Inhibition of miRNA-212/132 improves the reprogramming of fibroblasts into induced pluripotent stem cells by de-repressing important epigenetic remodelling factors.
MicroRNAs (miRNAs) repeatedly have been demonstrated to play important roles in the generation of induced pluripotent stem cells (iPSCs). To further elucidate the molecular mechanisms underlying transcription factor-mediated reprogramming we have established a model, which allows for the efficient screening of whole libraries of miRNAs modulating the generation of iPSCs from murine embryonic fibroblasts. Applying this model, we identified 14 miRNAs effectively inhibiting iPSC generation, including miR-132 and miR-212. Intriguingly, repression of these miRNAs during iPSC generation also resulted in significantly increased reprogramming efficacy. MiRNA target evaluation by qRT-PCR, Western blot, and luciferase assays revealed two crucial epigenetic regulators, the histone acetyl transferase p300 as well as the H3K4 demethylase Jarid1a (KDM5a) to be directly targeted by both miRNAs. Moreover, we demonstrated that siRNA-mediated knockdown of either p300 or Jarid1a recapitulated the miRNA effects and led to a significant decrease in reprogramming efficiency. Thus, conducting a full library miRNA screen we here describe a miRNA family, which markedly reduces generation of iPSC and upon inhibition in turn enhances reprogramming. These miRNAs, at least in part, exert their functions through repression of the epigenetic modulators p300 and Jarid1a, highlighting these two molecules as an endogenous epigenetic roadblock during iPSC generation.
Research-Group Reprogramming and Gene Therapy, Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; REBIRTH-Group Regenerative Gene Therapy, Hannover Medical School, Hannover, Germany.
Nils
N
Pfaff
Research-Group Reprogramming and Gene Therapy, Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; REBIRTH-Group Regenerative Gene Therapy, Hannover Medical School, Hannover, Germany.
Steffi
S
Liebhaber
REBIRTH-Group Translational Hepatology and Stem Cell Biology, Dept. of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany.
Selina
S
Möbus
REBIRTH-Group Translational Hepatology and Stem Cell Biology, Dept. of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany.
Abbas
A
Beh-Pajooh
Institute of Molecular and Translational Therapeutic Strategies, IFB-Tx, Hannover Medical School, Hannover, Germany.
Jan
J
Fiedler
Institute of Molecular and Translational Therapeutic Strategies, IFB-Tx, Hannover Medical School, Hannover, Germany.
Angelika
A
Pfanne
REBIRTH-Group Regenerative Gene Therapy, Hannover Medical School, Hannover, Germany; Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, USA.
Axel
A
Schambach
Institute of Molecular and Translational Therapeutic Strategies, IFB-Tx, Hannover Medical School, Hannover, Germany; National Heart and Lung Institute, Imperial College London, London, UK; REBIRTH-Group Translational Strategies, Hannover Medical School, Hannover, Germany.
Thomas
T
Thum
REBIRTH-Group Translational Hepatology and Stem Cell Biology, Dept. of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany. Electronic address: cantz.tobias@mh-hannover.de.
Tobias
T
Cantz
Research-Group Reprogramming and Gene Therapy, Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; REBIRTH-Group Regenerative Gene Therapy, Hannover Medical School, Hannover, Germany. Electronic address: moritz.thomas@mh-hannover.de.
Thomas
T
Moritz
D020413:3' Untranslated Regions; D000818:Animals; D000070416:Antagomirs / Q000378:metabolism; D001483:Base Sequence; D002454:Cell Differentiation; D002460:Cell Line; D065150:Cellular Reprogramming; D057890:Epigenomics / Q000379:methods*; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D056484:Jumonji Domain-Containing Histone Demethylases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D016415:Sequence Alignment; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D050880:p300-CBP Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D020413:3' Untranslated Regions; D000070416:Antagomirs; D035683:MicroRNAs; D034741:RNA, Small Interfering; D014157:Transcription Factors; D056484:Jumonji Domain-Containing Histone Demethylases; D050880:p300-CBP Transcription Factors
Cellular reprogramming; Epigenetic remodelling; Induced pluripotent stem cells; MicroRNAs