SWI/SNF-like chromatin remodeling factor Fun30 supports point centromere function in S. cerevisiae

Budding yeast centromeres are sequence-defined point centromeres and are, unlike in many other organisms, not embedded in heterochromatin. Here we show that Fun30, a poorly understood SWI/SNF-like chromatin remodeling factor conserved in humans, promotes point centromere function through the formation of correct chromatin architecture at centromeres. Our determination of the genome-wide binding and nucleosome positioning properties of Fun30 shows that this enzyme is consistently enriched over centromeres and that a majority of CENs show Fun30-dependent changes in flanking nucleosome position and/or CEN core micrococcal nuclease accessibility. Fun30 deletion leads to defects in histone variant Htz1 occupancy genome-wide, including at and around most centromeres. FUN30 genetically interacts with CSE4, coding for the centromere-specific variant of histone H3, and counteracts the detrimental effect of transcription through centromeres on chromosome segregation and suppresses transcriptional noise over centromere CEN3. Previous work has shown a requirement for fission yeast and mammalian homologs of Fun30 in heterochromatin assembly. As centromeres in budding yeast are not embedded in heterochromatin, our findings indicate a direct role of Fun30 in centromere chromatin by promoting correct chromatin architecture

Riociguat treatment in patients with chronic thromboembolic pulmonary hypertension: Final safety data from the EXPERT registry

Objective: The soluble guanylate cyclase stimulator riociguat is approved for the treatment of adult patients with pulmonary arterial hypertension (PAH) and inoperable or persistent/recurrent chronic thromboembolic pulmonary hypertension (CTEPH) following Phase

Fun30 is required for normal <i>CEN</i>-flanking nucleosome positioning and/or <i>CEN</i> core particle structure.

<p>A) Genome browser trace of Fun30 ChIP enrichment and nucleosome dyad frequency centred on and surrounding yeast <i>CEN1</i>. The upper trace shows Log₂ Fun30 ChIP-seq enrichment values binned at 10 bp intervals and smoothed with a 3 bin moving average. Wildtype (WT) and <i>Δfun30</i> chromatin was digested with MNase and nuclease-protected DNA species sequenced using paired-end mode Illumina technology. Nucleosome sequencing data (nuc) traces were plotted as mirror images in the lower panel. The graph shows a map of the centre point positions of paired sequence reads with end-to-end distances of 150 bp+/−20% wild-type and <i>Δfun30</i> mutant chromatin samples surrounding <i>CEN1</i>. The frequency distributions, which effectively map chromatin particle dyads, were binned at 10 bp intervals, and smoothed by applying a 3 bin moving average. Peaks in the dyad distributions correspond to translationally-positioned nucleosomes in the original genome. The <i>CEN</i> core particle is also mapped using this method and can be visualised as a small peak centred on the <i>CEN</i> region marked with a grey box. Pink bars show the positions of ORFs (B–D) Genome browser plots of Fun30 ChIP-seq and nucleosome sequence distributions as described above for <i>CEN10, 11</i> and <i>12</i> respectively. Fun30-dependent changes in the height of a nucleosome dyad or <i>CEN</i> core particle peak are marked with a red asterix. Fun30-dependent changes in the position of a <i>CEN</i>-flanking nucleosome dyad peak are marked with red arrows. Genome browser plots for all yeast <i>CENs</i> are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002974#pgen.1002974.s006" target="_blank">Figure S6</a>.</p

Loss of Fun30 leads to increased Transcription at centromere regions.

<p>Analysis of transcript levels at <i>CEN3</i> region by RT-qPCR in wildtype yeast (wt), the corresponding <i>Δfun30</i> mutant, <i>Δtrf4</i> mutant and the double mutant <i>Δfun30 Δtrf4</i> strains. Primers PM22/48 detecting transcripts directly over <i>CEN3</i> were used to amplify cDNA. The graph reports the relative amount of transcript compared to a control gene that is not regulated by Fun30. Similar results were obtained when we examined absolute amounts.</p

Fun30 affects Htz1 occupancy, including at centromeres.

<p>(A) Average occupancy analysis for histone Htz1 for divergent orientation genes relative to the 5′ Transcription start site (5TSS) position for wildtype cells (left panel), <i>Δfun30</i> cells (middle panel) and ratio <i>Δfun30</i> versus WT (right panel, <i>W303 3Myc-Htz1 versus W303 3Myc-Htz1 Δfun30</i>). (B) Average occupancy analysis for histone Htz1 for convergent orientation genes relative to the 3′ Transcription stop site (3TSS) position for wildtype cells (left panel), <i>Δfun30</i> cells (middle panel) and ratio <i>Δfun30</i> versus wildtype. (C) Effect of Fun30 on Htz1 occupancy 5 kbp up- and downstream of <i>CEN10</i> and <i>CEN11</i>. Shown is the Fun30 occupancy as measured by ChIP-seq in the top lane (dark blue, log₂ scale, expressed as ratio of normalized sequence tag counts from ChIP to input). Htz1 occupancy from wildtype (wt, red) and <i>Δfun30</i> (light blue) are shown in the two lanes below expressed as normalized sequence tag counts corrected for input in linear scale. The change in occupancy of Htz1 is indicated in the lane below as the values from the <i>Δfun30</i> cells minus the values from wt cells (black). Positions of ORFs and centromeres are indicated in the lowest lane, orange box: centromere, back and grey boxes: ORFs in the sense and antisense direction, respectively.</p

<i>FUN30</i> deletion counteracts viability defects upon formation of a dicentric chromosome.

<p>A) <i>Δfun30</i> cells show increased rates of loss of a circular minichromosome (pUG25), the left diagram illustrates the assay, the right panel shows % of plasmid loss in wildtype (wt) and f<i>un30-</i>deleted cells, shown is the average of two experiments, bars represent minimum and maximum values. B) Left panel: Diagram illustrating the dicentric chromosome breakage assay <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002974#pgen.1002974-Mythreye1" target="_blank">[46]</a>. In the presence of galactose, the ectopic formation of a second centromere on chromosome III is suppressed through transcription of the locus. In the presence of glucose, the suppression of transcription allows formation of a second centromere on the same chromosome, which ultimately leads to chromosome breakage and loss of viability. Right panel: Deletion of <i>FUN30</i> promotes viability on induction of a dicentric chromosome, to a comparable extent as centromere establishment factor <i>CHL4</i>. More than 500 colonies/plate were counted for cells grown in galactose and the corresponding number of colonies were established for cells grown in glucose. Shown is a representative experiment, error bars represent 10% confidence interval.</p

Summary of sequence libraries.

<p>Summary of sequence libraries.</p

Fun30 is required when Cse4 function is compromised.

<p>Growth of the double mutant <i>Δfun30 cse4-1</i> is strongly affected at semi-restrictive temperatures. Fivefold dilutions of wildtype (BY4741/Y00000), <i>Δfun30</i> (Y00389), <i>cse4-1</i> (AHY666) and <i>Δfun30 cse4-1</i> (SC53) cells were plated onto YPD plates and incubated at indicated temperatures for 3 days. Lower panels: Fun30 activity is restored by expressing wildtype Fun30 <i>in trans</i>, but not Fun30 with a point mutation in the ATPase domain; Cells were spotted on media with 2% glucose and grown for 3 days at 30°C or 35°C.</p

<i>S. cerevisiae</i> strains used in this study.

<p><i>S. cerevisiae</i> strains used in this study.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p