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. 2015 Jun;25(6):872-83.
doi: 10.1101/gr.188870.114. Epub 2015 Mar 16.

A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depends on histone recycling in transcribed chromatin

J Peter Svensson  1 , Manu Shukla  2 , Victoria Menendez-Benito  1 , Ulrika Norman-Axelsson  1 , Pauline Audergon  2 , Indranil Sinha  1 , Jason C Tanny  3 , Robin C Allshire  2 , Karl Ekwall  1
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A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depends on histone recycling in transcribed chromatin

J Peter Svensson et al. Genome Res. 2015 Jun.

"V体育2025版" Abstract

Nucleosome composition actively contributes to chromatin structure and accessibility. Cells have developed mechanisms to remove or recycle histones, generating a landscape of differentially aged nucleosomes. This study aimed to create a high-resolution, genome-wide map of nucleosome turnover in Schizosaccharomyces pombe. The recombination-induced tag exchange (RITE) method was used to study replication-independent nucleosome turnover through the appearance of new histone H3 and the disappearance or preservation of old histone H3. The genome-wide location of histones was determined by chromatin immunoprecipitation-exonuclease methodology (ChIP-exo). The findings were compared with diverse chromatin marks, including histone variant H2A. Z, post-translational histone modifications, and Pol II binding. Finally, genome-wide mapping of the methylation states of H4K20 was performed to determine the relationship between methylation (mono, di, and tri) of this residue and nucleosome turnover. Our analysis showed that histone recycling resulted in low nucleosome turnover in the coding regions of active genes, stably expressed at intermediate levels. High levels of transcription resulted in the incorporation of new histones primarily at the end of transcribed units VSports手机版. H4K20 was methylated in low-turnover nucleosomes in euchromatic regions, notably in the coding regions of long genes that were expressed at low levels. This transcription-dependent accumulation of histone methylation was dependent on the histone chaperone complex FACT. Our data showed that nucleosome turnover is highly dynamic in the genome and that several mechanisms are at play to either maintain or suppress stability. In particular, we found that FACT-associated transcription conserves histones by recycling them and is required for progressive H4K20 methylation. .

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Figures

Figure 1.
Figure 1.
The RITE system that was used to measure genome-wide nucleosome turnover in S. pombe. (A) Model of the RITE genetic switch system. Green triangles represent LoxP sites. (B,C) T7 ChIP-qPCR analysis of heterochromatic regions (B) and genes (C). (D) RNA levels at five loci in growth-arrested (3 h at 36°C) cells with the RITE system (Hu2549 with cdc25-22 allele). (E,F) HA ChIP-qPCR of heterochromatin regions (E) and genes (F). Three independent experiments were performed, and the error bars show SEM. (G) A browser view of HA and T7 ChIP-exo results for five genomic loci before (0 h) and after (2 h) the genetic switch. Data show the average signal from two independent experiments. (H) The running average (window 500) of ChIP-exo signals of 150-bp chromatin fragments from H3-HA versus H3-T7 (RITE). (I) The running average (window 500) of ChIP-exo signals of 150-bp chromatin fragments from H3-T7 (RITE) versus pInv-H3-HA.
Figure 2.
Figure 2.
Transcription-dependent nucleosome turnover is related to the position of nucleosomes within genes. (A,B) ChIP was used to measure nucleosome stability using HA epitope-tagged H3 (H3-HA; transcribed before the RITE switch) (A) or T7 epitope-tagged H3 (H3-T7; transcribed after the RITE switch) (B) in different regions of the transcript (5′ UTR, CDS, and 3′ UTR). Measurements were compared before (0 h) and after (2 h) β-estradiol addition. Bars show the average of two independent ChIP-exo experiments, and the error bars represent the range. (C,D) Heatmaps of nucleosomes at 2 h aligned at the transcription start site (TSS) for H3-HA (C) and H3-T7 (D). The top panels show genes that are expressed at intermediate levels (two to four transcripts per cell), and the bottom panels show genes with less than one transcript per cell. Short transcripts are found at the top of each panel and long transcripts are found at the bottom. The ChIP signal (RPKM for each 10 bp) is represented by color intensity. The white line marks the borders of the transcribed units. (E) Averaged ChIP signals (RPKM for each 10 bp) for epitope-tagged H3 aligned at the TSS and for all transcribed units in the genome. (F,G) The histone turnover score (normalized ΔΔRPKM) aligned at the TSS and transcription end site (TES) for all transcribed genes (F) and stratified according to transcription levels (G). (H) Average ChIP signal per transcribed unit for the H3-HA (old H3, top) and H3-T7 (new H3, bottom) ΔRPKM (2–0 h) according to the number of transcripts per cell. The running averages (window 500) are plotted against the average transcript levels within the window.
Figure 3.
Figure 3.
Correlation between chromatin modifications and nucleosome turnover. (A) Hierarchical clustering of the correlation coefficients from the ChIP signals for 150-bp genomic fragments. All ChIP-microarray samples were normalized to input (log2). In the ChIP-exo turnover data, i.e., H3-HA (RITE) and H3-T7 (RITE), data from the 2-h samples are relative to the 0-h time point. (B) Heatmaps of the ChIP signals (log2) from the antibody against indicated protein/protein modification at genes that were expressed at intermediate levels (two to four transcripts per cell).
Figure 4.
Figure 4.
H4K20 methylation is found in protein-coding regions of the genome. (A) Western blot analysis of wild-type (WT) cells (lane 1) and set9Δ cells (lanes 2,3 differ in the amount of chromatin loaded). (B) ChIP-qPCR analysis of H4K20me1 and H4K20me3 at five loci that have different transcription levels. Bars show the average and SEM of three independent experiments. (C) The average pattern of H4K20 methylation marks aligned with the TSS (left) or TES (right). (D) Patterns of H4K20me1 (left), H4K20me2 (middle), and H4K20me3 (right) aligned at the TSS, with genes sorted according to length (top, short genes; bottom, long genes). (E) Metagene analysis of the levels of H4K20me1 (top), H4K20me2 (middle), and H4K20me3 (bottom), stratified according to gene expression level.
Figure 5.
Figure 5.
Subsets of inducible genes are associated with H4K20me3. (A) The average H4K20me ChIP signals in genes returned using the gene ontology terms “Response to DNA Damage Stimulus” and (B) “Meiosis” aligned according to the TSS. (C) ChIP data of the different H4K20 methylations (me1/me2/me3) at the spd1 and rad50 loci. Gray boxes indicate annotated features transcribed to the right (above solid line) or left (below solid line). (D) Venn diagram shows the overlap between genes associated with H4K20me3 (log2 [WT/set9Δ] > 0.5) and genes that are down-regulated in pht1Δ and swr1Δ cells. (E) A model of the relationship between transcription and FACT-mediated nucleosome recycling. High-turnover nucleosomes are marked in yellow.
Figure 6.
Figure 6.
Transcription-mediated nucleosome conservation is required for the accumulation of H4K20 methylation. (A,B) Histone H3 density in WT cells (A) and changes in histone H3 density in spt16-18 cells relative to WT cells (B) and grown at permissive temperature (25°C) followed by 1 h at the restrictive temperature (36°). Data were stratified according to the average number of transcripts per cell during the vegetative growth phase. (C) ChIP-qPCR analysis of H4K20me1 at five genomic loci in spt16-18 cells and WT cells. (D) ChIP-qPCR analysis of H4K20me3 at five genomic loci in spt16-18 cells and WT cells. (E) Metagene analysis of Spt16 levels in WT cells. (F) ChIP-qPCR analysis of H4K20me1 at five genomic loci in htb1-K119R cells and WT cells. (C,D,F) Bars show the average of three independent experiments, and error bars show the SEM.
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