TY - JOUR
T1 - T396I mutation of mouse Sufu reduces the stability and activity of Gli3 repressor
AU - Makino, Shigeru
AU - Zhulyn, Olena
AU - Mo, Rong
AU - Puviindran, Vijitha
AU - Zhang, Xiaoyun
AU - Murata, Takuya
AU - Fukumura, Ryutaro
AU - Ishitsuka, Yuichi
AU - Kotaki, Hayato
AU - Matsumaru, Daisuke
AU - Ishii, Shunsuke
AU - Hui, Chi Chung
AU - Gondo, Yoichi
N1 - Funding Information:
We thank H. Sasaki for the 8xGliBS-luciferase vector, U. Ruther for the Gli3 mouse line, and P.T. Chuang for the Sufu cell line. We also thank A. Joyner, H. Masuya, M. Scott, and K. Imai for providing the in situ probes. NIH3T3 (RCB2767) and 293T (RCB2202) were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. Monoclonal antibodies against Pax7, Pax6, Nkx2.2, and FoxA2 developed by T. Jessell were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. ∆699 −/−
Publisher Copyright:
© 2015 Makino et al.
PY - 2015/3/11
Y1 - 2015/3/11
N2 - Hedgehog signaling is primarily transduced by two transcription factors: Gli2, which mainly acts as a full-length activator, and Gli3, which tends to be proteolytically processed from a full-length form (Gli3FL) to an N-terminal repressor (Gli3REP). Recent studies using a Sufu knockout mouse have indicated that Sufu is involved in regulating Gli2 and Gli3 activator and repressor activity at multiple steps of the signaling cascade; however, the mechanism of specific Gli2 and Gli3 regulation remains to be elucidated. In this study, we established an allelic series of ENU-induced mouse strains. Analysis of one of the missense alleles, SufuT396I, showed that Thr396residue of Sufu played a key role in regulation of Gli3 activity. SufuT396I/T396I embryos exhibited severe polydactyly, which is indicative of compromised Gli3 activity. Concomitantly, significant quantitative reductions of unprocessed Gli3 (Gli3FL) and processed Gli3 (Gli3REP) were observed in vivo as well as in vitro. Genetic experiments showed that patterning defects in the limb buds of SufuT396I/T396I were rescued by a constitutive Gli3REP allele (Gli3Δ699), strongly suggesting that SufuT396I reduced the truncated Gli3 repressor. In contrast, SufuT396I qualitatively exhibited no mutational effects on Gli2 regulation. Taken together, the results of this study show that the Thr396residue of Sufu is specifically required for regulation of Gli3 but not Gli2. This implies a novel Sufu-mediated mechanism in which Gli2 activator and Gli3 repressor are differentially regulated.
AB - Hedgehog signaling is primarily transduced by two transcription factors: Gli2, which mainly acts as a full-length activator, and Gli3, which tends to be proteolytically processed from a full-length form (Gli3FL) to an N-terminal repressor (Gli3REP). Recent studies using a Sufu knockout mouse have indicated that Sufu is involved in regulating Gli2 and Gli3 activator and repressor activity at multiple steps of the signaling cascade; however, the mechanism of specific Gli2 and Gli3 regulation remains to be elucidated. In this study, we established an allelic series of ENU-induced mouse strains. Analysis of one of the missense alleles, SufuT396I, showed that Thr396residue of Sufu played a key role in regulation of Gli3 activity. SufuT396I/T396I embryos exhibited severe polydactyly, which is indicative of compromised Gli3 activity. Concomitantly, significant quantitative reductions of unprocessed Gli3 (Gli3FL) and processed Gli3 (Gli3REP) were observed in vivo as well as in vitro. Genetic experiments showed that patterning defects in the limb buds of SufuT396I/T396I were rescued by a constitutive Gli3REP allele (Gli3Δ699), strongly suggesting that SufuT396I reduced the truncated Gli3 repressor. In contrast, SufuT396I qualitatively exhibited no mutational effects on Gli2 regulation. Taken together, the results of this study show that the Thr396residue of Sufu is specifically required for regulation of Gli3 but not Gli2. This implies a novel Sufu-mediated mechanism in which Gli2 activator and Gli3 repressor are differentially regulated.
UR - http://www.scopus.com/inward/record.url?scp=84924353437&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84924353437&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0119455
DO - 10.1371/journal.pone.0119455
M3 - Article
C2 - 25760946
AN - SCOPUS:84924353437
VL - 10
JO - PLoS One
JF - PLoS One
SN - 1932-6203
IS - 3
M1 - e0119455
ER -