Protein kinase A determines timing of early differentiation through epigenetic regulation with G9a.
Timing of cell differentiation is strictly controlled and is crucial for normal development and stem cell differentiation. However, underlying mechanisms regulating differentiation timing are fully unknown. Here, we show a molecular mechanism determining differentiation timing from mouse embryonic stem cells (ESCs). Activation of protein kinase A (PKA) modulates differentiation timing to accelerate the appearance of mesoderm and other germ layer cells, reciprocally correlated with the earlier disappearance of pluripotent markers after ESC differentiation. PKA activation increases protein expression of G9a, an H3K9 methyltransferase, along with earlier H3K9 dimethylation and DNA methylation in Oct3/4 and Nanog gene promoters. Deletion of G9a completely abolishes PKA-elicited acceleration of differentiation and epigenetic modification. Furthermore, G9a knockout mice show prolonged expressions of Oct3/4 and Nanog at embryonic day 7.5 and delayed development. In this study, we demonstrate molecular machinery that regulates timing of multilineage differentiation by linking signaling with epigenetics.
Department of Stem Cell Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan; Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
Kohei
K
Yamamizu
Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
Mayako
M
Fujihara
Experimental Research Center for Infectious Disease, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan.
Makoto
M
Tachibana
Department of Stem Cell Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan; Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
Shiori
S
Katayama
Cancer Institute, Japanese Foundation for Cancer Research, Tokyo 135-8550, Japan.
Akiko
A
Takahashi
Cancer Institute, Japanese Foundation for Cancer Research, Tokyo 135-8550, Japan.
Eiji
E
Hara
Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
Hiroshi
H
Imai
Experimental Research Center for Infectious Disease, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan.
Yoichi
Y
Shinkai
Department of Stem Cell Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan; Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan. Electronic address: juny@frontier.kyoto-u.ac.jp.
Jun K
JK
Yamashita
D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D047369:Chromatin Immunoprecipitation; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D004622:Embryo, Mammalian / Q000166:cytology* / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D044127:Epigenesis, Genetic; D005260:Female; D018507:Gene Expression Regulation, Developmental; D011495:Histone-Lysine N-Methyltransferase / Q000502:physiology*; D006657:Histones / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D017403:In Situ Hybridization; D051379:Mice; D018345:Mice, Knockout; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D006657:Histones; D018398:Homeodomain Proteins; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D012333:RNA, Messenger; C533711:G9a protein, mouse; D011495:Histone-Lysine N-Methyltransferase; D017868:Cyclic AMP-Dependent Protein Kinases