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Figure 4 The Mis18 Complex and CENP-C Coordinate HJURP Recruitment to CENP-A Chromatin (A) M18BP1 localization is not sufficient to promote new CENP-A assembly on H3 chromatin or in the absence of CENP-C. Quantification of V5-CENP-A assembly on LacO chromatin-coated beads in M18BP1-depleted extracts complemented with LacI-FLAG-M18BP1-2. Extracts were additionally depleted of CENP-C or mock-depleted to assess the role of CENP-C. Depletion of CENP-C or tethering to H3 rather than CENP-A chromatin reduced V5-CENP-A assembly to background levels, indicated by signal equivalent to that observed when HJURP was omitted from the assembly reaction (−HJ). Plot shows mean V5-CENP-A signal ± SEM of at least three experiments. (B) Representative western blot of egg extract samples used for (A). Depletion condition is indicated above; immunoblotted species is indicated at right. Efficient immunodepletion of M18BP1 and CENP-C are indicated by comparing lanes 2 and 3 with lane 1. (C) M18BP1 and CENP-C make distinct contributions to FLAG-HJURP localization at interphase sperm centromeres. FLAG-HJURP translated in rabbit reticulocyte lysate was added to interphase egg extract depleted of CENP-C, M18BP1, or both. Plot shows mean centromeric FLAG signal ± SEM of three experiments. (D) HJURP associates with CENP-C specifically in interphase. Metaphase or interphase extract was supplemented with rabbit reticulocyte lysate containing putative HJURP binding partners (indicated above) translated in the presence of [35S]methionine. Co-immunoprecipitation with endogenous HJURP was detected by autoradiography. Equivalent precipitation of HJURP in all reactions is indicated by anti-HJURP immunoblot below. (E) HJURP directly interacts with the CENP-C C terminus. Full-length CENP-C, CENP-C truncations, or Drosophila tropomyosin 2 (dTm2) as a negative control were translated in rabbit reticulocyte lysate in the presence of [35S]methionine and then mixed with in vitro translated FLAG-HJURP. Co-immunoprecipitation with FLAG-HJURP was detected by autoradiography. (Top) Schematic of CENP-C, with conserved domains indicated by colored bars and the region corresponding to amino acids 1,191–1,400 boxed. (Bottom) Plot shows fraction of input bound for each species normalized to full-length CENP-C ± SD from two experiments. Representative autoradiograph is shown in Figure S4C. (F) The functions of M18BP1 and CENP-C in CENP-A assembly are effectively bypassed by direct tethering of HJURP to CENP-A or H3 chromatin. Quantification of V5-CENP-A assembly on LacO chromatin-coated beads in extract (depletion condition indicated below) supplemented with LacI-FLAG-HJURP. Plot shows mean V5 signal on beads ± SEM of at least three experiments. Corresponding LacI-FLAG-HJURP localization quantification and retention of V5-CENP-A signal after 1 mM IPTG treatment shown in Figures S4D–S4F. (G) Possible models for coordinated HJURP recruitment by M18BP1 and CENP-C. See also Figure S4. Image published in: French BT et al. (2017) Copyright © 2017. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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