( d , e ) GFP : : LGG - 1 punctae were quantified in ( d ) hypodermal seam cells or ( e ) proximal intestinal cells of L3 larvae of WT and glp - 1 ( e2141 ) animals . Animals were fed bacteria expressing control or hlh - 30 dsRNA for two generations at 20 ° C . Eggs were then transferred to plates seeded with the appropriate dsRNA - expressing bacteria at 25 ° C and analysed at the L3 larval stage ( mean ± s . e . m . of ~ 300 seam cells and ~ 25 intestines , * * P0 . 01 , Student ' s t - test ) .

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(d,e) GFP::LGG-1 punctae were quantified in (d) hypodermal seam cells or (e) proximal intestinal cells of L3 larvae of WT and glp-1(e2141) animals. Animals were fed bacteria expressing control or hlh-30 dsRNA for two generations at 20 °C. Eggs were then transferred to plates seeded with the appropriate dsRNA-expressing bacteria at 25 °C and analysed at the L3 larval stage (mean±s.e.m. of ~300 seam cells and ~25 intestines, **P0.01, Student's t-test).


( f , g ) glp - 1 ( e2141 ) animals expressing ( f ) LMP - 1 : : GFP or ( g ) SQST - 1 : : GFP were raised at the non - permissive temperature ( 25 ° C ) and fed bacteria expressing control or hlh - 30 dsRNA from hatching . Micrographs of the posterior intestine were taken on day 1 of adulthood , and LMP - 1 : : GFP fluorescence ( mean ± s . d . of ~ 10 animals , * * P0 . 01 , Student ' s t - test ) and SQST - 1 : : GFP foci ( mean ± s . d . of ~ 30 animals , * * P0 . 01 , Student ' s t - test ) were quantified . Experiments were performed at least three times with similar results . See Supplementary Fig . S2a , c for images of whole animals and Supplementary Fig . S2b , d for replicates . Magnification , × 200 ; scale bar , 100 μm .

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(f,g) glp-1(e2141) animals expressing (f) LMP-1::GFP or (g) SQST-1::GFP were raised at the non-permissive temperature (25°C) and fed bacteria expressing control or hlh-30 dsRNA from hatching. Micrographs of the posterior intestine were taken on day 1 of adulthood, and LMP-1::GFP fluorescence (mean±s.d. of ~10 animals, **P0.01, Student's t-test) and SQST-1::GFP foci (mean±s.d. of ~30 animals, **P0.01, Student's t-test) were quantified. Experiments were performed at least three times with similar results. See Supplementary Fig. S2a,c for images of whole animals and Supplementary Fig. S2b,d for replicates. Magnification, × 200; scale bar, 100 μm.


( a ) Nuclear localization of HLH - 30 was quantified in day 1 adult animals expressing HLH - 30 : : GFP . Animals were fed bacteria expressing control or tor dsRNA from hatching and raised at 20 ° C ( three biological replicates , ~ 50 animals each , mean ± s . d . , * * P0 . 01 , Student ' s t - test ) .

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(a) Nuclear localization of HLH-30 was quantified in day 1 adult animals expressing HLH-30::GFP. Animals were fed bacteria expressing control or tor dsRNA from hatching and raised at 20 °C (three biological replicates, ~50 animals each, mean±s.d., **P0.01, Student's t-test).


b , c ) Expression of putative autophagy - related and lysosomal target genes was measured by quantitative PCR in day 1 ( b ) WT ( N2 ) and ( c ) hlh - 30 ( tm1978 ) animals fed bacteria expressing control or tor dsRNA from hatching ( 20 ° C ) . Data are mean ± s . d . of biological triplicates . * P0 . 05 , * * P0 . 01 ; Student ' s t - test . csta * is a cathepsin A orthologue ( cosmid C08H9 . 1 ( ref . 30 ) ) .

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b,c) Expression of putative autophagy-related and lysosomal target genes was measured by quantitative PCR in day 1 (b) WT (N2) and (c) hlh-30(tm1978) animals fed bacteria expressing control or tor dsRNA from hatching (20 °C). Data are mean±s.d. of biological triplicates. *P0.05, **P0.01; Student's t-test. csta* is a cathepsin A orthologue (cosmid C08H9.1 (ref. 30)).


( d ) Lifespan analysis of WT animals and hlh - 30 ( tm1978 ) mutants fed bacteria expressing control or tor dsRNA from day 1 of adulthood was carried out at 20 ° C . See Supplementary Table S3 for details of lifespan analyses and replicate experiments .

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(d) Lifespan analysis of WT animals and hlh-30(tm1978) mutants fed bacteria expressing control or tor dsRNA from day 1 of adulthood was carried out at 20 °C. See Supplementary Table S3 for details of lifespan analyses and replicate experiments.


Lifespan analyses of ( a ) WT ( N2 ) and ( b ) germline - less glp - 1 ( e2141 ) animals raised at the non - permissive temperature ( 25 ° C ) and fed bacteria expressing control or hlh - 30 dsRNA from day 1 of adulthood were carried out at 20 ° C . Lifespan analyses of ( c ) dietary - restricted eat - 2 ( ad1116 ) mutants , ( d ) insulin / IGF - 1 receptor daf - 2 ( e1370 ) mutants , ( e ) mitochondrial respiration clk - 1 ( e2519 ) mutants and ( f ) mRNA translation rsks - 1 ( sv31 ) mutants , fed bacteria expressing control or hlh - 30 dsRNA from day 1 of adulthood ( c , d , f ) or larval L4 stage ( e ) , were carried out at 20 ° C . See Supplementary Table S2 for details of lifespan analyses including at least three independent experiments .

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Lifespan analyses of (a) WT (N2) and (b) germline-less glp-1(e2141) animals raised at the non-permissive temperature (25 °C) and fed bacteria expressing control or hlh-30 dsRNA from day 1 of adulthood were carried out at 20 °C. Lifespan analyses of (c) dietary-restricted eat-2(ad1116) mutants, (d) insulin/IGF-1 receptor daf-2(e1370) mutants, (e) mitochondrial respiration clk-1(e2519) mutants and (f) mRNA translation rsks-1(sv31) mutants, fed bacteria expressing control or hlh-30 dsRNA from day 1 of adulthood (c,d,f) or larval L4 stage (e), were carried out at 20 °C. See Supplementary Table S2 for details of lifespan analyses including at least three independent experiments.


( a ) Nuclear localization of HLH - 30 was quantified in day 1 adult WT , eat - 2 , daf - 2 , clk - 1 and rsks - 1 mutants ( mean ± s . d . of four biological replicates , ~ 50 animals each , * * P0 . 01 , analysis of variance ) .

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(a) Nuclear localization of HLH-30 was quantified in day 1 adult WT, eat-2, daf-2, clk-1 and rsks-1 mutants (mean±s.d. of four biological replicates, ~50 animals each, **P0.01, analysis of variance).


( b ) Expression of hlh - 30 was measured by quantitative PCR ( qPCR ) in day 1 adult WT ( N2 ) , eat - 2 ( ad1116 ) ( dietary restriction , DR ) , daf - 2 ( e1370 ) ( insulin / IGF - 1 signaling ) , clk - 1 ( e2519 ) ( mitochondrial respiration ) and rsks - 1 ( sv31 ) ( mRNA translation ) animals ( mean ± s . d . of three biological replicates , * P0 . 05 , Student ' s t - test ) .

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(b) Expression of hlh-30 was measured by quantitative PCR (qPCR) in day 1 adult WT (N2), eat-2(ad1116) (dietary restriction, DR), daf-2(e1370) (insulin/IGF-1 signaling), clk-1(e2519) (mitochondrial respiration) and rsks-1(sv31) (mRNA translation) animals (mean±s.d. of three biological replicates, *P0.05, Student's t-test).


( c ) Lifespan analysis of WT and transgenic animals overexpressing HLH - 30 : : GFP , and raised and maintained on OP50 was carried out at 20 ° C . See Supplementary Table S7 for details of lifespan analyses and replicate experiments .

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(c) Lifespan analysis of WT and transgenic animals overexpressing HLH-30::GFP, and raised and maintained on OP50 was carried out at 20 °C. See Supplementary Table S7 for details of lifespan analyses and replicate experiments.


( d ) GFP : : LGG - 1 punctae were quantified in hypodermal seam cells of WT animals or animals overexpressing HLH - 30 ( n > 300 , ± s . e . m . , * * P0 . 01 , Student ' s t - test ) . The increase in GFP : : LGG - 1 punctae observed in animals overexpressing HLH - 30 could be reversed by atg - 18 RNAi treatment ( data not shown ) .

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(d) GFP::LGG-1 punctae were quantified in hypodermal seam cells of WT animals or animals overexpressing HLH-30 (n>300, ±s.e.m., **P0.01, Student's t-test). The increase in GFP::LGG-1 punctae observed in animals overexpressing HLH-30 could be reversed by atg-18 RNAi treatment (data not shown).


( e ) TFEB expression was measured by qPCR in the livers of 4 . 5 - month - old female ( F ) and male ( M ) mice fed AL or subjected to DR for 5 . 5 weeks starting at 3 months of age ( mean ± s . e . m . of ~ 20 mice per group , * P0 . 05 , Student ' s t - test ) .

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(e) TFEB expression was measured by qPCR in the livers of 4.5-month-old female (F) and male (M) mice fed AL or subjected to DR for 5.5 weeks starting at 3 months of age (mean±s.e.m. of ~20 mice per group, *P0.05, Student's t-test).


( f ) TFEB protein was detected by western blotting of nuclear fractions from the livers of five female and five male mice fed AL or subjected to DR . Actin was included as a loading control , see Supplementary Fig . S8 for additional controls . ( g ) TFEB protein levels in Fig . 4f were quantified by densitometry and normalized to actin ( mean ± s . d . , * P0 . 05 , Student ' s t - test ) .

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(f) TFEB protein was detected by western blotting of nuclear fractions from the livers of five female and five male mice fed AL or subjected to DR. Actin was included as a loading control, see Supplementary Fig. S8 for additional controls. (g) TFEB protein levels in Fig. 4f were quantified by densitometry and normalized to actin (mean±s.d., *P0.05, Student's t-test).


J , Ridge graph comparisons of cyclin expression across the U5 - hNSC defined cell cycle phases . The x - axis is relative expression of the cyclin , and the y - axis is counts of cells per phase . The cell cycle phase with the peak expression is denoted by an arrowhead at the top of the plot .

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J, Ridge graph comparisons of cyclin expression across the U5-hNSC defined cell cycle phases. The x-axis is relative expression of the cyclin, and the y-axis is counts of cells per phase. The cell cycle phase with the peak expression is denoted by an arrowhead at the top of the plot.


C , Number of putative stem - like cells in each ccAF cell cycle phase , and distribution of absolute gene expression across ccAF cell cycle phases for the genes used to identify putative stem - like cells in Bhaduri et al . , 2020 .

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C, Number of putative stem-like cells in each ccAF cell cycle phase, and distribution of absolute gene expression across ccAF cell cycle phases for the genes used to identify putative stem-like cells in Bhaduri et al., 2020.


D , Putative stem - like cells and expression of the genes used to identify the putative stem - like cells projected onto Neftel et al . , 2019 . Expression of genes are overlaid as the color of each cell ( white = 0 , dark blue = max expression ) . Quadrants are broken up by dashed grey lines : OPC = upper left ; NPC = upper right ; AC = lower left ; and MES = lower right . Percentages of gene expression greater than 0 are shown in the corners of the plot .

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D, Putative stem-like cells and expression of the genes used to identify the putative stem-like cells projected onto Neftel et al., 2019. Expression of genes are overlaid as the color of each cell (white = 0, dark blue = max expression). Quadrants are broken up by dashed grey lines: OPC = upper left; NPC = upper right; AC = lower left; and MES = lower right. Percentages of gene expression greater than 0 are shown in the corners of the plot.


C , Violin plot showing PLP1 expression across all cell types from Darmanis et al . , 2017 . Data are log 10 - transformed before plotting . Expression of PLP1 in single cells is much higher in oligodendrocytes ( Oligo ) than in Neural G0 or oligodendrocyte progenitor cells ( OPC ) . D , Violin plot showing GFAP expression across all cell types from Darmanis et al . , 2017 . Data are log 10 - transformed before plotting . Expression of GFAP in single cells is more highly expressed in Neural G0 relative to all other cell types . E , Violin plot showing AQP4 expression across all cell types from Darmanis et al . , 2017 . Data are log 10 - transformed before plotting . Neural G0 expression of AQP4 in single cells is similar to astrocytes but has a lower median expression and lower upper limit .

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C, Violin plot showing PLP1 expression across all cell types from Darmanis et al., 2017. Data are log 10-transformed before plotting. Expression of PLP1 in single cells is much higher in oligodendrocytes (Oligo) than in Neural G0 or oligodendrocyte progenitor cells (OPC). D, Violin plot showing GFAP expression across all cell types from Darmanis et al., 2017. Data are log 10-transformed before plotting. Expression of GFAP in single cells is more highly expressed in Neural G0 relative to all other cell types. E, Violin plot showing AQP4 expression across all cell types from Darmanis et al., 2017. Data are log 10-transformed before plotting. Neural G0 expression of AQP4 in single cells is similar to astrocytes but has a lower median expression and lower upper limit.


B , Representative contour maps of flow cytometry for FUCCI following the loss of NF2 , PTPN14 , TAOK1 , CREBBP , and TP53 .

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B, Representative contour maps of flow cytometry for FUCCI following the loss of NF2, PTPN14, TAOK1, CREBBP, and TP53.


D , G0 / G1 and S / G2 / M transit times using time - lapse microscopy and FUCCI . Differences in G0 / G1 are statistically significant with p < 0 . 001 for targeted U5 - NSCs and p = 0 . 0006 for GSC - 131 compared to NTC .

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D, G0/G1 and S/G2/M transit times using time-lapse microscopy and FUCCI. Differences in G0/G1 are statistically significant with p<0.001 for targeted U5-NSCs and p=0.0006 for GSC-131 compared to NTC.


A ) Ten - fold serial dilutions from overnight cultures of wild - type and cdc14 - 1 cells dropped and grown on solid rich media or media containing MMS at 25 , 28 , 30 or 33ºC . Note that cdc14 - 1 cells exhibit growth sensitivity to MMS at 28ºC and 30ºC compared to wild - type cells .

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A) Ten-fold serial dilutions from overnight cultures of wild-type and cdc14-1 cells dropped and grown on solid rich media or media containing MMS at 25, 28, 30 or 33ºC. Note that cdc14-1 cells exhibit growth sensitivity to MMS at 28ºC and 30ºC compared to wild-type cells.


B ) Ten - fold serial dilution from mid - log phase cultures of wild - type and cdc14 - 1 cells grown on solid rich media or media containing mock DMSO ( as non - treated control ) , 4NQO , HU , phleomycin and benomyl at 30ºC . Note that cdc14 - 1 cells present a market sensitivity to all DNA damaged agents tested .

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B) Ten-fold serial dilution from mid-log phase cultures of wild-type and cdc14-1 cells grown on solid rich media or media containing mock DMSO (as non-treated control), 4NQO, HU, phleomycin and benomyl at 30ºC. Note that cdc14-1 cells present a market sensitivity to all DNA damaged agents tested.


C ) Left panel : Schematic representation showing relevant genomic structure of the strain used to assess intra - chromosomal repair . The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated . Arrow depicts the localization of the double - strand break . Right panel : Physical analysis of intra - chromosomal repair in wild - type and cdc14 - 1 cultures at the semipermisive temperature . After DSB formation by the expression of the HO , glucose was added to repress it , thus allowing repair with HM donor sequences . Genomic DNA was digested with StyI , separated on agarose gel and blotted . Blots were hybridized with a probe corresponding to the MATa - distal sequence . A second probe for the actin gene was used to control the amount of genomic DNA loaded at each time - point . Immunoblot analysis of Rad53 during mating type switching experiments is shown . Coomassie blue staining is depicted as loading control . Graphs show quantification of gene conversion ( GC ) leading to the re - establishment of MATa or switching to MAT and DSB formation . The data was normalized with the actin loading control . Graphs show the mean ± S . D . from three independent experiments . Replicates were averaged and statistical significance of differences assessed by a 2 - tailed unpaired Student t test .

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C) Left panel: Schematic representation showing relevant genomic structure of the strain used to assess intra-chromosomal repair. The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated. Arrow depicts the localization of the double-strand break. Right panel: Physical analysis of intra-chromosomal repair in wild-type and cdc14-1 cultures at the semipermisive temperature. After DSB formation by the expression of the HO, glucose was added to repress it, thus allowing repair with HM donor sequences. Genomic DNA was digested with StyI, separated on agarose gel and blotted. Blots were hybridized with a probe corresponding to the MATa-distal sequence. A second probe for the actin gene was used to control the amount of genomic DNA loaded at each time-point. Immunoblot analysis of Rad53 during mating type switching experiments is shown. Coomassie blue staining is depicted as loading control. Graphs show quantification of gene conversion (GC) leading to the re-establishment of MATa or switching to MAT and DSB formation. The data was normalized with the actin loading control. Graphs show the mean ± S.D. from three independent experiments. Replicates were averaged and statistical significance of differences assessed by a 2-tailed unpaired Student t test.


A ) Left panel : Schematic diagram displaying relevant genomic structure of the strains used to measure inter - chromosomal DNA repair efficiency . The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated . Note that crossover and non - crossover product have different restriction fragment sizes that can be differentiated in a Southern blot assay . Arrow depicts the localization of the double - strand break . Right panel : Physical analysis of wild - type and cdc14 - 1 mutant strains carrying the inter - chromosomal gene conversion assay . Cells were grown overnight before adding galactose at semipermisive temperature to induce HO expression , thus producing a DSB on chromosome V . Samples were taken at different time - points , genomic DNA was extracted , digested with EcoRI and analysed by Southern blot . Blots were hybridized with a MATa - only DNA sequence and an actin probe was used as loading control . Asterisk denotes an overexposed film to visualize crossover formation . Immunoblot of Rad53 was performed as previously described . Coomassie staining is shown as a control for loading . Graphs show the mean ± S . D . of gene conversion , DSB induction and crossover vs non - crossover ratio from three independent experiments . All data was normalized using actin as loading control . Replicates were averaged and statistical significance of differences assessed by a 2 - tailed unpaired Student t test .

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A) Left panel: Schematic diagram displaying relevant genomic structure of the strains used to measure inter-chromosomal DNA repair efficiency. The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated. Note that crossover and non-crossover product have different restriction fragment sizes that can be differentiated in a Southern blot assay. Arrow depicts the localization of the double-strand break. Right panel: Physical analysis of wild-type and cdc14-1 mutant strains carrying the inter-chromosomal gene conversion assay. Cells were grown overnight before adding galactose at semipermisive temperature to induce HO expression, thus producing a DSB on chromosome V. Samples were taken at different time-points, genomic DNA was extracted, digested with EcoRI and analysed by Southern blot. Blots were hybridized with a MATa-only DNA sequence and an actin probe was used as loading control. Asterisk denotes an overexposed film to visualize crossover formation. Immunoblot of Rad53 was performed as previously described. Coomassie staining is shown as a control for loading. Graphs show the mean ± S.D. of gene conversion, DSB induction and crossover vs non-crossover ratio from three independent experiments. All data was normalized using actin as loading control. Replicates were averaged and statistical significance of differences assessed by a 2-tailed unpaired Student t test.


B ) Left panel : Schematic representation depicting the genomic structure of the strains used to evaluate repair pathway choice . The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated . Note that the use of NHEJ or HR generates different product that can be recognized in Southern blots by the different disposition of the restriction sites for the endonucleases used . Arrow depicts the localization of the double - strand break . Right panel : Physical analysis of wild - type and cdc14 - 1 cells harbouring the repair pathway choice assay . Cells were grown overnight and HO induction was attained through addition of galactose for 1 . 5 hour at semipermisive temperature . After induction of the HO , glucose was added to repress the HO expression and samples were taken to analyse repair efficiency . DNA was extracted , digested with EcoRV , separated on agarose gels and blotted . Blots were hybridized with a probe corresponding to the MATa - only DNA sequence . A second probe to the HIS3gene was used to control the amount of genomic DNA loaded at each time - point . Graphs show quantification of mating type switching by gene conversion ( HR ) , restoration of MATa ( NHEJ ) and the kinetics of the DSB induction . All data was normalized with the HIS3 gene . Graphs show the mean ± S . D . from three independent experiments . Replicates were averaged and statistical significance of differences assessed by a 2 - tailed unpaired Student t test .

O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B) Left panel: Schematic representation depicting the genomic structure of the strains used to evaluate repair pathway choice. The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated. Note that the use of NHEJ or HR generates different product that can be recognized in Southern blots by the different disposition of the restriction sites for the endonucleases used. Arrow depicts the localization of the double-strand break. Right panel: Physical analysis of wild-type and cdc14-1 cells harbouring the repair pathway choice assay. Cells were grown overnight and HO induction was attained through addition of galactose for 1.5 hour at semipermisive temperature. After induction of the HO, glucose was added to repress the HO expression and samples were taken to analyse repair efficiency. DNA was extracted, digested with EcoRV, separated on agarose gels and blotted. Blots were hybridized with a probe corresponding to the MATa-only DNA sequence. A second probe to the HIS3gene was used to control the amount of genomic DNA loaded at each time-point. Graphs show quantification of mating type switching by gene conversion (HR), restoration of MATa (NHEJ) and the kinetics of the DSB induction. All data was normalized with the HIS3 gene. Graphs show the mean ± S.D. from three independent experiments. Replicates were averaged and statistical significance of differences assessed by a 2-tailed unpaired Student t test.


A ) Live - cell imaging of Cdc14 - YFP and Cnm67 - RFP asynchronous cells treated with the DNA damage agent phleomycin . Cells were grown overnight and phleomycin was added to a final concentration of 1μM . Samples were taken at different intervals to determine Cdc14 localization . Cnm67 was used as SPB reporter to determine spindle length . Scale bar : 3μm . Graphs represent the average percentage ± SD from three independent experiments of cells arrested in metaphase ( left graph ) and with Cdc14 signal at the nucleoplasm ( right graph ) .

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A) Live-cell imaging of Cdc14-YFP and Cnm67-RFP asynchronous cells treated with the DNA damage agent phleomycin. Cells were grown overnight and phleomycin was added to a final concentration of 1μM. Samples were taken at different intervals to determine Cdc14 localization. Cnm67 was used as SPB reporter to determine spindle length. Scale bar: 3μm. Graphs represent the average percentage ± SD from three independent experiments of cells arrested in metaphase (left graph) and with Cdc14 signal at the nucleoplasm (right graph).


A ) Western blot analysis to analyse Rad53 and Net1 phosphorylation levels under these conditions are shown . Note that Rad53 is rapidly phosphorylated in response to phleomycin and maintained throughout the entire experiment . Detection of Net1 phosphorylation levels was achieved by using Phos - tag polyacrylamide gels . Coomassie staining is showed as loading control .

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A) Western blot analysis to analyse Rad53 and Net1 phosphorylation levels under these conditions are shown. Note that Rad53 is rapidly phosphorylated in response to phleomycin and maintained throughout the entire experiment. Detection of Net1 phosphorylation levels was achieved by using Phos-tag polyacrylamide gels. Coomassie staining is showed as loading control.


B ) Western blot analysis showing Net1 phosphorylation through the cell cycle is included . The separation of the phospho - bands was achieved by using Phos - tag polyacrylamide gels as in Fig 3A . Coomassie staining is depicted as loading control .

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B) Western blot analysis showing Net1 phosphorylation through the cell cycle is included. The separation of the phospho-bands was achieved by using Phos-tag polyacrylamide gels as in Fig 3A. Coomassie staining is depicted as loading control.


B ) Cell cycle profile of Cdc14 localization and Net1 phosphorylation in the absence of DNA damage . A strain containing Cdc14 - GFP and Net1 - HA was blocked in G1 by using the pheromone alpha - factor and released into the cell cycle . Samples were taken at different time - points to analyse Cdc14 spatial localization and Net1 phosphorylation levels . A representative picture of non - released ( T0 ) , partially released ( T60 ) and fully released ( T75 ) Cdc14 from the nucleolus is shown . Scale bar : 3μm . Graph represents the percentage ± SD from three independent experiments of cells with no released , partially released ( FEAR ) and totally released ( MEN ) Cdc14 from the nucleolus along the cell cycle .

O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O

B) Cell cycle profile of Cdc14 localization and Net1 phosphorylation in the absence of DNA damage. A strain containing Cdc14-GFP and Net1-HA was blocked in G1 by using the pheromone alpha-factor and released into the cell cycle. Samples were taken at different time-points to analyse Cdc14 spatial localization and Net1 phosphorylation levels. A representative picture of non-released (T0), partially released (T60) and fully released (T75) Cdc14 from the nucleolus is shown. Scale bar: 3μm. Graph represents the percentage ± SD from three independent experiments of cells with no released, partially released (FEAR) and totally released (MEN) Cdc14 from the nucleolus along the cell cycle.


C ) Cells expressing Cdc14 - YFP and Cnm67 - RFP were grown overnight and blocked in G2 / M by using nocodazole prior phleomycin treatment ( 1μM ) . Samples were taken every 30 minutes to follow Cdc14 re - localization . The efficiency of the arrest was determined as in Fig 3A . Scale bar : 3μm . Graphs represent the percentage ± SD of cells with Cdc14nucleolar exclusion ( left graph ) and cells arrested in metaphase ( right graph ) from three independent experiments .

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C) Cells expressing Cdc14-YFP and Cnm67-RFP were grown overnight and blocked in G2/M by using nocodazole prior phleomycin treatment (1μM). Samples were taken every 30 minutes to follow Cdc14 re-localization. The efficiency of the arrest was determined as in Fig 3A. Scale bar: 3μm. Graphs represent the percentage ± SD of cells with Cdc14nucleolar exclusion (left graph) and cells arrested in metaphase (right graph) from three independent experiments.


D ) Live - cell imaging of Cdc14 localization in the presence of a non - reparable DSB at the MAT locus . Cells were grown overnight and the HO endonuclease was expressed by adding galactose to the media , thus producing a DSB on chromosome III . Cdc14 , the SPB component Cnm67 and the DNA damage checkpoint protein Ddc2 ( used as DSB reporter ) were labelled with the green , red and cyan fluorescent proteins respectively . Micrographs display the maximum projection of 9 planes showing Cdc14 co - localizing with the DSB and the SPBs in response to DNA damage ( arrows ) . Scale bar : 3μm . Graphs represent the percentage ± SD from three independents experiment of cells with nucleoplasmic Cdc14 along the HO endonuclease induction ( left graph ) and the percentage of Cdc14 - SPB - DSB co - localization after 5h in galactose ( right graph ) .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

D) Live-cell imaging of Cdc14 localization in the presence of a non-reparable DSB at the MAT locus. Cells were grown overnight and the HO endonuclease was expressed by adding galactose to the media, thus producing a DSB on chromosome III. Cdc14, the SPB component Cnm67 and the DNA damage checkpoint protein Ddc2 (used as DSB reporter) were labelled with the green, red and cyan fluorescent proteins respectively. Micrographs display the maximum projection of 9 planes showing Cdc14 co-localizing with the DSB and the SPBs in response to DNA damage (arrows). Scale bar: 3μm. Graphs represent the percentage ± SD from three independents experiment of cells with nucleoplasmic Cdc14 along the HO endonuclease induction (left graph) and the percentage of Cdc14-SPB-DSB co-localization after 5h in galactose (right graph).


E ) Western blot showing Net1 - 6HA phosphorylation state before and after the generation of a DSB induced by the HO expression . A strain lacking the HO endonuclease under the galactose promoter was used as undamaged control . The phospho - bands were resolved by using Phos - tag polyacrylamide gels as in Fig 3A and Fig 3B . Coomassie staining is depicted as loading control .

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E) Western blot showing Net1-6HA phosphorylation state before and after the generation of a DSB induced by the HO expression. A strain lacking the HO endonuclease under the galactose promoter was used as undamaged control. The phospho-bands were resolved by using Phos-tag polyacrylamide gels as in Fig 3A and Fig 3B. Coomassie staining is depicted as loading control.


A ) Diagram showing the genomic background and the approximation used to measure DSB - SPB interaction . Cnm67 - RFP and Ddc2 - GFP were used as SPB and DSB markers respectively . D1 : inter - SPB distance ; D2 : DSB distance to proximal SPB ; D3 : DSB distance to distal SPB . Graph represents the mean ± SD of D1 , D2 and D3 distances from 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 of a wild - type strain depicting DSB - SPB interaction is shown . Scale bar : 3μm .

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A) Diagram showing the genomic background and the approximation used to measure DSB-SPB interaction. Cnm67-RFP and Ddc2-GFP were used as SPB and DSB markers respectively. D1: inter-SPB distance; D2: DSB distance to proximal SPB; D3: DSB distance to distal SPB. Graph represents the mean ± SD of D1, D2 and D3 distances from 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 of a wild-type strain depicting DSB-SPB interaction is shown. Scale bar: 3μm.


B ) Time - lapse experiments in living cells showing the efficiency of DSB - SPB interaction upon induction of a single DSB . Cnm67 - CFP and Ddc2 - RFP were used as SPB and DSB markers respectively . D1 , D2 and D3 distances in wild - type and cdc14 - 1 cells were tracked using the maximum projection of three z - planes images at 15 - seconds intervals over period of 7 . 5 minutes . A total of 32 time - lapses for each strain were measured . A representative picture is shown . Examples of cells defective in DSB - SPB interaction are marked with arrows . Scale bar : 3μm .

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B) Time-lapse experiments in living cells showing the efficiency of DSB-SPB interaction upon induction of a single DSB. Cnm67-CFP and Ddc2-RFP were used as SPB and DSB markers respectively. D1, D2 and D3 distances in wild-type and cdc14-1 cells were tracked using the maximum projection of three z-planes images at 15-seconds intervals over period of 7.5 minutes. A total of 32 time-lapses for each strain were measured. A representative picture is shown. Examples of cells defective in DSB-SPB interaction are marked with arrows. Scale bar: 3μm.


C ) An RFP - marked I - SceI recognition site is re - localized to the SPBs after expressing the endonuclease . Cells harbouring a TetO array adjacent to the I - SceI site and a plasmid containing the endonuclease under the control of the galactose promoter were grown overnight in SC - Ade prior galactose induction . To check reproducibility with previous results a Rad52 - CFP was used . Cnm67 was labelled with the GFP to be used as SPB reporter . D1 , D2 and D3 distances were measured in wild - type and cdc14 - 1 mutants before and after endonuclease expression by using both TetI - RFP and Rad52 - CFP foci using the maximum projection of nine z - planes images . Graphs represent the average of three independent experiments . P values were calculated using a 2 - tailed unpaired Student t test . A representative picture of a wild - type strain depicting TetI - Rad52 - SPB interaction is shown . Scale bar : 3μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O

C) An RFP-marked I-SceI recognition site is re-localized to the SPBs after expressing the endonuclease. Cells harbouring a TetO array adjacent to the I-SceI site and a plasmid containing the endonuclease under the control of the galactose promoter were grown overnight in SC-Ade prior galactose induction. To check reproducibility with previous results a Rad52-CFP was used. Cnm67 was labelled with the GFP to be used as SPB reporter. D1, D2 and D3 distances were measured in wild-type and cdc14-1 mutants before and after endonuclease expression by using both TetI-RFP and Rad52-CFP foci using the maximum projection of nine z-planes images. Graphs represent the average of three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. A representative picture of a wild-type strain depicting TetI-Rad52-SPB interaction is shown. Scale bar: 3μm.


D ) DSB - SPB tethering requires the N - terminus domain of Mps3 . Cells expressing a wild - type Mps3 , a mps3Δ2 - 64 and a mps3Δ75 - 150 fused to the RFP as the sole source of the protein were scored for their ability to enhance DSB - SPB tethering in both wild - type and cdc14 - 1 backgrounds . Ddc2 - YFP was used as DSB reporter . Measurements were carried out using a maximum projection of nine z - planes images . Graphs represent the average distribution of D1 , D2 and D3 of three independent experiments . At least 100 cells for each experiment were scored . P values were calculated using a 2 - tailed unpaired Student t test .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) DSB-SPB tethering requires the N-terminus domain of Mps3. Cells expressing a wild-type Mps3, a mps3Δ2-64 and a mps3Δ75-150 fused to the RFP as the sole source of the protein were scored for their ability to enhance DSB-SPB tethering in both wild-type and cdc14-1 backgrounds. Ddc2-YFP was used as DSB reporter. Measurements were carried out using a maximum projection of nine z-planes images. Graphs represent the average distribution of D1, D2 and D3 of three independent experiments. At least 100 cells for each experiment were scored. P values were calculated using a 2-tailed unpaired Student t test.


E ) DSB - SPB interaction involves a proficient DNA damage checkpoint activation . D1 , D2 and D3 distances were scored in wild - type , sml1Δ and sml1Δ rad53Δ backgrounds using Ddc2 - GFP and Spc110 - RFP as DSB and SPB markers respectively . Measurements were performed by using a maximum projection of nine z - planes images . Graphs denote the average distribution of D1 , D2 and D3 from three independent experiments where at least 100 cells for each sample were scored . P values were calculated using a 2 - tailed unpaired Student t test .

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E) DSB-SPB interaction involves a proficient DNA damage checkpoint activation. D1, D2 and D3 distances were scored in wild-type, sml1Δ and sml1Δ rad53Δ backgrounds using Ddc2-GFP and Spc110-RFP as DSB and SPB markers respectively. Measurements were performed by using a maximum projection of nine z-planes images. Graphs denote the average distribution of D1, D2 and D3 from three independent experiments where at least 100 cells for each sample were scored. P values were calculated using a 2-tailed unpaired Student t test.


A ) Identification of Cdc14 phospho - targets during the DNA damage response by mass spectrometry analysis . Wild - type and cdc14 - 1 cells were grown overnight and blocked in G2 / M by using nocodazole to avoid cell cycle - dependent changes in protein phosphorylation between both strains . After the block was attained , cells were transferred to 37ºC for 45 minutes to deplete Cdc14 activity prior HO induction by galactose addition for 4h . Differential phosphorylation of phospho - peptides detected between the wild - type and cdc14 - 1 were grouped into broad categories depending on the molecular function of the proteins . The table includes the DNA damage and checkpoint - related proteins with Cdc14 - dependent hyper - phosphorylated status and the relative ratio between the wild - type and cdc14 - 1 during the DNA damage response . Red and blue indicate relative amount of the residue phosphorylation between both strains ( Red , high ; Blue , low ) .

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A) Identification of Cdc14 phospho-targets during the DNA damage response by mass spectrometry analysis. Wild-type and cdc14-1 cells were grown overnight and blocked in G2/M by using nocodazole to avoid cell cycle-dependent changes in protein phosphorylation between both strains. After the block was attained, cells were transferred to 37ºC for 45 minutes to deplete Cdc14 activity prior HO induction by galactose addition for 4h. Differential phosphorylation of phospho-peptides detected between the wild-type and cdc14-1 were grouped into broad categories depending on the molecular function of the proteins. The table includes the DNA damage and checkpoint-related proteins with Cdc14-dependent hyper-phosphorylated status and the relative ratio between the wild-type and cdc14-1 during the DNA damage response. Red and blue indicate relative amount of the residue phosphorylation between both strains (Red, high; Blue, low).


B ) Cdc14 dephosphorylates Spc110 in response to a DSB . Spc110 was tagged with the 6HA epitope in wild - type , cdc14 - 1 and spc110S36 - 91A backgrounds . Cells were grown overnight in raffinose - containing media and supplemented with galactose to induce HO expression . Samples were collected at each time - point indicated . Proteins were TCA extracted , separated on Phos - Tag containing gels and blotted . Coomassie staining is shown as loading control .

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B) Cdc14 dephosphorylates Spc110 in response to a DSB. Spc110 was tagged with the 6HA epitope in wild-type, cdc14-1 and spc110S36-91A backgrounds. Cells were grown overnight in raffinose-containing media and supplemented with galactose to induce HO expression. Samples were collected at each time-point indicated. Proteins were TCA extracted, separated on Phos-Tag containing gels and blotted. Coomassie staining is shown as loading control.


C ) Lambda - phosphatase treatment of protein extracts in the absence of DNA damage reduces Spc110 phosphorylation levels to the same extent as samples taken during the DNA damage response . Cells were grown overnight in raffinose containing media ( line 1 ) and incubated with galactose for 2 . 5 hours to induce HO expression ( line 2 ) . Extracts from raffinose - containing cultures were treated with mock ( line 3 ) or λ - PPase ( line 4 ) . All samples were separated in a Phos - Tag containing gel and blotted . Coomassie staining is shown as loading control .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) Lambda-phosphatase treatment of protein extracts in the absence of DNA damage reduces Spc110 phosphorylation levels to the same extent as samples taken during the DNA damage response. Cells were grown overnight in raffinose containing media (line 1) and incubated with galactose for 2.5 hours to induce HO expression (line 2). Extracts from raffinose-containing cultures were treated with mock (line 3) or λ-PPase (line 4). All samples were separated in a Phos-Tag containing gel and blotted. Coomassie staining is shown as loading control.


D ) The canonical FEAR pathway is required for Spc110 dephosphorylation during the DNA damage response . Spc110 was labelled with the 3HA tag in both wild - type and esp1 - 1 background strains . Cells were grown overnight in raffinose - containing media and supplemented with galactose to induce the HO expression . Samples were collected at each time - point indicated . Proteins were TCA extracted , separated on Phos - Tag containing gels and blotted . Coomassie staining is shown as a control for loading .

O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) The canonical FEAR pathway is required for Spc110 dephosphorylation during the DNA damage response. Spc110 was labelled with the 3HA tag in both wild-type and esp1-1 background strains. Cells were grown overnight in raffinose-containing media and supplemented with galactose to induce the HO expression. Samples were collected at each time-point indicated. Proteins were TCA extracted, separated on Phos-Tag containing gels and blotted. Coomassie staining is shown as a control for loading.


A ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

A) 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 .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

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 .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


A ) 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 .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

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.


A ) 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 .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

A) 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 .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O

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 .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


A ) 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 .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

A) 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 .

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


Snapshots 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

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Snapshots 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 genes


Snapshots of ChIP - seq signals of Sen1 ( this study ) , RNAP3 ( this study ) and RNAP2 ( data from ( Larochelle et al , 2018 ) ) across the U6 snRNA

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Snapshots of ChIP-seq signals of Sen1 (this study), RNAP3 (this study) and RNAP2 (data from (Larochelle et al, 2018)) across the U6 snRNA snu6


Snapshots of ChIP - seq signals of Sen1 ( this study ) , RNAP3 ( this study ) and RNAP2 ( data from ( Larochelle et al , 2018 ) ) across the srp7 loci .

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Snapshots 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 .

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 ) .

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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 ) .

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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).


( 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 .

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(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 ) .

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(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 ) .

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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).


Cells 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 ) .

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Cells 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 .

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(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.


Snapshots 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 .

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Snapshots 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 .

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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 ) .

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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).


Cells 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 .

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Cells 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 .

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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 .

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Northern blot analysis of the tRNA SPATRNAPRO.02 using an intron-specific probe (TCTAAACTCAGCATACAAGTGGGG). U5 snRNA was used as a loading control.


ChIP - qPCR analysis of Rpc37 around SPCTRNAARG . 10 gene in the strong terminator mutant ( mean ± standard deviation from 4 biological replicates ) .

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ChIP-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 ) .

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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).


C . 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 ) .

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C. 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 .

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H. Heatmap of motif deviations from 3 biological replicates of B6 and D2 ESCs for TFs highlighted in G.


A - 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 ) .

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A-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).


D . 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 .

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D. 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 .

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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.


B . 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 ) .

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B. 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). D


G . 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 .

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G. 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.


F . 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 .

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F. 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 ) .

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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). I


I . 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 .

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I. 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 ) .

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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