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An ancient protein-DNA interaction underlying metazoan sex determination


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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Figure 1: DMRT1 binds similar sites in vivo and in vitro.
Figure 2: Major-groove interactions and use of multiple DNA binding modes by DMRT proteins.
Figure 3: DNA backbone contacts, protein-protein interactions and binding summary.
Figure 4: Confirmation of crucial protein-DNA contacts.
Figure 5: DMRT1 binds DNA with multiple stoichiometries in vitro and in vivo. (a) EMSA showing binding of DMRT1 tetramer, trimer and dimer to sites 2, 1 and 3, respectively.
Figure 6: Modeling DNA interaction by Dsx and MAB-3 suggests related but different binding modes.
Figure 7: Disruption of crucial DMRT1 DNA contacts by a sex-reversing human mutation.

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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




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.

Corresponding authors

Correspondence to Hideki Aihara or Vivian J Bardwell.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Conservation of DMRT1 and portion used in structural analysis.

(a) Top, similarity plot of twenty-two amniote DMRT1 proteins aligned with Clustal Omega1 and plotted with plotcon ( Bottom, amino acid alignment showing the portion of DMRT1 used in X-ray crystallization. Amino acids are colored according to their chemical properties: polar amino acids (G,S,T,Y,C) are green, basic (K,R,H) blue, acidic (D,E) red, hydrophobic (A,V,L,I,P,W,F,M) black and neutral amino acids (Q,N) are purple. Species compared were human, bonobo, orangutan, gorilla, macaque, mouse lemur, tree shrew, mouse, rat, pika, dolphin, dog, pig, rock hyrax, opossum, chicken, budgerigar, tibetan ground-jay, crocodile, soft shell turtle, painted turtle, green anole, python, yellow-green tree viper, Japanese striped snake. (b) Conservation among 29 metazoan DMRT proteins in the region used for crystallization. DMRT proteins were from Placozoa (XM_002114844.1, XM_002114842.1), Cnidaria (JX559769.1, JX559768.1, JX559766.1, XM_002158684), Nematoda (NM_062737.6), Arthropoda (NM_079825.2, BT128785.1, AY060257.1), Platyhelminthes (AGL61627.1 , KC736561), Lophotrochozoa (XM_005096874.1, JX908760.1, XM_005096875.1), Urochordata (NM_001078212.1, XM_009861109.1, XM_004226545.1), Vertebrata (NM_021951.2, NM_021240.3, NM_033067.1), Cephalochordata (XM_002610965.1, XM_002608929.1), Echinodermata (XM_781845.3, XM_003723501.1, XM_789986.3) and Hemichordata (XM_002734449.2, NM_001171217.1, XM_006821714.1).

(c) Diagram of portion of DMRT1 used for crystallization, summarizing functional domains, which include a minor groove recognition module containing an arginine residue (R72) that makes base pair contacts, a zinc module with two sets of zinc-chelating amino acids (Zn1 and Zn2), and a recognition helix containing amino acids that make sequence-specific contacts in the DNA major groove (see Fig. 3 for additional detail).

1. Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular systems biology 7, 539, doi:10.1038/msb.2011.75 (2011).

Supplementary Figure 2 Support for X-ray structure.

(a-d) Anomalous difference Fourier peaks confirming the model. Each map was calculated using model-derived phases. (a) Map calculated from a data set collected at the zinc absorption edge (orange density contoured at 7 s). The model was refined with the zinc ions omitted prior to the model-phase calculation. (b) Map (contoured at 6 s) calculated from a data set collected at the Br absorption edge on a crystal containing 5-Br-dU at -9 position on one strand (shown in gray: CGAGAXTTGATACATTGTTGCTCGA, X = 5-Br-dU). (c ,d), Maps (contoured at 5 s) calculated with a data set collected at the Se absorption edge on a selenomethionine-labeled crystal. Two areas including M115 from protomers A and B (c) and M115 from protomer C (d) are shown. (e, f), Composite omit 2Fo-Fc map for the DMRT1-DNA complex. The electron density calculated at 3.8 Å resolution using PHENIX2 is displayed contoured at 1.0 s for the area within 3 Å from any atom in the final model. Two different views are shown.

2. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta crystallographica. Section D, Biological crystallography 66, 213-221, doi:10.1107/S0907444909052925 (2010).

Supplementary Figure 3 Effect of DMRT1 and DNA mutations on DNA binding.

(a) Point mutations in DMRT1 tested by EMSA at different protein concentrations. (b) EMSA analysis of DMRT1 binding to Sites 3, 1 and 2. Titrations were a series of protein doublings starting at 39 nl in vitro translate in a total of 10 ul reticulocyte lysate. Dimer (di), trimer (tri), tetramer (tet) (c) Additional point mutations in DMRT1 amino acids tested by EMSA at different protein concentrations (0.66 ul, 2ul, or 6ul in vitro translate in a total volume of 6 ul reticulocyte lysate). Plots at right show phosphorimager quantification of the fraction of probe shifted by each protein. (d) Immunoblot of in vitro translated proteins showing that comparable amounts of wild-type and mutant proteins were used.

Supplementary Figure 4 ChIP-Exo analysis.

(a) ChIP-Exo and ChIP-Seq analysis of DMRT1 binding sites grouped as shown in Fig. 5d, showing distinct in vivo binding modes at lower magnificaiton. As in Fig. 5e and f, plots show mapping of 5’ ends of Illumina sequences separated by strand, with top strand in grey and bottom in blue; vertical line indicates center-point of DMRT1 binding consensus. Left two columns show lower magnification and right columns show higher magnification. In ChIP-Seq plots red arrows indicate positions of inferred crosslinks, 3’ of which ChIP becomes inefficient. In Chip-Seq trimer data, the peak on the right side (triangle) may reflect increased density of potential crosslinks (see below). Stars highlight prominent differences in ChIP-Exo pattern in putative trimer binding sites relative to tetramer sites. (b) Predicted crosslinking potential for DMRT1 bound as tetramer, trimer or dimer. Red ovals denote amino acids with primary amine groups within 5.5 Å of the amine group of either an adenine or cytosine in the major groove or a guanine in the minor groove in the crystal structure. Red lines indicate density of cross-linkable residues. DMRT1 trimer complex has increased density of potential crosslinks on the right side, consistent with stronger ChIP-Exo stops on right side shown in panel a. Nucleotide changes indicated in gold would block binding of tetramer or trimer. (c) Motif searches under ChIP-Exo peaks. ChIP-exo peaks all had the shared 7 bp DMRT1 core binding site. As a test for binding of other proteins near DMRT1, ChIP-Exo peaks were searched for patterns indicated, which bind putative tetramers, trimers, or dimers. The motifs returned are shown, all of which contain only predicted DMRT1 binding sites, with differences in flanking sequences at positions -6, +5, and +6 expected for the three binding stoichiometries. (d) Box plot showing predicted average minor groove width across tetramer, trimer, and dimer ChIP-exo pooled sites from panel a.

Supplementary Figure 5 DM-domain mutations in humans, flies and nematodes.

(a) Locations of point mutations causing loss of function in human DMRT1 (top), Drosophila Dsx (middle) and C. elegans MAB-3 (bottom). Extent of α-helices was predicted using PSIPRED ( In Dsx, stars indicate residues shown by mutagenesis to be important for DNA binding: open stars indicate site-directed mutations tested by EMSA by Burtis and colleagues3, black filled stars indicate alanine substitutions tested by EMSA by Weiss and colleagues4 and grey stars indicate residues tested by random mutagenesis and yeast one-hybrid assay by Weiss and colleagues5. DSX sequences used for alignment were from Drosophila (FBpp0303107), human body louse (PHUM334970-RA), red flour beetle (TC010597-A), Mediterranean fruit fly (XP_004529237.1), house fly (XP_005178159.1), jewel wasp (XP_008205424.1), honey bee (NP_001104725.1) and silk moth (NP_001036871.1). Nematode MAB-3 sequences were CEL-MAB-3, CRE-MAB-3, CBR-MAB-3.2, CBN-MAB-3 and CJP-MAB-3 of Caenorhabditis and Bm6751 of Brugia malayi. MAB-3 alignments are relative to C. elegans MAB-3, which lacks one amino acid in the linker region and has one extra amino acid in DMb relative to other species. (b) Sequence alignment of DM domains from DMRT1, Dsx and MAB-3. Fully conserved residues are highlighted with colors indicating chemical property (green for polar, blue for basic), and partially conserved amino acids are indicated by one or two dots.

3. Erdman, S. E. & Burtis, K. C. The Drosophila doublesex proteins share a novel zinc finger related DNA binding domain. The EMBO journal 12, 527-535 (1993).

4. Narendra, U., Zhu, L., Li, B., Wilken, J. & Weiss, M. A. Sex-specific gene regulation. The Doublesex DM motif is a bipartite DNA-binding domain. The Journal of biological chemistry 277, 43463-43473, doi:10.1074/jbc.M204616200 (2002).

5. Zhang, W. et al. Regulation of sexual dimorphism: mutational and chemogenetic analysis of the doublesex DM domain. Molecular and cellular biology 26, 535-547, doi:10.1128/MCB.26.2.535-547.2006 (2006).

Supplementary Figure 6 DMRT1R111G does not alter binding stoichiometry on a DMRT1-binding site from the Foxl2 gene.

EMSA comparing DMRT1 and DMRT1R111G. Left lanes, wild type DMRT1 alone; middle, DMRT1 plus DMRT1R111G; right, DMRT1R111G alone. EMSAs contained 2 ul in vitro translate of indicated DMRT1 protein and black wedges indicate increments of 1.5 ul added protein up to a total of 6 ul. This site is bound primarily as a tetramer and addition of DMRT1R111G has no effect on stoichiometry. DMRT1R111G can convert wild type trimers bound to Site 1 into slower-migrating tetramers (right-most four lanes), likely by occupying the right side of the binding site, as also shown in Figure 7.

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Murphy, M., Lee, J., Rojo, S. et al. An ancient protein-DNA interaction underlying metazoan sex determination. Nat Struct Mol Biol 22, 442–451 (2015).

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