Hbo1 is required for the chromatin association of Mcm2 and Mcm6. (A) Fractionation scheme. HeLa cell lysates were separated into soluble cytoplasmic (S2), soluble nucleoplasmic (S3), and chromatin-enriched (P3) fractions as described previously (32). (B) HeLa cells infected with Ad-GFP (Control) or Ad-antisense Hbo1 (Hbo1as) were fractionated as outlined in panel A. Whole-cell extracts (WCE) were prepared by lysis in sodium dodecyl sulfate. WCE, S2, S3, and P3 fractions from equivalent numbers of cells were analyzed by Western blotting using antibodies against the proteins, indicated to the left of each set. (C) Mock-transfected HeLa cells (Control) or HeLa cells transfected with a 21-bp double-stranded RNA oligonucleotide (siRNA) were treated with Triton X-100, fixed with paraformaldehyde, and costained with rabbit polyclonal anti-Hbo1 (Hbo1), mouse monoclonal anti-Mcm6 (Mcm6), and 4′,6′-diamidino-2-phenylindole (DAPI). (D) HeLa cells infected with adenoviruses expressing GFP (Control) or antisense Hbo1 (Hbo1as) were treated with Triton X-100 and then fixed with paraformaldehyde. Cells were stained with rabbit polyclonal anti-Hbo1 and DAPI or with rabbit polyclonal anti-Orc2 and DAPI.

Hbo1 is required for Mcm6 binding to chromatin during G₁. (A) Synchronous cultures infected with either GFP adenovirus (Control) or Hbo1 antisense adenovirus (Hbo1as) were established as outlined in Fig. 2A. At the times indicated, the cultures were sampled, and the cells were stained for DNA content with propidium iodide. Histograms of the DNA fluorescence distributions were determined by flow cytometry and are plotted for each condition. (B) S3 chromatin-enriched fractions were prepared from cells infected with either Ad-GFP (Control) or Ad-antisense Hbo1 (Hbo1as), as outlined in the legend to Fig. 1A. The chromatin factions were assayed by Western blotting with antibodies against Hbo1, Mcm6, and Orc2. The S2 cytoplasmic fraction was assayed for cyclin E (CycE), a cell cycle marker, using a rabbit polyclonal antibody. (C) The chromatin binding of Hbo1, Mcm6, and Orc2 were quantified for the experiment shown in panel B. The open circles indicate the chromatin binding for GFP adenovirus treatment (Control), and the solid squares indicate the binding for Hbo1 antisense adenovirus treatment (Hbo1as). The results for each set are expressed as the fraction of the control signal at 0 h.

Sequence alignments of human Hbo1 and Xenopus homologues. Xenopus cDNAs encoding putative Hbo1 homologues were cloned by the I.M.A.G.E. Consortium. The amino-terminal regions of these proteins were aligned with the sequence of human Hbo1. The highly conserved MYST family histone acetyltransferase domains are further C terminal and are not included in the figure. Boxed regions indicate identical (dark gray shading) or similar (light gray shading) amino acids. We refer to the sequence represented by AAH72987 as XHbo1a and that represented by AAH77173 as XHbo1b. The peptide sequences used for producing antibodies are colored with red for peptide 1 and green for peptide 2 (see Materials and Methods).

Depletion of XHbo1 inhibits the chromatin binding of Mcm2 and DNA replication. (A) Sperm chromatin was incubated in mock-depleted (ΔMock) or XHbo1-depleted (ΔXHbo1) extracts in the presence of aphidicolin for 45 min, and chromatin fractions were separated and analyzed by immunoblotting with specific antibodies for Mcm2, Cdt1, Orc2, and Cdc6. A negative control for the chromatin fractions was obtained by incubating the extracts in the absence of sperm chromatin (−sperm). (B) Pre-RC components remain in the egg extracts after immunodepletion of XHbo1. Mock-depleted (ΔMock) and XHbo1-depleted (ΔXHbo1) extracts prepared as described in the legend to Fig. 6 were analyzed for Mcm2, Cdc6, Cdt1, and Orc2 by immunoblotting. (C) Sperm chromatin was incubated with Cy3-dCTP in mock-depleted (ΔMock) or XHbo1-depleted (ΔXHbo1) extracts for 60 min, and chromatin fractions were isolated in the presence of 0.25% NP-40. After fixation, samples were visualized by indirect immunofluorescence with Hoechst (DNA), anti-XMcm3 antibody (Mcm3), or Cy3 (Replication). (D) The replication activity was measured as the incorporation of [α-³²P]dCTP into sperm DNA with various extracts: untreated (No treat.), mock depleted (ΔMock), XHbo1 depleted (ΔXHbo1), or XHbo1 depleted plus a 1/10 volume of untreated extracts (ΔXhbo1 + egg extract). Replication products were subjected to agarose gel electrophoresis, followed by autoradiography.

Acetylation of pre-RC proteins by Hbo1. Two micrograms of recombinant human Hbo1 protein was incubated with His₆-geminin (lanes 2 and 5), His₆-Cdc6 (lanes 3 and 6), GST-Orc2 (lanes 7 and 9), and His₆-Mcm2 (lanes 8 and 10) in the presence of [³H]acetyl-CoA; the proteins were separated on 10% SDS-PAGE gels. The gels were stained with Coomassie (CBB) stain (lanes 4 to 6, 9, and 10), followed by fluorography (Fluorogram) (lanes 1 to 3, 7, and 8). GST protein (lanes 1 and 4) served as a negative control. Bands marked by asterisks identify labeled Hbo1 proteins produced by its autoacetylation.

Hbo1 is required for Mcm6 binding to chromatin during G₁ phase. (A) Experimental protocol. Cells were infected with adenovirus for 10 h, arrested with nocodazole for 16 h, and then released. (B) At the time points indicated, HeLa cells infected with adenoviruses expressing GFP (Control) or antisense Hbo1 (Hbo1as) were sampled, treated with Triton X-100 to extract soluble proteins, and then fixed with paraformaldehyde for IMF. In the top three rows of each set, cells were costained with rabbit polyclonal anti-Hbo1 (Hbo1), mouse monoclonal anti-Mcm6 (Mcm6), and DAPI (magnification, ×100). In the bottom two rows, an independent sample of cells from the same cultures were stained with rabbit polyclonal anti-Orc2 (Orc2) and DAPI (magnification, ×40). (C) Relative chromatin association of Hbo1, Mcm6, and Orc2. The integrated fluorescence intensity of individual nuclei from the experiments shown in panel B was measured, and the averages are shown plotted versus time after release from the nocodazole block. The values shown in each set are normalized by the intensity of the control cells at the 4-h time point. Error bars indicate 1 standard deviation. The data for control cells treated with GFP adenovirus are plotted as open circles, and the data for cells treated with Hbo1 antisense adenovirus are plotted as solid squares.

The enzymatic activity of Hbo1 during the cell cycle. (A) A549 cells were synchronized by a double-thymidine block and release, and whole-cell extracts were prepared at the indicated time points. The whole lysates were probed with antiactin (Actin) as a loading control and anti-Hbo1 antibody (Hbo1). Anti-Hbo1 immunoprecipitates were assayed for HAT activity by the transfer of [³H]acetate to purified chicken histones (Fluorogram). The input histone levels were monitored by Coomassie staining (CBB Stain). (B) The cell cycle synchrony of the culture used for the data shown in panel A was followed by flow cytometry. At the times indicated, cells were sampled and stained with propidium iodide. Histograms of DNA fluorescence are shown. The histogram of the starting asynchronous culture is also shown (async). (C) The cell cycle progression of the culture was also followed by assaying the Cdc2-cyclin B1 kinase activity of cyclin-B1 immunoprecipitates using histone H1 as a substrate.

Xenopus egg extracts contain an Hbo1 protein (XHbo1) that is required for chromatin-bound histone H4 acetylation and is associated with Orc1, Orc2, Cdt1, and geminin. (A) Immunoprecipitation and immunodepletion of a 124-kDa protein in egg extracts with anti-XHbo1 peptide antibody. Interphase egg extracts were treated with preimmune (ΔMock) or anti-XHbo1 peptide 1 (ΔXHbo1) antibodies conjugated to recombinant protein A beads. The depleted extracts were then subjected to sodium dodecyl sulfate (SDS)-PAGE (egg extract). Similarly, the immunoprecipitates from the preimmune (Mock) and anti-peptide 1 (Xhbo1) beads were also subjected to SDS-PAGE (IP Beads). XHbo1 was detected by probing immunoblots with anti-XHbo1 peptide 2 antibody. (B) Acetylation of histone H4 bound to chromatin in Xenopus egg extracts. Sperm chromatin was incubated with 4.5 MBq of [³H]acetyl-coenzyme A in the absence (control) or the presence of GFP-geminin (+gem) or aphidicolin (+aphi). After 60 min, chromatin fractions were isolated and subjected to SDS-PAGE, followed by autoradiography to detect tritium-labeled proteins. A negative control for the chromatin factions was obtained by incubating the extracts in the absence of sperm chromatin (−sperm). Sperm chromatin was also analyzed for acetylation of histone H4 bound to sperm (+sperm). The acetylation of histone H4 was detected by immunoblotting with anti-acetyl-histone H4 antiserum. (C) Sperm chromatin was incubated in untreated (control), mock-depleted (ΔMock), or XHbo1-depleted (ΔXHbo1) extracts in the presence of aphidicolin for 45 min, and chromatin fractions were separated. A negative control for the chromatin factions is shown (−sperm). Histone H4 acetylation was detected by immunoblotting (acetyl-histone H4) and protein staining of histones with Ponceau S is shown as loading control. Both immunostaining and protein-staining intensities were quantified, and the relative amount of acetylated histone H4 in total histone H4 is shown (acetyl H4/total H4). (D) Treated extracts, prepared as in panel A, were immunoblotted with antibody against XHbo1, Orc1, Mcm3, Cdt1, Orc2, Cdc6, and geminin.

Recombinant Cdt1 restores the chromatin binding of Mcm2-7 in the absence of XHbo1. Sperm chromatin was incubated in untreated, mock-depleted (ΔMock), or Hbo1-depleted (ΔXHbo1) extracts with addition of buffer alone (control), recombinant Xenopus Cdt1 (+Cdt1) or GFP-tagged Xenopus geminin (+gem). The chromatin fractions were isolated and immunoblotted as described in the legend to Fig. 6D.