11 research outputs found
A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depends on histone recycling in transcribed chromatin
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. 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
DNA Topoisomerase III Localizes to Centromeres and Affects Centromeric CENP-A Levels in Fission Yeast
<div><p>Centromeres are specialized chromatin regions marked by the presence of nucleosomes containing the centromere-specific histone H3 variant CENP-A, which is essential for chromosome segregation. Assembly and disassembly of nucleosomes is intimately linked to DNA topology, and DNA topoisomerases have previously been implicated in the dynamics of canonical H3 nucleosomes. Here we show that <i>Schizosaccharomyces pombe</i> Top3 and its partner Rqh1 are involved in controlling the levels of CENP-A<sup>Cnp1</sup> at centromeres. Both <i>top3</i> and <i>rqh1</i> mutants display defects in chromosome segregation. Using chromatin immunoprecipitation and tiling microarrays, we show that Top3, unlike Top1 and Top2, is highly enriched at centromeric central domains, demonstrating that Top3 is the major topoisomerase in this region. Moreover, centromeric Top3 occupancy positively correlates with CENP-A<sup>Cnp1</sup> occupancy. Intriguingly, both <i>top3</i> and <i>rqh1</i> mutants display increased relative enrichment of CENP-A<sup>Cnp1</sup> at centromeric central domains. Thus, Top3 and Rqh1 normally limit the levels of CENP-A<sup>Cnp1</sup> in this region. This new role is independent of the established function of Top3 and Rqh1 in homologous recombination downstream of Rad51. Therefore, we hypothesize that the Top3-Rqh1 complex has an important role in controlling centromere DNA topology, which in turn affects the dynamics of CENP-A<sup>Cnp1</sup> nucleosomes.</p> </div
Top3 is enriched at IGRs, centromeres, and sub-telomeric regions.
<p>(A) ChIP-chip relative enrichment of Top3-myc along chromosome III at 30°C. The schematic picture shows the approximate position of the centromere and the subtelomeric regions. Telomeric repeats are not represented on the array. (B) Moving average for the relative enrichment of Top3, Top2 and Top1 after alignment of genes at the TSS and TTS, respectively. Error bars represent 99% confidence intervals. The bottom bar illustrates statistical significance by t-tests for the difference between the graphs at each point using a continuous spectrum from black (p = 1) via red to yellow (p = 0). (C) Overlaps between 5′IGRs with an average >1.5-fold and 3′IGRs with an average >2-fold relative enrichment of Top3, Top2 and Top1, respectively. The overlaps are statistically significant (p<0.001) by pair-wise hyper-geometric distribution tests. All data is an average of two independent experiments.</p
The <i>top3-105</i> mutant carries an Y209C amino acid substitution and displays impaired growth at 36°C.
<p>(A) Alignment of Top3 amino acid sequences from different species. The position corresponding to Y209 of <i>S. pombe</i> Top3 is highlighted in grey. The amino acids are colored according to their physiochemical properties. An asterisk (*) indicates a fully conserved residue, a colon (:) indicates conservation between groups of strongly similar properties, and a period (.) indicates conservation between groups of weakly similar properties between all species. (B) Spotting of the indicated strains in 5-fold serial dilutions on plates incubated at 25°C, 30°C and 36°C. (C) Growth kinetics of the indicated strains in liquid media after a shift from 25°C to 36°C at time zero.</p
Top3 and Rqh1 affect CENP-A<sup>Cnp1</sup> enrichment at 5′IGRs and centromeres.
<p>(A) Moving average for the relative enrichment of CENP-A<sup>Cnp1</sup> in wild type and the <i>top3-105</i> mutant after alignment of genes at the TSS. Error bars represent 99% confidence intervals. The bottom bar illustrates statistical significance for the difference between the graphs at each point using a continuous spectrum going from black (p = 1) via red to yellow (p = 0). (B) Same as in A for 198 genes with >1.5-fold relative enrichment of CENP-A<sup>Cnp1</sup> in wild type. (C) ChIP-chip relative enrichment of CENP-A<sup>Cnp1</sup> in wild type and the <i>top3-105</i> mutant and the ratio between these along centromere I after 8 hours at 36°C. Grey boxes represent genes. A schematic representation where arrows represent repeat elements and black lines represent tRNA genes is shown. A * indicates that the peak is higher than the maximum value of the axis. (D) ChIP-qPCR relative enrichment of CENP-A<sup>Cnp1</sup> in wild type and the indicated mutants after 8 hours at 36°C. (E) ChIP-qPCR average relative enrichment of HJURP<sup>Scm3</sup>-Pk/V5 in wild type and the indicated mutants after 8 hours at 36°C. (F) ChIP-qPCR average relative enrichment of H3 in wild type and the <i>top3-105</i> mutant after 8 hours at 36°C. ChIP-qPCR was performed using triplicate samples in two independent experiments. Relative enrichment at the <i>cnt</i> region of chromosome I was calculated using the ddCt method, normalizing to ChIP input and <i>act1</i>. Samples were also normalized to the average of the wild type samples in each experiment. Error bars represent the standard deviations between six samples.</p
Top3 has small effects on gene transcription.
<p>Average RNA levels in wild type and the <i>top3-105</i> mutant after 8 hours at 36°C when genes are aligned at the TSS and TTS, respectively. Error bars represent 99% confidence intervals. The bottom bar illustrates statistical significance for the difference between the graphs at each point using a continuous spectrum going from black (p = 1) via red to yellow (p = 0). All data is an average of two independent experiments.</p
Top3 and CENP-A<sup>Cnp1</sup> occupancies are positively correlated.
<p>(A) ChIP-chip relative enrichment of Top3-myc, Top2-myc, Top1-myc and CENP-A<sup>Cnp1</sup> along centromere I at 30°C. Grey boxes represent genes. A schematic representation where arrows represent repeat elements and black lines represent tRNA genes is shown. The vertical arrow indicates the position of primers used in D-F. The scale bar represents 2.5 kb. (B) Density scatter plot for all centromeric probes showing the correlations between the relative enrichment of Cnp1 and Top3, Top2, and Top1, respectively. Probes corresponding to central cores are shown in color. Pearson's correlation coefficients for all centromeric probes (R) are shown. (C) Overlaps between 5′IGRs with an average >3-fold relative enrichment of Cnp1 and >1.5-fold relative enrichment of Top3, Top2 and Top1, respectively. The overlaps are statistically significant (p<0.001) by pair-wise hyper-geometric distribution tests. (D) Scatter plot for 5′IGRs with high (>1.5-fold) relative enrichment of Cnp1 showing the correlations between the average relative enrichment of Cnp1 and Top3, Top2, and Top1, respectively. Pearson's correlation coefficients (R) are shown. All data is an average of two independent experiments.</p
Top3 and Rqh1 are required for chromosome segregation.
<p>(A) Light and fluorescence microscopy images of the indicated strains with DAPI staining of the DNA after 8 hours at 36°C. (B) Light and fluorescence microscopy images of wild type and <i>top3-105</i> mutant cells after 8 hours at 36°C. The table shows the numbers of cells displaying normal and defective chromosome segregation among 40 late anaphase cells (with a mitotic spindle and two separate foci of DNA) or 40 telophase cells (with a septum) for the indicated strains after 8 hours at 36°C. The scale bars represents 6.65 µM.</p