Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 22
Filter
Add more filters










Publication year range
1.
J Hepatol ; 2024 May 22.
Article in English | MEDLINE | ID: mdl-38782119

ABSTRACT

BACKGROUND & AIMS: Transcription termination fine-tunes gene expression and contributes to the specification of RNA function in eukaryotic cells. Transcription termination of HBV is subject to the recognition of the canonical polyadenylation signal (cPAS) common to all viral transcripts. However, the regulation of this cPAS and its impact on viral gene expression and replication is currently unknown. METHODS: To unravel the regulation of HBV transcript termination, we implemented a 3' RACE (rapid amplification of cDNA ends)-PCR assay coupled to single molecule sequencing both in in vitro-infected hepatocytes and in chronically infected patients. RESULTS: The detection of a previously unidentified transcriptional readthrough indicated that the cPAS was not systematically recognized during HBV replication in vitro and in vivo. Gene expression downregulation experiments demonstrated a role for the RNA helicases DDX5 and DDX17 in promoting viral transcriptional readthrough, which was, in turn, associated with HBV RNA destabilization and decreased HBx protein expression. RNA and chromatin immunoprecipitation, together with mutation of the cPAS sequence, suggested a direct role of DDX5 and DDX17 in functionally linking cPAS recognition to transcriptional readthrough, HBV RNA stability and replication. CONCLUSIONS: Our findings identify DDX5 and DDX17 as crucial determinants of HBV transcriptional fidelity and as host restriction factors for HBV replication. IMPACT AND IMPLICATIONS: HBV covalently closed circular (ccc)DNA degradation or functional inactivation remains the holy grail for the achievement of HBV cure. Transcriptional fidelity is a cornerstone in the regulation of gene expression. Here, we demonstrate that two helicases, DDX5 and DDX17, inhibit recognition of the HBV polyadenylation signal and thereby transcriptional termination, thus decreasing HBV RNA stability and acting as restriction factors for efficient cccDNA transcription and viral replication. The observation that DDX5 and DDX17 are downregulated in patients chronically infected with HBV suggests a role for these helicases in HBV persistence in vivo. These results open new perspectives for researchers aiming at identifying new targets to neutralise cccDNA transcription.

2.
JCI Insight ; 9(11)2024 May 16.
Article in English | MEDLINE | ID: mdl-38753465

ABSTRACT

Glycogen storage disease type III (GSDIII) is a rare metabolic disorder due to glycogen debranching enzyme (GDE) deficiency. Reduced GDE activity leads to pathological glycogen accumulation responsible for impaired hepatic metabolism and muscle weakness. To date, there is no curative treatment for GSDIII. We previously reported that 2 distinct dual AAV vectors encoding for GDE were needed to correct liver and muscle in a GSDIII mouse model. Here, we evaluated the efficacy of rapamycin in combination with AAV gene therapy. Simultaneous treatment with rapamycin and a potentially novel dual AAV vector expressing GDE in the liver and muscle resulted in a synergic effect demonstrated at biochemical and functional levels. Transcriptomic analysis confirmed synergy and suggested a putative mechanism based on the correction of lysosomal impairment. In GSDIII mice livers, dual AAV gene therapy combined with rapamycin reduced the effect of the immune response to AAV observed in this disease model. These data provide proof of concept of an approach exploiting the combination of gene therapy and rapamycin to improve efficacy and safety and to support clinical translation.


Subject(s)
Dependovirus , Disease Models, Animal , Genetic Therapy , Genetic Vectors , Liver , Sirolimus , Animals , Sirolimus/pharmacology , Sirolimus/therapeutic use , Dependovirus/genetics , Genetic Therapy/methods , Mice , Liver/metabolism , Genetic Vectors/genetics , Genetic Vectors/administration & dosage , Muscle, Skeletal/metabolism , Phenotype , Glycogen Debranching Enzyme System/genetics , Glycogen Debranching Enzyme System/metabolism , Humans , Male
3.
Nat Commun ; 15(1): 2487, 2024 Mar 21.
Article in English | MEDLINE | ID: mdl-38514619

ABSTRACT

The cellular mechanisms underlying axonal morphogenesis are essential to the formation of functional neuronal networks. We previously identified the autism-linked kinase NUAK1 as a central regulator of axon branching through the control of mitochondria trafficking. However, (1) the relationship between mitochondrial position, function and axon branching and (2) the downstream effectors whereby NUAK1 regulates axon branching remain unknown. Here, we report that mitochondria recruitment to synaptic boutons supports collateral branches stabilization rather than formation in mouse cortical neurons. NUAK1 deficiency significantly impairs mitochondrial metabolism and axonal ATP concentration, and upregulation of mitochondrial function is sufficient to rescue axonal branching in NUAK1 null neurons in vitro and in vivo. Finally, we found that NUAK1 regulates axon branching through the mitochondria-targeted microprotein BRAWNIN. Our results demonstrate that NUAK1 exerts a dual function during axon branching through its ability to control mitochondrial distribution and metabolic activity.


Subject(s)
AMP-Activated Protein Kinase Kinases , AMP-Activated Protein Kinases , Animals , Mice , AMP-Activated Protein Kinases/genetics , AMP-Activated Protein Kinases/metabolism , Axons/metabolism , Mitochondria/metabolism , Neurons/metabolism
4.
Database (Oxford) ; 20232023 Dec 21.
Article in English | MEDLINE | ID: mdl-38128543

ABSTRACT

One challenge faced by scientists from the alternative RNA splicing field is to decode the cooperative or antagonistic effects of splicing factors (SFs) to understand and eventually predict splicing outcomes on a genome-wide scale. In this manuscript, we introduce SplicingLore, an open-access database and web resource that help to fill this gap in a straightforward manner. The database contains a collection of RNA-sequencing-derived lists of alternative exons regulated by a total of 75 different SFs. All datasets were processed in a standardized manner, ensuring valid comparisons and correlation analyses. The user can easily retrieve a factor-specific set of differentially included exons from the database or provide a list of exons and search which SF(s) control(s) their inclusion. Our simple workflow is fast and easy to run, and it ensures a reliable calculation of correlation scores between the tested datasets. As a proof of concept, we predicted and experimentally validated a novel functional cooperation between the RNA helicases DDX17 and DDX5 and the heterogeneous nuclear ribonucleoprotein C (HNRNPC) protein. SplicingLore is available at https://splicinglore.ens-lyon.fr/. Database URL:  https://splicinglore.ens-lyon.fr/.


Subject(s)
Alternative Splicing , RNA Splicing , Humans , RNA Splicing Factors/genetics , RNA Splicing/genetics , Genome , Exons/genetics
5.
Stem Cell Reports ; 18(5): 1090-1106, 2023 05 09.
Article in English | MEDLINE | ID: mdl-37163979

ABSTRACT

Mitochondrial dysfunction involving mitochondria-associated ER membrane (MAM) dysregulation is implicated in the pathogenesis of late-onset neurodegenerative diseases, but understanding is limited for rare early-onset conditions. Loss of the MAM-resident protein WFS1 causes Wolfram syndrome (WS), a rare early-onset neurodegenerative disease that has been linked to mitochondrial abnormalities. Here we demonstrate mitochondrial dysfunction in human induced pluripotent stem cell-derived neuronal cells of WS patients. VDAC1 is identified to interact with WFS1, whereas loss of this interaction in WS cells could compromise mitochondrial function. Restoring WFS1 levels in WS cells reinstates WFS1-VDAC1 interaction, which correlates with an increase in MAMs and mitochondrial network that could positively affect mitochondrial function. Genetic rescue by WFS1 overexpression or pharmacological agents modulating mitochondrial function improves the viability and bioenergetics of WS neurons. Our data implicate a role of WFS1 in regulating mitochondrial functionality and highlight a therapeutic intervention for WS and related rare diseases with mitochondrial defects.


Subject(s)
Induced Pluripotent Stem Cells , Neurodegenerative Diseases , Wolfram Syndrome , Humans , Wolfram Syndrome/genetics , Wolfram Syndrome/metabolism , Induced Pluripotent Stem Cells/metabolism , Neurodegenerative Diseases/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Neurons/metabolism , Mitochondria/metabolism , Mutation
6.
Int J Mol Sci ; 24(7)2023 Mar 29.
Article in English | MEDLINE | ID: mdl-37047372

ABSTRACT

Melanocytes are essential for skin homeostasis and protection, and their loss or misfunction leads to a wide spectrum of diseases. Cell therapy utilizing autologous melanocytes has been used for years as an adjunct treatment for hypopigmentary disorders such as vitiligo. However, these approaches are hindered by the poor proliferative capacity of melanocytes obtained from skin biopsies. Recent advances in the field of human pluripotent stem cells have fueled the prospect of generating melanocytes. Here, we have developed a well-characterized method to produce a pure and homogenous population of functional and proliferative melanocytes. The genetic stability and potential transformation of melanocytes from pluripotent stem cells have been evaluated over time during the in vitro culture process. Thanks to transcriptomic analysis, the molecular signatures all along the differentiation protocol have been characterized, providing a solid basis for standardizing the protocol. Altogether, our results promise meaningful, broadly applicable, and longer-lasting advances for pigmentation disorders and open perspectives for innovative biotherapies for pigment disorders.


Subject(s)
Pigmentation Disorders , Pluripotent Stem Cells , Vitiligo , Humans , Pigmentation Disorders/therapy , Melanocytes/pathology , Skin/pathology , Vitiligo/therapy , Vitiligo/pathology , Skin Pigmentation
7.
Neuropathol Appl Neurobiol ; 49(1): e12876, 2023 02.
Article in English | MEDLINE | ID: mdl-36575942

ABSTRACT

AIMS: Myotonic dystrophy type I (DM1) is one of the most frequent muscular dystrophies in adults. Although DM1 has long been considered mainly a muscle disorder, growing evidence suggests the involvement of peripheral nerves in the pathogenicity of DM1 raising the question of whether motoneurons (MNs) actively contribute to neuromuscular defects in DM1. METHODS: By using micropatterned 96-well plates as a coculture platform, we generated a functional neuromuscular model combining DM1 and muscleblind protein (MBNL) knock-out human-induced pluripotent stem cells-derived MNs and human healthy skeletal muscle cells. RESULTS: This approach led to the identification of presynaptic defects which affect the formation or stability of the neuromuscular junction at an early developmental stage. These neuropathological defects could be reproduced by the loss of RNA-binding MBNL proteins, whose loss of function in vivo is associated with muscular defects associated with DM1. These experiments indicate that the functional defects associated with MNs can be directly attributed to MBNL family proteins. Comparative transcriptomic analyses also revealed specific neuronal-related processes regulated by these proteins that are commonly misregulated in DM1. CONCLUSIONS: Beyond the application to DM1, our approach to generating a robust and reliable human neuromuscular system should facilitate disease modelling studies and drug screening assays.


Subject(s)
Induced Pluripotent Stem Cells , Myotonic Dystrophy , Adult , Humans , Myotonic Dystrophy/pathology , RNA-Binding Proteins/metabolism , Neuromuscular Junction/pathology , Induced Pluripotent Stem Cells/metabolism , Motor Neurons/pathology
8.
Nucleic Acids Res ; 50(16): 9226-9246, 2022 09 09.
Article in English | MEDLINE | ID: mdl-36039747

ABSTRACT

DDX5 and DDX17 are DEAD-box RNA helicase paralogs which regulate several aspects of gene expression, especially transcription and splicing, through incompletely understood mechanisms. A transcriptome analysis of DDX5/DDX17-depleted human cells confirmed the large impact of these RNA helicases on splicing and revealed a widespread deregulation of 3' end processing. In silico analyses and experiments in cultured cells showed the binding and functional contribution of the genome organizing factor CTCF to chromatin sites at or near a subset of DDX5/DDX17-dependent exons that are characterized by a high GC content and a high density of RNA Polymerase II. We propose the existence of an RNA helicase-dependent relationship between CTCF and the dynamics of transcription across DNA and/or RNA structured regions, that contributes to the processing of internal and terminal exons. Moreover, local DDX5/DDX17-dependent chromatin loops spatially connect RNA helicase-regulated exons with their cognate promoter, and we provide the first direct evidence that de novo gene looping modifies alternative splicing and polyadenylation. Overall our findings uncover the impact of DDX5/DDX17-dependent chromatin folding on pre-messenger RNA processing.


Subject(s)
DEAD-box RNA Helicases , RNA , Humans , RNA/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , DEAD-box RNA Helicases/metabolism , Alternative Splicing , Chromatin/genetics
9.
Nat Commun ; 13(1): 3841, 2022 07 04.
Article in English | MEDLINE | ID: mdl-35789154

ABSTRACT

Brain dysfunction in myotonic dystrophy type 1 (DM1), the prototype of toxic RNA disorders, has been mainly attributed to neuronal RNA misprocessing, while little attention has been given to non-neuronal brain cells. Here, using a transgenic mouse model of DM1 that expresses mutant RNA in various brain cell types (neurons, astroglia, and oligodendroglia), we demonstrate that astrocytes exhibit impaired ramification and polarization in vivo and defects in adhesion, spreading, and migration. RNA-dependent toxicity and phenotypes are also found in human transfected glial cells. In line with the cell phenotypes, molecular analyses reveal extensive expression and accumulation of toxic RNA in astrocytes, which result in RNA spliceopathy that is more severe than in neurons. Astrocyte missplicing affects primarily transcripts that regulate cell adhesion, cytoskeleton, and morphogenesis, and it is confirmed in human brain tissue. Our findings demonstrate that DM1 impacts astrocyte cell biology, possibly compromising their support and regulation of synaptic function.


Subject(s)
Myotonic Dystrophy , Animals , Astrocytes/metabolism , Humans , Mice , Mice, Transgenic , Myotonic Dystrophy/metabolism , RNA/genetics , RNA-Binding Proteins/metabolism , Tissue Adhesions
11.
J Invest Dermatol ; 142(10): 2695-2705.e11, 2022 10.
Article in English | MEDLINE | ID: mdl-35490743

ABSTRACT

Epidermolysis bullosa simplex (EBS), an autosomal dominant skin disorder, is characterized by skin fragility. Genetically, the majority of cases are related to missense sequence variations in two keratin genes K5 or K14, leading to cytolysis of basal keratinocytes (KCs) and intraepidermal blistering. Progress toward the identification of treatments has been hampered by an incomplete understanding of the mechanisms underlying this disease and availability of relevant and reliable in vitro models recapitulating the physiopathological mechanisms. Recent advances in stem cell field have fueled the prospect that these limitations could be overcome, thanks to the availability of disease-specific human induced pluripotent stem cells (hiPSCs). In this study, we generated hiPSC-derived KCs from patients carrying keratin gene K5-dominant sequence variations and compared them with nonaffected hiPSC-derived KCs as well as their primary counterparts. Our results showed that EBS hiPSC-derived KCs displayed proliferative defects, increased capacity to migrate, alteration of extracellular signal‒regulated kinase signaling pathway, and cytoplasmic keratin filament aggregates as observed in primary EBS KCs. Of interest, EBS hiPSC-derived KCs exhibited downregulation of hemidesmosomal proteins, revealing the different effects of keratin gene K5 sequence variations on keratin cytoskeletal organization. With a combination of culture miniaturization and treatment with the chaperone molecule 4-phenylbutyric acid, our results showed that hiPSC-derived KCs represent a suitable model for identifying novel therapies for EBS.


Subject(s)
Epidermolysis Bullosa Simplex , Induced Pluripotent Stem Cells , Epidermolysis Bullosa Simplex/metabolism , Extracellular Signal-Regulated MAP Kinases , Humans , Keratin-14/genetics , Keratin-14/metabolism , Keratin-5/genetics , Keratin-5/metabolism , Keratinocytes/metabolism , Keratins/genetics , Keratins/metabolism , Mutation , Phenotype
12.
Am J Hum Genet ; 108(11): 2171-2185, 2021 11 04.
Article in English | MEDLINE | ID: mdl-34699745

ABSTRACT

Recent studies indicate that neurodegenerative processes that appear during childhood and adolescence in individuals with Wolfram syndrome (WS) occur in addition to early brain development alteration, which is clinically silent. Underlying pathological mechanisms are still unknown. We have used induced pluripotent stem cell-derived neural cells from individuals affected by WS in order to reveal their phenotypic and molecular correlates. We have observed that a subpopulation of Wolfram neurons displayed aberrant neurite outgrowth associated with altered expression of axon guidance genes. Selective inhibition of the ATF6α arm of the unfolded protein response prevented the altered phenotype, although acute endoplasmic reticulum stress response-which is activated in late Wolfram degenerative processes-was not detected. Among the drugs currently tried in individuals with WS, valproic acid was the one that prevented the pathological phenotypes. These results suggest that early defects in axon guidance may contribute to the loss of neurons in individuals with WS.


Subject(s)
Age of Onset , Induced Pluripotent Stem Cells/cytology , Neurites , Neurons/cytology , Wolfram Syndrome/pathology , CRISPR-Cas Systems , Case-Control Studies , Endoplasmic Reticulum Stress , Gene Expression Regulation , Humans , Neurites/drug effects , Valproic Acid/pharmacology , Wolfram Syndrome/genetics
13.
Nucleic Acids Res ; 49(18): 10657-10676, 2021 10 11.
Article in English | MEDLINE | ID: mdl-34530456

ABSTRACT

Epstein-Barr virus (EBV) is a human herpesvirus associated with human cancers worldwide. Ex vivo, the virus efficiently infects resting human B lymphocytes and induces their continuous proliferation. This process is accompanied by a global reprogramming of cellular gene transcription. However, very little is known on the impact of EBV infection on the regulation of alternative splicing, a pivotal mechanism that plays an essential role in cell fate determination and is often deregulated in cancer. In this study, we have developed a systematic time-resolved analysis of cellular mRNA splice variant expression during EBV infection of resting B lymphocytes. Our results reveal that major modifications of alternative splice variant expression appear as early as day 1 post-infection and suggest that splicing regulation provides-besides transcription-an additional mechanism of gene expression regulation at the onset of B cell activation and proliferation. We also report a role for the viral proteins, EBNA2 and EBNA-LP, in the modulation of specific alternative splicing events and reveal a previously unknown function for EBNA-LP-together with the RBM4 splicing factor-in the alternative splicing regulation of two important modulators of cell proliferation and apoptosis respectively, NUMB and BCL-X.


Subject(s)
Alternative Splicing , B-Lymphocytes/virology , Herpesvirus 4, Human/physiology , Viral Proteins/metabolism , Cells, Cultured , Exons , Humans , Membrane Proteins/genetics , RNA Splice Sites , RNA-Binding Proteins/metabolism , Viral Proteins/physiology
14.
iScience ; 24(7): 102767, 2021 Jul 23.
Article in English | MEDLINE | ID: mdl-34278269

ABSTRACT

Human pluripotent stem cells have ushered in an exciting new era for disease modeling, drug discovery, and cell therapy development. Continued progress toward realizing the potential of human pluripotent stem cells will be facilitated by robust data sets and complementary resources that are easily accessed and interrogated by the stem cell community. In this context, we present SISTEMA, a quality-controlled curated gene expression database, built on a valuable catalog of human pluripotent stem cell lines, and their derivatives for which transcriptomic analyses have been generated using a single experimental pipeline. SISTEMA functions as a one-step resource that will assist the stem cell community to easily evaluate the expression level for genes of interest, while comparing them across different hPSC lines, cell types, pathological conditions, or after pharmacological treatments.

15.
Hum Mol Genet ; 31(1): 41-56, 2021 12 17.
Article in English | MEDLINE | ID: mdl-34312665

ABSTRACT

Alternative splicing has emerged as a fundamental mechanism for the spatiotemporal control of development. A better understanding of how this mechanism is regulated has the potential not only to elucidate fundamental biological principles, but also to decipher pathological mechanisms implicated in diseases where normal splicing networks are misregulated. Here, we took advantage of human pluripotent stem cells to decipher during human myogenesis the role of muscleblind-like (MBNL) proteins, a family of tissue-specific splicing regulators whose loss of function is associated with myotonic dystrophy type 1 (DM1), an inherited neuromuscular disease. Thanks to the CRISPR/Cas9 technology, we generated human-induced pluripotent stem cells (hiPSCs) depleted in MBNL proteins and evaluated the consequences of their losses on the generation of skeletal muscle cells. Our results suggested that MBNL proteins are required for the late myogenic maturation. In addition, loss of MBNL1 and MBNL2 recapitulated the main features of DM1 observed in hiPSC-derived skeletal muscle cells. Comparative transcriptomic analyses also revealed the muscle-related processes regulated by these proteins that are commonly misregulated in DM1. Together, our study reveals the temporal requirement of MBNL proteins in human myogenesis and should facilitate the identification of new therapeutic strategies capable to cope with the loss of function of these MBNL proteins.


Subject(s)
Induced Pluripotent Stem Cells , Myotonic Dystrophy , Alternative Splicing , Gene Editing , Humans , Induced Pluripotent Stem Cells/metabolism , Muscle Development/genetics , Myotonic Dystrophy/pathology , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism
16.
Front Cell Neurosci ; 15: 662035, 2021.
Article in English | MEDLINE | ID: mdl-34025359

ABSTRACT

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a non-coding CTG repeat expansion in the DMPK gene. This mutation generates a toxic CUG RNA that interferes with the RNA processing of target genes in multiple tissues. Despite debilitating neurological impairment, the pathophysiological cascade of molecular and cellular events in the central nervous system (CNS) has been less extensively characterized than the molecular pathogenesis of muscle/cardiac dysfunction. Particularly, the contribution of different cell types to DM1 brain disease is not clearly understood. We first used transcriptomics to compare the impact of expanded CUG RNA on the transcriptome of primary neurons, astrocytes and oligodendrocytes derived from DMSXL mice, a transgenic model of DM1. RNA sequencing revealed more frequent expression and splicing changes in glia than neuronal cells. In particular, primary DMSXL oligodendrocytes showed the highest number of transcripts differentially expressed, while DMSXL astrocytes displayed the most severe splicing dysregulation. Interestingly, the expression and splicing defects of DMSXL glia recreated molecular signatures suggestive of impaired cell differentiation: while DMSXL oligodendrocytes failed to upregulate a subset of genes that are naturally activated during the oligodendroglia differentiation, a significant proportion of missplicing events in DMSXL oligodendrocytes and astrocytes increased the expression of RNA isoforms typical of precursor cell stages. Together these data suggest that expanded CUG RNA in glial cells affects preferentially differentiation-regulated molecular events. This hypothesis was corroborated by gene ontology (GO) analyses, which revealed an enrichment for biological processes and cellular components with critical roles during cell differentiation. Finally, we combined exon ontology with phosphoproteomics and cell imaging to explore the functional impact of CUG-associated spliceopathy on downstream protein metabolism. Changes in phosphorylation, protein isoform expression and intracellular localization in DMSXL astrocytes demonstrate the far-reaching impact of the DM1 repeat expansion on cell metabolism. Our multi-omics approaches provide insight into the mechanisms of CUG RNA toxicity in the CNS with cell type resolution, and support the priority for future research on non-neuronal mechanisms and proteomic changes in DM1 brain disease.

17.
J Cachexia Sarcopenia Muscle ; 12(1): 209-232, 2021 02.
Article in English | MEDLINE | ID: mdl-33586340

ABSTRACT

BACKGROUND: Duchenne muscular dystrophy (DMD) causes severe disability of children and death of young men, with an incidence of approximately 1/5000 male births. Symptoms appear in early childhood, with a diagnosis made mostly around 4 years old, a time where the amount of muscle damage is already significant, preventing early therapeutic interventions that could be more efficient at halting disease progression. In the meantime, the precise moment at which disease phenotypes arise-even asymptomatically-is still unknown. Thus, there is a critical need to better define DMD onset as well as its first manifestations, which could help identify early disease biomarkers and novel therapeutic targets. METHODS: We have used both human tissue-derived myoblasts and human induced pluripotent stem cells (hiPSCs) from DMD patients to model skeletal myogenesis and compared their differentiation dynamics with that of healthy control cells by a comprehensive multi-omic analysis at seven time points. Results were strengthened with the analysis of isogenic CRISPR-edited human embryonic stem cells and through comparisons against published transcriptomic and proteomic datasets from human DMD muscles. The study was completed with DMD knockdown/rescue experiments in hiPSC-derived skeletal muscle progenitor cells and adenosine triphosphate measurement in hiPSC-derived myotubes. RESULTS: Transcriptome and miRnome comparisons combined with protein analyses demonstrated that hiPSC differentiation (i) leads to embryonic/foetal myotubes that mimic described DMD phenotypes at the differentiation endpoint and (ii) homogeneously and robustly recapitulates key developmental steps-mesoderm, somite, and skeletal muscle. Starting at the somite stage, DMD dysregulations concerned almost 10% of the transcriptome. These include mitochondrial genes whose dysregulations escalate during differentiation. We also describe fibrosis as an intrinsic feature of DMD skeletal muscle cells that begins early during myogenesis. All the omics data are available online for exploration through a graphical interface at https://muscle-dmd.omics.ovh/. CONCLUSIONS: Our data argue for an early developmental manifestation of DMD whose onset is triggered before the entry into the skeletal muscle compartment, data leading to a necessary reconsideration of dystrophin roles during muscle development. This hiPSC model of skeletal muscle differentiation offers the possibility to explore these functions as well as find earlier DMD biomarkers and therapeutic targets.


Subject(s)
Muscle Development , Muscular Dystrophy, Duchenne , Dystrophin , Humans , Induced Pluripotent Stem Cells , Male , Muscle Development/genetics , Muscular Dystrophy, Duchenne/diagnosis , Muscular Dystrophy, Duchenne/genetics , Muscular Dystrophy, Duchenne/therapy , Proteomics
18.
Nat Commun ; 11(1): 3045, 2020 06 16.
Article in English | MEDLINE | ID: mdl-32546717

ABSTRACT

Chronic NF-κB activation in inflammation and cancer has long been linked to persistent activation of NF-κB-responsive gene promoters. However, NF-κB factors also massively bind to gene bodies. Here, we demonstrate that recruitment of the NF-κB factor RELA to intragenic regions regulates alternative splicing upon NF-κB activation by the viral oncogene Tax of HTLV-1. Integrative analyses of RNA splicing and chromatin occupancy, combined with chromatin tethering assays, demonstrate that DNA-bound RELA interacts with and recruits the splicing regulator DDX17, in an NF-κB activation-dependent manner. This leads to alternative splicing of target exons due to the RNA helicase activity of DDX17. Similar results were obtained upon Tax-independent NF-κB activation, indicating that Tax likely exacerbates a physiological process where RELA provides splice target specificity. Collectively, our results demonstrate a physical and direct involvement of NF-κB in alternative splicing regulation, which significantly revisits our knowledge of HTLV-1 pathogenesis and other NF-κB-related diseases.


Subject(s)
Alternative Splicing/physiology , Gene Products, tax/metabolism , NF-kappa B/metabolism , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Gene Expression Regulation , Gene Products, tax/genetics , Human T-lymphotropic virus 1/pathogenicity , Humans , Leukocytes, Mononuclear/virology , NF-kappa B/genetics , Oncogenes , Transcription Factor RelA/metabolism
19.
Genome Biol ; 20(1): 259, 2019 11 29.
Article in English | MEDLINE | ID: mdl-31783898

ABSTRACT

BACKGROUND: Nucleotide composition bias plays an important role in the 1D and 3D organization of the human genome. Here, we investigate the potential interplay between nucleotide composition bias and the regulation of exon recognition during splicing. RESULTS: By analyzing dozens of RNA-seq datasets, we identify two groups of splicing factors that activate either about 3200 GC-rich exons or about 4000 AT-rich exons. We show that splicing factor-dependent GC-rich exons have predicted RNA secondary structures at 5' ss and are dependent on U1 snRNP-associated proteins. In contrast, splicing factor-dependent AT-rich exons have a large number of decoy branch points, SF1- or U2AF2-binding sites and are dependent on U2 snRNP-associated proteins. Nucleotide composition bias also influences local chromatin organization, with consequences for exon recognition during splicing. Interestingly, the GC content of exons correlates with that of their hosting genes, isochores, and topologically associated domains. CONCLUSIONS: We propose that regional nucleotide composition bias over several dozens of kilobase pairs leaves a local footprint at the exon level and induces constraints during splicing that can be alleviated by local chromatin organization at the DNA level and recruitment of specific splicing factors at the RNA level. Therefore, nucleotide composition bias establishes a direct link between genome organization and local regulatory processes, like alternative splicing.


Subject(s)
Base Composition , RNA Splicing , Exons , Genome, Human , Humans
20.
Nucleic Acids Res ; 47(21): 11164-11180, 2019 12 02.
Article in English | MEDLINE | ID: mdl-31602465

ABSTRACT

The CFP1 CXXC zinc finger protein targets the SET1/COMPASS complex to non-methylated CpG rich promoters to implement tri-methylation of histone H3 Lys4 (H3K4me3). Although H3K4me3 is widely associated with gene expression, the effects of CFP1 loss vary, suggesting additional chromatin factors contribute to context dependent effects. Using a proteomics approach, we identified CFP1 associated proteins and an unexpected direct link between Caenorhabditis elegans CFP-1 and an Rpd3/Sin3 small (SIN3S) histone deacetylase complex. Supporting a functional connection, we find that mutants of COMPASS and SIN3 complex components genetically interact and have similar phenotypic defects including misregulation of common genes. CFP-1 directly binds SIN-3 through a region including the conserved PAH1 domain and recruits SIN-3 and the HDA-1/HDAC subunit to H3K4me3 enriched promoters. Our results reveal a novel role for CFP-1 in mediating interaction between SET1/COMPASS and a Sin3S HDAC complex at promoters.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/embryology , DNA-Binding Proteins/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Multiprotein Complexes/physiology , Sin3 Histone Deacetylase and Corepressor Complex/metabolism , Trans-Activators/metabolism , Animals , Animals, Genetically Modified , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , DNA-Binding Proteins/genetics , Embryo, Nonmammalian , Histone-Lysine N-Methyltransferase/physiology , Multiprotein Complexes/metabolism , Protein Binding
SELECTION OF CITATIONS
SEARCH DETAIL
...