Pax7 activates myogenic genes by recruitment of a histone methyltransferase complex.
Satellite cells purified from adult skeletal muscle can participate extensively in muscle regeneration and can also re-populate the satellite cell pool, suggesting that they have direct therapeutic potential for treating degenerative muscle diseases. The paired-box transcription factor Pax7 is required for satellite cells to generate committed myogenic progenitors. In this study we undertook a multi-level approach to define the role of Pax7 in satellite cell function. Using comparative microarray analysis, we identified several novel and strongly regulated targets; in particular, we identified Myf5 as a gene whose expression was regulated by Pax7. Using siRNA, fluorescence-activated cell sorting (FACS) and chromatin immunoprecipitation (ChIP) studies we confirmed that Myf5 is directly regulated by Pax7 in myoblasts derived from satellite cells. Tandem affinity purification (TAP) and mass spectrometry were used to purify Pax7 together with its co-factors. This revealed that Pax7 associates with the Wdr5-Ash2L-MLL2 histone methyltransferase (HMT) complex that directs methylation of histone H3 lysine 4 (H3K4, refs 4-10). Binding of the Pax7-HMT complex to Myf5 resulted in H3K4 tri-methylation of surrounding chromatin. Thus, Pax7 induces chromatin modifications that stimulate transcriptional activation of target genes to regulate entry into the myogenic developmental programme.
McKinnell
Iain W
IW
Sprott Centre for Stem Cell Research, Ottawa Health Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6.
Ishibashi
Jeff
J
Le Grand
Fabien
F
Punch
Vincent G J
VG
Addicks
Gregory C
GC
Greenblatt
Jack F
JF
Dilworth
F Jeffrey
FJ
Rudnicki
Michael A
MA
D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins; D008745:Methylation; D051379:Mice; D008810:Mice, Inbred C57BL; D051788:Myeloid-Lymphoid Leukemia Protein / Q000235:genetics / Q000378:metabolism; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism; D051787:Myogenic Regulatory Factor 5 / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D051763:PAX7 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011496:Protein Methyltransferases; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
C120115:Ash2l protein, mouse; D002843:Chromatin; D004268:DNA-Binding Proteins; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; C497418:Myf5 protein, mouse; D051787:Myogenic Regulatory Factor 5; D009687:Nuclear Proteins; D051763:PAX7 Transcription Factor; D011506:Proteins; D034741:RNA, Small Interfering; D014157:Transcription Factors; C443118:Wdr5 protein, mouse; D051788:Myeloid-Lymphoid Leukemia Protein; D000076983:Histone Methyltransferases; D011496:Protein Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; C510860:Kmt2d protein, mouse