EMBO/soda-nel huggingface dataset:
Phosphoproteomics reveals novel modes of function and inter-relationships among PIKKs in response to genotoxic stress (2020)
Figure 2

Figure 2. PIKK dependencies of DNA damage-induced dynamics of the phosphoproteome in WT cells.

(A) Relative share of the three PIKKs in NCS-responsive phosphorylations.

(B) Venn diagram depicting the relative share of the three PIKKs in NCS-induced phosphorylations occurring on S/TQ sites.

(C) The profile of a group of substrates targeted by ATM 20 min after neocarzinostatin (NCS) addition and by ATR 1 hr after hydroxyurea (HU) addition. Box plots depict 23 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).

(D) Western blotting analysis confirming that the ATM substrate, pS824/KAP1 is targeted by ATR in response to HU. pS345/CHK1 - an established ATR substrate - served as a positive control for ATR activation.
Phosphoproteomics reveals novel modes of function and inter-relationships among PIKKs in response to genotoxic stress (2020)
Figure 3

Figure 3. Chemical inhibition of DNA-PK enhances ATM-mediated phosphorylations in WT cells.

(A) A cluster of 71 neocarzinostatin (NCS)-induced phosphorylations that were enhanced upon continuous inhibition of DNA-PK. Untreated cells are marked UT. This cluster breaks down into two distinct groups according to their ATM dependence 240 min after NCS addition. Clusters were obtained using K-means algorithm implemented in Perseus on Z-scored intensities. Box plots depict 71 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).

(B) Depicted are phosphopeptides from (A) that responded to NCS treatment in A-T cells. No significant elevation was observed in these phosphorylations in A-T cells following continuous inhibition of DNA-PK. Box plots depict 71 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 time the interquartile range (IQR).

(C) The cluster depicted in (A) was enriched for ATM-dependent phosphorylations and the S/TQ phosphorylation motifs. Enrichment was tested using the Fisher exact test implemented in Perseus Tyanova et al., 2016b().

(D) STRING-Network representation of the proteins in the same cluster. Proteins phosphorylated on the S/TQ motif are marked with a yellow border, and the line thickness represents the combined score for interaction confidence according to STRING.

(E) Western blotting analysis of selected phosphorylations in the above cluster showing DNA-PKi-induced elevation.

(F) Temporal dynamics of DNA-PK-dependent attenuation of pS824/KAP-1.
Phosphoproteomics reveals novel modes of function and inter-relationships among PIKKs in response to genotoxic stress (2020)
Figure 4

Figure 4. PIKK-dependent compensation for ATM absence in A-T cells.

(A) A subset of exclusive ATM-dependent sites in WT cells, which responded to neocarzinostatin (NCS) also in A-T cells. Their dependencies in A-T cells are shown.

(B) Dependencies of S/TQ phosphorylations within the same subset.

(C) The first time points at which 'compensated sites' from (A) responded to NCS in WT and A-T cells are presented.

(D) Temporal kinase dependencies of the 'compensated sites' in A-T cells. Shown are the numbers of 'compensated sites' in A-T cells, which depended on each kinase at the 20 and 240 min time points. The early ones depend mainly on DNA-PK, while the later ones - on ATR.

(E) STRING-Network representation of the proteins that were included in the early, DNA-PK-dependent compensation (purple) or the late, ATR-dependent compensation (green). Proteins phosphorylated on the S/TQ motif are highlighted by yellow margins. The thickness of connecting lines represents the combined score for interaction confidence according to STRING.

(F) Western blotting analysis confirming high-throughput results for selected ATM substrates.
Phosphoproteomics reveals novel modes of function and inter-relationships among PIKKs in response to genotoxic stress (2020)
Figure 5

Figure 5. Compensation for chemical inhibition of ATM in WT cells.

(A) A group of exclusively ATM-dependent sites that were ATM-dependent one hr after NCS addition, were still phosphorylated or dephosphorylated 4 hr after treatment, but were not affected by ATMi at that time point. Box plots depict 18 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).

(B) This group was enriched for sites that were compensated in an ATR-dependent manner in A-T cells. Box plots depict 9 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).

(C) Western blotting analysis of pS343/NBS1, which is compensated upon ATM inhibition, and pS824/ KAP-1 and pS395/NUMA, which are not compensated under these conditions.
Major histocompatibility complex class II‐restricted presentation of a cytosolic antigen by autophagy (2003)
Figure 1

Endogenous presentation of a peptide derived from cytosolic NeoR on HLA‐DP3. (A) The T cell clone 20–4/A4 recognizes an epitope derived from the NeoR protein in the context of HLA‐DP3. MHC class II expression on RCC1.24‐NeoR cells following IFN‐γ treatment confers recognition by 20–4/A4. This recognition is almost completely blocked by the HLA‐DP‐specific antibody B7/21. EBV‐B cells transfected with pINCO‐NeoR are able to present endogenous NeoR on MHC class II. As exemplified by the cell lines EBV‐B1.11 (HLA‐DP3) and EBV‐B1.25 (HLA‐DP4), recognition by 20–4/A4 is dependent on HLA‐DP3 expression. Bacterially expressed NeoR protein and a synthetic peptide encompassing amino acids NeoR216–229 served as controls. (B) Cross‐presentation experiments to exclude release of NeoR and re‐uptake as exogenous protein. EBV‐B1.25 cells transfected with the neoR expression plasmid pINCO‐NeoR, were co‐cultured for 24 h with the same number of untransfected EBV‐B1.11 cells, or vice versa. Then, T cells were added and co‐cultured with the cells for additional 24 h. To test for NeoR release by neoR‐transfected EBV‐B1.11 cells, total supernatant (200 μl) of 1×105 EBV‐B1.11 cells was harvested 48 h post transfection by centrifugation, and was used as culture media for 1×105 untransfected EBV‐B1.11 cells. After 48 h, 1×105 20–4/A4 T cells were added and GM‐CSF concentration determined after additional 24 h.

WILEY‐VCH
Major histocompatibility complex class II‐restricted presentation of a cytosolic antigen by autophagy (2003)
Figure 2

Endogenous presentation of NeoR on MHC class II does not depend on components of the MHC class I presentation pathway. RCC1.24‐NeoR (A), pINCO‐NeoR transfected EBV‐B1.11 cells (B), and as control pINCO‐Tyrosinase‐transfected EBV‐B1.11 cells (C), were either left untreated or treated with 5 μM lactacystin or 50 μM AAF‐CMK for 20 h. Following fixation with 0.5% paraformaldehyde, cells were incubated with T cells, and 24 h later GM‐CSF release was determined by ELISA. (D) FACS analysis of EBV‐B1.11 cells (open histograms) and EBV‐B1.11 cells stably transfected with the ICP47 gene (shaded histograms). Inhibition of TAP by ICP47 down‐regulates HLA class I but not HLA‐DP expression. An isotype‐matched antibody was used as control (thin‐lined histogram). EBV‐B1.11‐ICP47 cells were transfected with pINCO‐NeoR (E), pINCO‐Tyrosinase (F), or mock transfected (pINCO) and MHC‐restricted presentation monitored with T cells.

WILEY‐VCH
Major histocompatibility complex class II‐restricted presentation of a cytosolic antigen by autophagy (2003)
Figure 3

Lysosomal processing is required for MHC class II‐restricted presentation of endogenous and exogenous NeoR. (A) RCC1.24‐NeoR incubated with IFN‐γ were either left untreated (w/o) or treated with chloroquine or leupeptin for 24 h to block lysosomal processing. Subsequently cells were fixed with 0.5% paraformaldehyde and tested with 20–4/A4 in a GM‐CSF release assay. EBV‐B1.11 cells transfected with pINCO‐NeoR (B) or pINCO‐Tyrosinase (C) were tested likewise. Both substances abrogated recognition of neoR‐transfected cells, while HLA‐A2‐restricted presentation of the tyrosinase peptide was not affected. (D) EBV‐B1.11 cells were incubated with 200 ng/ml of recombinant NeoR protein and treated with leupeptin or chloroquine as described in (A). Presentation of exogenous NeoR on HLA‐DP3 is blocked by both substances as well, indicating that NeoR presentation is dependent on lysosomal processing. The IFN‐γ‐treated, NeoR‐negative parental cell line RCC1.24, and non‐transfected EBV‐B1.11 cells were used to demonstrate specificity of T cell recognition.

WILEY‐VCH
Major histocompatibility complex class II‐restricted presentation of a cytosolic antigen by autophagy (2003)
Figure 4

NeoR accesses an early endosomal compartment. (A) 293T cells transfected with pINCO‐NeoR were harvested 6 and 96 h post transfection and NeoR localization within various cell fractions determined by Western blot. In addition, supernatant of pINCO‐NeoR transfected 293T cells (96 h post transfection) was included to test for release of NeoR into the culture supernatant. At 6 h post transfection, NeoR protein becomes detectable in the cytoplasm, while at 96 h post transfection a substantial amount of NeoR protein is also localized in the endosomal/lysosomal fraction. For further characterization of the various fractions, blots were stripped and re‐probed with antibodies specific for the cytoplasmic protein LDH, the nuclear protein c‐myc, early endosomes (Rab‐5), late endosomes and lysosomes (Lamp‐1) and mitochondria (cytochrome c), which co‐purify with endosomes/lysosomes 18. (B) Endosomal/lysosomal fractions were prepared from pINCO‐NeoR‐transfected cells 96 h post transfection as in (A). To prevent lysosomal degradation of NeoR, half of the cells were treated with leupeptin for the last 48 h. These fractions were separated on a 27% Percoll gradient and eight different fractions collected. Successful separation into fractions with increasing density was verified by Western blot analysis with antibodies directed against Lamp‐1, Rab‐5 and cytochrome c. Inhibition of NeoR degradation by leupeptin causes accumulation of NeoR protein in the late endosomal/lysosomal fractions 7 and 8. (C) To determine where NeoR gains access to the vacuolar system, 293T cells were transfected with pINCO‐NeoR, treated with leupeptin to prevent NeoR degradation, and the endosomal/lysosomal fractions prepared at various time points post transfection and separated by Percoll gradient centrifugation. Starting at 16 h post transfection, NeoR becomes detectable in the Rab‐5‐positive fraction 5, and then spreads into late endosomal/lysosomal fractions. At 26 h post transfection an almost identical NeoR distribution pattern is observed in cells treated with or without BfA.

WILEY‐VCH
Major histocompatibility complex class II‐restricted presentation of a cytosolic antigen by autophagy (2003)
Figure 5

NeoR accesses the endosomal/lysosomal compartment through autophagy. (A) RCC were transfected with the NeoR‐GFP expression construct and subcellular localization of the fusion protein monitored by UV fluorescence. As compared to untreated cells, cells treated with 3‐MA show a much brighter UV fluorescence. (B) Semiquantitative analysis of fluorescence intensity of cells transfected with the NeoR‐GFP fusion construct. Compared to untreated cells, cells treated for 24 h with 7.5 mM 3‐MA show an about threefold increase in mean fluorescence intensity. (C) Western blot analysis of neoR‐transfected cells. Treatment of cells with 7.5 mM 3‐MA causes a dramatic increase in NeoR protein levels in only 12 h. The ER‐resident protein BiP was used as gel loading control. (D) This increase in total NeoR protein in 3‐MA‐treated cells is associated with an increase in cytoplasmic, and a decrease in endosomal/lysosomal NeoR protein levels. (E) Western blot analysis of whole cell lysates prepared from neoR‐transfected cells. Inhibition of protein synthesis by cycloheximide treatment leads to a rapid decrease in NeoR protein levels. Simultaneous treatment of cells with 3‐MA prevents NeoR degradation. BiP was again used as a gel loading control.

WILEY‐VCH
Major histocompatibility complex class II‐restricted presentation of a cytosolic antigen by autophagy (2003)
Figure 6

Inhibition of autophagy blocks endogenous presentation of the NeoR‐derived peptide on MHC class II. (A) RCC1.24‐NeoR (endogenous), and RCC1.24 cells to which NeoR protein had been added (exogenous), were treated with different concentrations of 3‐MA and cell surface presentation of the NeoR‐derived peptide on HLA‐DP3 monitored with the NeoR‐specific T cell clone 20–4/A4. Inhibition of autophagy by 3‐MA causes an almost complete inhibition of endogenous antigen presentation, while presentation of exogenous NeoR is not affected. (B) EBV‐B1.11 cells transfected with pINCO‐NeoR (endogenous), or incubated with recombinant NeoR protein (exogenous), were treated with the inhibitors of autophagy, wortmannin and 3‐MA, and HLA‐DP3‐restricted presentation of the NeoR‐derived peptide determined. Inhibition of antigen presentation by leupeptin served as control. (C) Presentation of the HLA‐A2‐restricted tyrosinase peptide in EBV‐B1.11 cells transfected with pINCO‐Tyrosinase is not affected by these inhibitors of autophagy. FACS analysis of HLA‐class I and HLA‐DP expression on RCC1.24 (D) and EBV‐B1.11 cells (E). As compared to untreated cells (thick‐lined histogram), treatment of cells with 7.5 mM 3‐MA (shaded histogram) does not cause down‐regulation of HLA‐class I or HLA‐DP. Isotype control is shown as thin‐lined histogram.

WILEY‐VCH
Systematic genetics and single-cell imaging reveal widespread morphological pleiotropy and cell-to-cell variability (2020)
Figure 4

Figure 4 Predicting gene function from phenotype profiles (see also Figure EV4).

(A) Endocytic patch formation dynamics in the ydl176wΔ (ipf1Δ) strain. Patch dynamics were examined using time-lapse fluorescence microscopy of wild-type (WT) and ipf1Δ deletion strains carrying reporters for the coat (Sla1-GFP; green), and actin (Sac6-tdTomato; red) modules. Left: Representative kymographs for the WT and ipf1Δ strains. Right: Box plot illustrating the distribution of lifetimes of Sla1-GFP and Sac6-tdTomato patches. Significance was determined using unpaired t-tests. Scale bar: 10 seconds.

(B) Examples of gene clusters obtained with hierarchical clustering of phenotype profiles composed of the 17 specific phenotype fractions. Phenotypes 1-17: [1] coat: increased patch number; [2] coat: aggregate; [3] vacuole: class E; [4] late endosome: condensed; [5] actin: bright patches; [6] late endosome: membrane; [7] actin: aggregate; [8] coat: decreased patch number; [9] actin: decreased patch number; [10] late endosome: fragmented; [11] coat: depolarized patches; [12] actin: depolarized patches; [13] vacuole: multilobed; [14] vacuole: fragmented; [15] vacuole: enlarged; [16] vacuole: class G; [17] vacuole: V-ATPase defect.

(C) Interaction network of NNF2 and YER043W (GTA1). Genes with phenotype profiles with a correlation > 0.7 and genetic interaction profiles with a correlation > 0.2, and at least two significant correlations to NNF2 and/or GTA1 were included in the network.

(D) Analysis of phenotype profile similarity between mutants in genes encoding proteins in same or different protein complex structures. Box plot indicates distribution of PCCs between pairs of phenotype profiles for genes that encode protein pairs in direct contact in a protein complex experimental structure (Yes - Direct), code for protein pairs in the same protein complex structure but not in direct contact (Yes - Indirect), and code for protein pairs that do not belong to the same protein complex structure (No). The number of pairs evaluated in each set is shown on the x-axis. Significance was determined using one-sided Mann-Whitney U tests. ***p-value < 0.001.

(E, F) Differentiation of functionally related protein complexes and protein complex organization using phenotype profiles. Heatmaps showing PCCs between components of the ESCRT complexes (E) and the SPOTS complex (F). A more intense blue colour indicates a higher PCC (scale bar at the top left of each heat map).
Systematic genetics and single-cell imaging reveal widespread morphological pleiotropy and cell-to-cell variability (2020)
Figure 6

Figure 6 Factors contributing to incomplete penetrance (see also Table EV8).

(A) Penetrance as a function of replicative age. Top: Bar graph showing the fraction of outliers in populations of increasing replicative age (# of divisions) for wild-type (WT), and 5 mutant strains (rrd2Δ, rpl20bΔ, cka2Δ, vac8Δ and vac17Δ). Data are presented as mean of 3 biological replicates +/- SD. Bottom: Box plot with the distribution of cell sizes for the same populations of cells. Whiskers extend to the 5th and 95th percentile.

(B) Micrographs of young (top row of images) and older (bottom row of images) wild-type (WT) cells expressing Vph1-EGFP (green vacuole) and stained with CF640R WGA (magenta bud scars). Scale bar: 5 µm.

(C) Combined effect of replicative age and a vacuole inheritance defect on penetrance. Micrographs of wild-type and vac17Δ cells expressing Vph1-EGFP (green vacuole) and Hta2-mCherry (red nucleus), stained with CF640R WGA (magenta bud scars). Cells with increasing bud-scar staining (replicative age) are shown from left to right. Scale bar: 5 µm.

(D) Relationship between stress response and penetrance. Single-cell UPRE-RFP levels were measured in ~60 different mutant strains that we had identified as penetrance mutants with intermediate penetrance with defects in actin or vacuole morphology. Cells were binned into equal-sized bins, from low to high stress response, assessed as outlier or inlier, and clustered based on their penetrance profile (composed from the fraction of outliers in each stress-response bin). Each line plot represents a penetrance profile. * denotes the cluster with a profile most similar to wild-type. Insert pie charts show the proportion of mutant strains in each cluster.

ExPANDED VIEW FIGURE LEGENDS
Slx5/Slx8-dependent ubiquitin hotspots on chromatin contribute to stress tolerance (2019)
Figure 1

Figure 1. The SUMO-targeted ubiquitin ligase Slx5/Slx8 is required for the formation of seven ubiquitin hotspots across the yeast genome.

(A) Genome-wide ubiquitin binding profiles identify numerous regions of H2B-ubiquitylation in WT cells and distinct sites of non-H2B ubiquitylation ('ubiquitin-hotspot', ub-hotspot, ub-HS) which persist in h2b-K123R and rad6∆ strains, and increase in cdc48 mutants (cdc48-6). A 90 kb stretch of chromosome XIII (ChrXIII) is depicted. Chromatin immunoprecipitation was performed using the FK2 ubiquitin antibody (see also Appendix Fig. S1A) and enriched DNA was analyzed on NimbleGen arrays (Chip-chip). DNA from non-specific IgG-ChIP experiments served as background control. Significantly enriched regions are marked by bars above the respective ChIP-chip tracks and are summarized in Dataset EV1. Data represent means from two independent replicates, except for ubiquitin (FK2) in rad6∆ and IgG-ChIP in WT (n = 1). All experiments, including those using cdc48-6 and other temperature-sensitive (ts) alleles, were performed at 30°C (semipermissive temperature for ts-alleles) unless stated otherwise.

(B) Seven ub-HSs show strong correlation between ubiquitin binding and Slx8 enrichment (Slx8-9myc ChIP). 16-kb-windows of the indicated regions centered around the ub-HSs are depicted for ubiquitin and Slx8 binding profiles. Ubiquitin (FK2) data for WT and cdc48-6 are from the same experiment as depicted in (A). Data represent means from two independent replicates.

(C) Schematic representation of the 16 yeast chromosomes (I-XVI) with positions of the ub-HSs marked by red triangles. Vertical bars indicate positions of centromeres.

(D) Slx8 and Slx5 are recruited to ub-HSs. DNA from Slx8-9myc (top) or 3HA-Slx5 (bottom) ChIP experiments of the indicated strains was analyzed by quantitative real-time PCR (ChIP-qPCR) at selected ub-HSs. Data represent mean ± standard deviation (SD, n = 2). Consistent with a previous study, we did not observe any Slx8 enrichment at centromeres (van de Pasch et al, 2013), however, we note that we could not confirm the reported centromere binding of Slx5 (see also Appendix Fig. S2E).

(E) K48-linked ubiquitin chains accumulate in a cdc48 mutant (cdc48-3). Ub-K48 ChIP followed by qPCR for same loci as in (D) are depicted. See also Fig. EV1C-D. Data represent means ± SD (n = 2).

(F) Slx5 and Slx8 are required for ub-HS formation. Ub-K48 ChIP-qPCR in strains lacking one subunit of the Slx5/Slx8 complex (slx5∆, slx8∆). Data represent means ± SD (n = 3).

(G) Ufd1 and Npl4 act in concert with Cdc48 to remove ubiquitylated proteins from ub-HS sites. Ub-K48 ChIP for the indicated strains grown at the semi-permissive temperature of 30°C. Data represent means ± SD (n = 2).

Data information: All ChIP-qPCR data represent means ± SD from 2-5 independent experiments as indicated, with quantification in triplicates. Data were normalized to an unrelated control region on ChrII (see Materials and Methods).