HBO1 histone acetylase is a coactivator of the replication licensing factor Cdt1

Abstract

HBO1 histone acetylase is important for DNA replication licensing. In human cells, HBO1 associates with replication origins specifically during the G1 phase of the cell cycle in a manner that depends on the replication licensing factor Cdt1, but is independent of the Cdt1 repressor Geminin. HBO1 directly interacts with Cdt1, and it enhances Cdt1-dependent rereplication. Thus, HBO1 plays a direct role at replication origins as a coactivator of the Cdt1 licensing factor. As HBO1 is also a transcriptional coactivator, it has the potential to integrate internal and external stimuli to coordinate transcriptional responses with initiation of DNA replication.

Figure 1.

HBO1 associates with mammalian origins of replication. (A) Binding profile of HBO1 at the MCM4 origin (OR) locus in HEK293 kidney, HeLa adenocarcinoma, and CCL-156 lymphoblastoid cells. Control IgG (B), MOF (C), ORC1 (D), and MCM5 (E) binding profile at the MCM4 origin in the same chromatin sample. (F) ChIP analysis of HBO1 and ORC2 binding profile at the mice HPRT1 origin. (Insert) Western blot analysis of HBO1 antibody specificity against human and mice whole cellular extracts. (G) Re-ChIP analysis of HBO1 and ORC1 co-occupancy on MCM4 and LaminB2 origins in HeLa cells.

Figure 2.

HBO1 association with origins is restricted to G1. (A) Expression level of HBO1, ORC1, Cdt1, and MCM2 mRNAs in CCL-156 cells in culture (asynchronously), following serum deprivation and cell cycle exit (G0), and following readdition of 20% serum in the media for 6 h after serum deprivation (G0 + serum). The left panel illustrates the cell cycle profile of samples used in the experiment. “no RT” referrs to the control condition where cDNA synthesis was omitted prior to PCR analysis. (B) Expression level of HBO1, MCM2, and ORC2 proteins upon readdition of serum for indicated number of hours. (C) ChIP analysis of HBO1 binding at origins and control sites in growing and serum-starved cells. (D) ChIP analysis of HBO1 binding at origins in cells staged at different phase of the cycle.

Figure 3.

Licensing factor Cdt1 stabilizes HBO1 at origins. (A) Impact of proteasome inhibition on HBO1 association with the laminB2 origin in cells previously treated with DNA damaging agent actinomycin D. (B) Impact of proteasome inhibition on ORC1, HBO1, and Cdt1 association with the LaminB2 origin in cells previously G1/S staged with HU. (C) Impact of proteasome inhibition on Cdt1 and HBO1 expression level and chromatin association in HeLa cells treated with ActD. (D) Impact of proteasome inhibition on Cdt1 and HBO1 expression level and chromatin association in HeLa cells treated with HU. (E) Impact of Cdt1Δ32 overexpression on HBO1 association with LaminB2 origin in HeLa cells treated with HU. (F) Impact of Cdt1Δ32 overexpression on HBO1 association with LaminB2 origin in HeLa cells treated with ActD. (G) Impact of Cdt1 depletion on HBO1 association with replication origins in HeLa cells.

Figure 4.

Cdt1 directly interacts with HBO1. (A) Detection of HBO1 association with Cdt1 by reverse coimmunoprecipitation in HeLa cells overexpressing either HA-Cdt1 or Flag-HBO1. (B) Same experiment using antibodies directed against endogenous HBO1 and Cdt1. (C) Interaction of in vitro translated Flag-HBO1 with full-length His-Cdt1 and His-Geminin. (D) Interaction of purified in vitro translated Flag-HBO1 with His-Cdt1. (E) In vitro preformed Cdt1/HBO1 complex were challenged by increasing amount of bacterially produced Geminin. (F) Mix of Cdt1, Geminin, and HBO1 proteins were incubated with the indicated antibodies, and the purified complexes was determined by Western blot analysis. (G) Role of the MYST zinc finger. (Left panel) The indicated HBO1 derivatives were analyzed for interaction with Cdt1 or occupancy at the MCM4 and LaminB2 origins. (*) P < 0.05 ; (**) P < 0.15.

Figure 5.

HBO1 enhances Cdt1 licensing activity. Rereplication events in the indicated cells lines overexpressing HBO1, Cdt1, or Cdt1 + HBO1 as defined by the percentage of cells with >4N content as determined by FACS analysis.