.5- or 12-month-old mdx mice) could generate different profiles of genome-wide distribution of histone 3 (H3) acetylation at lysines 9/14 (H3K9/14ac) and lysine 27 (H3K27ac)—two major histone modifications associated with a chromatin conformation permissive for gene expression (Zentner & Henikoff, 2013; Tessarz & Kouzarides, 2014). We also investigated the effect of HDACi on these histone acetylation patterns, by exposing mdx mice to TSA, as described above. In parallel, we performed RNAseq analysis, in order to monitor the transcriptional output of FAPs in the same experimental conditions. Figure 1B illustrates the experimental strategy. ChIP-seq experiments with anti-H3K9/14ac and H3K27ac antibodies revealed distinct profiles of histone acetylation in FAPs isolated from hind limb muscles of young (1.5 month) or old (12 month) mdx mice, either untreated or treated with TSA for 15 days (Fig 1C–F). Global analysis of the cumulative genomic distribution of ChIP-seq peak signals for these histone modifications showed that H3K9/14ac was largely biased toward gene promoters, while H3K27ac signal was distributed between gene promoters (one half) and intronic or distal intergenic elements that typically harbor enhancers (Appendix Fig S1C). This signal is consistent with the enrichment of H3K27ac typically observed at active enhancers and promoters (Hnisz et al, 2013; Zentner & Henikoff, 2013; Tessarz & Kouzarides, 2014). Slightly increased genome-wide levels of H3K9/14ac were observed at gene promoters of old mdx FAPs, as compared to young mdx FAPs (Fig 1C and D, middle panel). Interestingly, TSA treatment increased H3K9/14ac signal at gene promoters in young mdx FAPs (Fig 1C and D, left panel), while did not significantly alter the global H3K9/14ac levels at gene promoters in old mdx FAPs (Fig 1C and D, right panel). Conversely, a dramatic loss of H3K27 acetylation at both gene promoters and outside was observed in old mdx FAPs, as compared to their younger counterpart (Fig 1E and F, middle panel). TSA treatment could recover global H3K27 acetylation in old mdx FAPs (Fig 1E and F, right panel) to levels comparable to those of young mdx FAPs (Fig 1F, compare middle and right panels). By contrast, TSA decreased H3K27ac signal in young mdx FAPs inside and outside gene promoters (Fig 1E and F, left panel). Combinatorial intersection of differentially acetylated loci in FAPs across all experimental conditions for both histone modifications shows that the large majority of the H3K9/14ac peaks detected was induced by TSA in young mdx mice, with more than half of them specific for this condition (Fig 1G). Most of the remainder H3K9/14ac peaks induced by TSA in young mdx FAPs coincided with H3K9/14 hyperacetylated loci in old mdx FAPs, with a subset of them also coinciding with H3K9/14 hypoacetylated loci induced by TSA in old mdx FAPs (Fig 1G). This pattern of combinatorial intersections identifies a putative common subset of gene promoters regulated by H3K9/14 acetylation in both young and old mdx FAPs, whereby common hyperacetylated loci detected in TSA-treated young mdx FAPs and untreated old mdx FAPs coincided with loci hypoacetylated in response to TSA treatment in old mdx FAPs. This specific combination suggests that TSA might differentially affect the H3K9/14ac status of a common subset of gene promoters in FAPs throughout DMD progression. Interestingly, another subset of H3K9/14 hyperacetylated loci uniquely detected in old mdx FAPs coincided with hypoacetylated loci in TSA-treated old mdx FAPs (Fig 1G), further indicating that reversal of H3K9/14 hyperacetylation at gene promoters paradoxically occurs in old mdx FAPs. By contrast, TSA-mediated H3K9/14 hyperacetylation in old mdx FAPs was a rare event and mostly occurred at loci that were also hyperacetylated by TSA in young mdx FAPs (Fig 1G). These data suggest that along with the disease progression in mdx mice, FAPs might develop resistance to HDACi-induced H3K9/14 hyperacetylation, while becoming vulnerable to HDAC-mediated reduction of H3K9/14ac signal at hyperacetylated loci in old mdx FAPs. Conversely, the most dominant combinatorial patterns of H3K27ac included the hypoacetylation at gene loci in old mdx FAPs, with about half of these loci in which H3K27 hyperacetylation was recovered by TSA (Fig 1H). The other half included loci that were also hypoacetylated in young mdx FAPs treated with TSA or loci uniquely detected in old mdx FAPs, in which H3K27ac signal was not recovered by TSA (Fig 1H). This pattern shows that the reduction of H3K27ac in old mdx FAPs can be recovered by TSA at certain loci, but not at others, thereby indicating that old mdx FAPs develop partial resistance to HDACi-mediated H3K27 hyperacetylation. Interestingly, in young mdx FAPs TSA could only reduce H3K27ac, both at unique loci and at loci that were also hypoacetylated in old mdx FAPs (Fig 1H). HDACi modulate different patterns of gene expression in FAPs of mdx mice at different stages of disease progression The alterations of the genome-wide histone acetylation profiles detected in FAPs of mdx mice at different stages of disease and in response to TSA predict that consensual alterations in gene expression profiles could also occur in mdx FAPs in the same experimental conditions. We therefore performed RNAseq to analyze the gene expression profile of FAPs isolated from the same experimental conditions described above (illustrated in Fig 1B). Differentially expressed (DE) genes between young and old mdx FAPs were almost equally distributed between up- or downregulated genes (Appendix Fig S2A, middle panel; Appendix Fig S2B). Likewise, TSA induced a similar number of up- and downregulated DE genes in young mdx FAPs (Appendix Fig S2A, left panel; Appendix Fig S2B). In contrast, TSA preferentially dowregulated gene expression in old mdx FAPs (Appendix Fig S2A, right panel; Appendix Fig S2B). Combinatorial intersection of DE genes in FAPs across all experimental conditions revealed that the large majority of them was accounted by genes either uniquely up-regulated or downregulated in old mdx FAPs, as compared to their young counterpart; however, while the expression of a proportion of genes upregulated in old mdx FAPs was recovered by TSA-mediated repression, only the expression of very few genes that were downregulated in old mdx FAPs was recovered by TSA-mediated activation (Appendix Fig S2C). Overall, TSA-modulated genes in young and old FAPs did not show any relevant overlap, suggesting that HDACi modulate different patterns of gene expression in dystrophic FAPs at different stages of disease, as also predicted by their acetylation profiles. Heatmap of top DE genes across all experimental conditions revealed specific patterns of gene expression that could discriminate 2 major clusters of DE genes during FAP transition from an early to a late stage of disease progression in mdx mice, as well as their differential response to TSA (Appendix Fig S3 and Fig 1I). Gene ontology analysis identified specific biological processes for each of these clusters of DE genes. Cluster 1 included a subset of genes whose expression was induced by TSA in young mdx FAPs, but was repressed, and was not recovered by TSA, in old mdx FAPs (Appendix Fig S3 and Fig 1I). Gene ontology assigned these genes to processes related to activation of cell proliferation and migration (Fig 1J). These genes encode cell cycle activators, such as cyclins and cyclin-dependent kinases, histone variants, components of the cytoskeleton as well as activators of glycolysis (Appendix Fig S3 and Fig 1K). Cluster 2 included a subset of genes whose expression increased in old mdx FAPs, as compared to young mdx FAPs; however, TSA treatment could downregulate the expression of these genes to levels comparable to those observed in young mdx FAPs (Appendix Fig S3 and Fig 1I). Gene ontology analysis indicates that this cluster was enriched in genes implicated in regulation of extracellular matrix (ECM), response to hypoxia and inflammation, as well as cytokine-mediated signaling pathways (Fig 1J). Indeed, these genes encode several ligands and receptors for activation of intracellular signaling as well as downstream nuclear transcription factors implicated in ECM remodeling, fibrosis and inflammation (Appendix Fig S3 and Fig 1K). Altered patterns of H3K9/14ac and H3K27/14ac at promoters of genes implicated in cell cycle arrest and activation of SASP in FAPs of late-stage mdx mice We next performed an integrated analysis of ChIPseq and RNAseq datasets generated across all experimental conditions to identify specific patterns of histone acetylation and gene expression that could discriminate young from old mdx FAPs and their different ability to respond to TSA. Specific patterns of histone acetylation were associated to the expression levels of the nearest gene (−1,500/+500 bp distance from the TSS). This analysis revealed two major trends, one consisting of genes that were upregulated in old mdx FAPs and marked by H3K9/14 hyperacetylation at their promoters, another consisting of genes that were downregulated in old mdx FAPs and marked by H3K27 hypoacetylation (Appendix Fig S4A). Gene ontology revealed that upregulated genes marked by H3K9/14 hyperacetylation were enriched in genes belonging to cluster 2 (Appendix Fig S3 and Fig 1I–K) and implicated in ECM remodeling (e.g., TGFbeta signaling) and cytokine-mediated signaling pathways (e.g., TNFalpha or NFkB signaling) (Appendix Fig S4B). Figure 2A illustrates examples of increased H3K9/14ac levels at promoters of representative genes that were upregulated in old mdx FAPs, such as the components of pro-fibrotic TGFbeta signaling, Smad3, Tgfb2 and Tgfbi (Transforming Growth Factor Beta Induced), (Fig 2A). The upregulation of these representative genes and the increased levels of H3K9/14ac at their promoters in old mdx FAPs, as compared to young mdx FAPs, was validated by independent qPCR (Fig 2B) and ChIP-qPCR (Fig 2C) analyses, respectively. Interestingly, genes upregulated in old mdx FAPs and marked by promoter H3K9/14 hyperacetylation were also enriched in aging process and negative regulation of cell cycle (Appendix Fig S4B). Conversely, genes downregulated in old mdx FAPs and marked by H3K27 hypoacetylation showed enrichment for biological processes related to activation of cell cycle progression, DNA replication and mitosis (Appendix Fig S4C). Figure 2D shows examples of H3K27 hypoacetylation at promoters of representative downregulated genes in old mdx FAPs, such as the cell cycle activators E2F1, Cdk4, and Check2 (Fig 2D). The downregulation of these genes and the decreased l
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