HJURP binds CENP-A via a highly conserved N-terminal domain and mediates its deposition at centromeres

Abstract

The human histone H3 variant, CENP-A, replaces the conventional histone H3 in centromeric chromatin and, together with centromere-specific DNA-binding factors, directs the assembly of the kinetochore. We purified the prenucelosomal e-CENP-A complex. We found that HJURP, a member of the complex, was required for cell cycle specific targeting of CENP-A to centromeres. HJURP facilitated efficient deposition of CENP-A/H4 tetramers to naked DNA in vitro. Bacterially expressed HJURP binds at a stoichiometric ratio to the CENP-A/H4 tetramer but not to the H3/H4 tetramer. The binding occurred through a conserved HJURP short N-terminal domain, termed CBD. The novel characteristic identified in vertebrates that we named TLTY box of CBD, was essential for formation of the HJURP-CENP-A/H4 complex. Our data identified HJURP as a vertebrate CENP-A chaperone and dissected its mode of interactions with CENP-A.

Fig. 1.

Purification of CENP-A preassembly complex. (A) Localization of e-CENP-A and e-H3.1. Stable cell lines expressing either e-CENP-A or e-H3.1 were immunostained with antiHA antibody (Green) to detect the epitope tagged proteins and DAPI staining (Blue). (Lower) Western blotting of total cell extract from control HeLa cells (Lane 1) and stable HeLa cell line (Lane 2) expressing e-CENP-A. An anti-CENP-A antibody was used to reveal the blot. (B) Silver staining of proteins associated with e-CENP-A. The preassembly e-CENP-A complex (CENP-A.com) was purified by tandem immuno-affinity and the associated polypeptides were identified by mass spectrometry. Lane M corresponds to a protein molecular mass marker. Lane Mock, corresponds to a mock purification from a nontagged HeLa cell line. (C) Silver staining of proteins associated with e-H3.1. The prenucelosomal e-H3.1 complex (H3.1.com) was purified by tandem immunoaffinity and the associated polypeptides (Left) were identified by mass spectrometry. Lane M corresponds to a protein molecular mass marker. (D). Western blot detection of HJURP and NPM1 in the e-CENP-A preassembly complex. e-Cenp-A and e-H3.1 complexes were run on 4–12% SDS PAGE and after transfer, the blot was revealed with an antiHJURP, an anti-NPM1, and antiFLAG (to detect e-CENP-A and e-H3.1). (E) Silver staining of proteins associated with e-HJURP. The specific partners of e-HJURP were purified by tandem immunoaffinity and identified by mass spectrometry analyses (Lane 2, Upper). The identified proteins are indicated on the right. Lane M corresponds to a protein molecular mass marker. (Lower) Western blot detection of CENP-A present in the preassembly e-CENP-A complex (Lane 1) and e-HJURP complex (Lane 2). Both complexes were run on 4–12% SDS PAGE and after transfer, the blot was revealed with an anti-CENP-A antibody. The higher molecular mass of e-CENP-A is due to the presence of the HA-FLAG peptide fused to CENP-A.

Fig. 2.

HJURP is required for CENP-A localization to centromeres. (A) The centromeric association of CENP-A is lost in cells depleted of HJURP. HeLa cells were transfected with either scrambled siRNA (SsiRNA) or with CENP-A siRNA or with HJURP siRNA1 and siRNA2. Seventy-two hr posttransfection cells were immunostained (Green) with anti-CENP-A antibody and DAPI staining (Blue). (B) Western blot analysis of the depletion of HJURP and CENP-A upon treatment with siRNA. HeLa cells were transfected with the respective siRNA and 72 hr posttransfection they were harvested, total cell extracts were prepared, and the presence of HJURP and CENP-A was detected by Western blotting using antiHJURP and anti-CENP-A antibodies. The blot was also revealed with an antiactin antibody as a control for equal loading. (Left) Depletion of both CENP-A and HJURP upon treatment with siRNA against HJURP. (Right) Depletion of CENP-A upon treatment with siRNA against CENP-A. Ctrl, nonsiRNA treated cells; Ssi, cells treated with scrumble siRNA; Si1 and Si2, cells treated with two distinct (Si1 or Si2) siRNAs against HJURP (Si1 and Si2 siRNA were used to suppress the expression of HJURP in the experiments presented in (A).

Fig. 3.

Identification of a short N-terminal domain of HJURP required for interaction with CENP-A. (A) Interaction of full-length and deletion mutants of HJURP with CENP-A. Full-length HJURP and its deletion mutants (Δ1–Δ4) fused to GST, together with CENP-A and H4, were coexpressed and purified from bacteria. The purified material was separated on a SDS-PAGE and stained with coomassie. (Lower) Western blot of either the eluted samples (PD) or the input (In) revealed with anti-CENP-A antibody. Note that a short amino acid sequence (1–80 AA) from the N-terminal of the protein recapitulates the main property of the full-length protein and was able to bind stoichiometrically to the CENP-A-H4 tetramer (compare lanes 2, 4, and 6). (B) Schematic representation of the different HJURP deletion mutants used as GST-fusions in (A). (C) The GST-fusion with CBD (CBD of HJURP, 1–80 AA) does not interact with the H3-H4 tetramer. GST-CBD was coexpressed with either H3.1/H4 or with CENP-A/H4. The purified material was run on a SDS-PAGE and stained with coomassie. U, unbound material. B, bound material. The bands designed with stars are degradation products of the fusions.

Fig. 4.

The identified novel TLTY box within the HJURP vertebrate homologs is essential for the interaction with CENP-A. (A) Identification of a conserved coiled-coil domain and a novel TLTY box in higher-eukaryote HJURP homologs. The sequence alignments for the indicated species are shown. Alignments were generated by MultAlin. The brackets highlight the coiled-coil and the TLTY motifs, which are conserved from birds to human. (B) The TLTY box is essential for the interaction of HJURP with CENP-A. The TLTY box of HJURP was deleted from the minimal CBD and coexpressed as a GST-fusion [GST-CBD (Δ-TLTY)] in bacteria together with CENP-A and H4. Increasing amounts (Lanes 2–4) of the eluted from the gluthatione column GST-CBD (Δ-TLTY) complex was analyzed by SDS PAGE. GST-CBD was used as a positive control (Lane 1). (Lower) Western blot revealed with anti-CENP-A antibody for the respective samples. (C) The CBD of HJURP recognizes and binds to CATD, the CENP-A centromere targeting domain. GST-CBD was coexpressed with either H3^CATD/H4 or CENP-A/H4 in bacteria, the GST-CBD complexes were purified as described above, run on SDS PAGE, and stained with coomassie. U, unbound material. B, bound material. M, protein molecular mass markers. (*), a degradation product of GST-CBD.

Fig. 5.

HJURP is able to deposit efficiently CENP-A/H4 tetramer on DNA. (A) Negatively supercoiled human alpha-satellite DNA corresponding to topoisomer -1 (Lane 1, D) was incubated with increasing amount of CENP-A/H4 (at the indicated histone/DNA ratio, rw) in the absence (Lanes 3–5) or presence (Lanes 6–8) of equimolar (to the tetramers) amount of HJURP. The reaction was carried out for 30 min at 37 °C. The reaction products were then analyzed on native 4.5% polyacrylamide gel. (Lane 1) topoisomer-1 DNA; (Lane 2) reconstituted CENP-A/H4 tetrasomes on topoisomer -1 by salt dialysis using the indicated histone/DNA ratio (rw). (Right) Drawings showing the naked topoisomer -1 DNA and the CENP-A/H4 tetrasome. The positions of the naked topoisomer -1 DNA and the salt dialysis reconstituted tetrasome are also indicated. (B) Quantification of the relative amount of Cenp-A/H4 tetrameres deposited by HJURP in Fig. 5 A. The tetrasome/DNA ratio was quantified using ImageJ software. S.D. indicates tetramers assembled by salt dialysis. (C) Model of CENP-A deposition. Two molecules of HJURP dimerize through their coiled-coil domains and bind, via their TLTY boxes, the CATD of two molecules of CENP-A (Left).