ABSTRACT
Myogenic regulatory factors of the MyoD family have the ability to reprogram differentiated cells toward a myogenic fate. In this study, we demonstrate that Six1 or Six4 are required for the reprogramming by MyoD of mouse embryonic fibroblasts (MEFs). Using microarray experiments, we found 761 genes under the control of both Six and MyoD. Using MyoD ChIPseq data and a genome-wide search for Six1/4 MEF3 binding sites, we found significant co-localization of binding sites for MyoD and Six proteins on over a thousand mouse genomic DNA regions. The combination of both datasets yielded 82 genes which are synergistically activated by Six and MyoD, with 96 associated MyoD+MEF3 putative cis-regulatory modules (CRMs). Fourteen out of 19 of the CRMs that we tested demonstrated in Luciferase assays a synergistic action also observed for their cognate gene. We searched putative binding sites on these CRMs using available databases and de novo search of conserved motifs and demonstrated that the Six/MyoD synergistic activation takes place in a feedforward way. It involves the recruitment of these two families of transcription factors to their targets, together with partner transcription factors, encoded by genes that are themselves activated by Six and MyoD, including Mef2, Pbx-Meis and EBF.
Subject(s)
Cellular Reprogramming/genetics , Genome , Homeodomain Proteins/metabolism , MyoD Protein/metabolism , Regulatory Sequences, Nucleic Acid/genetics , Trans-Activators/metabolism , Animals , Base Sequence , Binding Sites/genetics , Cells, Cultured , Chromatin Immunoprecipitation , Embryo, Mammalian/cytology , Fibroblasts/metabolism , Homeodomain Proteins/genetics , Humans , Luciferases/metabolism , Mice, Knockout , Muscle Development/genetics , Mutation/genetics , Nuclear Proteins/metabolism , Nucleotide Motifs/genetics , Reproducibility of Results , Trans-Activators/genetics , Transcription Factors/metabolismABSTRACT
Myotonic dystrophy type 1 (DM1) is an RNA-mediated disorder caused by a non-coding CTG repeat expansion that, in particular, provokes functional alteration of CUG-binding proteins. As a consequence, several genes with misregulated alternative splicing have been linked to clinical symptoms. In our search for additional molecular mechanisms that would trigger functional defects in DM1, we took advantage of mutant gene-carrying human embryonic stem cell lines to identify differentially expressed genes. Among the different genes found to be misregulated by DM1 mutation, one strongly downregulated gene encodes a transcription factor, ZNF37A. In this paper, we show that this defect in expression, which derives from a loss of RNA stability, is controlled by the RNA-binding protein, CUGBP1, and is associated with impaired myogenesis-a functional defect reminiscent of that observed in DM1. Loss of the ZNF37A protein results in changes in the expression of the subunit α1 of the receptor for the interleukin 13. This suggests that the pathological molecular mechanisms linking ZNF37A and myogenesis may involve the signaling pathway that is known to promote myoblast recruitment during development and regeneration.
Subject(s)
Alternative Splicing/genetics , Kruppel-Like Transcription Factors/genetics , Muscle Development/genetics , Myotonic Dystrophy/genetics , Trinucleotide Repeat Expansion/genetics , Cell Line , Cell Nucleus/genetics , Cell Nucleus/metabolism , Embryonic Stem Cells , Humans , Interleukin-13 Receptor alpha1 Subunit/genetics , Interleukin-13 Receptor alpha1 Subunit/metabolism , Mutation , Myotonic Dystrophy/physiopathology , Signal Transduction/geneticsABSTRACT
Patients with myotonic dystrophy type 1 exhibit a diversity of symptoms that affect many different organs. Among these are cognitive dysfunctions, the origin of which has remained elusive, partly because of the difficulty in accessing neural cells. Here, we have taken advantage of pluripotent stem cell lines derived from embryos identified during a pre-implantation genetic diagnosis for mutant-gene carriers, to produce early neuronal cells. Functional characterization of these cells revealed reduced proliferative capacity and increased autophagy linked to mTOR signaling pathway alterations. Interestingly, loss of function of MBNL1, an RNA-binding protein whose function is defective in DM1 patients, resulted in alteration of mTOR signaling, whereas gain-of-function experiments rescued the phenotype. Collectively, these results provide a mechanism by which DM1 mutation might affect a major signaling pathway and highlight the pertinence of using pluripotent stem cells to study neuronal defects.
Subject(s)
Embryonic Stem Cells/cytology , Myotonic Dystrophy/metabolism , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , TOR Serine-Threonine Kinases/metabolism , Apoptosis/genetics , Apoptosis/physiology , Blotting, Western , Cell Line , Cell Proliferation , Cellular Senescence/genetics , Cellular Senescence/physiology , Electrophoresis, Polyacrylamide Gel , Humans , Immunohistochemistry , In Situ Hybridization , Myotonic Dystrophy/genetics , Real-Time Polymerase Chain Reaction , TOR Serine-Threonine Kinases/geneticsABSTRACT
Considerable hope surrounds the use of disease-specific pluripotent stem cells to generate models of human disease allowing exploration of pathological mechanisms and search for new treatments. Disease-specific human embryonic stem cells were the first to provide a useful source for studying certain disease states. The recent demonstration that human somatic cells, derived from readily accessible tissue such as skin or blood, can be converted to embryonic-like induced pluripotent stem cells (hiPSCs) has opened new perspectives for modelling and understanding a larger number of human pathologies. In this review, we examine the opportunities and challenges for the use of disease-specific pluripotent stem cells in disease modelling and drug screening. Progress in these areas will substantially accelerate effective application of disease-specific human pluripotent stem cells for drug screening.
Subject(s)
Drug Discovery , Drug Evaluation, Preclinical/methods , Induced Pluripotent Stem Cells , Cell Differentiation , Cells, Cultured , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Epigenesis, Genetic , Genetic Variation , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolismSubject(s)
Genetic Diseases, Inborn/pathology , Pluripotent Stem Cells/cytology , Adult , Cell Differentiation/drug effects , Cell Line , Coculture Techniques , Genetic Diseases, Inborn/genetics , Hedgehog Proteins/pharmacology , Humans , Membrane Proteins/deficiency , Membrane Proteins/genetics , Membrane Proteins/physiology , Models, Biological , Motor Neurons/pathology , Myotonic Dystrophy/pathology , Myotonin-Protein Kinase , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/physiology , Neurites/pathology , Pluripotent Stem Cells/drug effects , Protein Serine-Threonine Kinases/genetics , Research Design , Research Embryo Creation , Stromal Cells/physiology , Synapses/ultrastructure , Tretinoin/pharmacology , Trinucleotide Repeat ExpansionABSTRACT
Considerable hope surrounds the use of disease-specific pluripotent stem cells, which can differentiate into any cell type, as starting materials to generate models of human disease that will allow exploration of pathological mechanisms and the search for new treatments. Disease-specific human embryonic stem cells have provided a useful source for studying certain disease states. However, reprogramming of human somatic cells that use readily accessible tissue, such as skin or blood, to generate embryonic-like induced pluripotent stem cells has opened new perspectives for modeling and understanding a larger number of human pathologies. Here, we examine the challenges in creating a disease model from human pluripotent stem cells, and describe their use to model both cell-autonomous and non-cell-autonomous mechanisms, the need for adequate control experiments and the genetic limitations of human induced pluripotent stem cells. Progress in these areas will substantially accelerate effective application of disease-specific human pluripotent stem cells for drug screening.
Subject(s)
Drug Evaluation, Preclinical/methods , Genetic Diseases, Inborn/etiology , Pluripotent Stem Cells/physiology , Cell Differentiation , Epigenomics , Genetic Diseases, Inborn/drug therapy , Humans , Pluripotent Stem Cells/drug effects , Pluripotent Stem Cells/pathologyABSTRACT
Myotonic dystrophy type 1 (DM1) is a multisystem disorder affecting a variety of organs, including the central nervous system. By using neuronal progeny derived from human embryonic stem cells carrying the causal DM1 mutation, we have identified an early developmental defect in genes involved in neurite formation and the establishment of neuromuscular connections. Differential gene expression profiling and quantitative RT-PCR revealed decreased expression of two members of the SLITRK family in DM1 neural cells and in DM1 brain biopsies. In addition, DM1 motoneuron/muscle cell cocultures showed alterations that are consistent with the known role of SLITRK genes in neurite outgrowth, neuritogenesis, and synaptogenesis. Rescue and knockdown experiments suggested that the functional defects can be directly attributed to SLITRK misexpression. These neuropathological mechanisms may be clinically significant for the functional changes in neuromuscular connections associated with DM1.