Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 May 1;436(1-2):108-14.
doi: 10.1016/j.gene.2009.01.020. Epub 2009 Feb 10.

Histone acetyltransferase Hbo1: catalytic activity, cellular abundance, and links to primary cancers

Masayoshi Iizuka  1 , Yoshihisa Takahashi, Craig A Mizzen, Richard G Cook, Masatoshi Fujita, C David Allis, Henry F Frierson Jr, Toshio Fukusato, M Mitchell Smith
Affiliations

Histone acetyltransferase Hbo1: catalytic activity, cellular abundance, and links to primary cancers

Masayoshi Iizuka et al. Gene. .

Abstract

In addition to the well-characterized proteins that comprise the pre-replicative complex, recent studies suggest that chromatin structure plays an important role in DNA replication initiation. One of these chromatin factors is the histone acetyltransferase (HAT) Hbo1 which is unique among HAT enzymes in that it serves as a positive regulator of DNA replication. However, several of the basic properties of Hbo1 have not been previously examined, including its intrinsic catalytic activity, its molecular abundance in cells, and its pattern of expression in primary cancer cells. Here we show that recombinant Hbo1 can acetylate nucleosomal histone H4 in vitro, with a preference for lysines 5 and 12. Using semi-quantitative western blot analysis, we find that Hbo1 is approximately equimolar with the number of active replication origins in normal human fibroblasts but is an order of magnitude more abundant in both MCF7 and Saos-2 established cancer cell lines. Immunohistochemistry for Hbo1 in 11 primary human tumor types revealed strong Hbo1 protein expression in carcinomas of the testis, ovary, breast, stomach/esophagus, and bladder.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Hbo1 per se is a nucleosomal H4 acetyltransferase
(A) Purified His-Hbo1 protein (arrow) was resolved by SDS-PAGE and stained with Coomassie Blue. The migration of molecular weight markers (kDa) is shown. (B) Chicken free core histone mixture (lanes 1–6) and chicken mononucleosomes (lanes 7–12) were incubated with [3H]-acetyl-CoA and no added enzyme (lanes 1, 4, 7, and 10) or plus purified His-Hbo1 (lanes 2, 5, 8, and 11) or plus human Hat1 (lanes 3, 6, 9, and 12). Reaction products were then resolved by 15% SDS-PAGE and detected by Coomassie Blue and fluorography.
Figure 2
Figure 2. Acetylation site mapping of nucleosomal H4 by Hbo1
(Chicken mononucleosomes were incubated with purified His-Hbo1 and [3H]-acetyl-CoA. Histones were then recovered from the reaction by acid extraction and purified by RP-HPLC. Purified H4 was then treated to deblock the α-amino group and microsequenced. The dashed line illustrates the experimental repetitive yield of 92% normalized to the recovery of [3H]-acetyl-Lys8.
Figure 3
Figure 3. Hbo1 is the sole acetyltransferase in the Hbo1 protein complex
A549 cells were infected with adenoviral vectors expressing either wild type (“Hbo1”) or catalytically dead (“Hbo1-G485A”) Hbo1 and immunoprecipitates were prepared using anti-Hbo1 antibody. The immunoprecipitates were assayed for HAT activity in reactions containing [3H]-acetylCoA and chicken core histones. (A) HAT activity. The incorporation of [3H]-acetate is plotted in the top panel. HAT reactions were spotted on P81 filters, washed, and assayed by scintillation counting. The recovery of Hbo1 in the immunoprecipitates, as assayed by western blot, is shown in the bottom panel. The left lane (“beads”) represents a mock immunoprecipitate prepared without primary antisera from cells infected with the wild type Hbo1 adenovirus. (B) Substrate specificity. HAT reactions were separated by electrophoresis on a 15% SDS-PAGE gel. The top panel shows the histone content of the reactions by Coomassie staining. The bottom panel shows the [3]-labeled histones visualized by fluorography.
Figure 4
Figure 4. Hbo1 regulates global H4 acetylation
(A) Whole cell extracts infected with Ad-GFP (“GFP”) and Ad-antisense Hbo1 (“AS”) for 48 hr were analyzed by western blotting with antibodies to Hbo1, acetylated histone H4, and α-tubulin. (B) Whole cell extracts infected with Ad-Hbo1 (“WT”) and Ad-catalytically inactivated Hbo1 (“Mut”) for 48 hr were analyzed by western blotting with antibodies to Hbo1, acetylated histone H4, and α-tubulin. (C) Chromatin fractions of virus-infected MCF7 cells were separated on a protein gel, transferred to a membrane, were assayed for Histone H2B by immunoblotting to examine integrity of core histones.
Figure 5
Figure 5. Estimation of the number of Hbo1 molecules in cells
Whole cell extracts derived from 2.9 × 106 HFF2/T cells (A), 2 × 105 MCF7 cells (B), and 2 × 105 Saos-2 cells (C), along with defined amounts of recombinant Hbo1 proteins, were separated in protein gels, and transferred onto nitrocellulose membrane. The filter was probed with anti-Hbo1 antibody. Signal intensities of the bands were counted and the amounts of Hbo1 protein were estimated by reference to the standard plot.
Figure 6
Figure 6. Overexpression of Hbo1 in human primary tumors
The immunohistochemical staining of Hbo1 is shown for the tumor samples listed in Table 1, tissue array number 2. (A) Renal cell carcinoma, negative; (B) gastric adenocarcinoma, staining in ~10% of tumor cells; (C) gastric adenocarcinoma, staining in >50% of tumor cells; (D) breast adenocarcinoma; (E) ovarian yolk sac tumor; and (F) seminoma.
See this image and copyright information in PMC

References

    1. Aggarwal B, Calvi B. Chromatin regulates origin activity in Drosophila folliclecells. Nature. 2004;430:372–376. - PubMed
    1. Aparicio J, Viggiani C, Gibson D, Aparicio O. The Rpd3-Sin3 histone deacetylase regulates replication timing and enables intra-S origin control in Saccharomyces cerevisiae. Mol Cell Biol. 2004;24:4769–4780. - PMC - PubMed
    1. Arias EE, Walter JC. Strength in numbers: preventing rereplication via multiplemechanisms in eukaryotic cells. Genes Dev. 2007;21:497–518. - PubMed
    1. Avvakumov N, Cote J. The MYST family of histone acetyltransferases and theirintimate links to cancer. Oncogene. 2007;26:5395–407. - PubMed
    1. Bird AW, Yu DY, Pray-Grant MG, Qiu Q, Harmon KE, Megee PC, et al. Acetylation of histone H4 by Esa1 is required for DNA double-strand break repair. Nature. 2002;419:411–5. - PubMed

Publication types

MeSH terms

LinkOut - more resources