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. 2014 Jan 27;9(1):e86948.
doi: 10.1371/journal.pone.0086948. eCollection 2014.

Silencing motifs in the Clr2 protein from fission yeast, Schizosaccharomyces pombe

Daniel Steinhauf  1 , Alejandro Rodriguez  1 , Dimitrios Vlachakis  2 , Gordon Virgo  1 , Vladimir Maksimov  1 , Carolina Kristell  1 , Ida Olsson  1 , Tomas Linder  3 , Sophia Kossida  2 , Erik Bongcam-Rudloff  4 , Pernilla Bjerling  1
Affiliations

Silencing motifs in the Clr2 protein from fission yeast, Schizosaccharomyces pombe

Daniel Steinhauf et al. PLoS One. .

Abstract

The fission yeast, Schizosaccharomyces pombe, is a well-established model for heterochromatin formation, but the exact sequence of events for initiation remains to be elucidated. The essential factors involved include RNA transcribed from repeated sequences together with the methyltransferase Clr4. In addition, histone deacetylases, like Clr3, found in the SHREC complex are also necessary for transcriptional silencing. Clr2 is another crucial factor required for heterochromatin formation found in the SHREC complex. The function of Clr2 has been difficult to establish due to the lack of conserved domains or homology to proteins of known molecular function. Using a bioinformatics approach, three conserved motifs in Clr2 were identified, which contained amino acids important for transcriptional repression. Analysis of clr2 mutant strains revealed a major role for Clr2 in mating-type and rDNA silencing, and weaker effects on centromeric silencing. The effect on mating-type silencing showed variegation in several of the strains with mutated versions of Clr2 indicating an establishment or maintenance defect. Moreover, the critical amino acids in Clr2 were also necessary for transcriptional repression in a minimal system, by the tethering of Clr4 upstream of a reporter gene, inserted into the euchromatic part of the genome. Finally, in silico modeling suggested that the mutations in Clr2 cause disruption of secondary structures in the Clr2 protein. Identification of these critical amino acids in the protein provides a useful tool to explore the molecular mechanism behind the role of Clr2 in heterochromatin formation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. A bioinformatics approach revealed three conserved motifs in Clr2.
(A) Sequence alignment between Clr2 and 11 other similar fungal proteins. Three conserved motifs, named C2SM1-3, always present in the same order were identified. C2SM1 (red) and C2SM2 (light blue) are present in the N-terminal part of Clr2 and C2SM3 (dark blue) in the C-terminal part. Height of the boxes reflects significance (p-value). Logos for the three motifs that highlight conserved residues (B) C2SM1 (C) C2SM2 (D) C2SM3
Figure 2
Figure 2. Point mutations in Clr2 resulted in derepression of reporter genes in the mating type and pericentromeric regions.
Cells were serially diluted in steps of five and 5 µl were spotted onto selective media. Media used were (+URA), counter-selective (FOA) and selective (-URA) plates. The amount of ura4+ transcript was quantified using RT-qPCR and the wild type expression was set to 1. Top lane is the wild type strain (Clr2) that grew poorly on the selective plates. Second lane is a strain lacking Clr2 (Δ), third contains the V5-Clr2 followed by strains expressing mutated versions of the Clr2 protein: V5-Clr2P137G, V5-Clr2Y140G, V5-Clr2L142G, V5-Clr2R170G, V5-Clr2H178G, V5-Clr2L182G, V5-Clr2A375G and V5-Clr2E376G. (A) The spotted strain carries the mat3-M::ura4+ reporter in the mating type region. (B) The strains have the pericentromeric reporter gene imr(NcoI)::ura4+.
Figure 3
Figure 3. Strains containing the reporter gene mat3-M::ade6+ and point mutations in Clr2 have red/white sectored colonies.
All the strains carried the mat3-M::ade6+. (A) PJ1044, the wild-type strain (Clr2), (B) PJ1085, lacking Clr2 (Δ), (C) PJ1335 contains the V5-Clr2, (D) PJ1349 with V5-Clr2P137G, (E) PJ1425 with V5-Clr2Y140G, (F) PJ1424 with V5-Clr2L142G, (G) PJ1347 with V5-Clr2R170G, (H) PJ1361 with V5-Clr2H178G, (I) PJ1362 with V5-Clr2L182G, (J) PJ1363 with V5-Clr2A375G and (K) PJ1353 V5-Clr2E376G.
Figure 4
Figure 4. Strains containing point mutations in Clr2 has mottled and sectored iodine stained yeast colonies.
(A) PJ1044, the wild-type strain (Clr2), (B) PJ1085, lacking Clr2 (Δ), (C) PJ1335 contains the V5-Clr2, (D) PJ1349 with V5-Clr2P137G, (E) PJ1425 with V5-Clr2Y140G, (F) PJ1424 with V5-Clr2L142G, (G) PJ1347 with V5-Clr2R170G, (H) PJ1361 with V5-Clr2H178G, (I) PJ1362 with V5-Clr2L182G, (J) PJ1363 with V5-Clr2A375G and (K) PJ1353 V5-Clr2E376G.
Figure 5
Figure 5. V5-Clr2 constructs were detected by Western blot.
All the proteins were fused to a V5 tag in the N-terminus and detected using an anti-V5 antibody. The wild type Clr2 protein, wtClr2, as well as most of the point mutated versions of Clr2 could be detected when expressed from an nmt promoter (np). In all strains except L142G a protein of predicted size (63.4 kDa) was detected between the 55 and 70 kDa size markers. The mutated versions of the Clr2 protein migrated to the same position as the wild type protein and were detected by the anti-V5 antibody. Equal loading was detected using an anti-actin antibody.
Figure 6
Figure 6. In silico analysis of the conserved motifs C2SM1-3 and the consequences of introducing the mutations.
3D molecular modeling study of the three conserved motifs in Clr2. C2SM1 (A), C2SM2 (B) and C2SM3 (C). Mutating the conserved amino acids in Clr2 was predicted to disrupt the secondary structure of the protein. Overlay between the predicted wild type structure in red and the simulated post-MD mutant protein conformation in green color.
Figure 7
Figure 7. Point mutations in Clr2 affected transcriptional silencing at several locations in the S. pombe genome.
Cells were diluted by a factor of 5 for each and spotted on +URA, selective –URA and counter-selective FOA plates. Top lane is the wild type strain (Clr2) that grows poorly on the –URA plates. Second lane is a strain lacking Clr2 (Δ), third contains the V5-Clr2 followed by the point mutations in Clr2; Clr2P137G, Clr2Y140G, Clr2L142G, Clr2R170G, Clr2H178G, Clr2L182G and Clr2E376G. The reporter gene is (A) rDNA::ura4+ (B) cc2(SphI)::ura4+ and in (C) the ade6+ reporter gene inserted into the ura4 locus and with Gal4DBD-Clr4ΔCD resulting in repression of the ade6+gene.
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The work was supported by the Swedish Cancer Society, grant number 2011/360, http://www.cancerfonden.se; the Swedish Research Council, grant number 521-2011-2437, http://www.vr.se; the Goran Gustafsson Fundation, http://www.gustafssonsstiftelse.se/uu_kth/ALLBIO, FP7, grant number 289452, http://www.allbioinformatics.eu/doku.php. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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