Modulation of histone deposition by the karyopherin kap114

Abstract

The nuclear import of histones is a prerequisite for the downstream deposition of histones to form chromatin. However, the coordinate regulation of these processes remains poorly understood. Here we demonstrate that Kap114p, the primary karyopherin/importin responsible for the nuclear import of histones H2A and H2B, modulates the deposition of histones H2A and H2B by the histone chaperone Nap1p. We show that a complex comprising Kap114p, histones H2A and H2B, and Nap1p is present in the nucleus and that the presence of this complex is specifically promoted by Nap1p. This places Kap114p in a position to modulate Nap1p function, and we demonstrate by the use of two different assay systems that Kap114p inhibits Nap1p-mediated chromatin assembly. The inhibition of H2A and H2B deposition by Kap114p results in the concomitant inhibition of RCC1 loading onto chromatin. Biochemical evidence suggests that the mechanism by which Kap114p modulates histone deposition primarily involves direct histone binding, while the interaction between Kap114p and Nap1p plays a secondary role. Furthermore, we found that the inhibition of histone deposition by Kap114p is partially reversed by RanGTP. Our results indicate a novel mechanism by which cells can regulate histone deposition and establish a coordinate link between histone nuclear import and chromatin assembly.

FIG. 1.

Kap114p associates with Nap1p and histones in the nucleus. (a) Kap114-PrA nuclear extract was incubated with IgG-Sepharose and then washed, and proteins were eluted with a MgCl₂ step gradient and then analyzed by Western blotting (WB) with an anti-Nap1p antibody. Molecular standards, in kilodaltons, are indicated on the left. (b) Equivalent amounts of cytosol and nuclear extracts from Kap114-PrA cells were analyzed by Western blotting with antibodies against Pgk1p or acetylated H4. H2A-PrA (c) and H4-PrA (d) and associated proteins were isolated from nuclear extracts and analyzed by Coomassie staining (CBB) or Western blotted with the indicated antibodies. The fractions shown in panels c and d were analyzed on the same gel and blot and were treated identically. Wash, final wash fraction; U/B, unbound fraction. (e) H2A-PrA and associated proteins were isolated from nuclear extracts as described for panel c, washed, incubated with 20 μM RanQ69L at room temperature for 1 h, washed again, eluted, and analyzed as described above. Wash-1, final wash fraction prior to RanQ69L addition; Wash-2, wash fraction prior to MgCl₂ elution. (f) Binding of MBP-Kap114p (250 nM) to GST-H2A_1-46 (1 μM) was assessed in the presence or absence of Nap1p (1 μM) and/or RanQ69L (20 μM).

FIG. 2.

Nap1p promotes the association of Kap114p with histones in the nucleus. H2A-PrA and associated proteins were isolated from nuclear extracts from the indicated strains with IgG-Sepharose. Fractions were eluted with a MgCl₂ step gradient and analyzed by Coomassie staining (CBB) or Western blotted (WB) and probed with an anti-Myc antibody.

FIG. 3.

Kap114p inhibits Nap1p-mediated chromatin assembly on plasmid templates. (a) Plasmid supercoiling assays were performed according to the schematic by the use of recombinant Nap1p (500 nM) in the presence of MBP-Kap114p or MBP-lacZα (625 nM, 1.25 μM, or 2.5 μM), as indicated. After deproteinization, the DNAs were analyzed in an agarose gel. (b) Reactions were performed with Nap1p according to the schematic, incubated with MBP-Kap114p or MBP-lacZα (625 nM, 1.25 μM, or 2.5 μM) for 1 h, and analyzed as described above. S, supercoiled DNA.

FIG. 4.

Kap114p inhibits histone H2A and H2B deposition onto Xenopus sperm chromatin. (a) Nap1p decondenses Xenopus sperm chromatin. Sperm chromatin was incubated with the indicated concentrations of Nap1p, fixed, stained with Hoechst, and analyzed by fluorescence microscopy. (b) Sperm chromatin was decondensed with 1 μM Nap1p as indicated. Reactions were then incubated with core histones and TRITC-labeled H2A and H2B as indicated. Excess unlabeled (XS U/L) core histones (5 μg) were added to the reaction on the right. The reactions were fixed and stained with Hoechst. (c) Reactions were performed as described above in the presence of RCC1-GFP (200 nM) and MBP-Kap114p or MBP-lacZα (1 μM each), as indicated. (d) Reactions were performed as described above, using only unlabeled histones in the presence of MBP-Kap114p (200 nM, 500 nM, or 1 μM) or MBP-lacZα (1 μM), as indicated. The chromatin was isolated and analyzed by Western blotting with an anti-H2B antibody.

FIG. 5.

Characterization of Nap1p point mutants. (a) Alanine mutations of Nap1p abolish its Kap114p binding. Binding assays were performed on glutathione-Sepharose with 2 μM GST or a 400 nM concentration of each GST-Nap1p mutant in the presence of 300 nM MBP-Kap114p. Eighty percent of the bound material was analyzed by SDS-PAGE and Coomassie blue (CBB) staining. The remaining material was analyzed by Western blotting (WB) with an anti-MBP antibody. The schematic shows the relative positions of alanine mutations in the Kap114p-binding domain (BD)of Nap1p. (b) Plasmid supercoiling assays were performed with 500 nM wild-type Nap1p or 250 and 500 nM concentrations of the indicated Nap1p mutants. (c) A Kap114-PrA-expressing Δnap1 strain was constructed and transformed with vector alone or with a plasmid expressing wild-type Nap1p or the indicated Nap1p mutant. The cytosol was prepared from each strain and analyzed for the expression of Nap1p and Kap114-PrA by Western blotting (input). The same cytosol was used for immunoisolation of Kap114-PrA and associated proteins (IP). After being washed, the associated proteins were eluted with a buffer containing the indicated concentrations of MgCl₂ and then analyzed by Western blotting for Nap1p and Kap114-PrA. (d) Binding between MBP-Kap114p and the indicated GST proteins was analyzed as described for panel a in the presence or absence of core histones (500 nM) and RanQ69L (20 μM), as indicated. (e and f) The localization of the indicated GFP-tagged proteins was analyzed in wild-type yeast. Coincident Hoechst staining is also shown.

FIG. 6.

Direct interaction between Nap1p and Kap114p contributes to but is not essential for inhibition of histone deposition. (a) Supercoiling assays were performed with wild-type Nap1p or the indicated Nap1p mutants (500 nM) in the presence of MBP-Kap114p (1 or 2.5 μM) as indicated. Histone deposition assays were performed on Xenopus sperm chromatin and were visualized by microscopy (b) or Western blotting (WB) (c) with 1 μM wild-type Nap1p or Nap1p(290-292 Ala). The concentration of MBP-Kap114p was 1 μM for panel b and 500 nM for panel c.

FIG. 7.

Kap114p contains multiple histone-binding domains. (a) The indicated MBP-tagged Kap114p deletions were expressed and purified from bacteria. Twenty picomoles of each (100% of the input for subsequent binding studies) was analyzed by SDS-PAGE. (b to d) Binding between each MBP-Kap114p deletion (200 nM) and GST (2 μM) or GST-H2A_1-46 (1 μM) was tested and analyzed as shown. (e) Schematic showing the histone-binding domains of Kap114p as determined by the deletion analysis shown in panels b to d. Solid gray lines represent fragments which lacked binding.

FIG. 8.

Multiple histone-binding domains contribute to the modulation of histone deposition by Kap114p. (a) Portion of schematic from Fig. 7e depicting Kap114p histone-binding domains (hatched areas). (b to d) Supercoiling assays were performed as described in the text by the use of 500 nM Nap1p in the presence of the indicated MBP-tagged fragments of Kap114p (1 μM).

FIG. 9.

Other histone-binding karyopherins can inhibit histone deposition in vitro. (a) Plasmid supercoiling assays were performed with 500 nM Nap1p and a 500 nM, 1 μM, or 2.5 μM concentration of the indicated MBP-Kaps. Histone deposition assays were performed on Xenopus sperm chromatin and were visualized by microscopy (b) or Western blotting (WB) (c) as described in the text by the use of 1 μM Nap1p and a 1 μM concentration (b) or 200 and 500 nM concentrations (c) of the indicated Kaps.

FIG. 10.

RanGTP can reverse the Kap114p-mediated inhibition of histone deposition. (a and b) Plasmid supercoiling assays were performed with 500 nM Nap1p, 1 and 2 μM MBP-Kap114p or MBP-Kap121p, and 20 μM RanQ69L as indicated. (c) Histone deposition assays were performed on sperm chromatin with 1 μM Nap1p, the indicated Kap (200 nM, 500 nM, or 1 μM), and 20 μM RanQ69L analyzed by Western blotting as shown.

FIG. 11.

Proposed model for the function of Kap114p and RanGTP in histone deposition. See Discussion for details.