Figure 5A . 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 . 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 . 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 .

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Figure 5A. 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.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.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.


Figure 1SUMO 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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Figure 1SUMO 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.


Figure 2SUMO 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 .

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Figure 2SUMO 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.


Figure 3Plot 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 .

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Figure 3Plot 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.


Figure 4SUMO 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 .

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Figure 4SUMO 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.


Figure 6SUMO 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 .

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Figure 6SUMO 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.


Figure 7SUMO 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 .

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Figure 7SUMO 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.


Figure 8Electrophoretic 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 .

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Figure 8Electrophoretic 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.


Figure 9Electrophoretic 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 .

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Figure 9Electrophoretic 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.


Figure 1C . 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 .

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Figure 1C. 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.


Figure 2D . 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 .

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Figure 2D. 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.


Figure 4A . 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 .

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Figure 4A. 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.


Figure 5A . 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 .

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Figure 5A. 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.


Figure 6C . 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 .

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Figure 6C. 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.


Figure 7C . 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 .

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Figure 7C. 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.


Figure 1 ( 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 ) . ( 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 .

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Figure 1(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).(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.


Figure 2 ( 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 ) ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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)(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.


Figure 4 ( 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 . ( 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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.(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).


Figure 5 ( 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 ) . ( 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 . ) ( 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 . ( 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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).(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.)(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.(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).


Figure 2B ) 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B) 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.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.


Figure 3B ) 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 . 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 . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B) 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.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.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.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.


Figure 4D ) 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4D) 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.


Figure 6A ) 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 . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A) 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.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.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.


Figure 1Plasma 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 ) . 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Plasma 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).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).


Figure 2Knockdown 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 ) . 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 ) . 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 ) . 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 . Serum corticosterone levels ( n = 6 animals per group ) .

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Figure 2Knockdown 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).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).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).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.Serum corticosterone levels (n = 6 animals per group).


Figure 3Body 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 ) . Blood glucose ( n = 6 per group ) . 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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Figure 3Body 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).Blood glucose (n = 6 per group).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).


Figure 4Body 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 ) . 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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Figure 4Body 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).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).


Figure 5Intraperitoneal 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 ) . 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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Figure 5Intraperitoneal 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).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).


Figure 6Body 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 ) . 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 . 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 ) . Representative immunohistochemistry for UCP1 in BAT . Scale bar 100 µm . 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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Figure 6Body 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).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.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).Representative immunohistochemistry for UCP1 in BAT. Scale bar 100 µm.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).


Figure 1 ( 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 . ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.(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.(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.(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.


Figure 2 ( 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 . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.(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.


Figure 3 ( 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 . ( C and D ) Metagene representations of KDM7A ( C ) and UTX ( D ) ChIP - seq signals in units of read count per million mapped reads at a meta composite of the genomic regions around the TNF - α - up - regulated genes . ( F ) Heat map of H3K27ac ( green ) , p65 ( yellow ) , KDM7A ( red ) , and UTX ( purple ) levels in resting and TNF - α - stimulated ECs . Each row shows ± 10 kb centered on the KDM7A ( top ) or UTX ( bottom ) peak . Rows are ordered by max KDM7A or UTX signal in each region . The ChIP - seq signal is depicted by color scaled intensities . ( G and H ) Gene tracks of ChIP - seq signals for H3K27Ac , p65 , KDM7A , and UTX , and RNA - seq signals around the VCAM1 ( G ) and SELE ( H ) loci in TNF - α ( - ) and TNF - α ( + ) ECs . ChIP - Seq and RNA - seq signals are visualized by Integrated Genome Viewer ( http : / / software . broadinstitute . org / software / igv / ) . ( I and J ) ChIP - qPCR of p65 ( I ) and KDM7A ( J ) at the VCAM1 TSS , normalized to input . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) . † P < 0 . 05 compared to TNF - α ( + ) siKDM7A ( - ) . Statistical differences were analyzed by the Tukey - Kramer test . ( K and L ) ChIP - qPCR of p65 ( K ) and UTX ( L ) at the SELE TSS , normalized to input . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) . Statistical differences were analyzed by the Tukey - Kramer test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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.(C and D) Metagene representations of KDM7A (C) and UTX (D) ChIP-seq signals in units of read count per million mapped reads at a meta composite of the genomic regions around the TNF-α-up-regulated genes.(F) Heat map of H3K27ac (green), p65 (yellow), KDM7A (red), and UTX (purple) levels in resting and TNF-α-stimulated ECs. Each row shows ± 10 kb centered on the KDM7A (top) or UTX (bottom) peak. Rows are ordered by max KDM7A or UTX signal in each region. The ChIP-seq signal is depicted by color scaled intensities.(G and H) Gene tracks of ChIP-seq signals for H3K27Ac, p65, KDM7A, and UTX, and RNA-seq signals around the VCAM1 (G) and SELE (H) loci in TNF-α (-) and TNF-α (+) ECs. ChIP-Seq and RNA-seq signals are visualized by Integrated Genome Viewer (http://software.broadinstitute.org/software/igv/).(I and J) ChIP-qPCR of p65 (I) and KDM7A (J) at the VCAM1 TSS, normalized to input. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). † P < 0.05 compared to TNF-α (+) siKDM7A (-). Statistical differences were analyzed by the Tukey-Kramer test.(K and L) ChIP-qPCR of p65 (K) and UTX (L) at the SELE TSS, normalized to input. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). Statistical differences were analyzed by the Tukey-Kramer test.


Figure 4 ( A and B ) Boxplots of the log2 fold change of ChIP - seq enrichment of H3K9me2 in KDM7A target genes ( A ) and that of H3K27me3 in UTX target genes ( B ) with TNF - α treatment ( TNF + ) and control ( TNF - ) . The horizontal line within the box represents the median sample value . The ends of the box represent the 3rd and 1st quantiles . The whiskers extend from the ends of the box to the outermost data point that falls within the distances computed as follows : 3rd quartile + 1 . 5 * ( interquartile range ) and 1st quartile - 1 . 5 * ( interquartile range ) . Each dot in the box plot shows data for one single target gene . Genes colored red represent the top five downregulated target genes with TNF - α - treatment , as compared to control . Graphs are representative of two independent experiments . ( C ) Gene tracks of ChIP - seq signals for H3K9me2 and H3K27me3 around the VCAM1 locus in TNF - α ( - ) and TNF - α ( + ) ECs . ( D and E ) ChIP - qPCR showing enrichment ( percent input ) of H3K9me2 ( D ) and H3K27me3 ( E ) around the VCAM1 locus . Primer pairs targeting distinct regions are listed on the X axis . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) . Statistical differences were analyzed by the Student ' s t test . ( F ) Gene tracks of ChIP - seq signals for H3K9me2 and H3K27me3 around the SELE locus in TNF - α ( - ) and TNF - α ( + ) ECs . ( G and H ) ChIP - qPCR showing enrichment ( percent input ) of H3K9me2 ( G ) and H3K27me3 ( H ) around the SELE locus . Primer pairs targeting distinct regions are listed on the X axis . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) . Statistical differences were analyzed by the Student ' s t test . ( I and J ) ChIP - qPCR showing enrichment ( percent input ) of H3K9me2 and H3K27me3 around the HOXA13 ( I ) and GATA4 ( J ) loci . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) . Statistical differences were analyzed by the Student ' s t test . ( K and L ) ChIP - qPCR showing enrichment ( percent input ) of H3K9me2 around VCAM1 - 11kb ( K ) and H3K27me3 around SELE - 12kb ( L ) . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) . † P < 0 . 05 compared to TNF - α ( + ) siKDM7A ( - ) or TNF - α ( + ) siUTX ( - ) . Statistical differences were analyzed by the Tukey - Kramer test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A and B) Boxplots of the log2 fold change of ChIP-seq enrichment of H3K9me2 in KDM7A target genes (A) and that of H3K27me3 in UTX target genes (B) with TNF-α treatment (TNF+) and control (TNF-). The horizontal line within the box represents the median sample value. The ends of the box represent the 3rd and 1st quantiles. The whiskers extend from the ends of the box to the outermost data point that falls within the distances computed as follows: 3rd quartile + 1.5*(interquartile range) and 1st quartile - 1.5*(interquartile range). Each dot in the box plot shows data for one single target gene. Genes colored red represent the top five downregulated target genes with TNF-α-treatment, as compared to control. Graphs are representative of two independent experiments.(C) Gene tracks of ChIP-seq signals for H3K9me2 and H3K27me3 around the VCAM1 locus in TNF-α (-) and TNF-α (+) ECs.(D and E) ChIP-qPCR showing enrichment (percent input) of H3K9me2 (D) and H3K27me3 (E) around the VCAM1 locus. Primer pairs targeting distinct regions are listed on the X axis. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). Statistical differences were analyzed by the Student's t test.(F) Gene tracks of ChIP-seq signals for H3K9me2 and H3K27me3 around the SELE locus in TNF-α (-) and TNF-α (+) ECs.(G and H) ChIP-qPCR showing enrichment (percent input) of H3K9me2 (G) and H3K27me3 (H) around the SELE locus. Primer pairs targeting distinct regions are listed on the X axis. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). Statistical differences were analyzed by the Student's t test.(I and J) ChIP-qPCR showing enrichment (percent input) of H3K9me2 and H3K27me3 around the HOXA13 (I) and GATA4 (J) loci. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). Statistical differences were analyzed by the Student's t test.(K and L) ChIP-qPCR showing enrichment (percent input) of H3K9me2 around VCAM1 -11kb (K) and H3K27me3 around SELE -12kb (L). Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). † P < 0.05 compared to TNF-α (+) siKDM7A (-) or TNF-α (+) siUTX (-). Statistical differences were analyzed by the Tukey-Kramer test.


Figure 5 ( A ) Plot of enhancers defined in TNF - α - treated ECs , ranked by increasing bromodomain - containing protein 4 ( BRD4 ) signal . Super enhancers ( SEs ) were defined by ROSE ( Loven et al , 2013 , Whyte et al , 2013 ) , based on the ChIP - Seq signals for BRD4 ( Brown et al , 2014 ) . SEs are indicated by dashed lines and are colored yellow . The numbers show the total SEs and their classification based on KDM7A and UTX binding . Representative SE - related genes are indicated in the graph . ( B and C ) Metagene representations of KDM7A ( B ) and UTX ( C ) ChIP - seq signals in units of read count per million mapped reads , at a meta composite of SEs and typical enhancers ( TEs ) . TEs were obtained from the pre - compiled HUVEC ngsplotdb ( https : / / github . com / shenlab - sinai / ngsplot ) . ( D ) Pirate plots showing ChIA - PET interaction changes at control - specific , common , and TNF - α - specific SEs . The Y axis indicates the log2 ratio of ChIA - PET signals ( TNF - α 60 min / 0 min ) . The central bar indicates the average . ( E and F ) Bean plots showing ChIA - PET interaction changes at KDM7A ( E ) and UTX ( F ) binding sites . The Y axis indicates the log2 ratio of ChIA - PET signals ( TNF - α 60 min / 0 min ) . The central bar indicates the median . ( G and H ) ChIA - PET interactions of active RNA pol II around the VCAM1 ( G ) and IL8 ( H ) loci integrated with the ChIP - seq profiles of KDM7A , UTX , and BRD4 , and RNA - seq profiles in TNF - α ( - ) and TNF - α ( + ) ECs . ChIA - PET interactions were visualized by the WashU Epigenome Browser ( http : / / epigenomegateway . wustl . edu / browser / ) . Interactions detected by ChIA - PET are depicted with purple lines . Red bars show TNF - α - specific SEs . ( I - K ) Gene tracks of ChIP - seq signals for H3K9me2 and H3K27me3 around VCAM1 ( I ) , IL8 ( J ) , and SELE ( K ) in TNF - α ( - ) and TNF - α ( + ) ECs . Red bars show TNF - α - specific SEs .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Plot of enhancers defined in TNF-α-treated ECs, ranked by increasing bromodomain-containing protein 4 (BRD4) signal. Super enhancers (SEs) were defined by ROSE (Loven et al, 2013, Whyte et al, 2013), based on the ChIP-Seq signals for BRD4 (Brown et al, 2014). SEs are indicated by dashed lines and are colored yellow. The numbers show the total SEs and their classification based on KDM7A and UTX binding. Representative SE-related genes are indicated in the graph.(B and C) Metagene representations of KDM7A (B) and UTX (C) ChIP-seq signals in units of read count per million mapped reads, at a meta composite of SEs and typical enhancers (TEs). TEs were obtained from the pre-compiled HUVEC ngsplotdb (https://github.com/shenlab-sinai/ngsplot).(D) Pirate plots showing ChIA-PET interaction changes at control-specific, common, and TNF-α-specific SEs. The Y axis indicates the log2 ratio of ChIA-PET signals (TNF-α 60 min/0 min). The central bar indicates the average.(E and F) Bean plots showing ChIA-PET interaction changes at KDM7A (E) and UTX (F) binding sites. The Y axis indicates the log2 ratio of ChIA-PET signals (TNF-α 60 min/0 min). The central bar indicates the median.(G and H) ChIA-PET interactions of active RNA pol II around the VCAM1 (G) and IL8 (H) loci integrated with the ChIP-seq profiles of KDM7A, UTX, and BRD4, and RNA-seq profiles in TNF-α (-) and TNF-α (+) ECs. ChIA-PET interactions were visualized by the WashU Epigenome Browser (http://epigenomegateway.wustl.edu/browser/). Interactions detected by ChIA-PET are depicted with purple lines. Red bars show TNF-α-specific SEs.(I-K) Gene tracks of ChIP-seq signals for H3K9me2 and H3K27me3 around VCAM1 (I), IL8 (J), and SELE (K) in TNF-α (-) and TNF-α (+) ECs. Red bars show TNF-α-specific SEs.


Figure 6 ( B ) Representative snapshot from the IVM analysis of leukocyte adhesive interactions in the femoral arteries . Margins of vessels are indicated with dashed lines . White spots represent fluorescently labeled leukocytes visualized by the intravenous injection of rhodamine 6G . ( C ) Quantitative analyses of leukocyte adhesive interactions in the femoral arteries . Data are shown as means ± SE ( n = 3 / group ) . * P < 0 . 05 compared to the vehicle + TNF - α group . Statistical differences were analyzed by the Student ' s t test . See also Movies EV1 and 2 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(B) Representative snapshot from the IVM analysis of leukocyte adhesive interactions in the femoral arteries. Margins of vessels are indicated with dashed lines. White spots represent fluorescently labeled leukocytes visualized by the intravenous injection of rhodamine 6G.(C) Quantitative analyses of leukocyte adhesive interactions in the femoral arteries. Data are shown as means ± SE (n=3/group). *P < 0.05 compared to the vehicle + TNF-α group. Statistical differences were analyzed by the Student's t test. See also Movies EV1 and 2.


Figure 1 ( D ) Representative immunoblots ( n = 3 ) showing phosphorylation state of Smad2 and JNK during TGF - β - induced EMT in NMuMG cells . Lamin B serves as loading control . ( E ) Quantification of one set of representative immunoblots ( in figure D ) and its graphical representation showing dynamic of Smad2 and JNK activation during TGF - β - induced EMT in NMuMG . ( F ) Representative immunohistochemistry image for pSmad2 and pJNK in non - invasive ( n = 34 ) and invasive ( n = 14 ) breast cancer samples . Scale bars for 10X and 40X images are 200 µm and 50 µm respectively . ( H ) Immunofluorescence microscopy for changes in the localization and expression levels of EMT markers in epithelialNMuMG cells ( d0 ) and cells treated with TGF - β for seven days in the presence of JNK inhibitor ( d7 JNKi ) or control ( d7 DMSO ) . Staining was performed with antibodies against Fibronectin 1 , with Phalloidin to visualize the actin cytoskeleton and against Paxillin to detect focal adhesion plaques . It is important to note that the untreated epithelial cells are highly proliferative and appear small in size , round in shape and adhere to surrounding cells whereas day 7 TGF - β - treated ( mesenchymal ) cells are much bigger in size , elongated in shape and detached from the nearby cells . Scale bar , 100 μm , 63X magnification . ( I ) mRNA levels for classical EMT markers in cells treated as in ( F ) were measured by qRT - PCRs relative to Ctcf and fold change as compare to untreated and plotted on the y - axis . Mean and SEM is plotted from three independent biological replicates . ( J ) Transwell migration assay for NMuMG cells treated as in G . Plotted Mean ± SEM of three biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

O O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(D) Representative immunoblots (n=3) showing phosphorylation state of Smad2 and JNK during TGF-β-induced EMT in NMuMG cells. Lamin B serves as loading control.(E) Quantification of one set of representative immunoblots (in figure D) and its graphical representation showing dynamic of Smad2 and JNK activation during TGF-β-induced EMT in NMuMG.(F) Representative immunohistochemistry image for pSmad2 and pJNK in non-invasive (n=34) and invasive (n=14) breast cancer samples. Scale bars for 10X and 40X images are 200 µm and 50 µm respectively.(H) Immunofluorescence microscopy for changes in the localization and expression levels of EMT markers in epithelialNMuMG cells (d0) and cells treated with TGF-β for seven days in the presence of JNK inhibitor (d7 JNKi) or control (d7 DMSO). Staining was performed with antibodies against Fibronectin 1, with Phalloidin to visualize the actin cytoskeleton and against Paxillin to detect focal adhesion plaques. It is important to note that the untreated epithelial cells are highly proliferative and appear small in size, round in shape and adhere to surrounding cells whereas day 7 TGF- β-treated (mesenchymal) cells are much bigger in size, elongated in shape and detached from the nearby cells. Scale bar, 100 μm, 63X magnification.(I) mRNA levels for classical EMT markers in cells treated as in (F) were measured by qRT-PCRs relative to Ctcf and fold change as compare to untreated and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates.(J) Transwell migration assay for NMuMG cells treated as in G. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.


Figure 2 . ( E ) ChIP assay using H3K27me3 - specific antibody in epithelial cells ( day 0 ) ) or NMuMG cells treated for seven days with TGF - β ( mesenchymal ) and DMSO or JNKi . qRT - PCRs were performed for indicated gene promoters and enrichments are plotted on the right y - axis as ratio of precipitated DNA ( bound ) to total input DNA . On the left y - axis RNA levels of analyzed genes derived from RNA - seq data ( fold change to untreated normalized tag counts ) are plotted . Mean and SEM is plotted from two independent biological replicates . ( F ) FAIRE assay was performed in cells treated as in ( D ) and qRT - PCRs were performed for the same gene promoters and plotted as in ( E ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2. (E) ChIP assay using H3K27me3-specific antibody in epithelial cells (day 0)) or NMuMG cells treated for seven days with TGF-β (mesenchymal) and DMSO or JNKi. qRT-PCRs were performed for indicated gene promoters and enrichments are plotted on the right y-axis as ratio of precipitated DNA (bound) to total input DNA. On the left y-axis RNA levels of analyzed genes derived from RNA-seq data (fold change to untreated normalized tag counts ) are plotted. Mean and SEM is plotted from two independent biological replicates.(F) FAIRE assay was performed in cells treated as in (D) and qRT-PCRs were performed for the same gene promoters and plotted as in (E).