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Comparative Study
. 2005 Mar 15;19(6):677-82.
doi: 10.1101/gad.1265205.

Replication-independent core histone dynamics at transcriptionally active loci in vivo

Christophe Thiriet  1 , Jeffrey J Hayes
Affiliations
Comparative Study

Replication-independent core histone dynamics at transcriptionally active loci in vivo

Christophe Thiriet et al. Genes Dev. .

Abstract

We used a novel labeling technique in the naturally synchronous organism Physarum polycephalum to examine the fate of core histones in G2 phase. We find rapid exchange of H2A/H2B dimers with free pools that is greatly diminished by treatment of the cells with alpha-amanitin. This exchange is enhanced in pol II-coding sequences compared with extragenic regions or inactive loci. In contrast, H3/H4 tetramers exhibit far lower levels of exchange in the pol II-transcribed genes tested, suggesting that tetramer exchange occurs via a distinct mechanism. However, we find that transcribed regions of the ribosomal RNA gene loci exhibit rapid exchange of H3/H4 tetramers. Thus, our data show that the majority of the pol II transcription-dependent histone exchange is due to elongation in vivo rather than promoter remodeling or other pol II-dependent alterations in promoter structure and, in contrast to pol I, pol II transcription through nucleosomes in vivo causes facile exchange of both H2A/H2B dimers while allowing conservation of epigenetic "marks" and other post-translational modifications on H3 and H4.

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Figures

Figure 1.
Figure 1.
Exogenous core histones H2A/H2B localize to Physarum nuclei and are assembled into nucleosomes when introduced during G2 phase. (A) Experimental strategy for incorporation of exogenous histones into Physarum cells. Solutions containing H2A/Flag-H2B were applied to the upper surface of macroplasmodia at the beginning of G2 phase, then cells were grown for 3 h prior to analysis. (B) Exogenous H2A/H2B localize to nuclei independent of transcription. Proteins were introduced at the beginning of G2 phase in the absence or presence of α-amanitin and subcellular distribution in nuclear (N) and cytoplasmic (C) fractions analyzed by SDS-PAGE and Western blots with anti-Flag antibodies. Duplicate experiments are shown. (C) Exogenous H2A/Flag-H2B is assembled into nucleosomes via an α-amanitin sensitive process. Nucleosomes were purified from macroplasmodia treated with exogenous H2A/H2B in the absence or presence of α-amanitin and protein content analyzed. (Top) Coomassie-stained gel. (Bottom) Western blot of the gel. (D) Exogenous H2A/H2B dimers colocalize with transcriptionally active chromatin. Incorporation of fluorescein-labeled dimers was carried out in G2 phase for 3 h, cell explants were squashed, fixed, and stained for acetylated H4 with antipenta-acetylated H4 peptide antibodies. Nuclei were imaged by confocal microscopy.
Figure 2.
Figure 2.
Exogenous H2A/H2B dimers are preferentially assembled into nucleosomes on transcribed sequences of active genes. (A) Diagram of profilin ProA and ProP loci, which are transcriptionally inactive and active, respectively, in Physarum macroplasmodia. Lines below the loci correspond to the fragments analyzed in B. (Right) The transcription state of the genes was confirmed by RT-PCR. (B) Incorporation of exogenous H2A/H2B into Physarum chromatin. Extract from an untreated control cell (-) or a single cell treated with exogenous H2A/H2B (+) as described in Figure 1A was analyzed by ChIP. The enrichment of fragments after the immunoprecipitation from the active ProP locus relative to the ProA locus is listed below the lanes (average of two experiments). PCR reaction products using the input extract (In) and the immunoprecipitate (ChIP) are shown. (C-E) Incorporation of exogenous histone complexes into Physarum chromatin throughout the ProP and ArdC loci. (C) DNA regions amplified by PCR in the ChIP assays (bars below lines). (D) Extent of incorporation of exogenous H2A/Flag-H2B into chromatin during G2 phase. Histones were introduced at the beginning of G2 phase, and cells harvested for ChIP 3 h later. Numbers correspond to individually amplified regions shown in C. The intensities of amplified bands were normalized to internal controls and the ln (ChIP band)/(input band) shown in the bar graph. (E) Incorporation of exogenous H3/H4 into chromatin throughout the ProP and ArdC loci during S and G2 phase. Exogenous (H3/H4)2 tetramers were introduced into Physarum cells at the beginning of S or G2 phase, and the extent of incorporation into chromatin throughout each locus was analyzed 3 h later as in D.
Figure 3.
Figure 3.
Kinetics of exchange of core histone proteins on active loci. (A) Experimental strategy. Nucleosomes were labeled in vivo by incorporation of Flag-tagged histones at the beginning of S phase for 3 h; the cells were then washed, and the loss of labeled proteins from the chromatin of the indicated loci monitored by ChIP immediately (0) and 1.5 and 3 h after washing. (B) Exchange of H2A/H2B on sequences within the inactive ProA gene (ProA) and the active ArdC promoter (ArdCp) and coding region (ArdCc). Primers used were, ProA, ArdC 2, and ArdC 6 from Figure 2. The enrichment of the target sequence relative to the 0.5% of the input is plotted with results from two independent experiments shown. (C) Exchange of H3/H4 was monitored as in B, except that cells were treated with H3/Flag-H4.
Figure 4.
Figure 4.
Exchange of core histone proteins throughout the AltB locus before and after activation of transcription. Core histones on endogenous sequences were labeled in vivo as in Figure 3; then, the relative amount of H2A/Flag-H2B and H3/Flag-H4 associated with each AltB sequence was determined before (Inactive) and after (Active) transcription activation of the gene. The ratio of Active/Inactive ChIP was plotted for each of the indicated sequences. Results are the average of two independent experiments with ≤5% average variation.
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