Sgo1 Regulates Both Condensin and Ipl1/Aurora B to Promote Chromosome Biorientation

<div><p>Correct chromosome segregation is essential in order to prevent aneuploidy. To segregate sister chromatids equally to daughter cells, the sisters must attach to microtubules emanating from opposite spindle poles. This so-called biorientation manifests itself by increased tension and conformational changes across kinetochores and pericentric chromatin. Tensionless attachments are dissolved by the activity of the conserved mitotic kinase Aurora B/Ipl1, thereby promoting the formation of correctly attached chromosomes. Recruitment of the conserved centromeric protein shugoshin is essential for biorientation, but its exact role has been enigmatic. Here, we identify a novel function of shugoshin (Sgo1 in budding yeast) that together with the protein phosphatase PP2A-Rts1 ensures localization of condensin to the centromeric chromatin in yeast <i>Saccharomyces cerevisiae</i>. Failure to recruit condensin results in an abnormal conformation of the pericentric region and impairs the correction of tensionless chromosome attachments. Moreover, we found that shugoshin is required for maintaining Aurora B/Ipl1 localization on kinetochores during metaphase. Thus, shugoshin has a dual function in promoting biorientation in budding yeast: first, by its ability to facilitate condensin recruitment it modulates the conformation of the pericentric chromatin. Second, shugoshin contributes to the maintenance of Aurora B/Ipl1 at the kinetochore during gradual establishment of bipolarity in budding yeast mitosis. Our findings identify shugoshin as a versatile molecular adaptor that governs chromosome biorientation.</p></div

Sgo1 and Rts1 are required for the maintenance of Ipl1 localization and activity at the centromere.

<p>(A) Localization of Ipl1-GFP during mitosis in wild type cells and in cells lacking Sgo1, Rts1 or Cdc55. SPBs are visualized with Spc29-RFP. Bar – 5 µm. (B) Quantification of Ipl1-GFP localization on pre-anaphase spindles in wild type and <i>sgo1</i>Δ, <i>sgo1-N51I</i>, <i>rts1</i>Δ and <i>cdc55</i>Δ mutants (SPB distance <2 µm). Mean values of three independent experiments are shown. At least 150 cells were scored in each experiment. Top – examples of scored categories. (C) Enrichment of Ipl1-FLAG on centromeric DNA (0.1 kb away from CEN1 and 1.1 kb away from CEN4) and on rDNA (NTS1-2) normalized to the levels of Ipl1-FLAG bound to the arm of chromosome 10 in mitotic cells. ChIP-qPCR experiments of Ipl1-FLAG were performed using wild type and <i>sgo1</i>Δ cells arrested with nocodazole. Error bars represent the standard error of the mean. (D) Enrichment of Ipl1-FLAG on centromeric DNA (0.1 kb away from CEN1 and 1.1 kb away from CEN4) and on rDNA (NTS1-2) normalized to the levels of Ipl1-FLAG bound to the arm of chromosome 10 in mitotic cells. ChIP-qPCR experiments of Ipl1-FLAG were performed using wild type and <i>rts1</i>Δ cells arrested with nocodazole. Error bars represent the standard error of the mean.</p

Sgo1-mediated PP2A recruitment to the centromere is essential for tension sensing.

<p>(A) Localization of the GFP-tagged Sgo1 and Sgo1-N51I mutant. Spindle pole bodies (SPBs) are visualized with Spc29-RFP. Only pre-anaphase spindles (SPB distance <2 µm, spindle located in the mother cell) were scored. Plots on the right: histogram of signal intensity across the white line in the insets and percentage of cells with localized GFP signal. Bar – 5 µm. (B) Localization of Rts1-GFP in wild type cells or in cells carrying the <i>sgo1-N51I</i> mutation. SPBs are visualized with Spc29-RFP. Only pre-anaphase spindles (SPB distance <2 µm, spindle located in the mother cell) were scored. Plots on the right: histogram of signal intensity across the white line in the insets and percentage of cells with localized GFP signal. Bar – 5 µm. (C) Sensitivity to the microtubule depolymerizing drug benomyl in cells lacking <i>SGO1</i>. Cell viability was scored upon artificial tethering of the PP2A regulatory subunit Rts1 and Cdc55, respectively, to the kinetochore. (D) Progression of the wild type, <i>sgo1Δ, sgo1-N51I</i> and <i>rts1Δ</i> mutants through cell cycle upon depletion of the cohesin subunit Mcd1 which leads to formation of tensionless kinetochores. (E) Sensitivity of the wild type, <i>sgo1Δ, rts1Δ</i> and <i>sgo1-N51I</i> mutants to microtubule poisons and to the overexpression of Cik1-cc which triggers the formation of syntelic attachments at high frequencies.</p

Centromeric localization of condensin does not require PP2A's phosphatase activity.

<p>(A) Sgo1-TAP pulls down the condensin subunit Smc2-6HA. Co-immunoprecipitation of Smc2-6HA is dependent on the presence of Rts1. (B) Example of Ycg1-GFP localization after 60 min OKA treatment. SPBs are visualized with Spc29-RFP. Bar – 5 µm. (C) Quantification of subcellular localization of the condensin subunit Ycg1-GFP in the presence of OKA.</p

Sgo1 and Rts1 are essential for the maintenance of condensin at centromeres.

<p>(A) Localization of the condensin subunit Ycg1-GFP in wild type cells and cells lacking Sgo1, centromeric Rts1 (<i>rts1Δ</i> and <i>sgo1-N51I</i>) or Cdc55. SPBs are visualized with Spc29-RFP. Bar – 5 µm. Yellow arrowhead: centromeric Ycg1-GFP signal; white arrowhead: Ycg1-GFP on rDNA. (B) Quantification of Ycg1-GFP localization. Only pre-anaphase spindles (SPB distance <2 µm, spindle located in the mother cell) were scored. Means with SD of three independent experiments are shown. At least 150 cells were scored in each experiment. (C) Enrichment of Smc2-FLAG on centromeric DNA (0.1 kb away from CEN1 and 1.1 kb away from CEN4) and on rDNA (NTS1-2) normalized to the levels of Smc2-FLAG bound to the arm of chromosome 10 in mitotic cells. Chromatin immunoprecipitation(ChIP)-qPCR experiments of Smc2-FLAG were performed using wild type and <i>sgo1</i>Δ cells arrested with nocodazole. Error bars represent the standard error of the mean. (D) Enrichment of Smc2-FLAG on centromeric DNA (0.1 kb away from CEN1 and 1.1 kb away from CEN4) and on rDNA (NTS1-2) normalized to the levels of Smc2-FLAG bound to the arm of chromosome 10 in mitotic cells. ChIP-qPCR experiments of Smc2-FLAG were performed using wild type and <i>rts1</i>Δ cells arrested with nocodazole. Error bars represent the standard error of the mean. (E) Stretching of centromeric DNA in wild type cells compared with cells that fail to localize Rts1 to the centromeric region (<i>rts1</i>Δ and <i>sgo1-N51I</i>) and with cells lacking Cdc55. CEN4 is visualized by TetR-GFP recruited to the TetO-repeats integrated 1 kb from the centromere. Only pre-anaphase spindles (SPB distance <2 µm, spindle located in the mother cell) were scored. Top – examples of scored categories. (F) Sensitivity of wild type cells and the indicated condensin mutants to microtubule poisons and to the overexpression of the Cik1-cc construct which triggers formation of syntelic attachments at high frequencies. Yeast strain D1225 expresses the non-posphorylatable mutants of the condensin subunits Ycg1, Brn1, and Ycs4 which impair anaphase-specific functions of condensin.</p

Model of Sgo1 function during establishment of biorientation.

<p>Connecting lines represent protein-protein interactions, dashed lines represent functional interactions. Full arrow depicts phosphorylation.</p

Dual role of Sgo1 in localization of condensin and Ipl1.

<p>(A) Localization of Ipl1-GFP in <i>rts1</i>Δ cells that overexpress <i>BIR1</i> or <i>SLI15</i>. SPBs are visualized with Spc29-RFP. Bar – 5 µm. (B) Quantification of Ipl1-GFP localization on pre-anaphase spindles. Means of three independent experiments are shown. At least 150 cells were scored in each experiment. (C) Growth of <i>sgo1</i>Δ and <i>rts1</i>Δ cells which overexpress <i>BIR1</i> or <i>SLI15</i> in the presence of microtubule poisons and under conditions leading to syntelic attachments. (D) Localization of the Ycg1-GFP signal in <i>sgo1</i>Δ cells which overexpress <i>BIR1</i> or <i>SLI15</i>. SPBs are visualized with Spc29-RFP. Yellow arrowheads indicate lack of centromeric localization. White arrowheads indicate Ycg1-GFP localized to rDNA. Bar – 5 µm. (E) Quantification of Ycg1-GFP localization. Only pre-anaphase spindles were scored (SPB distance <2 µm). Means with SD of three independent experiments are shown. At least 150 cells were scored in each experiment.</p

Transcriptome profiling of colorectal tumors from patients with sepsis reveals an ethnic basis for viral infection risk and sepsis progression

Abstract Mortality from cancer-associated sepsis varies by cancer site and host responses to sepsis are heterogenous. Native Hawaiians have the highest mortality risk from cancer-associated sepsis and colorectal cancer (CRC), even though they demonstrate lower CRC incidence compared to other ethnicities. We conducted a retrospective transcriptomic analysis of CRC tumors and adjacent non-tumor tissue from adult patients of Native Hawaiian and Japanese ethnicity who died from cancer-associated sepsis. We examined differential gene expression in relation to patient survival and sepsis disease etiology. Native Hawaiian CRC patients diagnosed with sepsis had a median survival of 5 (IQR 4–49) months, compared to 117 (IQR 30–146) months for Japanese patients. Transcriptomic analyses identified two distinct sepsis gene signatures classified as early response and late response sepsis genes that were significantly altered in the Native Hawaiian cohort. Analysis of canonical pathways revealed significant up and downregulation in mechanisms of viral exit from host cells (p = 4.52E−04) and epithelial junction remodeling (p = 4.01E−05). Key genes including elongation initiation factor pathway genes, GSK3B, and regulatory associated protein of mTOR (RPTOR) genes that protect cells from infection were significantly downregulated in Native Hawaiians. Genes promoting sepsis progression including CLOCK, PPBP and Rho family GTPASE 2 (RND2) were upregulated in Native Hawaiian patients. Our transcriptomic approach advances understanding of sepsis heterogeneity by revealing a role of genetic background and defining patient subgroups with altered early and late biological responses to sepsis. This study is the first to investigate differential gene expression in CRC-associated sepsis patients in relation to ethnicity. Our findings may lead to personalized approaches in stratifying patient mortality risk for sepsis and in the development of effective targeted therapies for sepsis