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First published online 16 November 2004
doi: 10.1242/jcs.01523

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The human histone gene expression regulator HBP/SLBP is required for histone and DNA synthesis, cell cycle progression and cell proliferation in mitotic cells

School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, UK

View larger version (42K): [in a new window]   Fig. 1. Inhibition of HBP expression by RNA interference. (A) Top: Schematic drawing showing the location of the shRNA target sequences in the 810 nucleotide HBP open reading frame. Boundaries of sequences coding for domains involved in histone RNA binding (RBD) and RNA processing (RP) and for the putative translation stimulation domain (T) are indicated (Wang et al., 1996; Dominski et al., 1999; Sanchez and Marzluff, 2002). The position of the sequences coding for residues responsible for the control of HBP stability (St) are also shown (Zheng et al., 2003). Note that the drawing is not to scale. Middle: Schematic diagram showing the structure of the RNA interference cassette inserted into the pGEM-T Easy cloning site. Shown are pGEM-T Easy flanking regions, Sp6 RNA polymerase promoter (SP6), U6 RNA promoter (U6P) and termination signal (TTTTT). U6 promoter and terminator flank the inverted repeat coding for the shRNAs. MluI: restriction sites used for the excision of the cassettes and the subcloning into pEGFP-C3. Bottom: Sequences of the shRNAs used to inhibit HBP expression by RNAi. The control hairpin cHP deviates at two positions (indicated in lower case letters) from HP1. (B) HeLa cell extract was analysed by western blotting using anti-HBP serum. Strips were probed with both primary and secondary antibody, secondary antibody only, or with primary antibody preincubated with the antigenic peptide, and the secondary antibody. The HBP position is marked by the arrow; * marks a major non-specific band with the mobility corresponding to a 38 kDa protein. The positions of molecular mass standards are indicated. (C) HeLa cells were transfected with either pGEM-U6pHP1 or pGEM-U6p or mock-transfected (no DNA). Protein samples prepared 24 hours after transfection were analysed by western blotting using anti-HBP and subsequently anti-tubulin antibodies as loading control. Blots were stripped between subsequent probings. *as in B. rHBP is recombinant human HBP. (D) HeLa cells were transfected with pGEM-U6pT, pGEMU6pHP1 or pGEM-U6HP3 and treated as described for B.

View larger version (52K): [in a new window]   Fig. 2. Inhibition of HBP expression inhibits histone gene expression. Asynchronous HeLa cells were transfected with either the U6pHP1, U6pHP2, U6pHP3, U6pcHP or U6pT cassette and RNA samples were prepared 24 hours or 48 hours after transfection and analysed by northern blotting for HBP mRNA and histone mRNA, and for 18S rRNA as loading control. (A) Effect of transfection with U6pT, U6pHP1, U6pHP2, U6pHP3 or U6pcHP on HBP and histone H3 expression 24 hours after transfection. mRNA levels were standardised with respect to 18S rRNA and expressed as the percentage of mRNA in the cells transfected with U6pT (=100%). (B) Effect of mock transfection (no DNA) and transfection with U6pHP1 or U6pT on histone H1, H2A, H2B, H3 and H4 mRNA levels 24 hours and 48 hours after transfection. (C) mRNA levels from B standardised with respect to 18S rRNA and expressed as a percentage of mRNA in the `no DNA' control (=100%) at 24 hours and at 48 hours after transfection.

View larger version (57K): [in a new window]   Fig. 3. HBP expression is essential for cell proliferation. (A) Schematic drawing showing the structure of the pEGFP-C3 derivatives used to determine the effect of HBP RNAi on cell proliferation. The cassettes expressing shRNAs were inserted in the unique MluI restriction site located outside the sequence elements involved in the expression of EGFP (expressed from the pCMV promoter and using the SV40 polyadenylation signal) or the neomycin phosphotransferase gene (expressed from the pSV40 promoter and using the Herpes simplex thymidine kinase (HSV TK) polyadenylation signal). Indicated are the U6 RNA promoter and terminator (U6P, TTTTT) flanking the inverted repeat coding for the shRNA. (B) HeLa cells were transfected with pEGFP-U6pT, pEGFPU6pcHP, pEGFP-U6pHP1, pEGFP-U6pHP2 or pEGFP-U6pHP3, or mock treated without DNA. Subsequently G418 was added and cells were analysed as described in Materials and Methods. Shown are cells prior to addition of G418 (0 days) and after addition of G418 (3 days and 7 days).

View larger version (29K): [in a new window]   Fig. 4. Inhibition of HBP expression inhibits cell cycle progression. HeLa cells were arrested by treatment with nocodazole and subsequently released. One hour after release, cells were transfected with either pGEM-U6pT or pGEM-U6pHP1 or mock transfected without DNA. Samples were prepared for FACS analysis by staining with propidium iodide at the indicated times after release. (A) FACS profiles 8, 16 and 26 hours after release from nocodazole block. The boundaries that define cells with sub-G1-, G0/G1-, S- and G2/M-phase DNA content were determined using asynchronous HeLa cells and are shown in the 8 hours/noDNA samples. Identical settings were used for the analysis of the other samples. (B) Cell cycle distribution. Shown is the percentage of cells with sub-G1-, G0/G1-, S- and G2/M-phase DNA content. Results are representative of two independent experiments.

View larger version (22K): [in a new window]   Fig. 5. Inhibition of HBP expression delays S-phase progression. HeLa cells were transfected with either pGEM-U6pHP1, pGEM-U6pHP3 or the control constructs pGEM-U6pcHP and pGEM-U6pT and subsequently arrested by HU treatment as described in Materials and Methods. Samples were prepared for FACS analysis by staining with propidium iodide immediately prior to the release from HU block (0 hours) or 7 hours after release. (A) FACS profiles. The boundaries that define cells with G0/G1, S- and G2/M-phase DNA content were determined as described in Fig. 4A. (B) Cell cycle distribution. Shown is the percentage of cells with G0/G1, S- and G2/M-phase DNA content. Results are representative of two independent experiments.

View larger version (31K): [in a new window]   Fig. 6. Inhibition of HBP expression results in delay at G1/S phase boundary and interferes with S phase progression. HeLa cells were transfected with either pGEM-U6pHP1 or pGEM-U6pT and arrested by HU treatment as described in Materials and Methods. Samples were prepared for FACS analysis by staining with propidium iodide at the indicated times after release from HU. (A) FACS profiles. Time points shown are hours after release from HU-treatment. Zero-hour samples were taken immediately before the release from HU block. The boundaries that define cells with G0/G1, S- and G2/M-phase DNA content were determined as described in Fig. 4A. (B) Cell cycle distribution. Shown is the percentage of cells with G0/G1, S- and G2/M-phase DNA content. (C) Analysis of protein samples by western blotting. Membranes were probed with anti-cyclin A, anti-HBP and anti-tubulin antibodies except for the 0-hour time point when samples from the same protein preparation were analysed on different gels. Note that therefore the 3, 8 and 16 hours anti-tubulin panels are identical. Results are representative of three independent experiments.