Global analysis of SUMO chain function reveals multiple roles in chromatin regulation.

Like ubiquitin, the small ubiquitin-related modifier (SUMO) proteins can form oligomeric chains, but the biological functions of these superstructures are not well understood. Here, we created mutant yeast strains unable to synthesize SUMO chains (smt3(allR)) and subjected them to high-content microscopic screening, synthetic genetic array (SGA) analysis, and high-density transcript profiling to perform the first global analysis of SUMO chain function. This comprehensive assessment identified 144 proteins with altered localization or intensity in smt3(allR) cells, 149 synthetic genetic interactions, and 225 mRNA transcripts (primarily consisting of stress- and nutrient-response genes) that displayed a \u3e1.5-fold increase in expression levels. This information-rich resource strongly implicates SUMO chains in the regulation of chromatin. Indeed, using several different approaches, we demonstrate that SUMO chains are required for the maintenance of normal higher-order chromatin structure and transcriptional repression of environmental stress response genes in budding yeast

The MMS22L-TONSL Complex Mediates Recovery from Replication Stress and Homologous Recombination

Genome integrity is jeopardized each time DNA replication forks stall or collapse. Here we report the identification of a complex composed of MMS22L (C6ORF167) and TONSL (NFKBIL2) that participates in the recovery from replication stress. MMS22L and TONSL are homologous to yeast Mms22 and plant Tonsoku/Brushy1, respectively. MMS22L-TONSL accumulates at regions of ssDNA associated with distressed replication forks or at processed DNA breaks, and its depletion results in high levels of endogenous DNA double-strand breaks caused by an inability to complete DNA synthesis after replication fork collapse. Moreover, cells depleted of MMS22L are highly sensitive to camptothecin, a topoisomerase I poison that impairs DNA replication progression. Finally, MMS22L and TONSL are necessary for the efficient formation of RAD51 foci after DNA damage, and their depletion impairs homologous recombination. These results indicate that MMS22L and TONSL are genome caretakers that stimulate the recombination-dependent repair of stalled or collapsed replication forks

β-catenin mediates growth defects induced by centrosome loss in a subset of APC mutant colorectal cancer independently of p53.

Colorectal cancer is the third most common cancer and the second leading cause of cancer-related deaths worldwide. The centrosome is the main microtubule-organizing center in animal cells and centrosome amplification is a hallmark of cancer cells. To investigate the importance of centrosomes in colorectal cancer, we induced centrosome loss in normal and cancer human-derived colorectal organoids using centrinone B, a Polo-like kinase 4 (Plk4) inhibitor. We show that centrosome loss represses human normal colorectal organoid growth in a p53-dependent manner in accordance with previous studies in cell models. However, cancer colorectal organoid lines exhibited different sensitivities to centrosome loss independently of p53. Centrinone-induced cancer organoid growth defect/death positively correlated with a loss of function mutation in the APC gene, suggesting a causal role of the hyperactive WNT pathway. Consistent with this notion, β-catenin inhibition using XAV939 or ICG-001 partially prevented centrinone-induced death and rescued the growth two APC-mutant organoid lines tested. Our study reveals a novel role for canonical WNT signaling in regulating centrosome loss-induced growth defect/death in a subset of APC-mutant colorectal cancer independently of the classical p53 pathway

Patient-derived cancer HCOs exhibit different sensitivity to centrosome loss independently of p53.

A) The indicated cancer HCO lines (control or p53 KO) were grown from single adult stem cells in the presence of DMSO or 0.5 μM centrinone B for 8 days. Organoids were then fixed, stained with DAPI (blue) and phalloidin (red) and imaged. Merged maximum intensity projection images are shown. B) The areas of individual organoids were quantified in the merged maximum intensity projection images from (A) and presented in the graph as relative values normalized to the respective DMSO control; every dot represents an organoid (n = 3, ***PC) p53 mutation status in the three cancer HCOs from the whole exome sequencing data. D) Western blot analysis of p53 and loading controls (GAPDH and actin) in lysates prepared from the indicated control and p53 KO cancer HCO lines. E) Quantification of (D). Protein levels from three independent experiments were quantified and presented in the graph (n = 3, **PPF) High-resolution maximum intensity projections of pericentrin (centrosome) and DNA (DAPI) immunofluorescence staining in POP-112 organoids treated with DMSO or 0.5 μM centrinone B for 8 days. G) The number of pericentrin foci and nuclei were automatically identified in z-stack sections spanning the entire organoid. The foci to nucleus ratio was determined for four organoids in each condition (***PH) POP-112 organoids were grown from single adult stem cells in the presence of DMSO (0) or the indicated centrinone B concentrations for 8 days. Organoid areas were quantified as in (B). Relative average organoid area from three independent experiments is presented in the graph (n = 3, **PP<0.001, all other treatments compared to DMSO are not significant, One-way ANOVA with Bonferroni post-hoc). Scale bars are (A) 500 μm and (F) 25 μm.</p

Centrosome loss represses normal HCOs growth in a p53-dependent manner.

A) Control and p53 KO normal HCOs were grown from single adult stem cells in the presence of DMSO or 0.5 μM centrinone B for 14 days. Organoids were then fixed, stained with DAPI to label DNA (blue) and phalloidin to label actin (red) and imaged. Merged maximum intensity projections of images are shown. B) The areas of individual organoids were quantified in the merged maximum intensity projections (MIPs) of images from (A) (n = 3) and presented in the graph as values relative to the DMSO control where every dot represents an organoid (***PC) Tide analysis of p53 gene indel efficiency in the normal HCO p53 KO line. The algorithm provides the R2 value as a goodness-of-fit measure and calculates the statistical significance for each indel. Red represents significant indels (PD) Representative maximum intensity projections of p53 immunofluorescence staining in control and p53 KO normal HCOs. E) Normal human organoids were extracted from Matrigel and lysed. p53 and the loading control (GAPDH) protein levels were assessed using western blot analysis. F) Quantification of (E). Protein levels from three independent experiments were quantified and presented in the graph (n = 3, **PG) High-resolution maximum intensity projection images of CEP192 (centrosome) and DNA (DAPI) immunofluorescence staining in selected normal HCOs from (A). H) The number of CEP192 foci and nuclear objects was assessed in independent z-sections spanning three (DMSO) or five (centrinone) independent organoids. The foci to nucleus ratio was determined for each organoid (*P<0.05). Scale bars are (A) 500 μm, (D) 25 μm and (G) 10 μm.</p

Centrosome loss-induced organoid growth defect/death is β-catenin dependent in CSC-406 cancer line.

A) APC mutational status in the three cancer HCOs obtained via whole exome sequencing data. B) CSC-406 cancer organoids were grown from an equal number of single cells in the presence of DMSO or the indicated concentrations of XAV939 with DMSO or 0.5 μM centrinone B for 8 days. Organoids were then fixed, stained with DAPI (blue) and phalloidin (green) and imaged. Merged maximum intensity projections of representative images are shown. C) Percentage of surviving organoids in different conditions from (B) was quantified and presented in the graph (n = 3, *PD) The areas of individual organoids from B were quantified in the merged maximum intensity projection images and the relative values are presented in the graph, every dot represents an organoid (n = 3, *PE) CSC-406 cancer organoids were treated with DMSO or 20 μM XAV939 for three days, extracted from Matrigel and lysed. b-catenin and the loading control (GAPDH) protein levels were assessed using western blot analysis. The same set of Western blots was used to generate Fig 4E. F) Protein levels from three independent experiments were quantified and presented in the graph (n = 3, ***PG) CSC-406 cancer organoids were grown from an equal number of single cells in the presence of DMSO or 3 μM ICG-001 and were treated with DMSO or 0.5 μM centrinone B for 8 days. Organoids were then fixed, stained with DAPI (blue) and phalloidin (red) and imaged. Merged maximum intensity projections of representative images are shown. H) Percentage of surviving organoids in different conditions from (G) was quantified and presented in the graph (n = 3, ***PI) The areas of individual organoids from (G) were quantified in the merged maximum intensity projection images and the relative values are presented in the graph, every dot represents an organoid (n = 3, ***P<0.001, ns = non-significant, One-way ANOVA with Bonferroni post-hoc). Scale bars are (B and G) 500 μm.</p

Unaltered original data for all Western blots.

Boxed regions indicate cropped area used for Figure preparation. (PDF)</p

β-catenin inhibition is essential for preventing centrosome loss-induced death of POP-092 cancer organoids.

A) POP-092 cancer HCOs were grown from an equal number of single adult stem cells in the presence of DMSO or the indicated concentrations of XAV939 and DMSO or 0.5 μM centrinone B for 8 days. Organoids were then fixed, stained with DAPI to label nuclei and phalloidin to label actin, and imaged. Merged maximum intensity projection images were used to quantify the percentage of surviving organoids in the different conditions (n = 3, ns: non-significant, One-way ANOVA with Bonferroni post-hoc). B) The areas of individual organoids from (A) were quantified in the merged maximum intensity projection images and the relative values are presented in the graph, every dot represents an organoid (n = 3, ns: non-significant, One-way ANOVA with Bonferroni post-hoc). C) CSC-406 and POP-092 cancer HCOs were treated with DMSO or 20 μM XAV939 for three days, extracted from Matrigel and lysed. b-catenin and the loading control (GAPDH) protein levels were assessed using western blot analysis. D) Protein levels from three independent experiments from (C) were quantified with Fiji and presented in the graph (n = 3, **PPE) POP-092 cancer HCOs were treated with DMSO or the indicated XAV939 concentrations for three days, extracted from Matrigel and lysed. b-catenin and the loading control (GAPDH) protein levels were assessed using western blot analysis. A representative immunoblot is shown (n = 2). The same set of Western blots was used to generate Fig 3E–3F) POP-092 cancer HCOs were grown from an equal number of single cells in the presence of DMSO or 3 μM ICG-001 and were treated with DMSO or 0.5 μM centrinone B for 8 days. Organoids were then fixed and stained with DAPI to label DNA (blue) and phalloidin to label actin (Red). Representative maximum intensity projection images are shown. G) Percentage of surviving organoids in the different conditions tested in (F) was quantified (n = 3, **PH) Organoid areas from (F) were quantified in the merged maximum intensity projection images and the relative values are presented in the graph, every dot represents an organoid (n = 3, ***PI) Single focal plane images of β-catenin and DAPI immunofluorescence staining in fixed CSC-406 and POP-092 cancer HCOs. J) The relative intensity of b-catenin within nuclear objects identified using the DAPI channel was determined for four independent z-sections from each organoid presented in panel I. (n = 4, ***P<0.001, Mann-Whitney U test). Scale bars are (F) 500 μm and (I) 25 μm.</p

ICG-001 reduces the level of survivin mRNA in MDA-MB-231 cells.

A) MDA-MB-231 cells were treated with DMSO or 3 or 6 μM ICG-001 for 72 h before collecting cells for mRNA extraction. qPCR for surviving was performed on the subsequent cDNA using RPLP0 as a control. Relative survivin mRNA was normalized using RPLP0 and compared to levels in DMSO-treated cells. (n = 2, ***P (TIF)</p

Cancer organoid whole exome sequencing.

Colorectal cancer is the third most common cancer and the second leading cause of cancer-related deaths worldwide. The centrosome is the main microtubule-organizing center in animal cells and centrosome amplification is a hallmark of cancer cells. To investigate the importance of centrosomes in colorectal cancer, we induced centrosome loss in normal and cancer human-derived colorectal organoids using centrinone B, a Polo-like kinase 4 (Plk4) inhibitor. We show that centrosome loss represses human normal colorectal organoid growth in a p53-dependent manner in accordance with previous studies in cell models. However, cancer colorectal organoid lines exhibited different sensitivities to centrosome loss independently of p53. Centrinone-induced cancer organoid growth defect/death positively correlated with a loss of function mutation in the APC gene, suggesting a causal role of the hyperactive WNT pathway. Consistent with this notion, β-catenin inhibition using XAV939 or ICG-001 partially prevented centrinone-induced death and rescued the growth two APC-mutant organoid lines tested. Our study reveals a novel role for canonical WNT signaling in regulating centrosome loss-induced growth defect/death in a subset of APC-mutant colorectal cancer independently of the classical p53 pathway.</div