EMBO/SourceData huggingface dataset:
Figure 6A) Time-lapse experiments of living cells harbouring a non-reparable DSB were carried out after expressing the HO endonuclease in wild-type and cdc14-1 mutant cells. Spc110-RFP was used as SPB reporter and Tub1-GFP was used as tubulin marker. Three z-planes images every 5-second intervals over a period of 5 minutes were captured and used to quantify the average SPB track length and velocity using Spc110-RFP as SPB marker. At least 100 cells were scored.A representative maximum projection image is depicted. Scale bar: 3μm.A) A Western blot is shown to confirm the equal amount of Tub1-GFP in both wild-type and cdc14-1 backgrounds. Coomassie blue staining is shown as loading control.B) The steady state phosphorylation of Spc110 is required to maintain spindle polarity in response to DNA damage. Wild-type, cdc14-1 and spc110S36-91A cells expressing Spc110-RFP were scored for their ability to maintain spindle polarity at the axial plane of the cell upon induction of a single DSB induced by the HO endonuclease. A maximum projection of nine z-planes was used to score the cells. At least 100 cells from three independent experiments were scored. Graph represents the percentage ± SD of cells with oriented/miss-oriented SPBs with respect to the axial plane of the cell. P value was calculated using a 2-tailed unpaired Student t test.C) The asymmetric loading of dynein into astral MTs emanating from the daughter SPB is affected in cdc14-1 and spc110S36-91A cells during the DNA damage response. Dynein distribution on astral MTs emerging from each SPB was scored by using Dyn1-GFP in strains containing Spc110-RFP. Graph represents the percentage ± SD of cells grouped into three categories depending on the pattern of dynein distribution on astral MTs: Type1, Dynein is exclusively loaded on astral MTs emerging from the daughter SPB; Type 2, Dynein is equally loaded on astral MTs emanating from both SPBs; Type 3, Dynein is absent from astral MTs. At least 100 cells from three independent experiments were scored. A representative picture showing the three patterns is included. Scale bar: 3μm.
Figure 7A) Cells treated with nocodazole are affected in DSB-SPB interaction. Cnm67-CFP and Ddc2-RFP were used as SPB and DSB markers respectively. Diagram depicts the genomic background used. D1: inter-SPB distance; D2: DSB distance to proximal SPB; D3: DSB distance to distal SPB. D1, D2 and D3 distances after inducing a DSB in the presence of nocodazole or mock dimethyl sulfoxide (DMSO) were scored and plotted. Graph represents the mean ± SD of D1, D2 and D3 distances of three independent experiments. At least 100 cells per experiment were scored using the maximum projection of three z-planes. P values were calculated using a 2-tailed unpaired Student t test. A representative picture is shown. Scale bar: 3μm.B) Physical analysis of wild-type cells containing the repair pathway choice assay described in Fig 2B. The diagram with the genomic information, the restriction enzymes used and the location of the probe are shown. Cells were synchronized in G1 by using the α-factor pheromone and released into fresh media for 1 hour. Induction of HO expression was attained by adding galactose for 2 hours. After formation of the DSB, glucose was added to repress the HO in the presence of nocodazole or mock DMSO and samples were taken to analyse the kinetics of the repair. DNA was extracted, digested with EcoRV, separated on agarose gels and blotted. Both probes for the MATa-distal sequence and the HIS3 gene (as loading control) were used. Graphs show the quantification of MATa restoration (NHEJ), mating type switching by gene conversion (HR), and DSB kinetics formation. All data were normalized with the HIS3 gene. Graphs show the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test.B) Physical analysis of wild-type cells containing the repair pathway choice assay described in Fig 2B. The diagram with the genomic information, the restriction enzymes used and the location of the probe are shown. Cells were synchronized in G1 by using the α-factor pheromone and released into fresh media for 1 hour. Induction of HO expression was attained by adding galactose for 2 hours. After formation of the DSB, glucose was added to repress the HO in the presence of nocodazole or mock DMSO and samples were taken to analyse the kinetics of the repair. FACS analyses are shown.
Figure 8A) Growth sensitivity to MMS and phleomycin of a Cdk phospho-deficient mutant of Spc110. Ten-fold serial dilutions from overnight cultures of wild-type, spc110S36-91A and a cdc14-1 cells grown on solid rich media containing mock DMSO, MMS or Phleomycin at 30ºC.B) Diagram showing the genomic background and the approximation used to measure DSB-SPB interaction. Spc110 and spc110S36-91A were labelled with the RFP in a background containing Ddc2-GFP as DSB reporter. D1: inter-SPB distance; D2: DSB distance to proximal SPB; D3: DSB distance to distal SPB. At least 100 cells from three independent experiments were scored using the maximum projection of nine z-planes. Graph shows the mean ± S.D. P values were calculated using a 2-tailed unpaired Student t test.C) Time-lapse experiments of living cells expressing the HO endonuclease in wild-type and spc110S36-91A mutants cells. Five z-planes every 5-second intervals over a period of 5 minutes were captured. Quantifications were made using the average SPB track length and velocity of at least 100 cells. Spc110 and spc110S36-91A were labelled with the RFP and used as SPB reporter.D) Southern blot of wild-type and spc110S36-91A mutant harbouring the inter-chromosomal gene conversion assay portrayed. An overnight culture was induced by adding galactose at 32ºC to express the HO endonuclease. Samples were collected at different time-points, genomic DNA was extracted, digested with EcoRI and analysed by Southern blot. Blots were probed with a MATa-only and actin (for loading control) DNA sequences. Graphs show quantification of gene conversion, DSB induction and crossover vs non-crossover ratio. All data were normalized using the actin signal. Graphs represent the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. Asterisk denotes an overexposed film to visualize the formation of crossover products.D) Southern blot of wild-type and spc110S36-91A mutant harbouring the inter-chromosomal gene conversion assay portrayed. An overnight culture was induced by adding galactose at 32ºC to express the HO endonuclease. Samples were collected at different time-points, genomic DNA was extracted, digested with EcoRI and analysed by Southern blot. Blots were probed with a MATa-only and actin (for loading control) DNA sequences. FACS profiles for DNA content are included. Graphs show quantification of gene conversion, DSB induction and crossover vs non-crossover ratio. All data were normalized using the actin signal. Graphs represent the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. Asterisk denotes an overexposed film to visualize the formation of crossover products.
Figure 9A) DNA damage sensitivity to MMS and phleomycin of a Cdk phospho-mimic mutant of Spc110. Ten-fold serial dilutions from mid-log phase cultures of wild-type, spc110S36-91A and spc110S36-91D cells grown on solid rich media containing mock DMSO, MMS or Phleomycin at 30ºC.B) Schematic representation of the genomic background and the approximation used to measure DSB-SPB tethering. Wild-type Spc110 and spc110S36-91D were labelled with the RFP. Ddc2 was tagged with the GFP to be used as DSB reporter. D1: inter-SPB distance; D2: DSB distance to proximal SPB; D3: DSB distance to distal SPB. At least 100 cells from three independent experiments were scored using the maximum projection of nine z-planes. Graph shows the mean ± S.D. P values were calculated using a 2-tailed unpaired Student t test.C) Time-lapse experiments of living cells after induction of the HO endonuclease in wild-type and spc110S36-91D cells. Three z-planes every 10-second intervals over a period of 5 minutes were captured to measure the average SPBs track length and velocity. Spc110 and spc110S36-91D were labelled with the RFP and used to measured SPBs kinetics. At least 100 cells from three independent experiments were scored.D) Southern blot of wild-type and spc110S36-91D mutant bearing the inter-chromosomal gene conversion assay depicted. Mid-log phase cells were HO-induced at 32ºC by adding galactose to the media. Samples were collected at different time-points and the genomic DNA was extracted. Preps were digested with EcoRI and analysed by Southern blot. Blots were hybridized with MATa-only and actin probes. Graphs show quantification of gene conversion, DSB induction and crossover vs non-crossover ratio. All data were normalized using the actin signal. Graphs represent the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. Asterisk denotes an overexposed film to visualize the formation of crossover products.D) Southern blot of wild-type and spc110S36-91D mutant bearing the inter-chromosomal gene conversion assay depicted. Mid-log phase cells were HO-induced at 32ºC by adding galactose to the media. Samples were collected at different time-points and the genomic DNA was extracted. Preps were digested with EcoRI and analysed by Southern blot. Blots were hybridized with MATa-only and actin probes. FACS profiles for DNA content are displayed. Graphs show quantification of gene conversion, DSB induction and crossover vs non-crossover ratio. All data were normalized using the actin signal. Graphs represent the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. Asterisk denotes an overexposed film to visualize the formation of crossover products.
Figure 1Snapshots of ChIP-seq signals of Sen1 (this study), RNAP3 (this study) and RNAP2 (data from (Larochelle et al, 2018)) across (A) a 25 kb region of chromosome 3 containing several tRNA and 5S rRNA genesSnapshots of ChIP-seq signals of Sen1 (this study), RNAP3 (this study) and RNAP2 (data from (Larochelle et al, 2018)) across the U6 snRNA snu6Snapshots of ChIP-seq signals of Sen1 (this study), RNAP3 (this study) and RNAP2 (data from (Larochelle et al, 2018)) across the srp7 loci.Genome-wide pairwise Pearson correlation coefficient matrix at a resolution of 10 bp.ChIP-qPCR analysis of Flag-tagged Sen1 at the indicated loci in a population of cycling cells (mean ± standard deviation from 4 biological replicates).ChIP-qPCR analysis of the Flag-tagged RNAP3 subunit Rpc37 and Flag-tagged Sen1 across the SPCTRNAARG.10 tRNA locus, whose TATA box was either mutated (TATA-) or not (TATA+). The mutations introduced to disrupt the putative TATA boxes are indicated in red above the graphs. The enrichment values were normalized to SPCTRNATHR.10 (mean ± standard deviation from 4 biological replicates; p-value obtained using the Wilcoxon-Mann Whitney statistical test).
Figure 2(left) ChIP-qPCR analysis of Rpc37 in the presence or absence of Sen1 at the indicated loci in a population of cycling cells (mean ± standard deviation from 4 biological replicates). (right) Western blot analysis of Rpc37 protein levels in the presence or absence of Sen1. Tubulin was used as a loading control.(left) Total RNA from the indicated strains were separated on a 2,8% agarose gel. (right) Quantification of overall tRNA levels (mean ± standard deviation from 3 biological replicates).Strand-specific RT-qPCR was used to quantify the indicated RNAP3 transcripts. Transcript levels were normalized to act1 (mean ± standard deviation from 3 biological replicates).
Figure 3Cells were grown in minimal medium during 18 hours to induce the strong expression of RnhA. R-ChIP using a catalytically-inactive RNase H1 (Rnh1-D129N) was used to quantify R-loop formation at RNAP3-transcribed genes (mean ± standard deviation from 4 biological replicates).(left) ChIP-qPCR of the 13myc-tagged RNAP3 subunit Rpc25 in the indicated strains at the indicated loci (mean ± standard deviation of 4 biological replicates). (right) Western blot analysis of Myc-tagged Rpc25 and Flag-tagged RnhA protein levels in the indicated strains. Tubulin was used as a loading control.
Figure 4Snapshots of ChIP-seq signals of the RNAP3 subunits Rpc1 and Rpc2 in the presence or absence of Sen1 across a representative (A) tRNA gene, (B) 5S rRNA gene and (C) srp7. Boxed regions highlight the increased density of reads in the downstream region of genes in the absence of Sen1.Average ChIP-seq profile of Rpc1 and Rpc2 across all isolated tRNA and 5S rRNA genes in the presence and absence of Sen1.Scanning of Rpc37-3flag occupancy at three different tDNA loci in the absence of Sen1 by ChIP-qPCR (mean ± standard deviation from 6 biological replicates).
Figure 5Cells of the indicated genotypes that carried or not the DRT5T dimeric tRNA construct (schematized on top) were grown either in the presence of the optimum concentration of adenine (left) or in the presence of a limiting concentration of adenine (right). Two independent clones of the same genotype (#1 and #2) were used. See text for details. 3F refers to the 3 Flag epitope tag at the C-terminus of Rpc37.Strand-specific RT-qPCR was used to quantify the levels of read-through transcripts (see Methods). The mean ± standard deviation from 4 biological replicates is represented here. P-values were obtained using the Wilcoxon-Mann Whitney statistical test.Northern blot analysis of the tRNA SPATRNAPRO.02 using an intron-specific probe (TCTAAACTCAGCATACAAGTGGGG). U5 snRNA was used as a loading control.
Figure 6ChIP-qPCR analysis of Rpc37 around SPCTRNAARG.10 gene in the strong terminator mutant (mean ± standard deviation from 4 biological replicates).Strand specific RT-qPCR was used to quantify read-through transcripts at SPCTRNAARG.10 (arg10) and SPCTRNASER.09 (ser09) in the strong terminator mutant (mean ± standard deviation from 4 biological replicates; p-value obtained using the Wilcoxon-Mann Whitney statistical test).
Figure 1C. MA plot highlighting genes differentially expressed between state independent of strain (glm state term, false discovery rate-adjusted P value (FDR) < 0.05 and log2FC >1). Core pluripotent TFs highlighted for each state (maroon = naive ESCs, teal = formative EpiLCs).H. Heatmap of motif deviations from 3 biological replicates of B6 and D2 ESCs for TFs highlighted in G.
Figure 2A-D. Example expression patterns for select genes identified by applying a general linear model (glm) including state, genotype, and interaction terms. Dots represent individual biological replicates (N = 3); lines highlight changes in mean values for replicates between each state (log2FC >1 and FDR < 0.05). A. State dependent expression is exemplified by Nanog which showed no difference between strains. B. Expression of Zfp277 exemplifies strain-dependence being consistently higher in B6. C & D. A significant genotype x state (GXS) interaction was identified for Nr5a2 (C, a marker for pluripotency) and Sox1 (D, a marker for neuronal differentiation).
Figure 3D. Feature plot of expression gradients for indicated gene overlaid on UMAP from a. Three lineage markers that help to distinguish cellular identity are shown for five cell clusters.E. Pseudocolor plots of FACS analysis on cells from EBs for both B6 and D2 strains. Cells were labeled with anti-EpCAM (top) or biological replicates with anti-SOX1 antibody (bottom). Gated population indicates percentages of EpCAM+ (Q3) or SOX1+ cells.
Figure 4B. Scatterplot of genomic locations of individual chromatin accessibility (ca)QTL (x-axis, LOD > 5) versus location of ATAC peak being regulated (y-axis) for ESCs. Genetic effects that act locally (n = 3,973), i.e. in cis, fall on the diagonal line, while distal acting QTL (n = 13,767) lie off the diagonal. Several prominent distal QTL hotspots appear as vertical lines. C. Similar to B, plotting expression (e)QTL position versus gene location (LOD > 5, n = 701 local-eQTL, n = 357 distal-eQTL). DG. Coverage profile for Gstp2 locus on Chr 19 showing H3K4me3, ATAC, and RNA read depth from merged replicates. Locations of Chr 7 trans caQTL targets are indicated with black bars and LOD scores listed below.
Figure 5F. Left - Coverage profiles (averaged biological replicates, N=3) for ChIP factor occupancy at Cd59a on Chr 2 under trans-regulation by the Chr 13d QTL. Distal ca-QTL target region used for quantification is indicated by a black box with LOD score below. Right - Scatterplots for ChIP factors comparing quantitative level of modification/binding for independent replicates between B6 and D2 ESCs.G. The difference in occupancy of P300 in the parental strains is correlated to difference in chromatin accessibility in the 33 BXDs based on the genotype of the BXD at the QTL for Chr 13 caQTL targets (LOD > 7, Pearson's r = -0.61). H. TRIM28 occupancy at the same targets of Chr 13d caQTL in F were negatively correlated with ATAC signal (Pearson's r = -0.64). II. Similar to F, coverage profiles for ChIP factor occupancy for a putative regulatory element on Chr 5 near Zfp932, targeted by Chr 12 trans caQTL, showed greater occupancy of POU5F1 and increased open chromatin compared to D2. Distal ca-QTL target region used for quantification is indicated by a black box with LOD score below.J. The difference in occupancy of POU5F1 in the parental strains is correlated to difference in chromatin accessibility in the 33 BXDs based on the genotype of the BXD at the QTL for Chr 12 caQTL targets (LOD > 7, Pearson's r = -0.66). K. Similar to J showing negative correlation between TRIM28 occupancy and chromatin accessibility (Pearson's r = -0.51). D
Figure 6A. Coverage profile for H3K4me3, ATAC, P300, and TRIM28 ChIP at a target locus for the QTL on Chr 4 for both chromatin accessibility (black boxes) and gene expression (Riok3 and Rmc1). LOD scores are listed under each target locus.E. Box and whisker plot indicating change in H3K9me3 levels between WT and Chr 4 KO mESCs grouped by whether chromatin accessibility is higher when the QTL is B6 (left) or D2 (right). Dots represent mean values from biological duplicate experiments and lines connect means. Orange indicates loci for which H3K9me3 signal decreases in the KO (p-value indicated on top, two-sided paired Wilcoxon test).H. MA-plot similar to D showing change in gene expression between WT and Chr 4 KO mESCs (n = 2). Chr 4 QTL targets are indicated in magenta and increase expression in the KO.
Figure 1Flag-INVS interacted with HA-Akt (lanes 1-3), but not with HA-PDK1 (lanes 4-6) or HA-PrKA (lanes 7-9) in HEK293T cells. Expression levels of HA-Akt, PDK1, PrKA, and Flag-INVS were similar, as determined by immunoblotting (HA: anti-HA antibody; F: anti-Flag antibody; C: control antibody). The results presented (A-E) are representative of at least two independent experiments.B. Flag-tagged INVS interacted with HA-tagged Akt1 (lane 1-3), Akt2 (lane 4-6), and Akt3 (lane 7-9) in co-immunoprecipitationassays. Expression of each Akt isoform and INVS were similar.C. INVS was co-immunoprecipitated with endogenous Akt from HEK293 cells, in which INVS is also endogenously expressed (lane 2).D. To determine the Akt domain responsible for interacting with INVS, Akt subfragments were generated. The C-terminus of Akt is the primary domain for INVS interaction (lanes 7-9). Similar levels of each Akt fragment were immunoprecipitated using anti-HA antibody.E. Schematic showing the structure and the functional domains of Flag-tagged full-length and fragmented INVS in mammalian expression vectors (N-Term: 1-421, Intermediate domain: 421-678, and C-Term: 678-1065) used in the current study. Intermediate (lanes 6-9) and C-terminal (lanes 10-12) fragments of INVS were important for interaction with Akt.
Figure 2A. The indicated recombinant INVS proteins (lanes 1-4) were used for IVK. Akt phosphorylates WT (lane5), 1-970 (lane 6), and 1-898 (lane 7) INVS, but failed to phosphorylate 1-670 (lane 8) INVS.The indicated recombinant INVS proteins (lanes 1-10) were used for IVK. Akt phosphorylates WT (lane 6) and 1-898 (lane7) INVS, but failed to phosphorylate other subfragments of INVS (1-822, 1-746, and 1-670, in lane 8, 9, and 10, respectively), as detected using a phospho-Akt substrate antibody..C. Using the indicated subfragments of INVS for IVK, we confirmed that Akt phosphorylates 675-1065 (lane 4), but not 675-822 (lane 5), or 675-746 (lane 6) INVS.E. Purified recombinant WT or 3A INVS were generated (top panel, lanes 1-4). In IVK, Akt phosphorylated the WT fragment (824-898, middle panel, lane 3), but failed to phosphorylate 3A INVS (middle panel, lane 4), as detected by using a phospho-Akt substrate antibody.F. Akt phosphorylated WT (lane 10), 1-898 (lane 12) and 675-1065 (lane 15) INVS, but failed to phosphorylate both full-length and 675-1065, ∆727-896, 1-857, or 1-822, 3A INVS (lanes 16, 11, 14, and 15, respectively), all of which lacked the T864/S865/T866 target amino acid.
Figure 3A. Confocal microscopy shows that active Akt (phospho-S473Akt) is localized in the basal body of primary cilia (right side panels show higher magnification).B. INVS is localized in the proximal region of the INVS compartment in primary cilia (INVS compartment, right side panels show higher magnification).C. INVS co-localizes with γ-tubulin, a centrosome marker.D. Anti-phospho-Akt substrates stain positively at the basal region of the primary cilia (arrows), as determined by anti-acetylated tubulin (right side panels show higher magnification).E. Silver-intensified immunogold electron microscopy shows that both phospho-Akt (left side panels) and INVS (right side panels) were localized to the ciliary pocket of primary cilia in the presence of PDGF-AA (white arrow).
Figure 4PDGF-AA stimulation enhances localization of active Akt (phospho-S473Akt) at the basal body of primary cilia (right side panels show higher magnification).Quantification of co-localization of phosphorylated Akt (green) and INVS (red) at the indicated time points after PDGF-AA stimulation revealed a time-dependent increase. Colocalization was measured by counting yellow pixels (colocalized area) using Imaris software (Bitplane AG). Results presented are means ±SE (n=31 at time 0, n=35 at 1 min., and n=33 at 3 min, respectively). Three independent experiments were analyzed with similar results. Statistical significance was determined by student's t test.Co-immunoprecipitation assays using HEK293T cells show that INVS preferentially binds to WT-Akt (second panel, lane1) or phospho-mimetic Akt (T308D/S473D, lane2), but fails to interact with phosphorylation-defective forms of Akt (T308A/S473A, lane 3).PDGF-AA stimulation of HEK293T cells results in augmented interaction of endogenous INVS with Akt (top panel, comparison between lane 1 and lane 2, untreated and PDGF-AA-stimulated conditions, respectively).Both WT and 3A-INVS, which are localized at the INVS compartment of primary cilia under serum-starved condition (left side panels, lower panels show higher magnification), translocate to the basal body of the cilium after PDGF-AA stimulation (right side panels, lower panels show higher magnification). Results presented are means ± SE of yellow pixels demonstrating colocalization of EGFP-INVS (green) with γ-tubulin (red) in hTERT-RPE1 cells (n=28). Three independent experiments showed similar results. Statistical significance was determined by student's t test. Fluorescence intensities of INVS (green), acetylated-α-tubulin (blue), and γ-tubulin (red) along the line (a-b) are plotted underneath.