B . RNAseq analysis was performed on whole blood and selected transcripts that are overexpressed in asymptomatic patients over symptomatic patients are shown . Genes with false discovery rate less than 0 . 05 ( * ) , 0 . 01 ( * * ) or 0 . 001 ( * * * ) are indicated .

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B. RNAseq analysis was performed on whole blood and selected transcripts that are overexpressed in asymptomatic patients over symptomatic patients are shown. Genes with false discovery rate less than 0.05 (*), 0.01 (**) or 0.001 (***) are indicated.


C . Cell counts of DCs , CD4 T cells , CD8 T cells and B cells that express specific chemokine markers between asymptomatic ( n = 19 ) , symptomatic patients ( n = 37 ) and healthy controls ( n = 10 ) are analyzed by CyTOF and shown . Data are presented as Mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Kruskal - Wallis test with Dunn ' s multiple comparison ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-EXP_ASSAY O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O 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. Cell counts of DCs, CD4 T cells, CD8 T cells and B cells that express specific chemokine markers between asymptomatic (n=19), symptomatic patients (n=37) and healthy controls (n=10) are analyzed by CyTOF and shown. Data are presented as Mean ± SD. *P<0.05; **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison).


A . Pull - down assays of yTBP ( 61 - 240 ) with Kap95p , Kap114p and Kap121p . GST - fused yTBP ( 61 - 240 ) was incubated with recombinant Kap95p , Kap114p and Kap121p . Unbound ( S ) and bound ( B ) samples were analyzed by SDS - PAGE and stained with Coomassie blue . B . Band intensities of Kap - βs from the SDS - PAGE gels in ( A ) were quantified and normalized by GST - yTBP intensity . Quantitative plots show percent Kap - βs binding on yTBP .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O

A. Pull-down assays of yTBP (61-240) with Kap95p, Kap114p and Kap121p. GST-fused yTBP (61-240) was incubated with recombinant Kap95p, Kap114p and Kap121p. Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. B. Band intensities of Kap -βs from the SDS-PAGE gels in (A) were quantified and normalized by GST-yTBP intensity. Quantitative plots show percent Kap-βs binding on yTBP.


C - E . ITC titration curves ( upper ) and binding isotherms ( lower ) of ( C ) Kap114p , ( D ) Kap95p , and ( E ) Kap121p with yTBP ( 61 - 240 ) . Salt concentration and Kd values are indicated .

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C-E. ITC titration curves (upper) and binding isotherms (lower) of (C) Kap114p, (D) Kap95p, and (E) Kap121p with yTBP (61-240). Salt concentration and Kd values are indicated.


A , B . Purified recombinant Kap114p was mixed with RanQ69L or yTBP and analyzed by SEC . SEC ( Superdex 200 ) elution profiles of the ( A ) Kap114p  RanQ69L and ( B ) Kap114p  yTBP complexes . Peak fractions were analyzed by SDS - PAGE and stained with Coomassie blue . The void volume ( Vo ) of the peak fraction and absorbance ( a . u . ) at 280 nm for each complex is indicated .

O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O

A, B. Purified recombinant Kap114p was mixed with RanQ69L or yTBP and analyzed by SEC. SEC (Superdex 200) elution profiles of the (A) Kap114pRanQ69L and (B) Kap114pyTBP complexes. Peak fractions were analyzed by SDS-PAGE and stained with Coomassie blue. The void volume (Vo) of the peak fraction and absorbance (a.u.) at 280 nm for each complex is indicated.


SV - AUC analysis of ( C ) yTBP The experimental data were analyzed using the Sedfit program and are presented above molecular weights calculated using standards ( in brackets ) .

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SV-AUC analysis of (C) yTBP The experimental data were analyzed using the Sedfit program and are presented above molecular weights calculated using standards (in brackets).


SV - AUC ( D ) Kap114p and ( E ) the Kap114p  yTBP complex . The experimental data were analyzed using the Sedfit program and are presented above molecular weights calculated using standards ( in brackets ) .

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SV-AUC (D) Kap114p and (E) the Kap114pyTBP complex. The experimental data were analyzed using the Sedfit program and are presented above molecular weights calculated using standards (in brackets).


F . SE - AUC analysis of the Kap114p  yTBP complex . The average molecular mass determined by the Sedfit program was ~ 133 . 5 kDa . The lower panel shows the residual difference ( standard deviation ) between experimental and fitted values .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. SE-AUC analysis of the Kap114pyTBP complex. The average molecular mass determined by the Sedfit program was ~133.5  kDa. The lower panel shows the residual difference (standard deviation) between experimental and fitted values.


Plots of the distance distribution function P ( r ) for the ( G ) Kap114p  RanQ69L complexes derived from experimental SAXS data using the GNOM program . Rg - based dimensionless Kratky plot of the Kap114p  RanQ69L ( H ) complexes . Intersection of the two dotted gray lines indicates features of a compact protein .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O

Plots of the distance distribution function P(r) for the (G) Kap114pRanQ69L complexes derived from experimental SAXS data using the GNOM program. Rg-based dimensionless Kratky plot of the Kap114pRanQ69L (H) complexes. Intersection of the two dotted gray lines indicates features of a compact protein.


The ab initio envelopes determined by GASBOR for the ( I ) Kap114p  RanQ69L complexes . Models of the ( J ) Kap114p  RanQ69L complexes derived from SAXS profiles using FoXSDock and docked into the ab initio envelopes obtained from GASBOR .

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The ab initio envelopes determined by GASBOR for the (I) Kap114pRanQ69L complexes. Models of the (J) Kap114pRanQ69L complexes derived from SAXS profiles using FoXSDock and docked into the ab initio envelopes obtained from GASBOR.


Plots of the distance distribution function P ( r ) for the ( K ) Kap114p  yTBP complexes derived from experimental SAXS data using the GNOM program . Rg - based dimensionless Kratky plot of the Kap114p  yTBP ( L ) complexes . Intersection of the two dotted gray lines indicates features of a compact protein .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

Plots of the distance distribution function P(r) for the (K) Kap114pyTBP complexes derived from experimental SAXS data using the GNOM program. Rg-based dimensionless Kratky plot of the Kap114pyTBP (L) complexes. Intersection of the two dotted gray lines indicates features of a compact protein.


B . Pull - down assays of yTBP with Kap114p , Kap114p ( Δ347 - 371 ) and Kap114p ( Δ899 - 956 ) . GST - fused yTBP ( 61 - 240 ) was incubated with recombinant Kap114p , Kap114p ( Δ347 - 371 ) and Kap114p ( Δ899 - 956 ) . Unbound ( S ) and bound ( B ) samples were analyzed by SDS - PAGE and stained with Coomassie blue . C . Band intensities of Kap - βs from the SDS - PAGE gels in ( B ) were quantified and normalized by GST - yTBP intensity . Quantitative plots show percent Kap - βs binding on yTBP .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O

B. Pull-down assays of yTBP with Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). GST-fused yTBP (61-240) was incubated with recombinant Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. C. Band intensities of Kap-βs from the SDS-PAGE gels in (B) were quantified and normalized by GST-yTBP intensity. Quantitative plots show percent Kap-βs binding on yTBP.


D , E . ITC titration curves ( upper ) and binding isotherms ( lower ) of ( D ) Kap114p ( Δ347 - 371 ) and ( E ) Kap114p ( Δ899 - 956 ) with yTBP ( 61 - 240 ) . Salt concentration and Kd values are indicated .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O

D, E. ITC titration curves (upper) and binding isotherms (lower) of (D) Kap114p (Δ347-371) and (E) Kap114p (Δ899-956) with yTBP (61-240). Salt concentration and Kd values are indicated.


F . Relative fluorescence GFP intensities in the nucleus and cytoplasm of wild type , KAP114 knockout strains , and KAP114 knockout strains rescued by KAP114 deletion mutants .

O O O O B-GENEPROD O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O

F. Relative fluorescence GFP intensities in the nucleus and cytoplasm of wild type, KAP114 knockout strains, and KAP114 knockout strains rescued by KAP114 deletion mutants.


G . His - tagged RanQ69L was incubated with recombinant Kap114p , Kap114p ( Δ347 - 371 ) and Kap114p ( Δ899 - 956 ) . Unbound ( S ) and bound ( B ) samples were analyzed by SDS - PAGE and stained with Coomassie blue . H . Band intensities of Kap - βs from the SDS - PAGE gels in ( G ) were quantified and normalized by His - Ran intensity . Quantitative plots show percent Kap - βs binding on His - Ran .

O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O

G. His-tagged RanQ69L was incubated with recombinant Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. H. Band intensities of Kap-βs from the SDS-PAGE gels in (G) were quantified and normalized by His-Ran intensity. Quantitative plots show percent Kap-βs binding on His-Ran.


I . GST pull - down assays of yTBP ( 61 - 240 ) with Kap114p or Kap114p ( Δ347 - 371 ) in the presence or absence of RanQ69L . GST - fused yTBP ( 61 - 240 ) was incubated with recombinant Kap114p and His - tagged RanQ69L at a molar ratio of 1 : 4 : 8 Kap114p : yTBP : RanQ69L . Unbound ( S ) and bound ( B ) samples were analyzed by SDS - PAGE and stained with Coomassie blue . J . Analysis of Kap114p dissociation from yTBP in the presence of RanQ69L . Band intensities of Kap114p and Kap114p ( Δ347 - 371 ) from the SDS - PAGE gels were used to determine the average fraction of bound protein .

O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O

I. GST pull-down assays of yTBP (61-240) with Kap114p or Kap114p (Δ347-371) in the presence or absence of RanQ69L. GST-fused yTBP (61-240) was incubated with recombinant Kap114p and His-tagged RanQ69L at a molar ratio of 1:4:8 Kap114p:yTBP:RanQ69L. Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. J. Analysis of Kap114p dissociation from yTBP in the presence of RanQ69L. Band intensities of Kap114p and Kap114p (Δ347-371) from the SDS-PAGE gels were used to determine the average fraction of bound protein.


B . EMSA analysis of yTBP binding to DNA . We incubated 5 nM FAM - labeled oligonucleotide ( TGTATGTATATAAAAC ) with indicated concentrations of purified yTBP ( 0 . 125 to 8 μM ) and then analyzed binding by 6 % non - denaturing PAGE . Band intensities of free DNA from the gels ( Top panel ) were quantified and plotted ( Bottom panel ) .

O O B-EXP_ASSAY O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

B. EMSA analysis of yTBP binding to DNA. We incubated 5 nM FAM-labeled oligonucleotide (TGTATGTATATAAAAC) with indicated concentrations of purified yTBP (0.125 to 8 μM) and then analyzed binding by 6% non-denaturing PAGE. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel).


C . Indicated concentrations ( 0 . 25 to 16 μM ) of purified recombinant Kap114p were incubated with yTBP ( 4 μM ) and FAM - labeled DNA ( 5 nM ) , followed by native PAGE analysis . Kap114p ( 16 μM ) was incubated with DNA without yTBP as a control . Band intensities of free DNA from the gels ( Top panel ) were quantified and plotted ( Bottom panel ) .

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C. Indicated concentrations (0.25 to 16 μM) of purified recombinant Kap114p were incubated with yTBP (4 μM) and FAM-labeled DNA (5 nM), followed by native PAGE analysis. Kap114p (16 μM) was incubated with DNA without yTBP as a control. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel).


D . Super - shift EMSA analysis of TFIIA binding to yTBP  DNA complex . We incubated 5 nM FAM - labeled DNA and yTBP ( 0 . 5 μM ) with indicated concentrations of purified TFIIA ( 0 . 08 to 5 μM ) and then analyzed binding by 6 % non - denaturing PAGE . Band intensities of free DNA from the gels ( Top panel ) were quantified and plotted ( Bottom panel ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

D. Super-shift EMSA analysis of TFIIA binding to yTBPDNA complex. We incubated 5 nM FAM-labeled DNA and yTBP (0.5 μM) with indicated concentrations of purified TFIIA (0.08 to 5 μM) and then analyzed binding by 6% non-denaturing PAGE. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel).


E . Indicated concentrations ( 0 . 06 to 4 μM ) of purified Kap114p were added to the reaction containing 0 . 5 μM of TFIIA , yTBP and FAM - labeled DNA ( TGTATGTATATAAAAC ) , before conducting native PAGE analysis . TFIIA and Kap114p ( 4 μM ) were incubated with DNA without yTBP as a control . Band intensities of free DNA from the gels ( Top panel ) were quantified and plotted ( Bottom panel ) . Electrophoretic bands containing protein - DNA complex and free DNA are indicated .

O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O

E. Indicated concentrations (0.06 to 4 μM) of purified Kap114p were added to the reaction containing 0.5 μM of TFIIA, yTBP and FAM-labeled DNA (TGTATGTATATAAAAC), before conducting native PAGE analysis. TFIIA and Kap114p (4 μM) were incubated with DNA without yTBP as a control. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel). Electrophoretic bands containing protein-DNA complex and free DNA are indicated.


A . Wild type and KAP114 knockout strains were diluted serially and spotted onto YPD plates in the presence of 1 . 5 M NaCl .

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A. Wild type and KAP114 knockout strains were diluted serially and spotted onto YPD plates in the presence of 1.5 M NaCl.


B . The KAP114 knockout strain was transformed with vector control ( lane 2 ) and a plasmid containing wild type KAP114 , KAP114 ( Δ347 - 371 ) , or KAP114 ( Δ899 - 956 ) ( lanes 3 - 5 ) . Wild type and KAP114 knockout strains containing plasmids were serially diluted and grown in minimal medium plates supplemented with 1 . 5 M NaCl . Growth temperatures are indicated .

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B. The KAP114 knockout strain was transformed with vector control (lane 2) and a plasmid containing wild type KAP114, KAP114 (Δ347-371), or KAP114 (Δ899-956) (lanes 3-5). Wild type and KAP114 knockout strains containing plasmids were serially diluted and grown in minimal medium plates supplemented with 1.5 M NaCl. Growth temperatures are indicated.


C . A heatmap generated by three independent biological replicates shows gene expression profiles of wild type and KAP114 knockout strains grown in the presence of 1 . 5 M NaCl . The Log2 ( fold - change ) values and the color scale are shown .

O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

C. A heatmap generated by three independent biological replicates shows gene expression profiles of wild type and KAP114 knockout strains grown in the presence of 1.5 M NaCl. The Log2 (fold-change) values and the color scale are shown.


E , F . ( E ) qPCR analysis of gene expression in the KAP114 knockout strains ( green bars ) compared to wild type ( black bars ) under regular conditions . ( F ) qPCR analysis of gene expression in the KAP114 knockout strain ( green bars ) compared to wild type ( black bars ) grown in high salt conditions . qPCR fold - changes in mRNA expression are relative to control ( YPD media , black bars ) . Actin was used as the internal control .

O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O

E, F. (E) qPCR analysis of gene expression in the KAP114 knockout strains (green bars) compared to wild type (black bars) under regular conditions. (F) qPCR analysis of gene expression in the KAP114 knockout strain (green bars) compared to wild type (black bars) grown in high salt conditions. qPCR fold-changes in mRNA expression are relative to control (YPD media, black bars). Actin was used as the internal control.


G . ChIP - qPCR assays showing yTBP - GFP binding to the promoter of indicated genes in wild type and KAP114 knockout strains grown in high salt conditions ( light green bars ) compared to under regular conditions ( black bars ) . Chip - qPCR fold - enrichment is relative to control ( YPD media , black bars ) .

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G. ChIP-qPCR assays showing yTBP-GFP binding to the promoter of indicated genes in wild type and KAP114 knockout strains grown in high salt conditions (light green bars) compared to under regular conditions (black bars). Chip-qPCR fold-enrichment is relative to control (YPD media, black bars).


H . Endogenous Ran , GFP - yTBP and proliferating cell nuclear antigen ( PCNA ; loading control ) in nuclear fractions isolated from wild type yeast grown in regular and high salt conditions were analyzed by Western blot using antibodies for Ran , GFP and PCNA . I , J . Analysis of Ran and GFP - yTBP in nuclear fractions isolated from wild type yeast grown in regular and high salt conditions . The combined bar and scatter plot shows the band intensity ratios of Ran ( I ) and GFP - yTBP ( J ) over PCNA . Each dot represents an individual data point .

O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O B-ORGANISM O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O

H. Endogenous Ran, GFP-yTBP and proliferating cell nuclear antigen (PCNA; loading control) in nuclear fractions isolated from wild type yeast grown in regular and high salt conditions were analyzed by Western blot using antibodies for Ran, GFP and PCNA. I, J. Analysis of Ran and GFP-yTBP in nuclear fractions isolated from wild type yeast grown in regular and high salt conditions. The combined bar and scatter plot shows the band intensity ratios of Ran (I) and GFP-yTBP (J) over PCNA. Each dot represents an individual data point.


SUMO conjugation assays performed under multiple turnover conditions using wild - type or mutant forms of RPA , SUMO and Siz2 . Only the 80 - minute time points are presented to highlight the formation of Rfa1 - SUMO and Siz2 - SUMO species . The complete time series are presented in Appendix Fig S1 . Bands for Mw marker are fully annotated in Figure 1B .

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SUMO conjugation assays performed under multiple turnover conditions using wild-type or mutant forms of RPA, SUMO and Siz2. Only the 80-minute time points are presented to highlight the formation of Rfa1-SUMO and Siz2-SUMO species. The complete time series are presented in Appendix Fig S1. Bands for Mw marker are fully annotated in Figure 1B.


SUMO conjugation assays performed under multiple turnover conditions using Siz2 ∆ CT and different RPA - DNA complexes . SDS - PAGE gels were stained with Coomassie . Bands for Mw marker are fully annotated in Figure 1B .

B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O O

SUMO conjugation assays performed under multiple turnover conditions using Siz2∆CT and different RPA-DNA complexes. SDS-PAGE gels were stained with Coomassie. Bands for Mw marker are fully annotated in Figure 1B.


Plot showing the initial rate of SUMO conjugation versus RPA - DNA concentration as obtained from single - turnover reactions to measure rates of SUMO conjugation to Rfa1 with different RPA - DNA complexes . Representative gels are shown in Appendix Fig S4 .

O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O

Plot showing the initial rate of SUMO conjugation versus RPA-DNA concentration as obtained from single-turnover reactions to measure rates of SUMO conjugation to Rfa1 with different RPA-DNA complexes. Representative gels are shown in Appendix Fig S4.


SUMO conjugation assays performed under multiple turnover conditions using RPA complexes with or without the WH domain of Rfa2 in presence of Siz2 ∆ CT lacking ( A ) or including ( B ) its SAP domain . In both cases , reactions were done in technical triplicate and SDS - PAGE gels were stained with SYPRO . Only one representative gel is shown . Bands for Mw marker are fully annotated in Figure 1B .

B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O B-GENEPROD O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

SUMO conjugation assays performed under multiple turnover conditions using RPA complexes with or without the WH domain of Rfa2 in presence of Siz2∆CT lacking (A) or including (B) its SAP domain. In both cases, reactions were done in technical triplicate and SDS-PAGE gels were stained with SYPRO. Only one representative gel is shown. Bands for Mw marker are fully annotated in Figure 1B.


SUMO conjugation assays performed under multiple turnover conditions as in Fig 4 . Experiments were performed in technical triplicates ( six times for WT ) . The structural models highlight the position of the mutations using a homology model of the SAP domain of Siz2 obtained using the structure of Siz1 ( PDB 2RNN ) as a template . A dashed box indicates the relative position of residues that undergo chemical shift perturbation upon DNA binding according to ( Suzuki et al , 2009 ) . Bands for Mw marker are fully annotated in Figure 1B .

B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

SUMO conjugation assays performed under multiple turnover conditions as in Fig 4. Experiments were performed in technical triplicates (six times for WT). The structural models highlight the position of the mutations using a homology model of the SAP domain of Siz2 obtained using the structure of Siz1 (PDB 2RNN) as a template. A dashed box indicates the relative position of residues that undergo chemical shift perturbation upon DNA binding according to (Suzuki et al, 2009). Bands for Mw marker are fully annotated in Figure 1B.


SUMO conjugation assays performed under multiple turnover conditions as in Fig 4 and using different Siz2 ∆ CT variants . The structural models highlight the position of the mutations using a homology model of the SAP domain of Siz2 obtained using the structure of Siz1 ( PDB 2RNN ) as a template . A dashed box indicates the relative position of residues that undergo chemical shift perturbation upon DNA binding according to ( Suzuki et al , 2009 ) . Bands for Mw marker are fully annotated in Figure 1B .

B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

SUMO conjugation assays performed under multiple turnover conditions as in Fig 4 and using different Siz2∆CT variants. The structural models highlight the position of the mutations using a homology model of the SAP domain of Siz2 obtained using the structure of Siz1 (PDB 2RNN) as a template. A dashed box indicates the relative position of residues that undergo chemical shift perturbation upon DNA binding according to (Suzuki et al, 2009). Bands for Mw marker are fully annotated in Figure 1B.


Electrophoretic mobility shift assays ( EMSA ) to assess binding of RPA or SUMO - RPA to 25nM single - stranded 6 - FAM - labeled 32dT DNA and displacement of SUMO - RPA from 25nM SUMO - RPA bound to single - stranded 6 - FAM - labeled 32dT DNA by RPA and vice versa . All titrations were done in technical triplicates . One representative gel is shown .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O

Electrophoretic mobility shift assays (EMSA) to assess binding of RPA or SUMO-RPA to 25nM single-stranded 6-FAM-labeled 32dT DNA and displacement of SUMO-RPA from 25nM SUMO-RPA bound to single-stranded 6-FAM-labeled 32dT DNA by RPA and vice versa. All titrations were done in technical triplicates. One representative gel is shown.


Electrophoretic mobility shift assays ( EMSA ) performed with 10 nM 6 - FAM - ds20ss32 , a 6 - FAM - labeled 20 - mer dsDNA with a 32 nucleotide 3 ' overhang and its complexes with RPA , RPA with K170 - SUMO modified Rfa1 or RPA reconstituted with ∆ WH - Rfa2 and serially diluted Siz2 . All titrations were done in technical triplicates . One representative gel is shown .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-SUBCELLULAR O B-GENEPROD O B-SUBCELLULAR O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O

Electrophoretic mobility shift assays (EMSA) performed with 10 nM 6-FAM-ds20ss32, a 6-FAM-labeled 20-mer dsDNA with a 32 nucleotide 3' overhang and its complexes with RPA, RPA with K170-SUMO modified Rfa1 or RPA reconstituted with ∆WH-Rfa2 and serially diluted Siz2. All titrations were done in technical triplicates. One representative gel is shown.


C . Total USV number in YFO and AFO . Data information : Data are presented in the box plot or pie chart format . The central band in each box represents the median ; boxes indicate the middle quartiles , and whiskers extend to the minimum and maximum values . * p < 0 . 05 , determined using Student ' s or Welch t - test .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Total USV number in YFO and AFO. Data information: Data are presented in the box plot or pie chart format. The central band in each box represents the median; boxes indicate the middle quartiles, and whiskers extend to the minimum and maximum values. *p<0.05, determined using Student's or Welch t-test.


D . A heat map of the 16 hyper - and 96 hypo - methylated DMRs in sperm from aged mice ordered by their chromosomal positions . M indicates the average .

O O O O O O O O O O O O O O O O O B-CELL_TYPE O O B-ORGANISM O O O B-SUBCELLULAR O O O O O O O

D. A heat map of the 16 hyper- and 96 hypo-methylated DMRs in sperm from aged mice ordered by their chromosomal positions. M indicates the average.


A . Volcano plot for DEGs in the embryonic brain of YFO and AFO at E14 . 5 ( n = 3 in each group ) . Green lines indicate a twofold change and p = 0 . 01 . Orange dots indicate REST / NRSF target genes . Data information : Data are presented in the volcano plot or line chart format . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , determined by nominal GSEA p - value .

O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Volcano plot for DEGs in the embryonic brain of YFO and AFO at E14.5 (n=3 in each group). Green lines indicate a twofold change and p=0.01. Orange dots indicate REST/NRSF target genes. Data information: Data are presented in the volcano plot or line chart format. *p<0.05, ** p<0.01, ***p<0.001, determined by nominal GSEA p-value.


A . Volcano plot for DEGs in the embryonic brain of YFO and AFO at E11 . 5 ( n = 3 in each group ) . Green lines indicate a twofold change and p = 0 . 01 . Orange dots indicate REST / NRSF target genes . Data information : Data are presented in the volcano plot or line chart format . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , determined by volcano or nominal GSEA P - value . NES , normalized enrichment score .

O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Volcano plot for DEGs in the embryonic brain of YFO and AFO at E11.5 (n=3 in each group). Green lines indicate a twofold change and p=0.01. Orange dots indicate REST/NRSF target genes. Data information: Data are presented in the volcano plot or line chart format. *p<0.05, ** p<0.01, ***p<0.001, determined by volcano or nominal GSEA P-value. NES, normalized enrichment score.


C . Immunostaining for cortical layer markers in the postnatal brain of YFO and AFO ( representative magnified images of the white boxes in panel A ) . Satb2 + / Ctip2 - : layer 2 - 4 , Ctip2high : layer 5 , Satb2 - / Ctip2 - : layer 6 . Scale bar : 100 μm . F . Cell number in each cortical layer in the postnatal brain of YFO and AFO . Data information : All data are presented as the mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , determined using Student t - test .

O O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-TISSUE I-TISSUE O O O B-GENEPROD O B-TISSUE I-TISSUE O B-GENEPROD O O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE O O O B-TISSUE O O O O O O O O O O O O O O O O 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. Immunostaining for cortical layer markers in the postnatal brain of YFO and AFO (representative magnified images of the white boxes in panel A). Satb2+/Ctip2-: layer 2-4, Ctip2high: layer 5, Satb2-/Ctip2-: layer 6. Scale bar: 100 μm. F. Cell number in each cortical layer in the postnatal brain of YFO and AFO. Data information: All data are presented as the mean ± SEM. *p<0.05, **p<0.01, determined using Student t-test.


D . Immunostaining for another cortical layer markers in the postnatal brain of YFO and AFO ( representative magnified images of the white boxes in panel A ) . Tbr1 - / Ctip2 - : layer 2 - 4 , Ctip2high : layer 5 , Tbr1 + / Ctip2 - : layer 6 . Scale bar : 100 μm . G . Cell density in each cortical layer in the postnatal brain of YFO and AFO . Data information : All data are presented as the mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , determined using Student t - test .

O O B-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-TISSUE I-TISSUE O O O B-GENEPROD O B-TISSUE I-TISSUE O B-GENEPROD O O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE O O O B-TISSUE O O O O O O O O O O O O O O O O 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. Immunostaining for another cortical layer markers in the postnatal brain of YFO and AFO (representative magnified images of the white boxes in panel A). Tbr1-/Ctip2-: layer 2-4, Ctip2high: layer 5, Tbr1+/Ctip2-: layer 6. Scale bar: 100 μm. G. Cell density in each cortical layer in the postnatal brain of YFO and AFO. Data information: All data are presented as the mean ± SEM. *p<0.05, **p<0.01, determined using Student t-test.


C . Representative image of 5 - mC immunofluorescence in the sperm of saline - and 5 - Aza - treated mice ( ST and AT , respectively ) . Scale bar : 100 μm . D . Quantification analysis of 5 - mC immunofluorescence in individual sperm cells from STO ( n = 1 , 673 sperm from 4 male mice ) and ATO ( n = 2 , 675 sperm from 4 male mice ) . Data information : All data are presented in the box plot format . The central band in each box represents the median , boxes indicate the middle quartiles , whiskers extended to the minimum and maximum values . * p < 0 . 05 and * * p < 0 . 01 , determined using Student ' s or Welch t - test .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O B-CELL_TYPE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O B-CELL_TYPE O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative image of 5-mC immunofluorescence in the sperm of saline- and 5-Aza-treated mice (ST and AT, respectively). Scale bar: 100 μm. D. Quantification analysis of 5-mC immunofluorescence in individual sperm cells from STO (n=1,673 sperm from 4 male mice) and ATO (n=2,675 sperm from 4 male mice).Data information: All data are presented in the box plot format. The central band in each box represents the median, boxes indicate the middle quartiles, whiskers extended to the minimum and maximum values. *p<0.05 and **p<0.01, determined using Student's or Welch t-test.


F . Overall USV number in STO and ATO . Data information : All data are presented in the box plot format . The central band in each box represents the median , boxes indicate the middle quartiles , whiskers extended to the minimum and maximum values . * p < 0 . 05 and * * p < 0 . 01 , determined using Student ' s or Welch t - test .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Overall USV number in STO and ATO. Data information: All data are presented in the box plot format. The central band in each box represents the median, boxes indicate the middle quartiles, whiskers extended to the minimum and maximum values. *p<0.05 and **p<0.01, determined using Student's or Welch t-test.


( A ) Distribution of antimycin A resistance scores for wild isolates , compared to standard laboratory strain ( red vertical line ) . Resistance scores are the ratio of fitness on rich glucose media with vs without 500 µg / L antimycin A . Fitness was estimated based on colony size on solid media , corrected for spatial and plate effects After quality control , we obtained quantitative fitness scores for 154 strains , with a signal - to - noise ratio of 29 . 8 and an unexplained variance of 0 . 12 .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O 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-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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) Distribution of antimycin A resistance scores for wild isolates, compared to standard laboratory strain (red vertical line). Resistance scores are the ratio of fitness on rich glucose media with vs without 500 µg/L antimycin A. Fitness was estimated based on colony size on solid media, corrected for spatial and plate effects After quality control, we obtained quantitative fitness scores for 154 strains, with a signal-to-noise ratio of 29.8 and an unexplained variance of 0.12.


( B ) Volcano plot of a genome - wide association using mixed - model linear regression of antimycin A resistance for 118 , 527 genetic variants . Variants with moderate or high impact are shown in blue . Red dot : the variant at locus I : 3845516 , which causes a T343A change in the Pyk1 amino - acid sequence . This variant was among the top scoring ( effect size = - 0 . 645 , rank 34 ; p = 0 . 0008 , rank 70 ) .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O 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) Volcano plot of a genome-wide association using mixed-model linear regression of antimycin A resistance for 118,527 genetic variants. Variants with moderate or high impact are shown in blue. Red dot: the variant at locus I:3845516, which causes a T343A change in the Pyk1 amino-acid sequence. This variant was among the top scoring (effect size=-0.645, rank 34; p=0.0008, rank 70).


( C ) Boxplot showing antimycin A resistance for 154 strains grouped by the two alleles at the pyk1 locus . Strains carrying a C at this genomic locus ( orange box ) generally have higher antimycin resistance than strains carrying the reference allele T ( blue box ) . As is standard , this boxplot and all other boxplots in this manuscript show the median of the data as central line , the quartiles as box and the extend of the rest of the distribution as whiskers . Points which are 1 . 5 times the inter - quartile range beyond the high and low quartiles are considered outliers and shown individually .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Boxplot showing antimycin A resistance for 154 strains grouped by the two alleles at the pyk1 locus. Strains carrying a C at this genomic locus (orange box) generally have higher antimycin resistance than strains carrying the reference allele T (blue box). As is standard, this boxplot and all other boxplots in this manuscript show the median of the data as central line, the quartiles as box and the extend of the rest of the distribution as whiskers. Points which are 1.5 times the inter-quartile range beyond the high and low quartiles are considered outliers and shown individually.


( B ) Left boxplot : two glycolytic intermediates directly upstream of PYK were strongly depleted in the A - strain ( nT - strain = 9 , nA - strain = 8 ) . Right barplot : PYK activity was directly measured using a lactate dehydrogenase - coupled colorimetric enzyme activity assay , showing the mean and standard deviation of substrate conversion rate for 3 biological replicates , each measured in technical duplicates . Data information : Significance keys : * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 ( Welch ' s t - test )

O O O O O O O O O O O O B-GENEPROD O 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-GENEPROD B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Left boxplot: two glycolytic intermediates directly upstream of PYK were strongly depleted in the A-strain (nT-strain=9, nA-strain=8). Right barplot: PYK activity was directly measured using a lactate dehydrogenase-coupled colorimetric enzyme activity assay, showing the mean and standard deviation of substrate conversion rate for 3 biological replicates, each measured in technical duplicates. Data information: Significance keys: * p<0.05, ** p<0.005, *** p<0.0005 (Welch's t-test)


( C ) Concentrations of fructose - 1 , 6 - bisphosphate , which correlate with glycolytic flux , are significantly higher in A - strain ( ratio = 1 . 35 , nT - strain = 9 , nA - strain = 8 ) . Data information : Significance keys : * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 ( Welch ' s t - test )

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Concentrations of fructose-1,6-bisphosphate, which correlate with glycolytic flux, are significantly higher in A-strain (ratio = 1.35, nT-strain=9, nA-strain=8). Data information: Significance keys: * p<0.05, ** p<0.005, *** p<0.0005 (Welch's t-test)


( D ) Boxplots for selected metabolomics data indicate differences in energy and redox status between T - and A - strains ( nT - strain = 9 , nA - strain = 8 ) . Data information : Significance keys : * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 ( Welch ' s t - test )

O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Boxplots for selected metabolomics data indicate differences in energy and redox status between T- and A-strains (nT-strain=9, nA-strain=8). Data information: Significance keys: * p<0.05, ** p<0.005, *** p<0.0005 (Welch's t-test)


( E ) Heatmap of the 432 genes that are differentially expressed at the RNA level between the T - and A - strains ( FDR < 10 % ) and are measured in all four conditions ( columns ) . First column : genes ordered by increasing fold - changes for RNAs ( computed as log2 [ T ] - log2 [ A ] ) . Second column : fold - changes for proteins ( computed as log2 [ T ] - log2 [ A ] ) . Third column : fold - changes for RNAs in cells treated with rapamycin & caffeine ( TORC1 inhibition ; computed as log2 [ treatment ] - log2 [ control ] ) ( data from ref . ( Rallis et al , 2013 ) ) . Fourth column : fold - changes for RNAs in cells treated with H2O2 ( oxidative stress ; computed as log2 [ treatment ] - log2 [ control ] ) ( data from Chen et al , 2003 ) . Log2 fold - changes are capped at absolute values of 1 for all columns .

O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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) Heatmap of the 432 genes that are differentially expressed at the RNA level between the T- and A-strains (FDR <10%) and are measured in all four conditions (columns). First column: genes ordered by increasing fold-changes for RNAs (computed as log2[T]-log2[A]). Second column: fold-changes for proteins (computed as log2[T]-log2[A]). Third column: fold-changes for RNAs in cells treated with rapamycin & caffeine (TORC1 inhibition; computed as log2[treatment]-log2[control]) (data from ref. (Rallis et al, 2013)). Fourth column: fold-changes for RNAs in cells treated with H2O2 (oxidative stress; computed as log2[treatment]-log2[control]) (data from Chen et al, 2003). Log2 fold-changes are capped at absolute values of 1 for all columns.


( A ) Growth measured by optical density ( OD ) at 600nm for 3 biological replicates each of the T - and A - strains over 6 hrs of exponential growth in YES media . The x - axis refers to the time since inoculation of the culture . ( B ) The same data as in ( A ) plotted on a log2 scale ( solid lines ) with lines fitted ( dotted lines ) . The doubling time is the inverse of the slope .

O O O B-EXP_ASSAY O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Growth measured by optical density (OD) at 600nm for 3 biological replicates each of the T- and A-strains over 6 hrs of exponential growth in YES media. The x-axis refers to the time since inoculation of the culture. (B) The same data as in (A) plotted on a log2 scale (solid lines) with lines fitted (dotted lines). The doubling time is the inverse of the slope.


( C ) Media samples were taken from the same cultures ( n = 3 for both strains ) at the 8 hr timepoint and the remaining glucose was quantified . The consumed glucose was calculated based on the amount of glucose measured in the same , fresh media and normalised to the OD of each culture at the time of sampling .

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(C) Media samples were taken from the same cultures (n=3 for both strains) at the 8 hr timepoint and the remaining glucose was quantified. The consumed glucose was calculated based on the amount of glucose measured in the same, fresh media and normalised to the OD of each culture at the time of sampling.


( D ) Oxygen consumption rates in A - and T - strains ( ratio of means = 0 . 65 , p = 0 . 0002 , Welch ' s T - test , 3 biological replicates , each measured in technical duplicates ) . Data information : Vertical bars show the mean of the data . Error bars and shaded areas in all cases denote standard deviation . Significance keys : * p < 0 . 05 , * * * p < 0 . 0005 ( Welch ' s t - test ) .

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(D) Oxygen consumption rates in A- and T-strains (ratio of means=0.65, p=0.0002, Welch's T-test, 3 biological replicates, each measured in technical duplicates). Data information: Vertical bars show the mean of the data. Error bars and shaded areas in all cases denote standard deviation. Significance keys: * p<0.05, *** p<0.0005 (Welch's t-test).


( E ) Culture dry weight after 24 hrs was measured reported as a fraction of the weight of glucose put into the media for 3 biological replicates per strain . Data information : Vertical bars show the mean of the data . Error bars and shaded areas in all cases denote standard deviation . Significance keys : * p < 0 . 05 , * * * p < 0 . 0005 ( Welch ' s t - test ) .

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(E) Culture dry weight after 24 hrs was measured reported as a fraction of the weight of glucose put into the media for 3 biological replicates per strain. Data information: Vertical bars show the mean of the data. Error bars and shaded areas in all cases denote standard deviation. Significance keys: * p<0.05, *** p<0.0005 (Welch's t-test).


( A ) Spot assays on solid media from a three - fold dilution series of exponential cultures at the same cell density in 96 - well plates ( 3 biological replicates of each strain ) and spotted in 16 technical replicates ( each dilution in 4x4 square ) . The A - strain is more resistant to antimycin A but less resistant to oxidative stress triggered by H2O2 or diamide . A control without toxin ( left ) was included in each batch of spot assays performed and a representative image is shown here .

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(A) Spot assays on solid media from a three-fold dilution series of exponential cultures at the same cell density in 96-well plates (3 biological replicates of each strain) and spotted in 16 technical replicates (each dilution in 4x4 square). The A-strain is more resistant to antimycin A but less resistant to oxidative stress triggered by H2O2 or diamide. A control without toxin (left) was included in each batch of spot assays performed and a representative image is shown here.


( B ) Fitness ( approximated by maximum slope of smoothed growth curves ) of A - and T - strains on 12 carbon sources , with either yeast extract ( YE ) or ammonium ( NH4 ) , with or without 0 . 1 % priming glucose to support initial growth . For all 48 conditions , two biological replicates of A - and T - strains were grown in technical quadruplicates each . Dotted lines in panels mark a fitness ratio of 1 ( i . e . , same fitness ) .

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(B) Fitness (approximated by maximum slope of smoothed growth curves) of A- and T-strains on 12 carbon sources, with either yeast extract (YE) or ammonium (NH4), with or without 0.1% priming glucose to support initial growth. For all 48 conditions, two biological replicates of A- and T-strains were grown in technical quadruplicates each. Dotted lines in panels mark a fitness ratio of 1 (i.e., same fitness).


( C ) Fitness of A - and T - strains on 95 nitrogen sources on Biolog Phenotype MicroArrays . Conditions with no substantial growth were excluded ( black circles , maximum slope < 0 . 015 ) . Red lines show arbitrary significance cut - off , put at | log2 ( A - strain / T - strain ) | > 0 . 75 .

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(C) Fitness of A- and T-strains on 95 nitrogen sources on Biolog Phenotype MicroArrays. Conditions with no substantial growth were excluded (black circles, maximum slope <0.015). Red lines show arbitrary significance cut-off, put at |log2(A-strain/T-strain)|>0.75.


( D ) Fitness of A - and T - strains on 72 different drugs and toxins , at 4 concentrations each , on Biolog Phenotype MicroArrays . ( Results for benzamidine were inconclusive , with both strains comparatively resistant in one concentration each . )

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(D) Fitness of A- and T-strains on 72 different drugs and toxins, at 4 concentrations each, on Biolog Phenotype MicroArrays. (Results for benzamidine were inconclusive, with both strains comparatively resistant in one concentration each.)


( E ) Spot assays to validate selected results Several assays were performed with both h - and h90 strains , and no mating - type specific differences were evident between the two sets of allele - swap strains .

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(E) Spot assays to validate selected results Several assays were performed with both h- and h90 strains, and no mating-type specific differences were evident between the two sets of allele-swap strains.


( F ) Chronological lifespan of A - and T - strains , i . e . the proportion of non - dividing cells in stationary phase that maintain proliferative potential after refeeding . The data show colony forming units ( CFUs ) per ml of culture over 7 days of stationary phase in glucose - depleted rich media . Three biological repeats were carried out for both strains , with each repeat measured as technical triplicates . Error bars represent standard error of the biological replicates .

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(F) Chronological lifespan of A- and T-strains, i.e. the proportion of non-dividing cells in stationary phase that maintain proliferative potential after refeeding. The data show colony forming units (CFUs) per ml of culture over 7 days of stationary phase in glucose-depleted rich media. Three biological repeats were carried out for both strains, with each repeat measured as technical triplicates. Error bars represent standard error of the biological replicates.


( G ) Boxplot showing resistance to 3mM H2O2 grouped by pyk1 allele for 156 strains from our collection . The T - strains had a higher mean fitness in H2O2 than the A - strains ( 1 . 01 ± 0 . 06 vs 0 . 95 ± 0 . 11 ; p = 0 . 0021 , Welch ' s t - test ) . The resistance score was obtained as for antimycin A .

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(G) Boxplot showing resistance to 3mM H2O2 grouped by pyk1 allele for 156 strains from our collection. The T-strains had a higher mean fitness in H2O2 than the A-strains (1.01±0.06 vs 0.95±0.11; p=0.0021, Welch's t-test). The resistance score was obtained as for antimycin A.


( H ) Boxplot showing growth fitness on rich media , grouped by pyk1 allele for 158 S . pombe strains ( 0 . 88 ± 0 . 16 vs 0 . 94 ± 0 . 24 ; p = 0 . 18 , Welch ' s t - test ) .

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(H) Boxplot showing growth fitness on rich media, grouped by pyk1 allele for 158 S. pombe strains (0.88±0.16 vs 0.94±0.24; p=0.18, Welch's t-test).


B ) Log ( odds enrichment ) scores for charged GKs in the flanks of the 30 % strongest versus the 30 % weakest APRs in the E . coli cytoplasmic proteome . Whiskers indicate 95 % confidence intervals for no enrichment as determined through bootstrapping analysis .

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B) Log(odds enrichment) scores for charged GKs in the flanks of the 30 % strongest versus the 30 % weakest APRs in the E. coli cytoplasmic proteome. Whiskers indicate 95 % confidence intervals for no enrichment as determined through bootstrapping analysis.


C ) Percentual difference in non - synonymous mutation rate in E . coli for GK versus background i . e . versus the corresponding residue in non - gatekeeper positions .

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C) Percentual difference in non-synonymous mutation rate in E. coli for GK versus background i.e. versus the corresponding residue in non-gatekeeper positions.


D ) Percentual difference in non - synonymous mutation rate in mammalian species for GK versus background i . e . versus the corresponding residue in non - gatekeeper positions .

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D) Percentual difference in non-synonymous mutation rate in mammalian species for GK versus background i.e. versus the corresponding residue in non-gatekeeper positions.


B ) Time - resolved Dynamic Light Scattering ( DLS ) analysis indicating average particle size over time for each of the peptides in the β - Gal peptide set ( aggregating core sequence SVIIWSLGN ) .

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B) Time-resolved Dynamic Light Scattering (DLS) analysis indicating average particle size over time for each of the peptides in the β-Gal peptide set (aggregating core sequence SVIIWSLGN).


C ) ThT binding kinetics of the β - Gal peptide set . Background - corrected fluorescence over time is shown for 3 independent repeats .

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C) ThT binding kinetics of the β-Gal peptide set. Background-corrected fluorescence over time is shown for 3 independent repeats.


D ) pFTAA binding kinetics of the β - Gal peptide set . Background - corrected fluorescence over time is shown for 3 independent repeats .

D) pFTAA binding kinetics of the β-Gal peptide set. Background-corrected fluorescence over time is shown for 3 independent repeats.


E ) End - point solubility analysis quantifying the concentration of peptide remaining in the supernatant following ultracentrifugation for the β - Gal peptide set . Bars depict mean concentration , whiskers indicate standard deviation ( n = 4 ) . Statistical significance was determined through two - way ANOVA with Bonferroni ' s correction for multiple comparisons . P - values for significant differences are indicated .

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E) End-point solubility analysis quantifying the concentration of peptide remaining in the supernatant following ultracentrifugation for the β-Gal peptide set. Bars depict mean concentration, whiskers indicate standard deviation (n=4). Statistical significance was determined through two-way ANOVA with Bonferroni's correction for multiple comparisons. P-values for significant differences are indicated.


F ) TEM images of the β - Gal peptide set after 3 days incubation . Top panels give an overview image of one square in a TEM grid , lower panel shows enlarged image ( magnification 12000X ) . Scale bars indicate 12 µm ( top row ) or 1 µm ( bottom row ) .

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F) TEM images of the β-Gal peptide set after 3 days incubation. Top panels give an overview image of one square in a TEM grid, lower panel shows enlarged image (magnification 12000X). Scale bars indicate 12 µm (top row) or 1 µm (bottom row).


H ) Violin and boxplots showing the distribution of end - point solubilities across 11 aggregating cores , grouped by GK . The dotted line indicates a concentration of µM , corresponding to completely insoluble peptides .

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H) Violin and boxplots showing the distribution of end-point solubilities across 11 aggregating cores, grouped by GK. The dotted line indicates a concentration of µM, corresponding to completely insoluble peptides.


I ) Solubility analysis of the in vitro solubility screen performed by Niwa et al ( Niwa et al , 2009 ) . Left panel shows combined box - and violin plots depicting the solubility distributions of proteins containing at least one strong APR ( TANGO score > 70 ) not protected by any negative GKs ( red ) versus proteins where all strong APRs are flanked by at least one negative charge ( blue ) . The right panel shows a control analysis , in which proteins were classified by the occurrence of at least one strong APR ( TANGO score > 70 ) not flanked by any positive GKs ( red ) versus proteins where all strong APRs are flanked by at least one positive charge ( blue ) . The upper boxplot whiskers indicate the largest value no further than 1 . 5 times the Inter - Quartile Range ( IQR ) from the upper hinge , the lower whiskers show the smallest value at least 1 . 5 * IQR from the lower hinge . Notches extend 1 . 58 * IQR / sqrt ( n ) from either side of the median and represent a 95 % confidence interval for the median value . Statistical significance was determined through Kruskal - Wallis test with a post - hoc Wilcoxon analysis for pairwise testing . P - values are indicated .

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I) Solubility analysis of the in vitro solubility screen performed by Niwa et al (Niwa et al, 2009). Left panel shows combined box- and violin plots depicting the solubility distributions of proteins containing at least one strong APR (TANGO score > 70) not protected by any negative GKs (red) versus proteins where all strong APRs are flanked by at least one negative charge (blue). The right panel shows a control analysis, in which proteins were classified by the occurrence of at least one strong APR (TANGO score > 70) not flanked by any positive GKs (red) versus proteins where all strong APRs are flanked by at least one positive charge (blue). The upper boxplot whiskers indicate the largest value no further than 1.5 times the Inter-Quartile Range (IQR) from the upper hinge, the lower whiskers show the smallest value at least 1.5 * IQR from the lower hinge. Notches extend 1.58 * IQR / sqrt(n) from either side of the median and represent a 95% confidence interval for the median value. Statistical significance was determined through Kruskal-Wallis test with a post-hoc Wilcoxon analysis for pairwise testing. P-values are indicated.


D ) Time - resolved Dynamic Light Scattering ( DLS ) analysis showing average particle radius over time for the β - Gal peptides flanked by both natural and non - natural GKs , at increasing NaCl concentrations ( indicated in µM ) . Structures of the amino acids flanking the aggregating core are indicated on the right - hand side , the sequence of the aggregating core is indicated below .

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D) Time-resolved Dynamic Light Scattering (DLS) analysis showing average particle radius over time for the β-Gal peptides flanked by both natural and non-natural GKs, at increasing NaCl concentrations (indicated in µM). Structures of the amino acids flanking the aggregating core are indicated on the right-hand side, the sequence of the aggregating core is indicated below.


A ) DnaK binding to cellulose - bound peptide array . Sequences of the aggregating cores are indicated in the top of the panel , the GKs flanking them in each instance are indicated above the blots .

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A) DnaK binding to cellulose-bound peptide array. Sequences of the aggregating cores are indicated in the top of the panel, the GKs flanking them in each instance are indicated above the blots.


C ) Representative Western blot of in vitro translation analysis of folding - incompetent model constructs . Bands show Total ( T ) and Soluble ( S ) fractions after centrifugation at 21 , 000 g for 30 mins . " + Dnak " indicates addition of either DnaK mix - containing DnaK , DnaJ and GrpE .

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C) Representative Western blot of in vitro translation analysis of folding-incompetent model constructs. Bands show Total (T) and Soluble (S) fractions after centrifugation at 21,000 g for 30 mins. "+ Dnak" indicates addition of either DnaK mix - containing DnaK, DnaJ and GrpE.


D ) Bar plots showing mean solubility as determined through quantification of western blot in ( C ) , whiskers indicate standard deviation ( n = 4 ) . Statistically significant differences between the - DnaK and + DnaK conditions were determined through two - way ANOVA with Bonferroni ' s correction for multiple comparisons . P - values for significant differences are indicated .

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D) Bar plots showing mean solubility as determined through quantification of western blot in (C), whiskers indicate standard deviation (n=4). Statistically significant differences between the -DnaK and +DnaK conditions were determined through two-way ANOVA with Bonferroni's correction for multiple comparisons. P-values for significant differences are indicated.


E ) Representative western blot of the solubility analysis of GK variants upon overexpression in E . coli K12 . Bands show Soluble ( Sol ) and Pellet fractions after centrifugation at 17 , 100 g for 15 mins . " - DnaK " indicates expression in E . coli K12 DnaK knockout line , " + Dnak " indicates co - expression of pKJE7 , encoding DnaK , DnaJ and GrpE .

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E) Representative western blot of the solubility analysis of GK variants upon overexpression in E. coli K12. Bands show Soluble (Sol) and Pellet fractions after centrifugation at 17,100 g for 15 mins. "-DnaK" indicates expression in E. coli K12 DnaK knockout line, "+ Dnak" indicates co-expression of pKJE7, encoding DnaK, DnaJ and GrpE.


F ) Barplots showing mean solubility as determined through quantification of western blot bands in ( E ) , whiskers indicate standard deviation ( n = 7 , except for the AA construct , where n = 4 ) . Statistical significance was determined through two - way ANOVA followed by Tukey ' s multiple comparisons test . P - values are indicated .

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F) Barplots showing mean solubility as determined through quantification of western blot bands in (E), whiskers indicate standard deviation (n=7, except for the AA construct, where n=4). Statistical significance was determined through two-way ANOVA followed by Tukey's multiple comparisons test. P-values are indicated.


G ) Structured Illumination Microsopy ( SIM ) images of E . coli after three hours of expression of one of the GK variants . GFP fluorescence is shown in green . " ΔDnaK " indicates expression in E . coli K12 DnaK knockout line , " + Dnak " indicates co - expression of pKJE7 , encoding DnaK , DnaJ and GrpE .

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G) Structured Illumination Microsopy (SIM) images of E. coli after three hours of expression of one of the GK variants. GFP fluorescence is shown in green. "ΔDnaK" indicates expression in E. coli K12 DnaK knockout line, "+ Dnak" indicates co-expression of pKJE7, encoding DnaK, DnaJ and GrpE.


Plasma corticosterone levels at baseline , 30 minutes after induction of stress by placing mice in a clean cage , and after 90 minutes upon return in the home cage . DEX mice received an injection of dexamethasone at 0 . 1 mg / kg body weight 150 minutes before collecting the first blood sample ( n = 5 - 6 animals per group ) . Flox and GRBATKO mice were injected with 0 . 02 mg / kg dexamethasone and the procedure described in ( A ) was followed to induce stress in order to detect potential differences between Flox and GRBATKO mice which could have been obscured by the higher concentration used in ( A ) ( n = 6 - 7 animals per group ) .

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Plasma corticosterone levels at baseline, 30 minutes after induction of stress by placing mice in a clean cage, and after 90 minutes upon return in the home cage. DEX mice received an injection of dexamethasone at 0.1 mg/kg body weight 150 minutes before collecting the first blood sample (n = 5-6 animals per group). Flox and GRBATKO mice were injected with 0.02 mg/kg dexamethasone and the procedure described in (A) was followed to induce stress in order to detect potential differences between Flox and GRBATKO mice which could have been obscured by the higher concentration used in (A) (n = 6-7 animals per group).


GR mRNA levels in BAT of Flox and GRBATKO mice . Organs were harvested in the stressed condition after placing mice for 30 minutes in a clean cage . Half of the animals received a dexamethasone injection 180 minutes before onset of the procedure , controls received an injection with saline ( n = 5 - 6 animals per group ) .

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GR mRNA levels in BAT of Flox and GRBATKO mice. Organs were harvested in the stressed condition after placing mice for 30 minutes in a clean cage. Half of the animals received a dexamethasone injection 180 minutes before onset of the procedure, controls received an injection with saline (n = 5-6 animals per group).


Serum corticosterone levels under stressed conditions in Flox and GRBATKO mice ( n = 5 - 6 animals per group ) . Blood glucose levels ( n = 5 - 6 animals per group ) .

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Serum corticosterone levels under stressed conditions in Flox and GRBATKO mice (n = 5-6 animals per group). Blood glucose levels (n = 5-6 animals per group).


Knockdown of GR in BAT assessed by immunoblotting . Β - Tubulin was used as loading control . Quantification of immunoblot in ( A ) using Image Lab ( n = 6 animals per group ) .

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Knockdown of GR in BAT assessed by immunoblotting. Β-Tubulin was used as loading control. Quantification of immunoblot in (A) using Image Lab (n = 6 animals per group).


Body temperature over the course of the cold exposure , determined daily ( n = 6 animals per group ) .

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Body temperature over the course of the cold exposure, determined daily (n = 6 animals per group).


Body weight at the end of the cold exposure ( n = 6 animals per group ) .

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Body weight at the end of the cold exposure (n = 6 animals per group).


BAT weight at the end of the cold exposure ( n = 6 animals per group ) .

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BAT weight at the end of the cold exposure (n = 6 animals per group).


Representative Hematoxilin and eosin staining image of BAT of cold - exposed Flox and GRBATKO mice . Scale bar 50 µm .

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Representative Hematoxilin and eosin staining image of BAT of cold-exposed Flox and GRBATKO mice. Scale bar 50 µm.


Triglyceride content in BAT from Flox and GRBATKO animals ( n = 5 - 6 animals per group ) .

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Triglyceride content in BAT from Flox and GRBATKO animals (n = 5-6 animals per group).


Heatmap of mean gene expression fold changes in thermogenic marker genes based on BAT microarray analysis comparing GRBATKO vs Flox control animals , indicating unchanged thermogenic marker gene expression . Color gradient ranges from 2 - fold higher expression in GRBATKO over Flox controls ( red ) to 0 . 1 - fold ( lower ) expression ( blue ) ( n = 4 animals per group ) .

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Heatmap of mean gene expression fold changes in thermogenic marker genes based on BAT microarray analysis comparing GRBATKO vs Flox control animals, indicating unchanged thermogenic marker gene expression. Color gradient ranges from 2-fold higher expression in GRBATKO over Flox controls (red) to 0.1-fold (lower) expression (blue) (n = 4 animals per group).


Volcano plot from microarray gene expression data as in ( I ) , with cut - off values for the adjusted p - value < 0 . 1 and fold change > 1 . 5 .

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Volcano plot from microarray gene expression data as in (I), with cut-off values for the adjusted p-value <0.1 and fold change >1.5.


Serum corticosterone levels ( n = 6 animals per group ) .

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Serum corticosterone levels (n = 6 animals per group).


Body weight after 16 h fasting and 6 h chow refeeding at 22 ° C ( n = 6 animals per group )

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Body weight after 16 h fasting and 6 h chow refeeding at 22°C (n = 6 animals per group)


Body temperature assessed after 16 h fasting and subsequent refeeding for 6 h ( n = 6 animals per group ) .

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Body temperature assessed after 16 h fasting and subsequent refeeding for 6 h (n = 6 animals per group).


Serum insulin and corticosterone levels determined by ELISA ( n = 6 animals per group ) .

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Serum insulin and corticosterone levels determined by ELISA (n = 6 animals per group).


Serum NEFA and triglycerides concentration in Flox and GRBATKO mice after fasting and refeeding ( n = 6 animals per group ) .

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Serum NEFA and triglycerides concentration in Flox and GRBATKO mice after fasting and refeeding (n = 6 animals per group).


Body weight gain over a 24 - week period of high - fat feeding ( n = 13 - 14 animals per group ) . Panel shows experimental design for Figures 4 - 6 .

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Body weight gain over a 24-week period of high-fat feeding (n = 13-14 animals per group). Panel shows experimental design for Figures 4-6.


Oxygen consumption over a 90 - hour period . Grays columns indicate dark phase ( n = 6 - 8 mice per group ) . Average oxygen consumption specified for light phase , dark phase , and total ( n = 6 - 8 animals per genotype ) .

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Oxygen consumption over a 90-hour period. Grays columns indicate dark phase (n = 6-8 mice per group). Average oxygen consumption specified for light phase, dark phase, and total (n = 6-8 animals per genotype).


Total oxygen consumption plotted against body weight indicating that lower energy expenditure in GRBATKO animals is independent of body weight ( n = 6 - 8 animals per group ) .

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Total oxygen consumption plotted against body weight indicating that lower energy expenditure in GRBATKO animals is independent of body weight (n = 6-8 animals per group).


Locomotor activity over a 90 - hour period ( n = 6 - 8 animals per group ) .

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Locomotor activity over a 90-hour period (n = 6-8 animals per group).


Intraperitoneal GTT after 19 weeks of HFD - feeding , glucose injections at 1 . 5 mg per g of body weight ( n = 11 - 13 animals per group ) . Area under the curve calculated from ( A ) ( n = 11 - 13 animals per group ) .

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Intraperitoneal GTT after 19 weeks of HFD-feeding, glucose injections at 1.5 mg per g of body weight (n = 11-13 animals per group). Area under the curve calculated from (A) (n = 11-13 animals per group).


Plasma insulin levels during GTT ( n = 11 - 13 animals per group ) .

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Plasma insulin levels during GTT (n = 11-13 animals per group).


Intraperitoneal ITT after 20 weeks of high - fat diet intervention . Insulin was adjusted to 1 mU per g of body weight ( n = 11 - 13 animals per group ) . Area under the curve determined from ( D ) ( n = 11 - 13 animals per group ) .

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Intraperitoneal ITT after 20 weeks of high-fat diet intervention. Insulin was adjusted to 1 mU per g of body weight (n = 11-13 animals per group). Area under the curve determined from (D) (n = 11-13 animals per group).


Plasma insulin levels after a 5 - hour fast , before injecting insulin ( n = 11 - 13 animals per group ) .

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Plasma insulin levels after a 5-hour fast, before injecting insulin (n = 11-13 animals per group).


Body weight in Flox and GRBATKO after 24 weeks of high - fat feeding ( n = 10 - 13 animals per group ) .

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Body weight in Flox and GRBATKO after 24 weeks of high-fat feeding (n = 10-13 animals per group).


BAT and iWAT weight in Flox and GRBATKO mice fed HFD for 24 weeks ( n = 10 - 13 animals per group ) .

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BAT and iWAT weight in Flox and GRBATKO mice fed HFD for 24 weeks (n = 10-13 animals per group).


GR levels in BAT after 24 weeks high - fat feeding ( n = 10 - 13 mice per group ) .

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GR levels in BAT after 24 weeks high-fat feeding (n=10-13 mice per group).


Representative H & E stained sections of BAT from Flox and GRBATKO animals . Scale bar 100 µm .

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Representative H&E stained sections of BAT from Flox and GRBATKO animals. Scale bar 100 µm.


Triglyceride content in BAT per gram of wet tissue ( n = 10 - 13 animals per group ) .

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Triglyceride content in BAT per gram of wet tissue (n = 10-13 animals per group).


qRT - PCR analysis of thermogenic marker gene expression in BAT of Flox and GRBATKO mice after high - fat feeding ( n = 9 - 13 animals per group ) .

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qRT-PCR analysis of thermogenic marker gene expression in BAT of Flox and GRBATKO mice after high-fat feeding (n = 9-13 animals per group).


Representative immunohistochemistry for UCP1 in BAT . Scale bar 100 µm .

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Representative immunohistochemistry for UCP1 in BAT. Scale bar 100 µm.


Serum insulin levels determined by ELISA ( n = 10 - 13 animals per group ) .

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Serum insulin levels determined by ELISA (n = 10-13 animals per group).


Blood glucose in Flox and GRBATKO animals after 24 weeks HFD ( n = 10 - 13 animals per group ) .

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Blood glucose in Flox and GRBATKO animals after 24 weeks HFD (n = 10-13 animals per group).


( A ) Heat map representation of the TNF - α - up - regulated genes . Genes were classified based on the TNF - α - treated time points , using the algorithm " HOPACH " .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O

(A) Heat map representation of the TNF-α-up-regulated genes. Genes were classified based on the TNF-α-treated time points, using the algorithm "HOPACH".


( B ) Heat map showing down - regulated miRNAs at 4 hrs , but not 24 hrs , after TNF - α treatment . The middle column indicates the fold - change values ( control / TNF ) at 4 hrs . The right column indicates the relative expression level of each miRNA from the FANTOM5 database ( Consortium et al , 2014 ) . Asterisks indicate the top three most down - regulated miRNAs which were reported to be expressed in human endothelial cells ( ECs ) according to the FANTOM5 database .

O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O 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-ORGANISM B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O

(B) Heat map showing down-regulated miRNAs at 4 hrs, but not 24 hrs, after TNF-α treatment. The middle column indicates the fold-change values (control/TNF) at 4 hrs. The right column indicates the relative expression level of each miRNA from the FANTOM5 database (Consortium et al, 2014). Asterisks indicate the top three most down-regulated miRNAs which were reported to be expressed in human endothelial cells (ECs) according to the FANTOM5 database.


( C ) Bar plots showing mean mRNA levels of vascular cell adhesion molecule - 1 ( VCAM1 ) , intracellular adhesion molecule - 1 ( ICAM1 ) , and E - selectin ( SELE ) , measured 4 hrs after stimulation of human ECs with or without TNF - α ± miR - 3679 - 5p . Data are shown as means ± SE ( n = 3 ) . * P < 0 . 05 compared to TNF - α ( - ) mimics ( - ) . † P < 0 . 05 and ‡ P = 0 . 203 compared to TNF - α ( + ) mimics ( - ) . Statistical differences were analyzed by the Tukey - Kramer test .

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(C) Bar plots showing mean mRNA levels of vascular cell adhesion molecule-1 (VCAM1), intracellular adhesion molecule-1 (ICAM1), and E-selectin (SELE), measured 4 hrs after stimulation of human ECs with or without TNF-α ± miR-3679-5p. Data are shown as means ± SE (n=3). *P < 0.05 compared to TNF-α (-) mimics (-). † P < 0.05 and ‡ P = 0.203 compared to TNF-α (+) mimics (-). Statistical differences were analyzed by the Tukey-Kramer test.


( D ) Western blots for VCAM1 , ICAM , SELE , and β - actin ( ACTB ) in lysates from ECs treated with or without TNF - α ( 4 hrs ) ± miR - 3679 - 5p .

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(D) Western blots for VCAM1, ICAM, SELE, and β-actin (ACTB) in lysates from ECs treated with or without TNF-α (4 hrs) ± miR-3679-5p.


( E ) Representative images ( top ) and bar plot quantification ( bottom ) showing the adhesion of calcein - labeled U937 monocytes to ECs treated with or without TNF - α ± miR - 3679 - 5p . Scale bar represents 250 μm . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) mimics ( - ) . † P < 0 . 05 compared to TNF - α ( + ) mimics ( - ) . Statistical differences were analyzed by the Tukey - Kramer test .

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(E) Representative images (top) and bar plot quantification (bottom) showing the adhesion of calcein-labeled U937 monocytes to ECs treated with or without TNF-α ± miR-3679-5p. Scale bar represents 250 μm. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-) mimics (-). † P < 0.05 compared to TNF-α (+) mimics (-). Statistical differences were analyzed by the Tukey-Kramer test.


( F ) The scatter plot shows the correlation of RNA immunoprecipitation ( RIP ) followed by microarray data from two replicate screens for the identification of miR - 3679 - 5p target genes . Genes up - and down - regulated by miR - 3679 - 5p treatment ( | average Log2FC | > 1 ) are indicated in red and blue , respectively .

O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O

(F) The scatter plot shows the correlation of RNA immunoprecipitation (RIP)　followed by microarray data from two replicate screens for the identification of miR-3679-5p target genes. Genes up- and down-regulated by miR-3679-5p treatment (|average Log2FC|>1) are indicated in red and blue, respectively.


( J and K ) Effect of miR - 3679 - 5p mimics ( J ) or miR - 3679 - 5p inhibitor ( K ) on luciferase activity in pmirGLO - transfected ECs expressing the 3 ′ UTR of KDM7A . Data are shown as means ± SE ( n = 3 ) . * P < 0 . 05 compared to control . Statistical differences were analyzed by the Student ' s t test .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J and K) Effect of miR-3679-5p mimics (J) or miR-3679-5p inhibitor (K) on luciferase activity in pmirGLO-transfected ECs expressing the 3′ UTR of KDM7A. Data are shown as means ± SE (n=3). *P < 0.05 compared to control. Statistical differences were analyzed by the Student's t test.


( M and N ) The effect of miR - 3679 - 5p mimics ( M ) or miR - 3679 - 5p inhibitor ( N ) on luciferase activity in pmirGLO - transfected ECs expressing the 3 ′ UTR of UTX was measured . Data are shown as means ± SE ( n = 3 ) . * P < 0 . 05 compared to control . Statistical differences were analyzed by the Student ' s t test .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(M and N) The effect of miR-3679-5p mimics (M) or miR-3679-5p inhibitor (N) on luciferase activity in pmirGLO-transfected ECs expressing the 3′ UTR of UTX was measured. Data are shown as means ± SE (n=3). *P < 0.05 compared to control. Statistical differences were analyzed by the Student's t test.


( A ) Heat map representation of RNA - seq results . The p65 - dependent genes ( total 123 ; defined as genes up - regulated more than 2 - fold via TNF - α treatment and down - regulated more than 0 . 75 - fold via BAY 11 - 7082 treatment ) were classified based on the alteration patterns by the siRNA knockdown of KDM7A , UTX , and both , using the algorithm " HOPACH " .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O

(A) Heat map representation of RNA-seq results. The p65-dependent genes (total 123; defined as genes up-regulated more than 2-fold via TNF-α treatment and down-regulated more than 0.75-fold via BAY 11-7082 treatment) were classified based on the alteration patterns by the siRNA knockdown of KDM7A, UTX, and both, using the algorithm "HOPACH".


( C - E ) Bar plot showing mean mRNA levels of VCAM1 , ICAM1 , and SELE measured 4 hrs after stimulation of ECs with or without TNF - α ± ( siKDM7A + siUTX ) . Data are shown as means ± SE ( n = 3 ) . * P < 0 . 05 compared to TNF - α ( - ) siKDM7A + siUTX ( - ) . † P < 0 . 05 compared to TNF - α ( + ) siKDM7A + siUTX ( - ) . Statistical differences were analyzed by the Tukey - Kramer test .

O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O

(C-E) Bar plot showing mean mRNA levels of VCAM1, ICAM1, and SELE measured 4 hrs after stimulation of ECs with or without TNF-α ± (siKDM7A+siUTX). Data are shown as means ± SE (n=3). *P < 0.05 compared to TNF-α (-) siKDM7A+siUTX (-). † P < 0.05 compared to TNF-α (+) siKDM7A+siUTX (-). Statistical differences were analyzed by the Tukey-Kramer test.


( F ) Western blots for VCAM1 , ICAM , SELE , and ACTB in lysates from ECs treated with or without TNF - α ( 4 hrs ) ± ( siKDM7A + siUTX ) .

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(F) Western blots for VCAM1, ICAM, SELE, and ACTB in lysates from ECs treated with or without TNF-α (4 hrs) ± (siKDM7A+siUTX).


( G ) Representative images ( top ) and bar plot quantification ( bottom ) showing the adhesion of calcein - labeled U937 monocytes to ECs treated with or without TNF - α ± ( siKDM7A + siUTX ) . Scale bar represents 250 μm . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) siKDM7A + siUTX ( - ) . † P < 0 . 05 compared to TNF - α ( + ) siKDM7A + siUTX ( - ) . Statistical differences were analyzed by the Tukey - Kramer test .

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(G) Representative images (top) and bar plot quantification (bottom) showing the adhesion of calcein-labeled U937 monocytes to ECs treated with or without TNF-α ± (siKDM7A+siUTX). Scale bar represents 250 μm. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-) siKDM7A+siUTX (-). † P < 0.05 compared to TNF-α (+) siKDM7A+siUTX (-). Statistical differences were analyzed by the Tukey-Kramer test.


( A and B ) Scatter plots showing the connection between KDM7A or UTX binding and RNA expression in TNF - α - stimulated ECs . The X and Y axes indicate the log2 ratio of fragments per kilobase of exon per million mapped sequence reads ( FPKM ) values and the ChIP - seq signals ( TNF - α 60min / 0min ) for KDM7A ( A ) and UTX ( B ) , respectively . Genes with more than a 1 . 5 - fold increase in FPKM values ( light red dots ) satisfying a 0 . 5 - fold increase in ChIP - seq signals are indicated as dark red dots . Genes with a 1 . 5 - fold decrease in FPKM values ( light blue dots ) satisfying a 0 . 5 - fold decrease in ChIP - seq signals are indicated as dark blue dots . These genes are defined as the target genes for KDM7A ( A ) and UTX ( B ) , respectively . The names of representative genes are shown in the graphs .

O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

(A and B) Scatter plots showing the connection between KDM7A or UTX binding and RNA expression in TNF-α-stimulated ECs. The X and Y axes indicate the log2 ratio of fragments per kilobase of exon per million mapped sequence reads (FPKM) values and the ChIP-seq signals (TNF-α 60min/0min) for KDM7A (A) and UTX (B), respectively. Genes with more than a 1.5-fold increase in FPKM values (light red dots) satisfying a 0.5-fold increase in ChIP-seq signals are indicated as dark red dots. Genes with a 1.5-fold decrease in FPKM values (light blue dots) satisfying a 0.5-fold decrease in ChIP-seq signals are indicated as dark blue dots. These genes are defined as the target genes for KDM7A (A) and UTX (B), respectively. The names of representative genes are shown in the graphs.