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Regulation of a remote Shh forebrain enhancer by the Six3 homeoprotein


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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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



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).

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