Histone H3.3 deposition at E2F-regulated genes is linked to transcription - PubMed (original) (raw)

Histone H3.3 deposition at E2F-regulated genes is linked to transcription

Laetitia Daury et al. EMBO Rep. 2006 Jan.

Abstract

The histone variant H3.3 can be incorporated in chromatin independently of DNA synthesis. By imaging using green fluorescent protein-tagged histones, H3.3 deposition has been found to be linked with transcriptional activation. Here, we investigated H3.3 incorporation during G1 progression on cell-cycle-regulated E2F-dependent genes and on some control loci. We transiently transfected resting cells with an expression vector for tagged H3.3 and we analysed its presence by chromatin immunoprecipitation. We found that replication-independent H3.3 deposition occurred on actively transcribed genes, but not on silent loci, thereby confirming its link with transcription. Interestingly, we observed similar levels of H3.3 occupancy on promoters and on the coding regions of the corresponding genes, indicating that H3.3 deposition is not restricted to promoters. Finally, H3.3 occupancy correlated with the presence of transcription-competent RNA polymerase II. Taken together, our results support the hypothesis that H3.3 is incorporated after disruption of nucleosomes mediated by transcription elongation.

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Figures

Figure 1

Figure 1

Histone H3 is not depleted from the dihydrofolate reductase promoter during G1 progression. (A) Schematic representation of the mouse dhfr (dihydrofolate reductase) locus, with the position of probes detected by quantitative PCR (Q-PCR) in chromatin immunoprecipitation (ChIP) experiments. Note the presence of the msh3 gene 600 bp upstream of the dhfr transcription start site and transcribed in the opposite direction. (B) NIH3T3 cells were starved of serum for 48 h and then induced for 8 h with 20% FCS. Cells were then subjected to a ChIP assay using an anti-H3 antibody or no antibody as a control (NA). The amounts of the indicated sequences were measured by Q-PCR. A representative experiment out of three is shown. Ab, antibody.

Figure 2

Figure 2

Study of histone H3.3 deposition during G1 progression. (A) Description of the experimental procedure. NIH3T3 cells (400,000 cells/10 cm dish, six dishes per immunoprecipitation) were starved in 0.5% FCS for 48 h and then transiently transfected with 30 μg of the histone H3.3 expression vector per dish. At 34 h after transfection, cells were induced using 20% FCS and collected for chromatin immunoprecipitation (ChIP) 8 h later. (B) NIH3T3 cells, treated as described in (A), were subjected to a ChIP assay using either the anti-haemagglutinin antibody (HA) or the anti-Flag antibody (Irr) as a control. The amounts of the dhfr (dihydrofolate reductase) promoter (DHFR-P), and P0 sequences were measured by quantitative PCR (Q-PCR). A representative experiment out of ten is shown. Ab, antibody. (C) Same as (B), except that NIH3T3 cells were transfected with either the H3.3–HA or the H3.1–HA expression vector. A representative experiment out of two is shown. (D) Same as (B), except that the amounts of the indicated sequences were measured by Q-PCR. A representative experiment out of three is shown.

Figure 3

Figure 3

Analysis of histone H3.3 presence in stably expressing cells. NIH3T3 cells stably expressing tagged H3.3 were starved of serum for 48 h and then re-induced for 8 h with 20% FCS and subjected to a chromatin immunoprecipitation (ChIP) assay using the anti-haemagglutinin antibody (HA) or no antibody as a control. The amounts of the indicated sequences were measured by quantitative PCR. A representative experiment out of three is shown. Ab, antibody.

Figure 4

Figure 4

Deposition of H3.3 correlates with transcription. NIH3T3 cells were starved of serum for 48 h and then re-induced for 8 h with 20% FCS and subjected to a chromatin immunoprecipitation (ChIP) assay using an anti-pol II antibody (A) or an anti-S5-phosphorylated pol II antibody (B). The amounts of the indicated sequences were measured by quantitative PCR (Q-PCR). A representative experiment out of three is shown. (C) Left panel: NIH3T3 cells were starved of serum for 48 h and then re-induced or not, as indicated, with 20% FCS for 8 h. Right panel: NIH3T3 cells were transfected with the H3.3–haemagglutinin (HA) expression vector and re-induced for 2 or 8 h with 20% FCS, as indicated. Cells were then subjected to a ChIP assay using an anti-RNA polymerase II antibody (RNAP), anti-S5 phosphorylated RNAP antibody (S5P-RNAP; left panel) or an anti-HA antibody (right panel). The amount of dhfr (dihydrofolate reductase) promoter was measured by Q-PCR. A representative experiment out of three is shown. Also shown is a western blot monitoring H3.3–HA expression. Note that in the control we induced cells with serum for 2 h because exogenous histone expression is very low in uninduced cells (data not shown). Ab, antibody.

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