MyoD reprogramming requires Six1 and Six4 homeoproteins: genome-wide cis-regulatory module analysis.
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.
Institut Cochin, Université Paris-Descartes, Centre National de la Recherche Scientifique (CNRS), UMR 8104, Paris, France Institut National de la Santé et de la Recherche Médicale (INSERM) U1016, Paris, France Ecole Normale Supérieure, CNRS, Laboratoire de Physique Statistique, PSL Research University, Université Pierre-et-Marie Curie, Paris, France.
Marc
M
Santolini
Institut Cochin, Université Paris-Descartes, Centre National de la Recherche Scientifique (CNRS), UMR 8104, Paris, France Institut National de la Santé et de la Recherche Médicale (INSERM) U1016, Paris, France Division of Integrative Pathophysiology, Proteo-Science Center, Graduate School of Medicine, Ehime University, Ehime, Japan.
Iori
I
Sakakibara
Institut Cochin, Université Paris-Descartes, Centre National de la Recherche Scientifique (CNRS), UMR 8104, Paris, France Institut National de la Santé et de la Recherche Médicale (INSERM) U1016, Paris, France.
Morgane
M
Gauthier
Institut Cochin, Université Paris-Descartes, Centre National de la Recherche Scientifique (CNRS), UMR 8104, Paris, France Institut National de la Santé et de la Recherche Médicale (INSERM) U1016, Paris, France.
Francesc
F
Ribas-Aulinas
Proteo-Science Center, Ehime University, Ehime 791-8577, Japan.
Hirotaka
H
Takahashi
Proteo-Science Center, Ehime University, Ehime 791-8577, Japan.
Tatsuya
T
Sawasaki
Sorbonne Universités, UPMC Univ Paris 06, INSERM UMRS974, CNRS FRE3617, Center for Research in Myology, 75013 Paris, France.
Vincent
V
Mouly
Institut Cochin, Université Paris-Descartes, Centre National de la Recherche Scientifique (CNRS), UMR 8104, Paris, France Institut National de la Santé et de la Recherche Médicale (INSERM) U1016, Paris, France.
Jean-Paul
JP
Concordet
University Paris Diderot, Sorbonne Paris Cité, UMR 7216 CNRS, 75013 Paris, France.
Pierre-Antoine
PA
Defossez
Ecole Normale Supérieure, CNRS, Laboratoire de Physique Statistique, PSL Research University, Université Pierre-et-Marie Curie, Paris, France hakim@lps.ens.fr.
Vincent
V
Hakim
Institut Cochin, Université Paris-Descartes, Centre National de la Recherche Scientifique (CNRS), UMR 8104, Paris, France Institut National de la Santé et de la Recherche Médicale (INSERM) U1016, Paris, France pascal.maire@inserm.fr.
Pascal
P
Maire
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000235:genetics*; D047369:Chromatin Immunoprecipitation; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000378:metabolism; D016678:Genome; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D018345:Mice, Knockout; D024510:Muscle Development / Q000235:genetics; D009154:Mutation / Q000235:genetics; D017570:MyoD Protein / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D059372:Nucleotide Motifs / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D015203:Reproducibility of Results; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism
D018398:Homeodomain Proteins; D017570:MyoD Protein; D009687:Nuclear Proteins; C490947:Six1 protein, mouse; C098490:Six4 protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; D008156:Luciferases