A Novel Epigenetic Silencing Pathway Involving the Highly Conserved 5 '-3 ' Exoribonuclease Dhp1/Rat1/Xrn2 in Schizosaccharomyces pombe

Epigenetic gene silencing plays a critical role in regulating gene expression and contributes to organismal development and cell fate acquisition in eukaryotes. In fission yeast, Schizosaccharomyces pombe, heterochromatin-associated gene silencing is known to be mediated by RNA processing pathways including RNA interference (RNAi) and a 3’-5’ exoribonuclease complex, the exosome. Here, we report a new RNA-processing pathway that contributes to epigenetic gene silencing and assembly of heterochromatin mediated by 5’-3’ exoribonuclease Dhp1/Rat1/Xrn2. Dhp1 mutation causes defective gene silencing both at peri-centromeric regions and at the silent mating type locus. Intriguingly, mutation in either of the two well-characterized Dhp1-interacting proteins, the Din1 pyrophosphohydrolase or the Rhn1 transcription termination factor, does not result in silencing defects at the main heterochromatic regions. We demonstrate that Dhp1 interacts with heterochromatic factors and is essential in the sequential steps of establishing silencing in a manner independent of both RNAi and the exosome. Genomic and genetic analyses suggest that Dhp1 is involved in post-transcriptional silencing of repetitive regions through its RNA processing activity. The results describe the unexpected role of Dhp1/Rat1/Xrn2 in chromatin-based silencing and elucidate how various RNA-processing pathways, acting together or independently, contribute to epigenetic regulation of the eukaryotic genom

The fission yeast MTREC complex targets CUTs and unspliced pre-mRNAs to the nuclear exosome

Cryptic unstable transcripts (CUTs) are rapidly degraded by the nuclear exosome. However, the mechanism by which they are recognized and targeted to the exosome is not fully understood. Here we report that the MTREC complex, which has recently been shown to promote degradation of meiotic mRNAs and regulatory ncRNAs, is also the major nuclear exosome targeting complex for CUTs and unspliced pre-mRNAs in Schizosaccharomyces pombe. The MTREC complex specifically binds to CUTs, meiotic mRNAs and unspliced pre-mRNA transcripts and targets these RNAs for degradation by the nuclear exosome, while the TRAMP complex has only a minor role in this process. The MTREC complex physically interacts with the nuclear exosome and with various RNA-binding and RNA-processing complexes, coupling RNA processing to the RNA degradation machinery. Our study reveals the central role of the evolutionarily conserved MTREC complex in RNA quality control, and in the recognition and elimination of CUTs

A Novel Epigenetic Silencing Pathway Involving the Highly Conserved 5'-3' Exoribonuclease Dhp1/Rat1/Xrn2 in Schizosaccharomyces pombe.

Epigenetic gene silencing plays a critical role in regulating gene expression and contributes to organismal development and cell fate acquisition in eukaryotes. In fission yeast, Schizosaccharomyces pombe, heterochromatin-associated gene silencing is known to be mediated by RNA processing pathways including RNA interference (RNAi) and a 3'-5' exoribonuclease complex, the exosome. Here, we report a new RNA-processing pathway that contributes to epigenetic gene silencing and assembly of heterochromatin mediated by 5'-3' exoribonuclease Dhp1/Rat1/Xrn2. Dhp1 mutation causes defective gene silencing both at peri-centromeric regions and at the silent mating type locus. Intriguingly, mutation in either of the two well-characterized Dhp1-interacting proteins, the Din1 pyrophosphohydrolase or the Rhn1 transcription termination factor, does not result in silencing defects at the main heterochromatic regions. We demonstrate that Dhp1 interacts with heterochromatic factors and is essential in the sequential steps of establishing silencing in a manner independent of both RNAi and the exosome. Genomic and genetic analyses suggest that Dhp1 is involved in post-transcriptional silencing of repetitive regions through its RNA processing activity. The results describe the unexpected role of Dhp1/Rat1/Xrn2 in chromatin-based silencing and elucidate how various RNA-processing pathways, acting together or independently, contribute to epigenetic regulation of the eukaryotic genome

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 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

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 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

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