HBO1 histone acetylase activity is essential for DNA replication licensing and inhibited by Geminin

Abstract

HBO1, an H4-specific histone acetylase, is a coactivator of the DNA replication licensing factor Cdt1. HBO1 acetylase activity is required for licensing, because a histone acetylase (HAT)-defective mutant of HBO1 bound at origins is unable to load the MCM complex. H4 acetylation at origins is cell-cycle regulated, with maximal activity at the G1/S transition, and coexpression of HBO1 and Jade-1 increases histone acetylation and MCM complex loading. Overexpression of the Set8 histone H4 tail-binding domain specifically inhibits MCM loading, suggesting that histones are a physiologically relevant target for licensing. Lastly, Geminin inhibits HBO1 acetylase activity in the context of a Cdt1-HBO1 complex, and it associates with origins and inhibits H4 acetylation and licensing in vivo. Thus, H4 acetylation at origins by HBO1 is critical for replication licensing by Cdt1, and negative regulation of licensing by Geminin is likely to involve inhibition of HBO1 histone acetylase activity.

Figure 1

HBO1 acetylase activity is essential for DNA licensing. DNA fragments bound by Myc-HBO1 and HAT inactive Myc-HBO1^G485 were analyzed by sequential chromatin immunoprecipitation analysis for ORC and MCM complexes co-occupancy at the MCM4 and Chr16/Axin origin (n=2). The genomic regions around these replication origins (OR) are shown with neighboring genes (arrows) and positions of primer pairs (black bars) are indicated (coordinates flanking the origin are in kb). Values are expressed as relative occupancy over the control background region (mean ± SD).

Figure 2

HBO1 controls H4 acetylation at origins. (A) Bulk histones from control and HBO1 depleted HeLa cells were analyzed by Western blots with antibodies directed against the indicated modifications of H4 and H3. Total H3 serves as a loading control. The relative acetylation level in HBO1-depleted cells as compared to control cells is indicated to the right of each panel. (B) Acetylation levels (mean ± SD) of the indicated H4 lysine at origins and surrounding regions (n=3). (C) Acetylation levels (mean ± SD) of H3 and H4 at the indicated origins and constitutively hyper-acetylated promoters in HeLa cells depleted of HBO1 expressed as a percent of the levels in control cells (n=3). (D) DNA fragments bound by Myc-HBO1 and HAT inactive Myc-HBO1^G485 were analyzed by sequential chromatin immunoprecipitation analysis for H4-K12 acetylation (H4-K12Ac) co-occupancy at the MCM4 and Chr16/Axin origin (n=2).

Figure 3

H4 acetylation at origins is cell cycle regulated. (A) H4 acetylation levels (mean ± SD) at replication origins and flanking regions (indicated in kb from the origin) in cells at the indicated stages of the cell cycle (n=3). The genomic regions around these replication origins (OR) are shown with neighboring genes (arrows) and positions of primer pairs (black bars) are indicated (coordinates flanking the origin are in kb). (B) Same as above, except that the origins were isolated from annotated promoters.

Figure 4

Co-expression of HBO1 and Jade-1 stimulates H4 acetylation and MCM complex loading. (A) Western blot analysis showing H4 acetylation status (extracted histones; H3 levels serves as the internal control) and presence of the indicated proteins with in chromatin in cells over-expressing Jade-1L or HBO1 or both. (B) Similar experiment in cells co-expressing Jade-1L with either HBO1 or HAT-defective derivative HBO1^G485.

Figure 5

Expression of the Set8 histone tail-binding domain inhibits H4 acetylation and MCM complex loading. (A) Western blot analysis to measure acetylation at the indicated residues in acid extracted histones prepared from HeLa cells that do or do not express the histone-binding domain (HBD) of Set8. (B) Association of ORC2, HBO1 and MCM5 (mean ± SD) at the Chr16/Axin1 origin detected by ChIP in cell that do or do not express Set8-HBD (n=3). (C) Comparison of full-length Set8 and Set8-HBD effects on H4 acetylation and levels of MCM3, ORC1, and HBO1 in chromatin and nuclear extract. (D) Western blot analysis to examine the effect of mimosine and Set8-HBD (blocks in early S-phase) on H4 acetylation and levels of MCM3, Cdt1 and ORC2 in the chromatin.

Figure 6

Geminin inhibits H4 acetylase activity of HBO1 in vitro. (A) Effect of recombinant full-length Geminin on the H4 acetylase activity of immunoprecipitated Flag-HBO1, Flag-HBO1^G485, and purified yeast piccolo NuA4 complex. (B) Same experiment as in (A) except that Flag-HBO1 was purified from cells over-expressing Flag-HBO1 and HA-Cdt1. (C) Flag-HBO1 immunoprecipitates prepared from cells that do or do not co-express HA-Cdt1 were analyzed by Western blotting with antibodies against HBO1, HA-Cdt1, and ING4. HBO1 complexes in the presence or absence of Cdt1 were also tested for their HAT activity on H4-K12. (D) Effect of Geminin on the H4 activity of HA-Cdt1 complexes purified from cells co-expressing either HA-Cdt1+Flag-HBO1 or HA-Cdt1+Flag-HBO1^G485. Numbers under the panels indicate the relative amount of acetylated H4-K12 HAT as compared to cells in the absence of Geminin. Note that weak H4-K12 acetylase activity observed in the HA-Cdt1+Flag-HBO1^G485 immunoprecipitates is insensitive to Geminin.

Figure 7

Geminin associates with replication origins and inhibits H4 acetylation in vivo. (A) Relative H4 tetra-acetylation level (mean ± SD) at origins in cells expressing the non-degradable Flag-Gem^L26A derivative (n=3). (B) Association of Flag-Gem^L26A (mean ± SD) at origins (MCM4, Myc, Chr16, Chr11) and control regions (Luc7L, PIP5K1A)(n=3).