9 research outputs found
Centromere Binding and a Conserved Role in Chromosome Stability for SUMO-Dependent Ubiquitin Ligases
<div><p>The <i>Saccharomyces cerevisiae</i> Slx5/8 complex is the founding member of a recently defined class of SUMO-targeted ubiquitin ligases (STUbLs). Slx5/8 has been implicated in genome stability and transcription, but the precise contribution is unclear. To characterise Slx5/8 function, we determined genome-wide changes in gene expression upon loss of either subunit. The majority of mRNA changes are part of a general stress response, also exhibited by mutants of other genome integrity pathways and therefore indicative of an indirect effect on transcription. Genome-wide binding analysis reveals a uniquely centromeric location for Slx5. Detailed phenotype analyses of <i>slx5</i>Δ and <i>slx8</i>Δ mutants show severe mitotic defects that include aneuploidy, spindle mispositioning, fish hooks and aberrant spindle kinetics. This is associated with accumulation of the PP2A regulatory subunit Rts1 at centromeres prior to entry into anaphase. Knockdown of the human STUbL orthologue RNF4 also results in chromosome segregation errors due to chromosome bridges. The study shows that STUbLs have a conserved role in maintenance of chromosome stability and links SUMO-dependent ubiquitination to a centromere-specific function during mitosis.</p></div
Additional file 1: of A high-resolution gene expression atlas of epistasis between gene-specific transcription factors exposes potential mechanisms for genetic interactions
List of putative GSTFs and strains used. (XLSX 27 kb
Additional file 3: of A high-resolution gene expression atlas of epistasis between gene-specific transcription factors exposes potential mechanisms for genetic interactions
Contribution of selection criteria to genetic interaction types. (A) Hierarchical clustering of GSTF pairs selected on growth-based genetic interaction scores, represented as in Fig. 3. Clustering was performed on the epistatic effects. GSTF pairs marked with a solid circle were also selected based on similarity in DNA binding. Colored branches depict example groups described in the text. (B) Hierarchical clustering of GSTF pairs selected based on similarity in DNA binding, represented as in A. GSTF pairs marked with a solid circle also exhibit a genetic interaction as derived by growth on agar plates [11]. (PDF 339 kb
Slx5 binding to centromeres is largely kinetochore-dependent.
<p>(A) Live cell microscopy of cells coexpressing Slx5-GFP and kinetochore protein Nnf1-mCherry. Right-hand panel shows a magnification of two nuclei, indicated in the merged image. Scale bars, 5 µm. (B) ChIP-qPCR of Slx5-GFP and Slx8-GFP in wt, <i>slx5</i>Δ or <i>slx8</i>Δ strains. Binding ratios (BR) at <i>CEN1</i> and <i>CEN2</i> are represented as enrichment over the control gene <i>POL1</i> (± s.d., n = 4), based on two independent biological replicate experiments. (C–D) ChIP-qPCR of Slx5-GFP in wt and <i>ndc10-1</i> strains at permissive (25°C) and nonpermissive (37°C) temperatures. Data is represented as enrichment at <i>CEN2</i> over <i>POL1</i> (Slx5-GFP and Slx5-GFP <i>ndc10-1</i>: ± s.d., n = 2) (wt and <i>ndc10-1</i>: n = 1, no enrichment).</p
Slx5 resides at centromeres.
<p>(A) ChIP-chip analysis of Slx5. Binding ratios (BR) of individual probes are mapped to the 16 chromosomes of the <i>S. cerevisiae</i> genome. BR is expressed as log<sub>2</sub> value of αGFP ChIP/input with subtraction of the mock/input signal. The positions of the centromeres are marked I to XVI. (B) Binding profile of Slx5 and Slx8 at chromosome 1. The genomic region shown in grey is magnified in (C). (D) Average centromeric binding profiles of Slx5, Slx8 and Ndc10. Signals from microarray probes are mapped relative to their position to the centromere and all probes are included that map to within 5000 bp of all 16 centromeres. Probes with a BR (log<sub>2</sub>) >2 are shown in red (Slx5) or blue (Ndc10). (E–H) ChIP-qPCR of Slx5 (E), Ndc10 (F), Slx8 (G) and wt (H). BRs at centromere 1, 2, and 5 are normalised to the control gene <i>POL1</i> (± s.d., n = 3).</p
RNF4 depletion causes chromosome segregation errors.
<p>(A) Time-lapse video microscopy of H2B-EYFP HeLa cells transfected with RNF4 siRNA or mock. Arrow indicates a chromosome bridge. Time (minutes) is given relative to the first time frame in prometaphase. Scale bars, 10 µm. (B) RNF4 knockdown efficiency determined by reverse transcription qPCR. cDNA was prepared from H2B-EYFP HeLa cells transfected with siRNAs targeting RNF4 or mock siRNA. mRNA levels of <i>RNF4</i> and <i>β-ACTIN</i> were analysed by qPCR. Graph represents a single experiment, showing three technical replicates (± s.d.). (C) Quantification of chromosome segregation defects of H2B-EYFP HeLa cells, as shown in (A), transfected with different siRNAs as indicated. Graph represents the average of two independent experiments per siRNA (± s.d.) and at least 72 cells per siRNA. (D) Three examples of anaphase cells with lagging chromosomes in fixed HeLa cells transfected with RNF4 siRNA oligos. Inset (negative stain) shows a persistent chromatin bridge. Scale bars, 10 µm.</p
Rts1 foci are partially Shugoshin (Sgo1)-dependent.
<p>(A) Growth rate assay of cells spotted in five-fold serial dilutions on YPD plates. Images are after two days growth at 25°C. (B) Live cell fluorescence microscopy of asynchronous wt, <i>slx5</i>Δ, <i>sgo1</i>Δ and <i>slx5</i>Δ <i>sgo1</i>Δ cells expressing Rts1-GFP. Scale bars, 5 µm. (C) Quantification of Rts1 foci in cells, shown in (B). Quantification is based on an asynchronous cell population (n = 89–203), that was morphologically divided in a small-budded and large-budded cell population.</p
Mitotic spindle defects in <i>slx5</i>Δ and <i>slx8</i>Δ mutants.
<p>(A) Time-lapse video microscopy of wt, <i>slx5</i>Δ and <i>slx8</i>Δ cells expressing GFP-Tub1. The upper panel shows a metaphase spindle in a wt cell at 2 minute intervals. Spindle elongation is initiated at t = 18′. The two panels below show examples of aberrant positioning of metaphase spindles in <i>slx5</i>Δ and <i>slx8</i>Δ cells during a temporary metaphase arrest. Arrows indicate spindle dislocation from the bud neck. Contours of cells are marked with a white line and are based on the DIC image. Scale bars, 5 µm. (B) Example of a spindle in <i>slx8</i>Δ, followed from metaphase to late anaphase. Colours are inverted to increase visibility of the astral microtubules at the outer tips of the spindle. The cell has a prolonged metaphase (t = 0′–52′) during which the spindle dislocates into the bud (t = 4′). Entry into anaphase is initiated at t = 52′, followed by spindle extension (t = 54′–80′). Formation of a fish hook spindle is apparent during late anaphase (t = 76′–80′). Scale bar, 5 µm. (C) Examples of fish hook spindles in <i>slx5</i>Δ and <i>slx8</i>Δ and a normal elongated spindle in wt during late anaphase. Scale bars, 5 µm. (D) Benomyl sensitivity assay. Growth rate of yeast cells is measured on YPD plates complemented with benomyl or DMSO (control). Images are after two days growth at 30°C. The benomyl-sensitive SAC mutant <i>mad2</i>Δ is included as control. (E) Quantification of spindle length, defined as the distance between two spindle pole bodies in wt, <i>slx5</i>Δ and <i>slx8</i>Δ expressing Spc42-GFP. Spindle length (n >100) was quantified from 20 minutes before anaphase onset to completion of anaphase. Grey lines depict the spindle length of individual cells. Black lines represent the average wt spindle length ± s.d., which is also shown as reference in the <i>slx5</i>Δ and <i>slx8</i>Δ plots. (F) Quantifications of spindle phenotypes in wt, <i>slx5</i>Δ and <i>slx8</i>Δ during metaphase and anaphase.</p
<i>Slx5</i>Δ and <i>slx8</i>Δ mutants accumulate Rts1 at kinetochores during metaphase.
<p>(A) Growth rate of cells spotted in five-fold serial dilutions on YPD plates. Images are after two days of growth at 25°C. (B) Growth rate of yeast in liquid YPD media. Relative growth rate (mutant/wt) was quantified during mid-log phase (± s.d., n = 6). Expected relative growth rates (RGR) of the double deletion mutants are calculated by multiplying the observed RGRs of the single deletion mutants. (C) Live cell microscopy of large-budded wt, <i>slx5</i>Δ and <i>slx8</i>Δ cells expressing Rts1-GFP. Nuclear Rts1 foci are detected in <i>slx5</i>Δ and <i>slx8</i>Δ, which are absent in large-budded wt cells. Scale bars, 5 µm. (D–E) Quantification of subcellular Rts1 location in wt, <i>slx5</i>Δ and <i>slx8</i>Δ cells. An asynchronous cell population (n >200 cells) was morphologically divided in small-budded (G1-S phase) and large-budded (G2/M phase) cells. Diffuse Rts1 location in nucleus and cytoplasm is schematically indicated in grey. Presence of one or two Rts1 foci is schematically indicated as black nuclear dots. Enrichment of Rts1 at the bud neck is indicated as a black bar. Rts1 foci were not detected in nonbudded cells. (F–G) Live cell fluorescence microscopy of wt (F) and <i>slx5</i>Δ <i>mad2</i>Δ cells (G), expressing Rts1-GFP and kinetochore protein Nnf1-mCherry. The small-budded wt has a normal centromeric Rts1 focus. Note that Rts1 is also enriched at the bud membrane. The left panel in (G) shows two small-budded <i>slx5</i>Δ <i>mad2</i>Δ cells with normal Rts1 foci that colocalise with kinetochores. The right panel in (G) shows a large-budded <i>slx5</i>Δ <i>mad2</i>Δ cell with an aberrant, mislocalised centromeric Rts1 focus during metaphase. Scale bars, 5 µm.</p