Coupling of DNA synthesis and histone synthesis in S phase independent of cyclin/cdk2 activity

Abstract

DNA and histone synthesis are both triggered at the beginning of S phase by cyclin/cdk2 activity. Previous studies showed that inhibition of DNA synthesis with hydroxyurea or cytosine arabinoside (AraC) triggers a concerted repression of histone synthesis, indicating that sustained histone synthesis depends on continued DNA synthesis. Here we show that ectopic expression of HIRA, the likely human ortholog of two cell cycle-regulated repressors of histone gene transcription in yeast (Hir1p and Hir2p), represses transcription of histones and that this, in turn, triggers a concerted block of DNA synthesis. Thus, in mammalian cells sustained DNA synthesis and histone synthesis are mutually dependent on each other during S phase. Although cyclin/cdk2 activity drives activation of both DNA and histone synthesis at the G1/S transition of cycling cells, concerted repression of DNA or histone synthesis in response to inhibition of either one of these is not accompanied by prolonged inhibition of cyclin A/cdk2 or E/cdk2 activity. Therefore, during S phase coupling of DNA and histone synthesis occurs, at least in part, through a mechanism that is independent of cyclin/cdk2 activity. Coupling of DNA and histone synthesis in S phase presumably contributes to the prompt and orderly assembly of newly replicated DNA into chromatin.

FIG. 1.

Ectopic expression of HIRA represses histone gene transcription. (a) U2OS cells were transiently transfected with pcDNA3 or pcDNA3 HA-HIRA as indicated together with pCMV-CD19. Transfected CD19⁺ cells were collected, and total RNA was prepared and Northern blotted to determine abundances of the indicated mRNAs. EtBr, ethidium bromide. (b) U2OS cells were transiently transfected with pcDNA3 (lanes 1 to 4), pcDNA3 HA-HIRA (lane 5 to 8), or pcDNA3 HA-HIRA plus pcDNA3 HA-SLBP (lanes 9 to 12) together with pCMV-CD19. Thirty-six hours later 10 μg of actinomycin D/ml was added, and at the indicated times afterwards the CD19⁺ transfected cells were collected and the abundances of histone and GAPDH mRNAs were determined by Northern blotting. Total-cell lysates were Western blotted with anti-HA. (c) The histone mRNA signals from panel b were quantitated on a phosphorimager and expressed as a percentages of control (100% = signal intensity at time zero). (d) U2OS cells were infected with an adenovirus encoding LacZ (lanes 1 and 2) or HIRA(421-729) (lanes 3 and 4). Twenty-four hours later nuclei were harvested and the rate of histone H2B transcription was determined in a runoff assay. ³²P-labeled mRNA (5 × 10⁶ ethanol-precipitable cpm per membrane) was used to probe a membrane slot blotted with pcDNA3Δ (lanes 2 and 4) or pcDNA3ΔH2B (lanes 1 and 3). (e) The signals in panel d were quantitated on a phosphorimager, corrected for the background hybridization on vector alone, and expressed as arbitrary units.

FIG. 2.

Endogenous HIRA forms subnuclear foci that contain HDAC4 in a subpopulation of primary cells. Primary human WI38 cells grown on coverslips were stained with anti-HIRA (a and d), anti-HDAC4 antibodies (b), and DAPI (c and e) to visualize the DNA. Bars, 10 μM.

FIG. 3.

Endogenous HIRA forms subnuclear foci in G₁ phase of the cell cycle. Primary human WI38 cells were stained with antibodies to HIRA (a, d, and g), NPAT (b), CENP-F (e), and PCNA (h) and with DAPI (c, f, and i) to visualize the DNA. (j) The NPAT-containing foci in 60 randomly selected DAPI-stained nuclei (hatched bars) and 60 nuclei containing HIRA foci (shaded bars) were counted, and the numbers of cells with zero, two, and four foci were expressed as percentages of the total. (k) The cells staining positive for CENP-F-containing foci in 100 randomly selected DAPI-stained nuclei (hatched bars) and 100 nuclei containing HIRA foci (shaded bars) were counted, and the numbers of positive and negative cells were expressed as percentages of the total. (l) Same as panel k except that cells stained with antibodies to PCNA were counted. Bars, 10 μM.

FIG. 4.

S-phase arrest and histone mRNA repression depend on the evolutionarily conserved B domain of HIRA. (a) U2OS cells were transiently transfected with a plasmid encoding wild-type (WT) HA-HIRA (lane 2) or a series of deletion mutants (lanes 3 to 9) or the empty vector (pcDNA3; lane 1) together with pCMV-CD19. The cells were harvested, and cell cycle distribution and abundances of histone H2B and GAPDH mRNAs in the transfected CD19⁺ cells were determined. (b) Total-cell lysates were Western blotted with anti-HA. (c) U2OS cells were transiently transfected with pcDNA3 (lane 1), 3, 10, or 28 μg of pcDNA3 HA-HIRA WT (lanes 2 to 4), or 3, 10, or 28 μg of pcDNA3 HA-HIRAΔB (lanes 5 to 7) together with pCMV-CD19. The cells were harvested, the cell cycle distribution of the CD19⁺ transfected cells was determined, and an aliquot of total-cell lysate was Western blotted with anti-HA. (d) U2OS cells were transiently transfected with pcDNA3 (lane 1), pcDNA3 HA-HIRA WT (lane 2), or pcDNA3 HA-HIRA ΔB (lane 3) together with pCMV-CD19. Transfected CD19⁺ cells were collected, and total RNA was Northern blotted to determine expression of GAPDH and histone H2B mRNAs.

FIG. 5.

HIRA-induced histone gene repression precedes S-phase arrest and generates incompletely assembled chromatin. (a) U2OS cells were transiently transected with pcDNA3 or pcDNA3 HA-HIRA WT as indicated together with pCMV-CD19. The cells were arrested at the G₁/S transition with HU and then released into S phase. Cells were harvested at the indicated times afterwards, and the cell cycle distribution of the CD19⁺ cells (top) and abundances of histone H2B, GAPDH, and E2F1 mRNAs (indicated at the right) in the CD19⁺ cells were determined. Total-cell lysates were Western blotted with anti-HA (HIRA). (b) U2OS cells were infected with an adenovirus expressing LacZ (lanes 3, 5, 7, and 9) or HIRA(421-729) (lanes 2, 4, 6, and 8). Twenty-four hours later the cells were permeabilized and treated without MNase or with 0.25, 2.5, or 25 Worthington units of MNase/ml (shaded boxes). Genomic DNA was purified, fractionated by 0.6% agarose gel electrophoresis, and visualized with ethidium bromide. The size markers in lane 1 are a 100-bp ladder. (c) U2OS cells were infected with an adenovirus expressing LacZ or HIRA(421-729). After 4 h 2 mM HU was added to the cells, and 20 h later it was washed off and the cells were allowed to progress into S phase for 4 h. The cells were harvested, permeabilized, and treated without MNase or with 0.25 or 2.5 Worthington units of MNase/ml (shaded boxes). Genomic DNA was purified, fractionated by 0.6% agarose gel electrophoresis, and visualized with ethidium bromide. The size markers in lane 1 are a 100-bp ladder. (d) Samples from cells harvested for panel c were also processed for FACS analysis to determine the cell cycle distribution. Ad, adenovirus.

FIG. 6.

HIRA-induced S-phase arrest is overridden by the histone mRNA-stabilizing protein, SLBP. (a) U2OS cells were transiently transected with pcDNA3 (lanes 1 and 9) or pcDNA3 HA-HIRA WT (lanes 2 to 8) in the absence or presence of pcDNA3 HA-SLBP (0.01, 0.1, 0.25, 0.5, 1, or 3 μg [triangle]) together with pCMV-CD19. Total-cell lysates were prepared and Western blotted with antibodies to SLBP or HA-HIRA. (b) Same as panel a except that the cell cycle distribution of CD19⁺ cells was determined. (c) RNA was prepared from transfected CD19⁺ cells and Northern blotted to determine abundance of histone H2B. (d) U2OS cells were transiently transfected with pcDNA3 or pcDNA3 HA-HIRA WT in the absence or presence of pcDNA3 HA-SLBP (3, 8, or 18 μg [triangle]) together with pCMV-CD19. The cells were arrested in G₁ phase with mimosine and then released into S phase. The cell cycle distribution of the CD19⁺ cells was determined 12 h after release. (e) Cell lysates from panel d were Western blotted with anti-HA antibodies.

FIG. 7.

Override of HIRA-induced S-phase arrest by SLBP requires binding of SLBP to 3′ UTR of histone mRNA. (a) U2OS cells were transiently transfected with pcDNA3 or pcDNA3 HA-HIRA WT in the absence or presence of pcDNA3 HA-SLBP WT, pcDNA3 HA-SLBP RR/KK, or pcDNA3 HA-SLBP QKQ (0.2 or 1 μg [triangles]) together with pCMV-CD19. Cells were harvested, and the cell cycle distribution was determined by FACS. (b) Lysates were Western blotted with antibodies to HA.

FIG. 8.

HIRA-induced S-phase arrest is independent of changes in cyclin/cdk2 activity. (a) U2OS cells were transiently transfected with pcDNA3 (lane 1), pcDNA3 encoding the indicated HIRA deletion mutants (lanes 2 to 4), or pRC-CMVp21cip1 (lane 5) together with pCMV-CD19. The transfected CD19⁺ cells were collected, and lysates were prepared and Western blotted with anti-PCNA, anti-cyclin A, and anti-cdk2 as indicated. (b) Same as panel a except lysates were used to assay cdk2, cyclin A, and cyclin E by immunoprecipitation-kinase assay. (c) Same as panel a except cell lysates were Western blotted with anti-pRB.

FIG. 9.

HU-induced histone mRNA repression is independent of changes in cyclin/cdk2 activity. (a) U2OS cells were treated with 2 mM HU for 0, 2, or 22 h as indicated and then harvested and processed for FACS analysis to determine the cell cycle distribution of the cells. (b) Cells were treated as in panel a, and total cellular RNA was prepared and Northern blotted to determine the abundances of the histone H2B and GAPDH mRNAs. (c) Cells were treated as in panel a, lysates were prepared and processed for immunoprecipitation-kinase assay with antibodies to cyclin E (HE111; top), cyclin A (C160; middle), and cdk2 (M2; bottom) as indicated. In each case, lane 1 is derived from extract immunoprecipitated with an appropriate negative-control antibody (mouse 9E10 for HE111 and C160 and purified rabbit anti-mouse polyclonal for M2).