Genome-Wide Mapping of Yeast Histone Chaperone Anti-Silencing Function 1 Reveals Its Role in Condensin Binding with Chromatin

Genome-wide participation and importance of the histone chaperone Asf1 (Anti-Silencing Function 1) in diverse DNA transactions like replication, repair, heterochromatic silencing and transcription are well documented. Yet its genome-wide targets have not been reported. Using ChIP-seq method, we found that yeast Asf1 associates with 590 unique targets including centromeres, telomeres and condensin-binding sites. It is found selectively on highly transcribed regions, which include replication fork pause sites. Asf1 preferentially associates with the genes transcribed by RNA polymerase (pol) III where its presence affects RNA production and replication-independent histone exchange. On pol II-transcribed genes, a negative correlation is found between Asf1 and nucleosome occupancy. It is not enriched on most of the reported sites of histone exchange or on the genes, which are misregulated in the asf1Δ cells. Interestingly, chromosome-wide distributions of Asf1 and one of the condensin subunits, Brn1 show a nearly identical pattern. Moreover, Brn1 shows reduced occupancy at various condensin-binding sites in asf1Δ cells. These results along with high association of Asf1 with heterochromatic centromeres and telomeres ascribe novel roles to Asf1 in condensin loading and chromatin dynamics

Oncogene expression from extrachromosomal DNA is driven by copy number amplification and does not require spatial clustering in glioblastoma stem cells

Extrachromosomal DNA (ecDNA) are frequently observed in human cancers and are responsible for high levels of oncogene expression. In glioblastoma (GBM), ecDNA copy number correlates with poor prognosis. It is hypothesized that their copy number, size, and chromatin accessibility facilitate clustering of ecDNA and colocalization with transcriptional hubs, and that this underpins their elevated transcriptional activity. Here, we use super-resolution imaging and quantitative image analysis to evaluate GBM stem cells harbouring distinct ecDNA species (EGFR, CDK4, PDGFRA). We find no evidence that ecDNA routinely cluster with one another or closely interact with transcriptional hubs. Cells with EGFR-containing ecDNA have increased EGFR transcriptional output, but transcription per gene copy is similar in ecDNA compared to the endogenous chromosomal locus. These data suggest that it is the increased copy number of oncogene-harbouring ecDNA that primarily drives high levels of oncogene transcription, rather than specific interactions of ecDNA with each other or with high concentrations of the transcriptional machinery

Asf1 association correlates with transcription activity of pol II.

<p>Average occupancy was calculated for (A) Asf1 ChIP and mock, (B) pol II, and (C) nucleosomes in the 2 kb region surrounding TSS or TTS of different classes of genes and plotted for the five categories based on the transcript abundance <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108652#pone.0108652-Holstege1" target="_blank">[42]</a>. Legends box shows the color code, numbers indicate mRNA molecules/cell in the category. Vertical pink bar on the graphs represents the break in the middle. Panel A shows that the ChIP signals are well above the mock on highly transcribed genes. (D) Heat map depicting the Asf1 ChIP-Seq signal 500 bp upstream and downstream of the TSS (bent arrow) at snoRNA genes. Color gradient code is shown at the bottom. (E) Asf1 association at pol II-transcribed snoRNA genes, 1 kb upstream and 1 kb downstream of the TSS is compared with pol II and nucleosome occupancy profiles at the gene loci. Averages of Asf1 ChIP- and mock-Seq signals for 39 out of 77 genes are plotted. The vertical arrow denotes the gene 3′-end. (F) Venn intersections of Asf1-occupied (this study) genes with those misregulated in <i>asf1Δ</i> cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108652#pone.0108652-Minard1" target="_blank">[28]</a>. <i>P</i> value of the overlap is 3.9×10⁻¹¹ (significant overlap). (G) Venn intersections of Asf1-occupied (this study) genes with those misregulated in <i>asf1Δ</i> cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108652#pone.0108652-Zabaronick1" target="_blank">[25]</a> or after Asf1 depletion <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108652#pone.0108652-Zabaronick1" target="_blank">[25]</a>, with <i>P</i> values 0.118 and 0.42 respectively (insignificant overlaps).</p

Replication-independent H3 exchange on pol III-transcribed genes.

<p>(A) and (B) Time-course analysis of histone H3 exchange at different loci in wild-type (A) versus <i>asf1Δ</i> (B) cells is shown. Histone exchange assay by ChIP-qPCR analysis was made to follow the H3 exchange <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108652#pone.0108652-Jamai1" target="_blank">[34]</a> at various pol III-transcribed genes. Averages of ChIP data for three independent experiments with error bars are shown.</p

Chromosomal features enriched with Asf1.

<p>(A) Yeast chromosomes show similar density of Asf1 peaks. Total number of assigned Asf1-peaks for each chromosome when plotted against the chromosome length shows linear relationship. (B) Pie-chart showing different categories of genome-wide chromosomal features targeted by Asf1, number next to a category indicates total number of features occupied by Asf1 in that category. Out of 32, occupancy could be ascertained only on 10 telomeres. (C) Heat map shows Asf1 occupancy from blue (low) to yellow (high) at 500 bp upstream and downstream of the 5′ end (marked with a short bar) of 16 yeast centromeres. The brown bar marks the position of centromeres. (D) Comparison of average Asf1 occupancy on different genomic regions. Asf1 ChIP and mock sequencing tag counts on 1 kb region on both sides of a reference point on four genomic features were binned and the bin-wise average is plotted. Reference point is denoted by ‘0’ on the X-axis, which is TSS for tRNAs and pol II ORFs but SGD start co-ordinates for the others.</p