Histone chaperones represent a structurally and functionally diverse family of histone-binding proteins that prevent promiscuous interactions of histones before their assembly into chromatin. DAXX is a metazoan histone chaperone specific to the evolutionarily conserved histone variant H3.3. Here we report the crystal structures of the DAXX histone-binding domain with a histone H3.3–H4 dimer, including mutants within DAXX and H3.3, together with in vitro and in vivo functional studies that elucidate the principles underlying H3.3 recognition specificity. Occupying 40% of the histone surface-accessible area, DAXX wraps around the H3.3–H4 dimer, with complex formation accompanied by structural transitions in the H3.3–H4 histone fold. DAXX uses an extended α-helical conformation to compete with major inter-histone, DNA and ASF1 interaction sites. Our structural studies identify recognition elements that read out H3.3-specific residues, and functional studies address the contributions of Gly 90 in H3.3 and Glu 225 in DAXX to chaperone-mediated H3.3 variant recognition specificity.
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Protein Data Bank
Atomic structures of the DAXX–H3.3–H4 complex have been deposited in the RCSB Protein Data Bank with accession codes 4H9N (five-substituent native complex at 1.95A˚ ), 4H9S (seven-substituent native complex at 2.60A˚ ), 4H9O (five-substituent H3.3(G90M) mutant complex at 2.05A˚ ), 4H9P (five-substituent H3.3(G90A) mutant complex at 2.20A˚ ), 4H9Q (five-substituent DAXX(E225A) mutant complex at 1.95A˚ ) and 4H9R (five-substituent DAXX(E225A)–H3.3(G90A) mutant complex at 2.20A˚ ). Supplementary Video 1 shows the ternary five-substituent native complex of DAXX–H3.3–H4.
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We thank the personnel of synchrotron beam lines 24-I/D-C/E at the Advanced Photon Source (Argonne National Laboratory) and beam line X29 at the Brookhaven National Laboratory for their assistance, and the Center for Synchrotron Biosciences grant, P30-EB-009998, from the National Institute of Biomedical Imaging and Bioengineering (NIBIB) for funding. The use of the Rigaku/MSC microMax 007HF and Formulator in the Rockefeller University Structural Biology Resource Center was made possible by grant numbers 1S10RR022321-01 and 1S10RR027037-01 from the National Center for Research Resources of the NIH. We thank B. Black for sharing data before publication, A. Ruthenburg, J. Song and Z. Cheng for advice and discussions, and E. Datan for help in expressing DAXX protein. D.J.P. was supported by funds from the Abby Rockefeller Mauze Trust, and the Maloris and STARR Foundations. C.D.A. also acknowledges support from the STARR Foundation and The Rockefeller University. J.W.C. acknowledges support from UK Medical Research Council (MRC) (grants U105181009 and UD99999908). S.J.E. was supported by a Boehringer Ingelheim Funds fellowship and the David Rockefeller Graduate Program and holds an EMBO ALTF 1232-2011.
The authors declare no competing financial interests.
This file contains Supplementary Figures 1-18 and Supplementary Tables 1 and 2. (PDF 17204 kb)
Video of the ternary complex of DAXX (ribbon representation in magenta), H3.3 and H4 (surface representations in blue and green, respectively). The DAXX chaperone in an all alpha-helical conformation envelops the histone H3.3-H4 dimer for H3.3-specific recognition. (AVI 6541 kb)
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Elsässer, S., Huang, H., Lewis, P. et al. DAXX envelops a histone H3.3–H4 dimer for H3.3-specific recognition. Nature 491, 560–565 (2012) doi:10.1038/nature11608
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