The spatial regulation of condensin activity in chromosome condensation

Abstract

Condensin mediates chromosome condensation, which is essential for proper chromosome segregation during mitosis. Prior to anaphase of budding yeast, the ribosomal DNA (RDN) condenses to a thin loop that is distinct from the rest of the chromosomes. We provide evidence that the establishment and maintenance of this RDN condensation requires the regulation of condensin by Cdc5p (polo) kinase. We show that Cdc5p is recruited to the site of condensin binding in the RDN by cohesin, a complex related to condensin. Cdc5p and cohesin prevent condensin from misfolding the RDN into an irreversibly decondensed state. From these and other observations, we propose that the spatial regulation of Cdc5p by cohesin modulates condensin activity to ensure proper RDN folding into a thin loop. This mechanism may be evolutionarily conserved, promoting the thinly condensed constrictions that occur at centromeres and RDN of mitotic chromosomes in plants and animals.

Keywords: cohesin; cohesion; condensation; condensin; polo kinase.

Figure 1.

The polo kinase Cdc5p is essential for mid-M chromosome condensation. (A) Schematic of cohesin and condensin. Cohesin is comprised of a Smc1p/Smc3p heterodimer joined at their head domain through Mcd1p. Mcd1p is likewise bound to Scc3p, resulting in the cohesin holocomplex. Condensin is comprised of a Smc2p/Smc4p heterodimer linked by Brn1p. Brn1p is bound to Ycg1p and Ycs4p. Together, these proteins form the pentameric condensin complex. (B, top) Schematic of the auxin-induced degron (AID) system to study functions of essential proteins. Proteins of interest, like Cdc5p, were fused to a 3V5-AID tag that allows for wild-type functions in the absence of auxin. Upon addition of auxin, 3V5-AID-tagged proteins were rapidly degraded in a proteasome-mediated manner. (Bottom) “Staged mid-M” regimen used to assess strains function in mid-M. Cultures were synchronized in G1 and released in media containing auxin and nocodazole, to depleted AID-tagged protein and arrest cells in mid-M. (C) Depletion of Cdc5p results in a moderate cohesion defect. Cells were treated as in 1B and cohesion assayed in mid-M through a GFP spot assay. In these experiments, LacO arrays are integrated at LYS4, a centromere-distal arm site. Wild-type, Mcd1p-depleted, and Cdc5p-depleted cells were scored for sister chromatid cohesion. The average percentage of separated sister chromatids from three independent experiments (100 cells) are reported, and error bars represent standard deviation. (D) Depletion of Cdc5p results in a moderate cohesion maintenance defect. Wild-type, Pds5p-depleted, Cdc5p-depleted, and Cdc5 kinase-dead (kd)-expressing in combination with depletion of Cdc5p-AID cells were treated as in B and cohesion assayed at 15-min intervals after release from G1 to mid-M. The average percentage of separated sister chromatids from two independent experiments (100 cells) are reported. (E) Depletion of Cdc5p results in condensation loss. (Top) Example of rDNA locus (RDN) morphologies assayed. RDN is either assayed as a condensed loop or decondensed puff. The percentage of cells displaying decondensed RDN puffs is quantified. (Bottom) Wild-type, Cdc5p-depleted, Cdc5 kinase-dead (kd)-expressing in combination with depletion of Cdc5p-AID, and Brn1p-depleted cells were treated as in B and processed to make chromosome spreads to score RDN condensation (see the Materials and Methods). Average of three experiments scoring 200 cells are shown and error bars represent the standard deviation. (F) Depletion of Cdc5p results in loss of maintenance of condensation. (Top) Cells were arrested in mid-M using nocodazole. The arrest was confirmed by analysis of bud morphology (>95% large budded cells). Auxin was added to ensure Cdc5p-AID degradation. (Bottom) Wild-type, Cdc5p-depleted, and Brn1p-depleted cells were processed to assess condensation as described in E.

Figure 2.

Cdc5p stimulates condensin-dependent RDN condensation. (A) Schematic of defects in condensation. RDN is in a puffed unfolded state in G1. Depletion of condensation factors results in a puffed RDN that can either be in an unfolded state or in a misfolded state. The unfolded state is reversible, once the factor is expressed again the RDN can be folded. Misfolded state is irreversible, even after expressing again the condensation factor the RDN remains misfolded. (B) Schematic of “add later” genetic test setup. (Top) Indicated cultures were arrested in G1 (>95% schmooed morphology), and AID-tagged proteins (Mcd1p-AID, Brn1p-AID, and Cdc5p-AID) were depleted by the addition of auxin. G1 arrest was relieved by resuspending in media with auxin and nocodazole to arrest cells in mid-M. Cells were then resuspended in media containing only nocodazole for 2 h to allow the expression of the previously depleted AID-tagged proteins. (Bottom) Depletion of condensin resulted in unfolded RDN that was folded upon later expression of condensin. Depletion of cohesin resulted in misfolded RDN that stayed misfolded upon later expression of cohesin. (C) Cdc5p depletion results in a reversible unfolded RDN state. “Add later” genetic test results from MCD1-AID, BRN1-AID, and CDC5-AID strains were treated as described in B and scored for RDN condensation. The percentage of cells displaying puffed RDN (decondensed) is quantified. Average of two experiments scoring 300 cells are shown and error bars represent standard deviation. (D) Condensin and Cdc5p promote misfolding of the RDN in the absence of cohesin. “Add later” genetic test results from MCD1-AID/BRN1-AID and MCD1-AID/CDC5-AID strains were treated as described in B and scored for RDN condensation. The percentage of cells displaying decondensed puffed RDN (decondensed) is quantified. Average of three experiments scoring 300 cells is shown and error bars represent standard deviation.

Figure 3.

Genome-wide localization of condensin, cohesin and Cdc5p. (A) Cohesin recruits Cdc5p at unique sequences genome-wide. ChIP-seq signals from a representative portion of chromosome XVI are presented with bar charts. The region presented includes the centromere (CEN; black circle) and the pericentric region (peri-CEN; black box). Signal coming from condensin subunit Ycg1p-Myc in wild-type strain is in green, signal from cohesin subunit Mcd1p in wild-type strain is in blue, signal coming from Cdc5p-Flag is in orange, and signal coming from GFP-control is in gray. Cdc5p ChIP-seq signal from wild type is in the first orange lane, from cells depleted of condensin subunit Brn1p-AID is in the second orange lane, and from cells depleted of cohesin subunit Mcd1p-AID is in the third orange lane. All strains were depleted of the indicated AID protein from G1 to mid-M and arrested in mid-M (staged mid-M). The scale is 0–15 for Ycg1p, 0–21 for Mcd1p, and 0–17 for Cdc5p. (B) Cohesin recruits Cdc5p at the repetitive RDN locus. ChIP-seq signals from a portion of chromosome XII are presented with bar charts. ChIP-seq signal from strains as in A. The region presented includes one copy of the rDNA repeat (9.1 kb) made of the transcribed regions RND37 and RND5 (black box with white arrows indicating the direction of transcription) and the nontranscribed regions NTS1 and NTS2 (black line). Region of enrichment of cohesin signal is marked by two blue dashed lines (from NTS2 to NTS1). Region of enrichment of condensin signal is marked by two green dashed lines (end of NTS1). The scale is 0–5200 for Ycg1p, 0–2300 for Mcd1p, and 0–1700 for Cdc5p.

Figure 4.

Cdc5p is recruited by cohesin and brought to condensin at centromeres and rDNA. (A) Cdc5p is recruited by cohesin and brought to condensin at centromeres. ChIP-seq signals from a representative portion of chromosome XVI are presented with bar charts. The region presented includes the centromere (CEN black circle) and the pericentric region (peri-CEN black box). Signal coming from condensin subunit Ycg1p-Myc in wild-type strain is in green, signal from cohesin subunit Mcd1p in wild-type strain is in blue, signal coming from Cdc5p-V5 is in orange, and signal coming from GFP-control is in gray. Cdc5p ChIP-seq signal from wild type is in the first orange lane, from cells depleted of cohesin subunit Mcd1p-AID from G1 to mid-M is in the second orange lane, and from cells depleted of cohesin subunit Mcd1p-AID in mid-M is in the third orange lane. The black arrow indicates enrichment of Cdc5p at the centromere when cohesin is transiently associated with chromosomes. The scale is 0–15 for Ycg1p, 0–21 for Mcd1p, and 0–17 for Cdc5p. (B) Cdc5p is recruited by cohesin and brought to condensin at RDN. ChIP-seq signals from a portion of chromosome XII are presented with bar charts. ChIP-seq signal from strains as in A. The region presented includes one copy of the rDNA repeat (9.1 kb) made of the transcribed regions RND37 and RND5 (black box with white arrows indicating the direction of transcription) and the nontranscribed regions NTS1 and NTS2 (black line). Region of enrichment of cohesin signal is marked by two blue dashed lines (from NTS2 to NTS1). Region of enrichment of condensin signal is marked by two green dashed lines (end of NTS1). Black arrow indicates enrichment of Cdc5p at the condensin-bound region of the RDN when cohesin is transiently associated with chromosomes. The scale is 0–5200 for Ycg1p, 0–2300 for Mcd1p, and 0–1700 for Cdc5p.

Figure 5.

Localization of Cdc5p with CRISPR–Cas9 to the condensin-bound RDN partially restores the RDN condensation defect of cohesin-depleted cells. (A) Galactose addition to the media drives the expression of a catalytically dead dCas9 fused to Cdc5p. Guide RNAs recruit dCas9-Cdc5p fusion protein either to the condensin-bound region NTS1 (RDN5 gRNA) or 500 bp to the left at RDN5 (RDN5 gRNA) where only cohesin is bound. (B, top) “Staged mid-M” regimen used to assess condensation in mid-M. Cultures were synchronized in G1 and released in media containing galactose and auxin to drive the expression of dCas9-Cdc5p concomitantly to the depletion of Mcd1p-AID. Media also contained nocodazole to arrest cells in mid-M and assess condensation. (Bottom) Wild-type, Mcd1p-depleted, Mcd1p-depleted with dCas9-Cdc5p and RDN5 gRNA-expressing, and Mcd1p-depleted with dCas9-Cdc5p and NTS1 gRNA-expressing cells were treated with “staged mid-M” regimen and processed to score RDN condensation. Average of three experiments scoring 100 cells are shown and error bars represent the standard deviation.