Dhp1 does not affect the binding of RNAPII to repeat transcripts.

<p>(A) Cross-linking and analysis of cDNA (CRAC) followed by expression profiling reveals RNAPII targets on a genome-wide scale. (B) CRAC was carried out using a carboxyl-terminal HTP-tagged subunit of RNAPII, Rpb2-HTP. The difference plot maps the relative enrichments (Mutant/wt) of RNAPII substrates averaged across the genome for each mutant in relation to the transcriptional termination site (TTS). RNAPII substrates are relatively enriched after the normal termination site of transcripts in <i>dhp1-1</i> due to a transcriptional termination defect. (C) No dramatic changes in enrichment of RNAPII substrates were observed across the centromeric regions and the mating type locus between wt and <i>dhp1-1</i> cells, suggesting that Dhp1 does not affect silencing by TGS, but likely through a PTGS mechanism. The chromosome position of the genomic region is displayed below the figure (Fwd, forward strand; Rev, reverse strand).</p

Dhp1-mediated silencing occurs independently of RNAi and the exosome.

<p>(A-B) qRT-PCR analysis of silenced repeat regions in the centromere (A) and mating type locus (B) shows negative genetic interactions between <i>dhp1-1</i> and <i>ago1Δ</i> or <i>rrp6Δ</i>. (C-D) H3K9me₂ ChIPs show the enrichments of H3K9me mark at indicated strains. Relative enrichments of H3K9me₂ at indicated genomic regions were normalized to <i>leu1</i>^<i>+</i>. * <i>p</i> ≤ 0.05 as determined by Student’s <i>t</i> test comparing the indicated sample values with the wt values for qRT-PCR and qChIP. Significance between the single mutants and double mutants is indicated by horizontal lines linking the compared samples.</p

Dhp1 is required for effective heterochromatin maintenance.

<p>(A) A map of the mating type locus shows the <i>cenH</i>-containing <i>K</i> region, a nucleation site from which repressive chromatin is initiated and spread. After the initial establishment of heterochromatin, deletion of the <i>K</i> region does not affect the chromatin structure and the reporter gene (<i>ade6</i>^<i>+</i>) remains silenced due to maintenance. Disruption of maintenance results in derepression of the reporter gene. (B) Representative images of grid-based assays from which the percent of colonies grouped by <i>ade6</i>^<i>+</i> expression were calculated. (C) Graph displays the percent of colonies grouped by relative expression of <i>ade6</i>^<i>+</i> as visually determined by colony color. (D) qRT-PCR shows the relative expression compared to wt of the <i>ade6</i>^<i>+</i> reporter gene in <i>dhp1-1</i> and <i>swi6Δ</i>. (E) H3K9me₂ qChIP was carried out on the indicated strains. * <i>p</i> ≤ 0.05 as determined by Student’s <i>t</i> test comparing the indicated sample values with the wt values for qRT-PCR and qChIP.</p

The catalytic activity of Dhp1 is required for silencing.

<p>(A) Sequence alignment of the catalytic domains between <i>K</i>. <i>lactis</i> and <i>S</i>. <i>pombe</i>. The position of D55 and E207 of Dhp1 in <i>S</i>. <i>pombe</i> are highlighted. (B) A ten-fold serial dilution assay shows <i>dhp1-1</i> cells carrying indicated plasmids grown on standard SC-leucine medium at 30°C or 37°C. (C-D) qRT-PCR showing relative <i>dg</i> (C) and <i>cenH</i> (D) transcript levels in <i>dhp1-1</i> cells carrying indicated plasmids. *<i>P</i> ≤ 0.05 as determined by student’s <i>t</i> test comparing the indicated sample values with the WT for qRT-PCR.</p

Dhp1 contributes to heterochromatin spreading.

<p>(A) Insertion of the <i>cenH</i> nucleation site adjacent to the <i>ade6</i>^<i>+</i> reporter gene in an ectopic euchromatic locus (<i>ura4</i>^<i>+</i>) will result in the silencing of the reporter gene through heterochromatin spreading. Gray circles: H3K9me. (B) Above, representative images of grid-based assays used to calculate the percentage of cells exhibiting distinct <i>ade6</i>^<i>+</i> expression on low adenine medium. Below: 100% stacked column graph represents the observed phenotypes of these colonies. (C) H3K9me₂ qChIP shows relative enrichment at the <i>ade6</i>^<i>+</i> reporter gene and its surrounding regions normalized to <i>leu1</i>^<i>+</i>. The genomic positions for oligos used for qPCR are indicated by gray boxes. * <i>p</i> ≤ 0.05 as determined by Student’s <i>t</i> test comparing the indicated sample values with wt values.</p

Dhp1 represents a novel RNA processing mechanism which mediates epigenetic silencing independent of both RNAi and the exosome.

<p>(A) Aberrant transcripts produced by RNAPII are subject to degradation via three distinct PTGS pathways. RNAi involves the degradation of the transcript by Dcr1 or Triman (Tri1), producing siRNAs. The exosome degrades transcripts in a 3’ to 5’ direction with guidance from its cofactors, the TRAMP and MTREC complexes. Dhp1 mediates a distinct third pathway of post-transcriptional gene silencing via its 5’ to 3’ exoribonuclease activity. Together, these mechanisms function to initiate and maintain the epigenetic silencing of repeat RNAs. (B) RNA processing-mediated mechanisms in epigenetic silencing. We propose that Dhp1/Xrn2-mediated silencing is a novel RNAi- and exosome-independent processing mechanism in gene silencing.</p

Transcriptional gene silencing (TGS) is not reduced in <i>dhp1-1</i>.

<p>(A-B) Mit1-Myc ChIPs were carried out using the indicated strains and show that recruitment of SHREC is not decreased in <i>dhp1-1</i>. (C-D) RNAPII ChIPs were performed using the indicated strains and reveal no change of RNAPII occupancy in <i>dhp1</i> mutant. Relative enrichments of Mit1-Myc (A-B) or RNAPII (C-D) at the centromere (A and C) and the mating type locus (B and D) were normalized to <i>leu1</i>^<i>+</i>. * <i>p</i> ≤ 0.05 as determined by Student’s <i>t</i> test comparing the indicated sample values with the wt values.</p

Defective heterochromatin in <i>dhp1</i> mutants is marked by reduced H3K9 methylation at reporter genes.

<p>(A) Ten-fold serial dilution on media containing 20 μg/ml TBZ demonstrates chromosome segregation defects in <i>dhp1-1</i> mutants. (B-C) Chromatin immunoprecipitation (ChIP) performed with an anti-H3K9me₂ antibody shows reduced H3K9me₂ enrichment in <i>dhp1-1</i> at the reporter genes located at pericentromeric region (<i>otr</i>, B) and <i>mat2</i> (C). (D-E) The same reduction was not observed at the centromeric (D) and mating type locus (E) repeat regions by H3K9me2 ChIP. Relative enrichment (normalized to input) gives the approximate ratio between H3K9me₂ levels on the indicated heterochromatic regions versus the euchromatic control <i>leu1</i>^<i>+</i>. * <i>p</i> ≤ 0.05 as determined by Student’s <i>t</i> test comparing the indicated sample values with wt values.</p

Dhp1 is required for efficient <i>de novo</i> establishment of heterochromatin.

<p>(A) A role for a gene of interest (<i>goi</i>) in <i>de novo</i> establishment of heterochromatin is identified by deleting or mutating the <i>goi</i> in the <i>clr4Δ</i> background in which heterochromatin domains will be erased (derepressed). Re-introduction of functional <i>clr4</i>^<i>+</i> allows the formation of repressive chromatin provided that the <i>goi</i> is dispensable for heterochromatin establishment. (B) Left: dilution assay on TBZ shows completely resumed repression in the wt control following the re-introduction of <i>clr4</i>^<i>+</i>, but only partially resumed repression in <i>dhp1</i> mutants. Right: qRT-PCR showing relative <i>dg</i> and <i>cenH</i> transcript levels demonstrates the loss of repression in all strains with the <i>clr4Δ</i> background and completely resumed repression in wt cells only following complementation of <i>clr4</i>^<i>+</i>. Slight resumption of repression of <i>dg</i> and <i>cenH</i> repeats is observed in <i>dhp1-1</i> mutants. (C-D) H3K9me₂ qChIPs were performed using indicated strains. Relative enrichments at indicated genomic regions were normalized to <i>leu1</i>^<i>+</i>. * <i>p</i> ≤ 0.05 as determined by Student’s <i>t</i> test comparing the indicated sample values with the corresponding wt values (for example: <i>clr4Δ→ clr4</i>^<i>+</i> mutants were compared to <i>clr4Δ→ clr4</i>^<i>+</i> wt) for qRT-PCR and qChIP.</p

The UTP-A complex from <i>Chaetomium thermophilum</i>.

<p><b>(A)</b> Scheme showing the spatial assembly of the fungal UTP-A complex including the Utp4 X-ray structure and the EM-modeled 5'-ETS. The propellers of remaining UTP-A proteins (Utp8, Utp15, and 2× Utp17) are placed according previous biochemical and EM-studies. The α-solenoidal parts (including whole Utp5) are not included. The entire Utp10 molecule turning around Utp4 is interpreted as also the very C-terminus (atomic model) of Utp8 next to the Velcro-closure of Utp4. The position of the disease-modified arginine in human Utp4 in the interface to Utp10 is highlighted within a red sphere. <b>(B)</b> Comparison of the UTP-A complexes from <i>Chaetomium thermophilum</i> (left panel; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178752#pone.0178752.ref009" target="_blank">9</a>]) and <i>Saccharomyces cerevisiae</i> (right panel; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178752#pone.0178752.ref027" target="_blank">27</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178752#pone.0178752.ref028" target="_blank">28</a>]). While the overall architecture is conserved, the 5'-end of the RNA shows a different arrangement. In addition, the Upt8-Utp4 contact is not visible in the yeast structures.</p