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An ancient protein-DNA interaction underlying metazoan sex determination

Nature Structural & Molecular Biology volume 22, pages 442451 (2015) | Download Citation

Abstract

DMRT transcription factors are deeply conserved regulators of metazoan sexual development. They share the DM DNA-binding domain, a unique intertwined double zinc-binding module followed by a C-terminal recognition helix, which binds a pseudopalindromic target DNA. Here we show that DMRT proteins use a unique binding interaction, inserting two adjacent antiparallel recognition helices into a widened DNA major groove to make base-specific contacts. Versatility in how specific base contacts are made allows human DMRT1 to use multiple DNA binding modes (tetramer, trimer and dimer). Chromatin immunoprecipitation with exonuclease treatment (ChIP-exo) indicates that multiple DNA binding modes also are used in vivo. We show that mutations affecting residues crucial for DNA recognition are associated with an intersex phenotype in flies and with male-to-female sex reversal in humans. Our results illuminate an ancient molecular interaction underlying much of metazoan sexual development.

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Acknowledgements

We thank K. Shi and J. Nix for help with crystallization trials and data collection; the University of Minnesota Supercomputing Institute for computational resources; L. Amble, the University of Minnesota Tissue Procurement Facility and anonymous donors for human testis tissue; D. Greenstein, C. Kim, M. Slattery, B.F. Pugh, J. Simon, H. Towle and members of our laboratories for helpful comments on the manuscript; and T. Gamble for help with phylogenetic analysis. X-ray data were collected at the Advanced Photon Source (APS) NE-CAT beamlines, which are supported by the US National Institute of General Medical Science (P41 GM103403). APS is a US Department of Energy Office of Science User Facility operated by Argonne National Laboratory under contract DE-AC02-06CH11357. This work was funded by the US National Institutes of Health (GM59152 and GM50399 to D.Z.; AI087098 and GM095558 to H.A.), European Cooperation in Science and Technology (COST) (Action DSDnet BM1303 to K.M.) and Program Blanc Assistance-Publique-Institut Pasteur (to K.M.).

Author information

Author notes

    • Mark W Murphy
    • , John K Lee
    • , Sandra Rojo
    •  & Micah D Gearhart

    These authors contributed equally to this work.

Affiliations

  1. Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota, USA.

    • Mark W Murphy
    • , Micah D Gearhart
    • , David Zarkower
    •  & Vivian J Bardwell
  2. Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA.

    • John K Lee
    • , Kayo Kurahashi
    •  & Hideki Aihara
  3. Unit of Human Developmental Genetics, Institut Pasteur, Paris, France.

    • Sandra Rojo
    • , Anu Bashamboo
    •  & Kenneth McElreavey
  4. Northeastern Collaborative Access Team, Cornell University, Argonne, Illinois, USA.

    • Surajit Banerjee
  5. Service de Pédiatrie, Centre Hospitalier de Dunkerque, Dunkerque, France.

    • Guy-André Loeuille
  6. Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA.

    • David Zarkower
    • , Hideki Aihara
    •  & Vivian J Bardwell
  7. Developmental Biology Center, University of Minnesota, Minneapolis, Minnesota, USA.

    • David Zarkower
    •  & Vivian J Bardwell

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Contributions

M.W.M. performed and, with V.J.B., D.Z. and M.D.G. analyzed in vitro and in vivo DNA binding studies. M.D.G. performed bioinformatic analysis of ChIP data. J.K.L., K.K. and H.A. performed protein purification and crystallization. J.K.L., S.B. and H.A. collected X-ray diffraction data. J.K.L. processed the X-ray data and built and refined the atomic model. M.W.M., J.K.L., M.D.G., D.Z., H.A. and V.J.B. analyzed the structure and prepared the figures. G.-A.L. coordinated the clinical studies. A.B. and K.M. designed the human genetic studies and with S.R. analyzed the exome data sets. A.B. and S.R. performed Sanger sequencing. D.Z. and V.J.B. wrote the manuscript. M.W.M., M.D.G., H.A., A.B., K.M. and S.R. edited the manuscript. The first four authors made equivalent contributions.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Hideki Aihara or Vivian J Bardwell.

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DOI

https://doi.org/10.1038/nsmb.3032

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