( f ) Tissue lysates prepared from same mice without insulin injection were subjected to immunoblotting . Numbers below immunoblot bands indicate fold changes normalized to control bands . ( ' * ' indicates comparison between imatinib control : ' # ' indicates comparison between trehalose control ) .

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(f) Tissue lysates prepared from the same mice without insulin injection were subjected to immunoblotting. Numbers below immunoblot bands indicate the fold changes normalized to control bands. ('*' indicates comparison between imatinib and control: '#' indicates comparison between trehalose and control).


B Western blot for Hwa performed with lysates from treated as indicated in ( A ) . 1cs , 1 - stage ; 2cs , 2 - stage ; st6 , 32 - stage ; st8 . 5 , blastula stage . Blots are representative of 3 independent experiments . C Densitometry of Hwa bands from western blot in ( B ) for corresponding lanes normalized to α - tubulin . n = 3 independent experiments , mean ± , * p < 0 . 05 , * * p < 0 . 01 by unpaired t .

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B Western blot analysis for Hwa performed with lysates from embryos treated as indicated in (A). 1cs, 1-cell stage; 2cs, 2-cell stage; st6, 32-cell stage; st8.5, blastula stage. Blots are representative of 3 independent experiments. C Densitometry analysis of Hwa bands from the western blot in (B) for corresponding lanes normalized to α-tubulin. n = 3 independent experiments, mean ± SEM, *p < 0.05, **p < 0.01 by unpaired t test.


D Western blot of exogenous Hwa in HEK293T . Treatment of with lysosome inhibitors bafilomycin A1 ( Baf - A1 ) NH4Cl for 24 hours , DMSO was used as control . Dosage of Baf - A1 : 10 nM , 50 nM , 100 nM ; Dosage of NH4Cl : 2 . 5 mM , 5 mM , 10 mM . Hwa with modification is indicated by arrow ; Degraded Hwa is indicated by black arrow . Blots are representative of 3 independent experiments . E Densitometry of Hwa bands from western blot in ( D ) for corresponding lanes normalized to α - tubulin . n = 3 independent experiments , mean ± , * * p < 0 . 01 by unpaired t .

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

D Western blot analysis of exogenous Hwa in HEK293T cells. Treatment of cells with lysosome inhibitors bafilomycin A1 (Baf-A1) and NH4Cl for 24 hours, DMSO was used as control. Dosage of Baf-A1: 10 nM, 50 nM, 100 nM; Dosage of NH4Cl: 2.5 mM, 5 mM, 10 mM. Hwa with modification is indicated by white arrow; Degraded Hwa is indicated by black arrow. Blots are representative of 3 independent experiments. E Densitometry analysis of Hwa bands from the western blot in (D) for corresponding lanes normalized to α-tubulin. n = 3 independent experiments, mean ± SEM, **p < 0.01 by unpaired t test.


C Co - IP with HEK293T co - transfected with plasmids encoding Hwa - Flag ZNRF3 - HA or ZNRF3ΔRING . Lysates immunoprecipitated with anti - Flag were immunoblotted with HA Flag antibodies . Blots are representative of 3 independent experiments . D Co - IP with HEK293T co - transfected with plasmids encoding Hwa - Flag ZNRF3 - HA or ZNRF3ΔRING . Lysates immunoprecipitated with anti - HA were immunoblotted with Flag HA antibodies . Blots are representative of 3 independent experiments .

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C Co-IP with HEK293T cells co-transfected with plasmids encoding Hwa-Flag and ZNRF3-HA or ZNRF3ΔRING. Lysates immunoprecipitated with anti-Flag were immunoblotted with HA and Flag antibodies. Blots are representative of 3 independent experiments. D Co-IP with HEK293T cells co-transfected with plasmids encoding Hwa-Flag and ZNRF3-HA or ZNRF3ΔRING. Lysates immunoprecipitated with anti-HA were immunoblotted with Flag and HA antibodies. Blots are representative of 3 independent experiments.


E Colocalization of Hwa - Flag ( red ) ZNRF3 - HA ( green ) or ZNRF3ΔRING - HA ( green ) in Hela is indicated by yellow dots . Baf - A1 , 50 nM , 12 hrs . Magnification : 100 × ; scale bar : 10 μm .

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E Colocalization of Hwa-Flag (red) and ZNRF3-HA (green) or ZNRF3ΔRING-HA (green) in Hela cells is indicated by yellow dots. Baf-A1, 50 nM, 12 hrs. Magnification: 100 ×; scale bar: 10 μm.


B Whole mount in situ hybridization of Spemann organizer markers chrd , gsc , xnr3 in gastrula ( stage 10 . 5 ) . Oocytes were injected with AS or mRNAs as indicated , Dosage : znrf3 AS ( 3 ng AS4 + 3 ng AS9 ) , znrf3 mRNA 100 pg , znrf3ΔRING mRNA 50 pg . were obtained through Host transfer technology . Scale bars : 1 mm .

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B Whole mount in situ hybridization of Spemann organizer markers chrd, gsc, and xnr3 in gastrula embryos (stage 10.5). Oocytes were injected with AS or mRNAs as indicated, Dosage: znrf3 AS (3 ng AS4 + 3 ng AS9), znrf3 mRNA 100 pg, znrf3ΔRING mRNA 50 pg. embryos were obtained through Host transfer technology. Scale bars: 1 mm.


E Relative of β - catenin ( left ) Axin ( right ) in ( D ) normalized to GAPDH . n = 3 independent experiments , mean ± , * p < 0 . 05 , * * * p < 0 . 005 , * * * * p < 0 . 0001 by unpaired t .

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E Relative levels of β-catenin (left) and Axin (right) in (D) normalized to GAPDH. n = 3 independent experiments, mean ± SEM, *p < 0.05, ***p < 0.005, ****p < 0.0001 by unpaired t test.


H Whole mount in situ hybridization of Spemann organizer markers chrd gsc in gastrula ( stage 10 . 5 ) . were injected obtained as descripted in ( G ) . Scale bars : 1 mm .

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H Whole mount in situ hybridization of Spemann organizer markers chrd and gsc in gastrula embryos (stage 10.5). Embryos were injected and obtained as descripted in (G). Scale bars: 1 mm.


I Representative of tailbud stage Xenopus leavis injected with 500 pg mRNAs of ctrl ( gfp mRNA ) , znrf3FL , znrf3 truncations as indicated at 4 - stage . Duplicated axes ( red arrowhead ) . znrf3FL , full - znrf3 ; znrf3ΔRING , znrf3 lacking RING domain ; znrf31 - 320 , 1 - 320 of znrf3 ; znrf3320 - 606 , 320 - 606 of znrf3 ; znrf3606 - 854 , 606 - 854 of znrf3 . Scale bars : 1 mm .

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I Representative phenotypes of tailbud stage Xenopus leavis embryos injected with 500 pg mRNAs of ctrl (gfp mRNA), znrf3FL, and znrf3 truncations as indicated at 4-cell stage. Duplicated axes (red arrowhead). znrf3FL, full-length znrf3; znrf3ΔRING, znrf3 lacking the RING domain; znrf31-320, amino acids 1-320 of znrf3; znrf3320-606, amino acids 320-606 of znrf3; znrf3606-854, amino acids 606-854 of znrf3. Scale bars: 1 mm.


B Hwa ubiquitination was analyzed under denaturing conditions . HEK293T co - transfected with wildtype His - HA - Ubiquitin ( His - HA - Ub WT ) or lysine - less mutant ( K0 ) , Hwa - Flag , ZNRF3 - Myc . lysates were immunoblotted with Myc , Flag , α - tub ( α - tubulin ) . Lysates immunoprecipitated with anti - Flag were immunoblotted with HA Flag antibodies . K0 , lysine sites on ubiquitin were mutated . Blots are representative of 3 independent experiments .

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B Hwa ubiquitination was analyzed under denaturing conditions. HEK293T cells co-transfected with wildtype His-HA-Ubiquitin (His-HA-Ub WT) or lysine-less mutant (K0), Hwa-Flag, and ZNRF3-Myc. The lysates were immunoblotted with Myc, Flag, and α-tub (α-tubulin). Lysates immunoprecipitated with anti-Flag were immunoblotted with HA and Flag antibodies. K0, all lysine sites on ubiquitin were mutated. Blots are representative of 3 independent experiments.


D Screen of ZNRF3 - mediated Hwa ubiquitination sites under denaturing conditions . HEK293T were co - transfected with His - HA - Ub , Hwa - Flag or Hwa10KR - Flag , ZNRF3 or ZNRF3ΔRING . lysate was immunoblotted with Myc , Flag , α - tub ( α - tubulin ) . Lysates immunoprecipitated with anti - Flag were immunoblotted with HA Flag antibodies . Hwa10KR - Flag mutant , lysine sites in Hwa were replaced with arginine . Denaturing conditions : before immunoprecipitation , lysates were denatured by adding SDS to a final at 1 % boiling at 95 ℃ for 15 min . Blots are representative of 3 independent experiments .

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D Screen of ZNRF3-mediated Hwa ubiquitination sites under denaturing conditions. HEK293T cells were co-transfected with His-HA-Ub, Hwa-Flag or Hwa10KR-Flag, and ZNRF3 or ZNRF3ΔRING. The lysate was immunoblotted with Myc, Flag, and α-tub (α-tubulin). Lysates immunoprecipitated with anti-Flag were immunoblotted with HA and Flag antibodies. Hwa10KR-Flag mutant, all lysine sites in Hwa sequence were replaced with arginine. Denaturing conditions: before immunoprecipitation, lysates were denatured by adding SDS to a final concentration at 1% and boiling at 95℃ for 15 min. Blots are representative of 3 independent experiments.


A Western blot of Hwa in HEK293T co - transfected with Hwa - Flag , ZNRF3 - HA ( 5 μg ) or ZNRF3ΔRING ( 2 . 5 μg ) . Lysate was immunoblotted with Flag , HA , GAPDH antibodies . Blots are representative of 3 independent experiments . B Densitometry of Hwa bands from western blot in ( A ) for corresponding lanes normalized to GAPDH . n = 3 independent experiments , mean ± , ns p > 0 . 05 , * * p < 0 . 01 by unpaired t .

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A Western blot analysis of Hwa in HEK293T cells co-transfected with Hwa-Flag, and ZNRF3-HA (5 μg) or ZNRF3ΔRING (2.5 μg). Lysate was immunoblotted with Flag, HA, and GAPDH antibodies. Blots are representative of 3 independent experiments. B Densitometry analysis of Hwa bands from the western blot in (A) for corresponding lanes normalized to GAPDH. n = 3 independent experiments, mean ± SEM, ns p > 0.05, **p < 0.01 by unpaired t test.


C Western blot of Hwa in HEK293T co - transfected with Hwa - Flag ZNRF3 - HA . were treated with or without Baf - A1 ( 50 nM ) or MG132 ( 20 μM ) for about 12 hours . Lysates were immunoblotted with Flag , HA , α - tub ( α - tubulin ) antibodies . Blots are representative of 3 independent experiments . D Densitometry of Hwa bands from western blot in ( C ) for corresponding lanes normalized to α - tubulin . n = 3 independent experiments , mean ± , ns p > 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 by unpaired t .

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C Western blot analysis of Hwa in HEK293T cells co-transfected with Hwa-Flag and ZNRF3-HA. Cells were treated with or without Baf-A1 (50 nM) or MG132 (20 μM) for about 12 hours. Lysates were immunoblotted with Flag, HA, and α-tub (α-tubulin) antibodies. Blots are representative of 3 independent experiments. D Densitometry analysis of Hwa bands from the western blot in (C) for corresponding lanes normalized to α-tubulin. n = 3 independent experiments, mean ± SEM, ns p > 0.05, **p < 0.01, ****p < 0.0001 by unpaired t test.


F Colocalization by immunofluorescence of Hwa ( green signals ) with lysosome ( LAMP1 , red signals ) in Hela . were co - transfected with Hwa - Flag , ZNRF3 - HA or ZNRF3ΔRING - HA . Magnification , 100 × ; scale bars : 10 μm .

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F Colocalization by immunofluorescence of Hwa (green signals) with lysosome (LAMP1, red signals) in Hela cells. Cells were co-transfected with Hwa-Flag, and ZNRF3-HA or ZNRF3ΔRING-HA. Magnification, 100 ×; scale bars: 10 μm.


I Surface biotinylation streptavidin pulldown of Hwa - Myc - Flag . HEK293T were co - transfected with Hwa - MF ( Hwa - Myc - Flag ) , ZNRF3 - Myc or ZNRF3ΔRING - Myc . Lysate was immunoblotted with Myc , Flag , GAPDH antibodies . Lysates immunoprecipitated with anti - Flag ( 2nd elution ) were immunoblotted with Flag HA antibodies .

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I Surface biotinylation and streptavidin pulldown of Hwa-Myc-Flag. HEK293T cells were co-transfected with Hwa-MF (Hwa-Myc-Flag), and ZNRF3-Myc or ZNRF3ΔRING-Myc. Lysate was immunoblotted with Myc, Flag, and GAPDH antibodies. Lysates immunoprecipitated with anti-Flag (2nd elution) were immunoblotted with Flag and HA antibodies.


C Whole mount in situ hybridization of Spemann organizer markers chrd gsc in gastrula . were obtained as descripted in ( B ) . Scale bars : 1mm .

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C Whole mount in situ hybridization of Spemann organizer markers chrd and gsc in gastrula embryos. Embryos were obtained as descripted in (B). Scale bars: 1mm.


E Western blot of Hwa or Hwa10KR in at blastula stage . 1 - stage were co - injected with hwa ( 100 pg ) or hwa10KR ( 100 pg ) znrf3 ( 1 ng ) or znrf3ΔRING ( 100 pg ) as indicated . Lysates were immunoblotted with HA , Flag , α - tub ( α - tubulin ) .

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E Western blot analysis of Hwa or Hwa10KR in embryos at blastula stage. 1-cell stage embryos were co-injected with hwa (100 pg) or hwa10KR (100 pg) and znrf3 (1 ng) or znrf3ΔRING (100 pg) as indicated. Lysates were immunoblotted with HA, Flag, and α-tub (α-tubulin).


( a ) localization of HLH - 30 was visualized by microscopy in day 1 adult wild - type ( WT ) ( upper panel ) glp - 1 ( e2141 ) ( lower panel ) expressing HLH - 30 : : GFP raised at non - permissive ( 25 ° C ) . Inserts show enlarged intestinal . Graph shows of with HLH - 30 in nuclei of intestinal ( four biological replicates , ~ 50 each , mean ± s . d . , * * P0 . 01 , Student ' s t - ) . Magnification × 100 ; scale bar , 100 μm .

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(a) Nuclear localization of HLH-30 was visualized by fluorescence microscopy in day 1 adult wild-type (WT) (upper panel) and glp-1(e2141) (lower panel) animals expressing HLH-30::GFP raised at the non-permissive temperature (25°C). Inserts show enlarged intestinal cells. Graph shows percentage of animals with HLH-30 in the nuclei of intestinal cells (four biological replicates, ~50 animals each, mean±s.d., **P0.01, Student's t-test). Magnification × 100; scale bar, 100 μm.


( b ) Expression of hlh - 30 putative autophagy - related lysosomal target genes was measured by quantitative PCR ( qPCR ) in day 1 adult WT glp - 1 ( e2141 ) raised at 25 ° C ( mean ± s . d . of three biological replicates , * P0 . 05 , * * P0 . 01 , Student ' s t - ) .

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(b) Expression of hlh-30 and putative autophagy-related and lysosomal target genes was measured by quantitative PCR (qPCR) in day 1 adult WT and glp-1(e2141) animals raised at 25°C (mean±s.d. of three biological replicates, *P0.05, **P0.01, Student's t-test).


( c ) Expression of hlh - 30 putative autophagy - related lysosomal target genes was measured by qPCR in day 1 adult glp - 1 ( e2141 ) glp - 1 ( e2141 ) ; hlh - 30 ( tm1978 ) raised at 25 ° C ( mean ± s . d . of three biological replicates , * P0 . 05 , * * P0 . 01 , Student ' s t - ) . ctsa * is a cathepsin A orthologue ( cosmid C08H9 . 1 ( ref . 30 ) ) . See Supplementary Fig . 1 for qPCR analyses of hlh - 30 ( tm1978 ) ( control for c ) WT glp - 1 ( e2141 ) fed expressing hlh - 30 dsRNA .

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(c) Expression of hlh-30 and putative autophagy-related and lysosomal target genes was measured by qPCR in day 1 adult glp-1(e2141) and glp-1(e2141); hlh-30(tm1978) animals raised at 25°C (mean±s.d. of three biological replicates, *P0.05, **P0.01, Student's t-test). ctsa* is a cathepsin A orthologue (cosmid C08H9.1 (ref. 30)). See Supplementary Fig. 1 for qPCR analyses of hlh-30(tm1978) (control for c) and WT and glp-1(e2141) animals fed bacteria expressing hlh-30 dsRNA.


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

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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 ) expressing ( f ) LMP - 1 : : GFP or ( g ) SQST - 1 : : GFP were raised at non - permissive ( 25 ° C ) fed expressing control or hlh - 30 dsRNA from hatching . of posterior intestine were taken on day 1 of adulthood , LMP - 1 : : GFP ( mean ± s . d . of ~ 10 , * * P0 . 01 , Student ' s t - ) SQST - 1 : : GFP foci ( mean ± s . d . of ~ 30 , * * P0 . 01 , Student ' s t - ) were quantified . Experiments were performed at least three times with similar results . See Supplementary Fig . S2a , c for of whole 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 ) localization of HLH - 30 was quantified in day 1 adult expressing HLH - 30 : : GFP . were fed expressing control or tor dsRNA from hatching raised at 20 ° C ( three biological replicates , ~ 50 each , mean ± s . d . , * * P0 . 01 , Student ' s t - ) .

(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 lysosomal target genes was measured by quantitative PCR in day 1 ( b ) WT ( N2 ) ( c ) hlh - 30 ( tm1978 ) fed expressing control or tor dsRNA from hatching ( 20 ° C ) . Data are mean ± s . d . of biological triplicates . * P0 . 05 , * * P0 . 01 ; Student ' s t - . 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 of WT hlh - 30 ( tm1978 ) mutants fed 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 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 ) ( b ) germline - less glp - 1 ( e2141 ) raised at non - permissive ( 25 ° C ) fed 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 ( f ) mRNA translation rsks - 1 ( sv31 ) mutants , fed 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 .

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 ) localization of HLH - 30 was quantified in day 1 adult WT , eat - 2 , daf - 2 , clk - 1 rsks - 1 mutants ( mean ± s . d . of four biological replicates , ~ 50 each , * * P0 . 01 , 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 ) rsks - 1 ( sv31 ) ( mRNA translation ) ( mean ± s . d . of three biological replicates , * P0 . 05 , Student ' s t - ) .

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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 of WT transgenic overexpressing HLH - 30 : : GFP , raised maintained on OP50 was carried out at 20 ° C . See Supplementary Table S7 for details of lifespan analyses 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 were quantified in hypodermal seam of WT or overexpressing HLH - 30 ( n > 300 , ± s . e . m . , * * P0 . 01 , Student ' s t - ) . increase in GFP : : LGG - 1 observed in 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 livers of 4 . 5 - month - old ( F ) ( M ) mice fed AL or subjected to DR for 5 . 5 starting at 3 months of ( mean ± s . e . m . of ~ 20 mice per group , * P0 . 05 , Student ' s t - ) .

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

(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 U5 - hNSC defined cycle phases . x - axis is relative expression of cyclin , y - axis is of per phase . cycle phase with peak expression is denoted by an arrowhead at top of 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 - like in each ccAF cycle phase , distribution of absolute gene expression across ccAF cycle phases for genes used to identify putative - like 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 - like expression of genes used to identify putative - like projected onto Neftel et al . , 2019 . Expression of genes are overlaid as color of each ( = 0 , blue = max expression ) . Quadrants are broken up by dashed grey lines : OPC = upper left ; NPC = upper right ; AC = lower left ; MES = lower right . Percentages of gene expression greater than 0 are shown in corners of 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 types from Darmanis et al . , 2017 . Data are log 10 - transformed before plotting . Expression of PLP1 in single is much higher in oligodendrocytes ( Oligo ) than in Neural G0 or oligodendrocyte progenitor ( OPC ) . D , Violin plot showing GFAP expression across types from Darmanis et al . , 2017 . Data are log 10 - transformed before plotting . Expression of GFAP in single is more highly expressed in Neural G0 relative to other types . E , Violin plot showing AQP4 expression across types from Darmanis et al . , 2017 . Data are log 10 - transformed before plotting . Neural G0 expression of AQP4 in single is similar to astrocytes but has a lower median expression 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 loss of NF2 , PTPN14 , TAOK1 , CREBBP , 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 S / G2 / M transit times using time - lapse microscopy FUCCI . Differences in G0 / G1 are statistically significant with p < 0 . 001 for targeted U5 - NSCs 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 cdc14 - 1 dropped grown on solid rich media or media containing MMS at 25 , 28 , 30 or 33ºC . Note that cdc14 - 1 exhibit growth sensitivity to MMS at 28ºC 30ºC compared to wild - type .

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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 cdc14 - 1 grown on solid rich media or media containing mock DMSO ( as non - treated control ) , 4NQO , HU , phleomycin benomyl at 30ºC . Note that cdc14 - 1 present a market sensitivity to 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 used to assess intra - chromosomal repair . location of a MAT specific probe restriction endonuclease cleavage sites used for Southern blot to detect repair product formation are indicated . Arrow depicts localization of double - strand break . Right panel : Physical of intra - chromosomal repair in wild - type cdc14 - 1 cultures at semipermisive . After DSB formation by expression of HO , glucose was added to repress it , thus allowing repair with HM donor sequences . Genomic was digested with StyI , separated on agarose gel blotted . Blots were hybridized with a probe corresponding to MATa - distal . A second probe for actin gene was used to control amount of genomic loaded at each time - point . Immunoblot of Rad53 during mating type switching experiments is shown . Coomassie blue is depicted as loading control . Graphs show of gene conversion ( GC ) leading to re - establishment of MATa or switching to MAT DSB formation . data was normalized with actin loading control . Graphs show mean ± S . D . from three independent experiments . Replicates were averaged statistical significance of differences assessed by a 2 - unpaired Student t .

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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 strains used to measure inter - chromosomal repair efficiency . location of a MAT specific probe restriction endonuclease cleavage sites used for Southern blot to detect repair product formation are indicated . Note that crossover non - crossover product have different restriction fragment sizes that can be differentiated in a Southern blot . Arrow depicts localization of double - strand break . Right panel : Physical of wild - type cdc14 - 1 mutant strains carrying inter - chromosomal gene conversion . were grown overnight before adding galactose at semipermisive to induce HO expression , thus producing a DSB on chromosome V . Samples were taken at different time - points , genomic was extracted , digested with EcoRI analysed by Southern blot . Blots were hybridized with a MATa - only 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 is shown as a control for loading . Graphs show mean ± S . D . of gene conversion , DSB induction crossover vs non - crossover from three independent experiments . data was normalized using actin as loading control . Replicates were averaged statistical significance of differences assessed by a 2 - unpaired Student t .

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

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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 - imaging of Cdc14 - YFP Cnm67 - RFP asynchronous treated with damage agent phleomycin . were grown overnight phleomycin was added to a final of 1μM . Samples were taken at different intervals to determine Cdc14 localization . Cnm67 was used as SPB reporter to determine spindle . Scale bar : 3μm . Graphs represent average ± SD from three independent experiments of arrested in metaphase ( left graph ) with Cdc14 signal at 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 to analyse Rad53 Net1 phosphorylation under these conditions are shown . Note that Rad53 is rapidly phosphorylated in response to phleomycin maintained throughout entire experiment . Detection of Net1 phosphorylation was achieved by using Phos - tag polyacrylamide gels . Coomassie 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 showing Net1 phosphorylation through cycle is included . separation of phospho - bands was achieved by using Phos - tag polyacrylamide gels as in Fig 3A . Coomassie is depicted 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.


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

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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 ) expressing Cdc14 - YFP Cnm67 - RFP were grown overnight blocked in G2 / M by using nocodazole prior phleomycin treatment ( 1μM ) . Samples were taken every 30 minutes to follow Cdc14 re - localization . efficiency of arrest was determined as in Fig 3A . Scale bar : 3μm . Graphs represent ± SD of with Cdc14nucleolar exclusion ( left graph ) 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 - imaging of Cdc14 localization in presence of a non - reparable DSB at MAT locus . were grown overnight HO endonuclease was expressed by adding galactose to media , thus producing a DSB on chromosome III . Cdc14 , SPB component Cnm67 damage checkpoint Ddc2 ( used as DSB reporter ) were labelled with green , red cyan respectively . display maximum projection of 9 planes showing Cdc14 co - localizing with DSB SPBs in response to damage ( arrows ) . Scale bar : 3μm . Graphs represent ± SD from three independents experiment of with nucleoplasmic Cdc14 along HO endonuclease induction ( left graph ) of Cdc14 - SPB - DSB co - localization after 5h in galactose ( right graph ) .

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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 after generation of a DSB induced by HO expression . A lacking HO endonuclease under galactose promoter was used as undamaged control . phospho - bands were resolved by using Phos - tag polyacrylamide gels as in Fig 3A Fig 3B . Coomassie 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 genomic background approximation used to measure DSB - SPB interaction . Cnm67 - RFP Ddc2 - GFP were used as SPB DSB markers respectively . D1 : inter - SPB ; D2 : DSB to proximal SPB ; D3 : DSB to distal SPB . Graph represents mean ± SD of D1 , D2 D3 distances from three independent experiments . At least 100 per experiment were scored using maximum projection of three z - planes . P values were calculated using a 2 - unpaired Student t . A representative picture of a wild - type 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 showing efficiency of DSB - SPB interaction upon induction of a single DSB . Cnm67 - CFP Ddc2 - RFP were used as SPB DSB markers respectively . D1 , D2 D3 distances in wild - type cdc14 - 1 were tracked using maximum projection of three z - planes at 15 - seconds intervals over period of 7 . 5 minutes . A total of 32 time - lapses for each were measured . A representative picture is shown . Examples of 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 SPBs after expressing endonuclease . harbouring a TetO array adjacent to I - SceI site a plasmid containing endonuclease under control of 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 GFP to be used as SPB reporter . D1 , D2 D3 distances were measured in wild - type cdc14 - 1 mutants before after endonuclease expression by using both TetI - RFP Rad52 - CFP foci using maximum projection of nine z - planes . Graphs represent average of three independent experiments . P values were calculated using a 2 - unpaired Student t . A representative picture of a wild - type 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 N - terminus domain of Mps3 . expressing a wild - type Mps3 , a mps3Δ2 - 64 a mps3Δ75 - 150 fused to RFP as sole source of were scored for their ability to enhance DSB - SPB tethering in both wild - type cdc14 - 1 backgrounds . Ddc2 - YFP was used as DSB reporter . Measurements were carried out using a maximum projection of nine z - planes . Graphs represent average distribution of D1 , D2 D3 of three independent experiments . At least 100 for each experiment were scored . P values were calculated using a 2 - unpaired Student t .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 damage response by mass spectrometry . Wild - type cdc14 - 1 were grown overnight blocked in G2 / M by using nocodazole to avoid cycle - dependent changes in phosphorylation between both strains . After block was attained , 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 wild - type cdc14 - 1 were grouped into broad categories depending on molecular function of . table includes damage checkpoint - related with Cdc14 - dependent hyper - phosphorylated status relative between wild - type cdc14 - 1 during damage response . Red blue indicate relative amount of residue phosphorylation between both strains ( Red , high ; Blue , low ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 6HA epitope in wild - type , cdc14 - 1 spc110S36 - 91A backgrounds . were grown overnight in raffinose - containing media supplemented with galactose to induce HO expression . Samples were collected at each time - point indicated . were TCA extracted , separated on Phos - Tag containing gels blotted . Coomassie is shown as loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 extracts in absence of damage reduces Spc110 phosphorylation to same extent as samples taken during damage response . were grown overnight in raffinose containing media ( 1 ) incubated with galactose for 2 . 5 hours to induce HO expression ( 2 ) . Extracts from raffinose - containing cultures were treated with mock ( 3 ) or λ - PPase ( 4 ) . samples were separated in a Phos - Tag containing gel blotted . Coomassie 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.


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 harbouring a non - reparable DSB were carried out after expressing HO endonuclease in wild - type cdc14 - 1 mutant . Spc110 - RFP was used as SPB reporter Tub1 - GFP was used as tubulin marker . Three z - planes every 5 - second intervals over a period of 5 minutes were captured used to quantify average SPB track velocity using Spc110 - RFP as SPB marker . At least 100 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 equal amount of Tub1 - GFP in both wild - type cdc14 - 1 backgrounds . Coomassie blue is shown as loading control .

O O O O O O O O O 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) 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 ) steady state phosphorylation of Spc110 is required to maintain spindle polarity in response to damage . Wild - type , cdc14 - 1 spc110S36 - 91A expressing Spc110 - RFP were scored for their ability to maintain spindle polarity at axial plane of upon induction of a single DSB induced by HO endonuclease . A maximum projection of nine z - planes was used to score . At least 100 from three independent experiments were scored . Graph represents ± SD of with oriented / miss - oriented SPBs with respect to axial plane of . P value was calculated using a 2 - unpaired Student t .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) asymmetric loading of dynein into astral MTs emanating from daughter SPB is affected in cdc14 - 1 spc110S36 - 91A during damage response . Dynein distribution on astral MTs emerging from each SPB was scored by using Dyn1 - GFP in strains containing Spc110 - RFP . Graph represents ± SD of grouped into three categories depending on pattern of dynein distribution on astral MTs : Type1 , Dynein is exclusively loaded on astral MTs emerging from 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 from three independent experiments were scored . A representative picture showing 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) treated with nocodazole are affected in DSB - SPB interaction . Cnm67 - CFP Ddc2 - RFP were used as SPB DSB markers respectively . Diagram depicts genomic background used . D1 : inter - SPB ; D2 : DSB to proximal SPB ; D3 : DSB to distal SPB . D1 , D2 D3 distances after inducing a DSB in presence of nocodazole or mock dimethyl sulfoxide ( DMSO ) were scored plotted . Graph represents mean ± SD of D1 , D2 D3 distances of three independent experiments . At least 100 per experiment were scored using maximum projection of three z - planes . P values were calculated using a 2 - unpaired Student t . A representative picture is shown . Scale bar : 3μm .

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 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

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 of wild - type containing repair pathway choice described in Fig 2B . diagram with genomic information , restriction enzymes used location of probe are shown . were synchronized in G1 by using α - factor pheromone released into fresh media for 1 hour . Induction of HO expression was attained by adding galactose for 2 hours . After formation of DSB , glucose was added to repress HO in presence of nocodazole or mock DMSO samples were taken to analyse kinetics of repair . was extracted , digested with EcoRV , separated on agarose gels blotted . Both probes for MATa - distal HIS3 gene ( as loading control ) were used . Graphs show of MATa restoration ( NHEJ ) , mating type switching by gene conversion ( HR ) , DSB kinetics formation . data were normalized with HIS3 gene . Graphs show mean ± S . D . from three independent experiments . P values were calculated using a 2 - unpaired Student t .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

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 of wild - type containing repair pathway choice described in Fig 2B . diagram with genomic information , restriction enzymes used location of probe are shown . were synchronized in G1 by using α - factor pheromone released into fresh media for 1 hour . Induction of HO expression was attained by adding galactose for 2 hours . After formation of DSB , glucose was added to repress HO in presence of nocodazole or mock DMSO samples were taken to analyse kinetics of 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 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) 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 phleomycin of a Cdk phospho - deficient mutant of Spc110 . Ten - fold serial dilutions from overnight cultures of wild - type , spc110S36 - 91A a cdc14 - 1 grown on solid rich media containing mock DMSO , MMS or Phleomycin at 30ºC .

O O O O O B-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 O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 genomic background approximation used to measure DSB - SPB interaction . Spc110 spc110S36 - 91A were labelled with RFP in a background containing Ddc2 - GFP as DSB reporter . D1 : inter - SPB ; D2 : DSB to proximal SPB ; D3 : DSB to distal SPB . At least 100 from three independent experiments were scored using maximum projection of nine z - planes . Graph shows mean ± S . D . P values were calculated using a 2 - unpaired Student t .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 expressing HO endonuclease in wild - type spc110S36 - 91A mutants . Five z - planes every 5 - second intervals over a period of 5 minutes were captured . Quantifications were made using average SPB track velocity of at least 100 . Spc110 spc110S36 - 91A were labelled with RFP used as SPB reporter .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 spc110S36 - 91A mutant harbouring inter - chromosomal gene conversion portrayed . An overnight culture was induced by adding galactose at 32ºC to express HO endonuclease . Samples were collected at different time - points , genomic was extracted , digested with EcoRI analysed by Southern blot . Blots were probed with a MATa - only actin ( for loading control ) sequences . Graphs show of gene conversion , DSB induction crossover vs non - crossover . data were normalized using actin signal . Graphs represent mean ± S . D . from three independent experiments . P values were calculated using a 2 - unpaired Student t . Asterisk denotes an overexposed film to visualize formation of crossover products .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 spc110S36 - 91A mutant harbouring inter - chromosomal gene conversion portrayed . An overnight culture was induced by adding galactose at 32ºC to express HO endonuclease . Samples were collected at different time - points , genomic was extracted , digested with EcoRI analysed by Southern blot . Blots were probed with a MATa - only actin ( for loading control ) sequences . FACS profiles for content are included . Graphs show of gene conversion , DSB induction crossover vs non - crossover . data were normalized using actin signal . Graphs represent mean ± S . D . from three independent experiments . P values were calculated using a 2 - unpaired Student t . Asterisk denotes an overexposed film to visualize formation of crossover products .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) damage sensitivity to MMS phleomycin of a Cdk phospho - mimic mutant of Spc110 . Ten - fold serial dilutions from mid - log phase cultures of wild - type , spc110S36 - 91A spc110S36 - 91D grown on solid rich media containing mock DMSO , MMS or Phleomycin at 30ºC .

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 B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 genomic background approximation used to measure DSB - SPB tethering . Wild - type Spc110 spc110S36 - 91D were labelled with RFP . Ddc2 was tagged with GFP to be used as DSB reporter . D1 : inter - SPB ; D2 : DSB to proximal SPB ; D3 : DSB to distal SPB . At least 100 from three independent experiments were scored using maximum projection of nine z - planes . Graph shows mean ± S . D . P values were calculated using a 2 - unpaired Student t .

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 after induction of HO endonuclease in wild - type spc110S36 - 91D . Three z - planes every 10 - second intervals over a period of 5 minutes were captured to measure average SPBs track velocity . Spc110 spc110S36 - 91D were labelled with RFP used to measured SPBs kinetics . At least 100 from three independent experiments were scored .

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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 spc110S36 - 91D mutant bearing inter - chromosomal gene conversion depicted . Mid - log phase were HO - induced at 32ºC by adding galactose to media . Samples were collected at different time - points genomic was extracted . Preps were digested with EcoRI analysed by Southern blot . Blots were hybridized with MATa - only actin probes . Graphs show of gene conversion , DSB induction crossover vs non - crossover . data were normalized using actin signal . Graphs represent mean ± S . D . from three independent experiments . P values were calculated using a 2 - unpaired Student t . Asterisk denotes an overexposed film to visualize formation of crossover products .

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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 spc110S36 - 91D mutant bearing inter - chromosomal gene conversion depicted . Mid - log phase were HO - induced at 32ºC by adding galactose to media . Samples were collected at different time - points genomic was extracted . Preps were digested with EcoRI analysed by Southern blot . Blots were hybridized with MATa - only actin probes . FACS profiles for content are displayed . Graphs show of gene conversion , DSB induction crossover vs non - crossover . data were normalized using actin signal . Graphs represent mean ± S . D . from three independent experiments . P values were calculated using a 2 - unpaired Student t . Asterisk denotes an overexposed film to visualize formation of crossover products .

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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 ) RNAP2 ( data from ( Larochelle et al , 2018 ) ) across ( A ) a 25 kb region of chromosome 3 containing several tRNA 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 ) RNAP2 ( data from ( Larochelle et al , 2018 ) ) across U6 snRNA

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 ) RNAP2 ( data from ( Larochelle et al , 2018 ) ) across 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 of Flag - tagged Sen1 at indicated loci in a population of cycling ( 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 of Flag - tagged RNAP3 subunit Rpc37 Flag - tagged Sen1 across SPCTRNAARG . 10 tRNA locus , whose TATA box was either mutated ( TATA - ) or not ( TATA + ) . mutations introduced to disrupt putative TATA boxes are indicated in red above graphs . enrichment values were normalized to SPCTRNATHR . 10 ( mean ± standard deviation from 4 biological replicates ; p - value obtained using Wilcoxon - Mann Whitney statistical ) .

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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 of Rpc37 in presence or absence of Sen1 at indicated loci in a population of cycling ( mean ± standard deviation from 4 biological replicates ) . ( right ) Western blot of Rpc37 in 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 from indicated strains were separated on a 2 , 8 % agarose gel . ( right ) of overall tRNA ( 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 indicated RNAP3 transcripts . Transcript 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).


were grown in minimal medium during 18 hours to induce 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 13myc - tagged RNAP3 subunit Rpc25 in indicated strains at indicated loci ( mean ± standard deviation of 4 biological replicates ) . ( right ) Western blot of Myc - tagged Rpc25 Flag - tagged RnhA in 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 RNAP3 subunits Rpc1 Rpc2 in presence or absence of Sen1 across a representative ( A ) tRNA gene , ( B ) 5S rRNA gene ( C ) srp7 . Boxed regions highlight increased density of reads in downstream region of genes in absence of Sen1 .

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 Rpc2 across isolated tRNA 5S rRNA genes in presence 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 absence of Sen1 by ChIP - qPCR ( mean ± standard deviation from 6 biological replicates ) .

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).


of indicated genotypes that carried or not DRT5T dimeric tRNA construct ( schematized on top ) were grown either in presence of optimum of adenine ( left ) or in presence of a limiting of adenine ( right ) . Two independent clones of same genotype ( # 1 # 2 ) were used . See text for details . 3F refers to 3 Flag epitope tag at 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 of read - through transcripts ( see Methods ) . mean ± standard deviation from 4 biological replicates is represented here . P - values were obtained using Wilcoxon - Mann Whitney statistical .

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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 of 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 of Rpc37 around SPCTRNAARG . 10 gene in 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 ) SPCTRNASER . 09 ( ser09 ) in strong terminator mutant ( mean ± standard deviation from 4 biological replicates ; p - value obtained using Wilcoxon - Mann Whitney statistical ) .

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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 ( glm state term , false discovery rate - adjusted P value ( FDR ) < 0 . 05 log2FC > 1 ) . pluripotent TFs highlighted for each state ( maroon = naive ESCs , teal = formative EpiLCs ) .

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

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 , interaction terms . Dots represent biological replicates ( N = 3 ) ; lines highlight changes in mean values for replicates between each state ( log2FC > 1 FDR < 0 . 05 ) . A . State dependent expression is exemplified by Nanog which showed no difference between strains . B . Expression of Zfp277 exemplifies - dependence being consistently higher in B6 . C & D . A significant genotype x state ( GXS ) interaction was identified for Nr5a2 ( C , a marker for pluripotency ) Sox1 ( D , a marker for neuronal differentiation ) .

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 identity are shown for five clusters .

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 on from EBs for both B6 D2 strains . were labeled with anti - EpCAM ( top ) or biological replicates with anti - SOX1 antibody ( bottom ) . Gated population indicates percentages of EpCAM + ( Q3 ) or SOX1 + .

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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.