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Regulation of a remote Shh forebrain enhancer by the Six3 homeoprotein

Abstract

In humans, SHH haploinsufficiency results in holoprosencephaly (HPE), a defect in anterior midline formation1,2. Despite the importance of maintaining SHH transcript levels above a critical threshold, we know little about the upstream regulators of SHH expression in the forebrain. Here we describe a rare nucleotide variant located 460 kb upstream of SHH in an individual with HPE that resulted in the loss of Shh brain enhancer-2 (SBE2) activity in the hypothalamus of transgenic mouse embryos. Using a DNA affinity-capture assay, we screened the SBE2 sequence for DNA-binding proteins and identified members of the Six3 and Six6 homeodomain family as candidate regulators of Shh transcription. Six3 showed reduced binding affinity for the mutant compared to the wild-type SBE2 sequence. Moreover, Six3 with HPE-causing alterations failed to bind and activate SBE2. These data suggest a direct link between Six3 and Shh regulation during normal forebrain development and in the pathogenesis of HPE.

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Figure 1: SBE2 activity in the rostral hypothalamus is compromised by a sequence variant found in an individual with HPE.
Figure 2: Six3 and Six6 proteins bind directly to SBE2.
Figure 3: Overlap of Shh and Six3 and Six6 expression in the ventral diencephalon.
Figure 4: Six3 binds SBE2(C) with higher affinity than SBE2(T).
Figure 5: HPE-causing mutations in Six3 alter binding and activation of SBE2.

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References

  1. Roessler, E. et al. Mutations in the human Sonic Hedgehog gene cause holoprosencephaly. Nat. Genet. 14, 357–360 (1996).

    Article  CAS  Google Scholar 

  2. Dubourg, C. et al. Holoprosencephaly. Orphanet J. Rare Dis. 2, 8 (2007).

    Article  Google Scholar 

  3. Helms, J.A., Cordero, D. & Tapadia, M.D. New insights into craniofacial morphogenesis. Development 132, 851–861 (2005).

    Article  CAS  Google Scholar 

  4. Fuccillo, M., Joyner, A.L. & Fishell, G. Morphogen to mitogen: the multiple roles of hedgehog signalling in vertebrate neural development. Nat. Rev. Neurosci. 7, 772–783 (2006).

    Article  CAS  Google Scholar 

  5. Shimamura, K. & Rubenstein, J.L. Inductive interactions direct early regionalization of the mouse forebrain. Development 124, 2709–2718 (1997).

    CAS  PubMed  Google Scholar 

  6. Fuccillo, M., Rallu, M., McMahon, A.P. & Fishell, G. Temporal requirement for hedgehog signaling in ventral telencephalic patterning. Development 131, 5031–5040 (2004).

    Article  CAS  Google Scholar 

  7. Marcucio, R.S., Cordero, D.R., Hu, D. & Helms, J.A. Molecular interactions coordinating the development of the forebrain and face. Dev. Biol. 284, 48–61 (2005).

    Article  CAS  Google Scholar 

  8. Hu, D., Marcucio, R.S. & Helms, J.A. A zone of frontonasal ectoderm regulates patterning and growth in the face. Development 130, 1749–1758 (2003).

    Article  CAS  Google Scholar 

  9. Chiang, C. et al. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383, 407–413 (1996).

    Article  CAS  Google Scholar 

  10. Cordero, D. et al. Temporal perturbations in sonic hedgehog signaling elicit the spectrum of holoprosencephaly phenotypes. J. Clin. Invest. 114, 485–494 (2004).

    Article  CAS  Google Scholar 

  11. Jeong, Y., El-Jaick, K., Roessler, E., Muenke, M. & Epstein, D.J. A functional screen for Sonic hedgehog regulatory elements across a 1 Mb interval identifies long range ventral forebrain enhancers. Development 133, 761–772 (2006).

    Article  CAS  Google Scholar 

  12. Ming, J.E. & Muenke, M. Multiple hits during early embryonic development: digenic diseases and holoprosencephaly. Am. J. Hum. Genet. 71, 1017–1032 (2002).

    Article  CAS  Google Scholar 

  13. Treier, M. et al. Hedgehog signaling is required for pituitary gland development. Development 128, 377–386 (2001).

    CAS  PubMed  Google Scholar 

  14. Roessler, E. et al. Loss-of-function mutations in the human GLI2 gene are associated with pituitary anomalies and holoprosencephaly-like features. Proc. Natl. Acad. Sci. USA 100, 13424–13429 (2003).

    Article  CAS  Google Scholar 

  15. Park, S.S., Ko, B.J.a & Kim, B.G. Mass spectrometric screening of transcriptional regulators using DNA affinity capture assay. Anal. Biochem. 344, 152–154 (2005).

    Article  CAS  Google Scholar 

  16. Jean, D., Bernier, G. & Gruss, P. Six6 (Optx2) is a novel murine Six3-related homeobox gene that demarcates the presumptive pituitary/hypothalamic axis and the ventral optic stalk. Mech. Dev. 84, 31–40 (1999).

    Article  CAS  Google Scholar 

  17. Wallis, D.E. et al. Mutations in the homeodomain of the human SIX3 gene cause holoprosencephaly. Nat. Genet. 22, 196–198 (1999).

    Article  CAS  Google Scholar 

  18. Lagutin, O.V. et al. Six3 repression of Wnt signaling in the anterior neuroectoderm is essential for vertebrate forebrain development. Genes Dev. 17, 368–379 (2003).

    Article  CAS  Google Scholar 

  19. Zhu, C. et al. Six3-mediated auto repression and eye development requires its interaction with members of the Groucho-related family of co-repressors. Development 129, 2835–2849 (2002).

    CAS  PubMed  Google Scholar 

  20. Conte, I., Morcillo, J. & Bovolenta, P. Comparative analysis of Six3 and Six6 distribution in the developing and adult mouse brain. Dev. Dyn. 234, 718–725 (2005).

    Article  CAS  Google Scholar 

  21. Li, X., Perissi, V., Liu, F., Rose, D.W. & Rosenfeld, M.G. Tissue-specific regulation of retinal and pituitary precursor cell proliferation. Science 297, 1180–1183 (2002).

    CAS  PubMed  Google Scholar 

  22. Geng, X. et al. Haploinsufficiency of Six3 fails to activate Sonic hedgehog expression in the ventral forebrain and causes holoprosencephaly. Dev. Cell 15, 236–247 (2008).

    Article  CAS  Google Scholar 

  23. Liu, W., Lagutin, O.V., Mende, M., Streit, A. & Oliver, G. Six3 activation of Pax6 expression is essential for mammalian lens induction and specification. EMBO J. 25, 5383–5395 (2006).

    Article  CAS  Google Scholar 

  24. Carl, M., Loosli, F. & Wittbrodt, J. Six3 inactivation reveals its essential role for the formation and patterning of the vertebrate eye. Development 129, 4057–4063 (2002).

    CAS  PubMed  Google Scholar 

  25. Lopez-Rios, J., Tessmar, K., Loosli, F., Wittbrodt, J. & Bovolenta, P. Six3 and Six6 activity is modulated by members of the groucho family. Development 130, 185–195 (2003).

    Article  CAS  Google Scholar 

  26. Del Bene, F., Tessmar-Raible, K. & Wittbrodt, J. Direct interaction of geminin and Six3 in eye development. Nature 427, 745–749 (2004).

    Article  CAS  Google Scholar 

  27. Cheung, V.G. et al. Mapping determinants of human gene expression by regional and genome-wide association. Nature 437, 1365–1369 (2005).

    Article  CAS  Google Scholar 

  28. Emison, E.S. et al. A common sex-dependent mutation in a RET enhancer underlies Hirschsprung disease risk. Nature 434, 857–863 (2005).

    Article  CAS  Google Scholar 

  29. Haiman, C.A. et al. A common genetic risk factor for colorectal and prostate cancer. Nat. Genet. 39, 954–956 (2007).

    Article  CAS  Google Scholar 

  30. Cawley, S. et al. Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread regulation of noncoding RNAs. Cell 116, 499–509 (2004).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank the families for their participation in these studies. We also thank J. Richa and his staff at the University of Pennsylvania Transgenic and Mouse Chimeric Facility for their assistance in transgenic mouse production. We are grateful to V. Cheung, D. Kessler and T. Kadesch for their helpful comments on the manuscript. We are also grateful to K. Ewens and W. Ankener (R. Spielman laboratory) for the control human genotyping data and P. Bovolenta (Instituto Cajal, CSIC, Madrid, Spain) for kindly providing the human Six3 and Six6 expression constructs. This work was supported by NIH grants R01 NS39421 from NINDS (D.J.E.), R01 NS052386 (G.O.), March of Dimes grant #1-FY05-112 (D.J.E.), a Pew Scholar Award in the Biomedical Sciences (D.J.E.), Cancer Center Support CA-21765 (G.O.), the American Lebanese Syrian Associated Charities (ALSAC) (G.O.) and the Division of Intramural Research, National Human Genome Research Institute, National Institutes of Health (M.M.).

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Authors and Affiliations

Authors

Contributions

Y.J. performed the transgenic, EMSA, transfection and ChIP assays. F.C.L. designed and performed the DNA affinity capture assay, competitive EMSA and in situ hybridization. K.E.-J., E.R., C.D. and M.M. sequenced SBE2 from individuals with HPE. A.Y. generated the mass spectrometry data. X.L. provided the Six6−/− embryos. X.G. and G.O. generated the Six3 expression constructs. D.J.E. conceived and supervised the project and wrote the paper.

Corresponding author

Correspondence to Douglas J Epstein.

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Jeong, Y., Leskow, F., El-Jaick, K. et al. Regulation of a remote Shh forebrain enhancer by the Six3 homeoprotein. Nat Genet 40, 1348–1353 (2008). https://doi.org/10.1038/ng.230

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