Abstract
Hedgehog (Hh) signaling plays vital roles in animal development and tissue homeostasis, and its misregulation causes congenital diseases and several types of cancer. Suppressor of Fused (Su(fu)) is a conserved inhibitory component of the Hh signaling pathway, but how it is regulated remains poorly understood. Here we demonstrate that in Drosophila Hh signaling promotes downregulation of Su(fu) through its target protein HIB (Hh-induced BTB protein). Interestingly, although HIB-mediated downregulation of Su(fu) depends on the E3 ubiquitin ligase Cul3, HIB does not directly regulate Su(fu) protein stability. Through an RNAi-based candidate gene screen, we identify the spliceosome factor Crooked neck (Crn) as a regulator of Su(fu) level. Epistasis analysis indicates that HIB downregulates Su(fu) through Crn. Furthermore, we provide evidence that HIB retains Crn in the nucleus, leading to reduced Su(fu) protein level. Finally, we show that SPOP, the mammalian homologue of HIB, can substitute HIB to downregulate Su(fu) level in Drosophila. Our study suggests that Hh regulates both Ci and Su(fu) levels through its target HIB, thus uncovering a novel feedback mechanism that regulates Hh signal transduction. The dual function of HIB may provide a buffering mechanism to fine-tune Hh pathway activity.
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Acknowledgements
We thank Dr Yun Zhao (Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China), Fly Stocks of National Institute of Genetics of Japan (NIG-Fly), Vienna Drosophila RNAi Center (VDRC), the Bloomington Stock Center and Developmental Studies Hybridoma Bank at the University of Iowa for providing fly stocks and reagents. This work was supported by grants from the National Natural Science Foundation of China (30971679, 31071264 and 31271531) and the National Key Scientific Program of China (2011CB943902, 2010CB945102) to Qing Zhang. Jin Jiang is supported by grants from NIH (GM061269 and GM06745), Welch foundation (I-1603) and NSFC (31328017).
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Supplementary information
Supplementary information, Figure S1
HIB neither binds Su(fu) nor affects the stability of Su(fu) protein in S2 cells. (PDF 717 kb)
Supplementary information, Figure S2
HIB doesn't bind Crn in S2 cells. HA-Crn and Myc-HIB were cotransfected in S2 cells. (PDF 263 kb)
Supplementary information, Figure S3
SPOP downregulates endogenous Su(fu) in wing discs but does not change the stabililty of exogenous HA-Su(fu) in S2 cells. (PDF 618 kb)
Supplementary information, Figure S4
Hh doesn't affect su(fu) mRNA level or the stability of Su(fu) in the S2 cells. (PDF 344 kb)
Supplementary information, Figure S5
Hh affects Su(fu) level in S2 cells. (PDF 369 kb)
Supplementary information, Figure S6
HIB-Crn affects Su(fu) level in the S2 cells. (PDF 572 kb)
Supplementary information, Figure S7
Loss of Su(fu) alters Hh signaling in a genetic sensitized background. (PDF 327 kb)
Supplementary information, Figure S8
HIB doesn't affect su(fu) mRNA nuclear export. (PDF 194 kb)
Supplementary information, Figure S9
HIB may affect su(fu) translation. (PDF 369 kb)
Supplementary information, Figure S10
Endogenous crn is ubiquitously expressed in wing (A, B) and eye (C, D) discs detected by in situ hybridization. (PDF 275 kb)
Supplementary information, Table S1
The expression of Su(fu) affected by knockdown of the candidate genes in wing discs. (PDF 63 kb)
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Liu, C., Zhou, Z., Yao, X. et al. Hedgehog signaling downregulates Suppressor of Fused through the HIB/SPOP-Crn axis in Drosophila. Cell Res 24, 595–609 (2014). https://doi.org/10.1038/cr.2014.29
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DOI: https://doi.org/10.1038/cr.2014.29
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