![]() Western blots representative of n = 2 biological replicates. (E) Pull-downs of FLAGHA-tagged histone H3 variants induced by doxycycline (Dox+) compared with control purifications (Dox−) from soluble cell extracts. (D) Pull-downs of full-length GST-DNAJC9 mixed with pre-assembled H3.3-H4 (top) and MCM2 HBD-H3.3-H4 complexes (bottom). ![]() Proteins referred to by human UniProt protein identification code. (B and C) Mass spectrometry analysis of SILAC labeled pull-downs of wild-type (WT) and histone binding mutant (HBM) forms of MCM2 (B) and TONSL (C) from soluble cell extracts n = 2 biological replicates. Hypothetical histone-dependent interactors (colored light green and orange for MCM2 and TONSL, respectively) and histone-independent interactors (gray) are depicted. Identification of DNAJC9 as a dual histone chaperone and heat shock co-chaperone (A) Schematic representations of the histone H3-H4 (red and cyan, respectively) binding mode of MCM2 (dark green) and TONSL (yellow) highlighting histone binding mutants (Huang et al., 2015 Saredi et al., 2016) and the experimental strategy for the identification of histone-dependent interactors. With its dual functionality, DNAJC9 integrates ATP-resourced protein folding into the histone supply pathway to resolve aberrant intermediates throughout the dynamic lives of histones.ĭNAJC9 HSP40 HSP70 MCM2 TONSL chromatin replication heat shock co-chaperone histone chaperone nucleosome assembly transcription.Ĭopyright © 2021 The Authors. We show that DNAJC9 recruits HSP70-type enzymes via its J domain to fold histone H3-H4 substrates: upstream in the histone supply chain, during replication- and transcription-coupled nucleosome assembly, and to clean up spurious interactions. We elucidate the structure of DNAJC9, in a histone H3-H4 co-chaperone complex with MCM2, revealing how this dual histone and heat shock co-chaperone binds histone substrates. Using structure-guided and functional proteomics, we identify and characterize a histone chaperone function of DNAJC9, a heat shock co-chaperone that promotes HSP70-mediated catalysis. Whether mechanisms exist to safeguard the histone fold during histone chaperone handover events or to release trapped intermediates is unclear. Electronic address: biosynthesis to assembly into nucleosomes, histones are handed through a cascade of histone chaperones, which shield histones from non-specific interactions. Electronic address: 9 Novo Nordisk Foundation Center for Protein Research (CPR), University of Copenhagen, Copenhagen, Denmark Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark. Electronic address: 8 Key Laboratory of Molecular Design for Plant Cell Factory of Guangdong Higher Education Institutes, Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518055, China. 7 Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.6 Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany.5 Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.4 Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark.3 Novo Nordisk Foundation Center for Protein Research (CPR), University of Copenhagen, Copenhagen, Denmark.2 Key Laboratory of Molecular Design for Plant Cell Factory of Guangdong Higher Education Institutes, Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518055, China.1 Novo Nordisk Foundation Center for Protein Research (CPR), University of Copenhagen, Copenhagen, Denmark Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark.
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