The EF-hand calcium-binding protein calmyrin inhibits the transcriptional and DNA-binding activity of Pax3.
Pax3 is a member of the paired class homeodomain family of transcription factors and has been demonstrated to be an early marker in myogenic differentiation. To gain a better understanding of how protein-protein interactions regulate Pax3 transcriptional activity, we performed a yeast two-hybrid analysis to identify proteins that interact with Pax3. Screening of two cDNA libraries isolated nine independent clones that contained the complete encoding sequence of the EF-hand calcium-binding protein calmyrin. In this report, we demonstrate that calmyrin specifically interacts with Pax3 in vitro. In addition, we demonstrate that the interaction between Pax3 and calmyrin is mediated by the region of the Pax3 paired domain that is involved in making DNA contacts and the Pax3 octapeptide domain and its surrounding amino acid sequences. We also demonstrate that endogenous Pax3 and calmyrin are co-expressed in undifferentiated primary myoblasts and that calmyrin expression levels increase in the nucleus upon myogenic differentiation. Finally, we demonstrate that co-expression of calmyrin with Pax3 inhibits the transcriptional activity of Pax3 by inhibiting Pax3 from binding to its recognition DNA sequences. These results therefore suggest potential ways in which calcium, through its regulation of the EF-hand calcium-binding protein calmyrin, can alter the DNA-binding activity and subsequent transcriptional activity of Pax3.
Hollenbach
Andrew D
AD
Department of Genetics, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA.
McPherson
Craig J
CJ
Lagutina
Irina
I
Grosveld
Gerard
G
D016475:3T3 Cells; D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D002454:Cell Differentiation; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D020832:EF Hand Motifs; D015723:Gene Library; D007668:Kidney / Q000378:metabolism; D008074:Lipoproteins / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D051379:Mice; D024510:Muscle Development; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D000072041:PAX3 Transcription Factor; D051761:Paired Box Transcription Factors; D010766:Phosphorylation; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics; D020798:Two-Hybrid System Techniques
D002135:Calcium-Binding Proteins; C491605:Cib1 protein, mouse; D004268:DNA-Binding Proteins; D008074:Lipoproteins; D000072041:PAX3 Transcription Factor; D051761:Paired Box Transcription Factors; D012333:RNA, Messenger; D014157:Transcription Factors; C068686:Pax3 protein, mouse