Inactivation of the budding yeast cohesin loader Scc2 alters gene expression both globally and in response to a single DNA double strand break

Abstract

Genome integrity is fundamental for cell survival and cell cycle progression. Important mechanisms for keeping the genome intact are proper sister chromatid segregation, correct gene regulation and efficient repair of damaged DNA. Cohesin and its DNA loader, the Scc2/4 complex have been implicated in all these cellular actions. The gene regulation role has been described in several organisms. In yeast it has been suggested that the proteins in the cohesin network would effect transcription based on its role as insulator. More recently, data are emerging indicating direct roles for gene regulation also in yeast. Here we extend these studies by investigating whether the cohesin loader Scc2 is involved in regulation of gene expression. We performed global gene expression profiling in the absence and presence of DNA damage, in wild type and Scc2 deficient G2/M arrested cells, when it is known that Scc2 is important for DNA double strand break repair and formation of damage induced cohesion. We found that not only the DNA damage specific transcriptional response is distorted after inactivation of Scc2 but also the overall transcription profile. Interestingly, these alterations did not correlate with changes in cohesin binding.

Keywords: DI-cohesion, damage induced cohesion; DNA double strand break; DSB DNA, double strand break; FDR, false discovery rate; HO, homothallic switching endonuclease; S, supplementary; SCC, sister chromatid cohesion; SMC, structural maintenance of chromosomes; Scc2; WT, wild type; cohesin network; microarray; transcription profile.

Figure 1.

Experimental set up. (A) Schematic illustration of the experimental system used throughout this study. Pairs of S. cerevisiae strains, either WT or harboring the scc2–4 ts allele, genetically identical in all other aspects except for the presence or absence of the recognition site for the HO enzyme, were grown in YEP media supplemented with 2% raffinose at 21°C, and arrested in G2/M. A temperature raise to 32°C for 30 minutes, renders Scc2 dysfunctional before galactose addition, to induce one DSB or not. After 90 minutes break induction, cells were collected, total RNA prepared, cDNA synthesized and fragmented before hybridization to GeneChip Yeast genome 2.0 Array. (B) Pulse-field gel electrophoresis (PFGE) for verification of pGAL-HO break induction. The arrow points at Chr. VI. (C) FACS profiles of indicated yeast strains at indicated time points.

Figure 2.

Inactivation of Scc2 causes global changes in gene expression. (A) Correlation plot illustrating correlations between WT and Scc2 deficient cells and induction of DSB. White color represents perfect correlation (ρ = 1) and black represents no correlation (ρ=0). Higher correlation is seen within each cell type, independent of DSB induction. (B) Summary of the number of probes/genes significantly affected when comparing WT and scc2–4 cells in the absence and presence of DSB. (C and D) Bioinformatic analysis using SGD GO slim mapping. Up- and downregulated genes were sorted according to biological process. Processes were regarded as significantly enhanced if FDR ≤ 0.05 (*, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.005). (C) Biological processes of upregulated genes in Scc2 deficient cells compared to WT. (D) Biological processes of downregulated genes in Scc2 deficient cells compared to WT.

Figure 3.

For figure legend, see page 3651.Figure 3 (See previous page). Transcriptional alterations induced by a single DSB on Chr. VI. (A) Heatmap showing the 143 probe sets transcriptionally altered in response to a single DSB on Chr. VI (FDR ≤ 0.05) in WT cells. (B) Heatmap showing the 1189 probe sets transcriptionally altered in response to a single DSB on Chr. VI after inactivation of Scc2 (FDR ≤ 0.05). (A and B) Red represents genes that are upregulated and green those that are downregulated. (C) Relative gene expression of indicated genes was measured by qRT-PCR. Expression of respective gene for both WT and scc2–4 in the presence of break was related to its own absence of break sample that was set to 1. Data are mean values from 3 independent experiments with the respective deviation. Statistical significances are indicated by *, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.005.

Figure 4.

A single DSB evokes different transcriptional programs in WT and scc2–4 cells. (A and B) SGD GO slim mapping was used to sort up- and downregulated genes according to biological process. Processes were regarded as significantly enhanced if FDR ≤ 0.05 (*, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.005). (A) Frequencies for enhanced processes in the group of upregulated genes in response to DSB in WT and scc2–4 cells, in comparison with the frequency of the same processes in scc2–4 and WT cells, respectively. (B) Frequencies for enhanced processes in the group of downregulated genes in response to DSB in WT and scc2–4 cells, in comparison with the frequency of the same processes in scc2–4 and WT cells, respectively.

Figure 5.

Scc2 is important for repression of DSB proximal genes. (A) Schematic illustration of the region immediately surrounding the DSB on Chr. VI (adapted from Sacchcaromyces Genome Database). Gray arrow points at the HO recognition site. (B) Relative gene expression of indicated genes was measured by qRT-PCR. Expression of respective gene for both WT and scc2–4 in the presence of break was related to its own absence of break sample that was set to 1. (C) Relative gene expression of indicated genes was measured by qRT-PCR. Expression of respective gene for scc2–4 cells in the absence of break was related to the WT absence of break sample that was set to 1. (B and C) Data are mean values from 3 independent experiments with the respective deviation (*, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.005).

Figure 6.

Cohesin binding genome wide and at the Chr. VI DSB. (A and B) Chromosomal association of Flag-tagged Scc1 analyzed by ChIP sequencing in WT and scc2–4 cells in the presence and absence of DSB induction at Chr. VI as indicated. Orange peaks display significant chromosomal binding sites where the x-axes show chromosomal positions and the y-axes show linear fold enrichment. (A) Shown is a representative undamaged region of Chr. III. (B) Shown is the region immediately surrounding the DSB on Chr. VI. Gray arrow points at the HO break site.