Representative GO terms enriched in genes differentially expressed upon FOXA2 deletion in PANC1 cells . Terms are ranked by the negative log10 p - value from the hypergeometric test . Panels on the right show the effects of FOXA2 deletion on cell morphology and adhesion to multiple substrates . Three different bulk populations were used as biological replicates : circularity index was measured on 60 different cells per bulk population while adhesion is reported as mean of six different fields per bulk for a total of eighteen observations per condition .

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Representative GO terms enriched in genes differentially expressed upon FOXA2 deletion in PANC1 cells. Terms are ranked by the negative log10 p-value from the hypergeometric test. Panels on the right show the effects of FOXA2 deletion on cell morphology and adhesion to multiple substrates. Three different bulk populations were used as biological replicates: circularity index was measured on 60 different cells per bulk population while adhesion is reported as mean of six different fields per bulk for a total of eighteen observations per condition.


Scatter plots showing the correlation between FOXA2 ChIP - seq biological duplicates in CFPAC1 ( top panel ) and PANC1 cells ( bottom panel ) . Pearson ' s correlation ( r ) between samples is indicated .

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Scatter plots showing the correlation between FOXA2 ChIP-seq biological duplicates in CFPAC1 (top panel) and PANC1 cells (bottom panel). Pearson's correlation (r) between samples is indicated.


Scatter plot showing the correlation between FOXA2 ChIP - seq peaks in CFPAC1 and PANC1 cells . Green and blue dots indicate FOXA2 peaks that are significantly ( FDR < 0 . 001 ) different between the two cell types and with log2 fold enrichment ≥ 2 as determined by DiffBind . All other peaks are indicated as grey dots . The boxplots on the right show the intensity of CFPAC1 - specific , PANC1 - specific and other FOXA2 peaks . Central values represent the median , the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers .

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Scatter plot showing the correlation between FOXA2 ChIP-seq peaks in CFPAC1 and PANC1 cells. Green and blue dots indicate FOXA2 peaks that are significantly (FDR < 0.001) different between the two cell types and with log2 fold enrichment ≥ 2 as determined by DiffBind. All other peaks are indicated as grey dots. The boxplots on the right show the intensity of CFPAC1-specific, PANC1-specific and other FOXA2 peaks. Central values represent the median, the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers.


Representative FOXA2 ChIP - seq snapshot showing FOXA2 peaks classified as CFPAC1 - specific , shared or PANC1 - specific .

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Representative FOXA2 ChIP-seq snapshot showing FOXA2 peaks classified as CFPAC1-specific, shared or PANC1-specific.


The five top transcription factor motifs over - represented in TSS - distal CFPAC1 - specific FOXA2 peaks . TFs were grouped into families or subfamilies and ranked based on the best PWM q - value . The POU domain TF family includes HNF1β . Top scoring motifs are shown on the right .

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The five top transcription factor motifs over-represented in TSS-distal CFPAC1-specific FOXA2 peaks. TFs were grouped into families or subfamilies and ranked based on the best PWM q-value. The POU domain TF family includes HNF1β. Top scoring motifs are shown on the right.


Bar plot showing the percentage of different sets of FOXA2 peaks overlapping with HNF1β ChIP - seq peaks .

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Bar plot showing the percentage of different sets of FOXA2 peaks overlapping with HNF1β ChIP-seq peaks.


Bar plot showing the percentage of different sets of FOXA2 peaks overlapping with FOS ( left ) and JUNB ( right ) ChIP - seq peaks .

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Bar plot showing the percentage of different sets of FOXA2 peaks overlapping with FOS (left) and JUNB (right) ChIP-seq peaks.


Two representative ChIP - seq snapshots indicating the genomic distribution of FOXA2 and partner transcription factors in CFPAC1 peaks . The FOXA2 ChIP - seq track in PANC1 cells is also shown for comparison .

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Two representative ChIP-seq snapshots indicating the genomic distribution of FOXA2 and partner transcription factors in CFPAC1 peaks. The FOXA2 ChIP-seq track in PANC1 cells is also shown for comparison.


Representative areas from human PDAC tissue showing expression of HNF1β in G1 , G2 and G3 areas . The red dotted line indicates the borders of a G3 area . The percentage of HNF1β - positive cells in areas of different grade from a PDAC tissue microarray is shown .

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Representative areas from human PDAC tissue showing expression of HNF1β in G1, G2 and G3 areas. The red dotted line indicates the borders of a G3 area. The percentage of HNF1β-positive cells in areas of different grade from a PDAC tissue microarray is shown.


Colocalization of HNF1β and FOXA2 in human PDAC areas . PDAC sections from three different patients were stained with the anti - HNF1β antibody , stripped and re - stained with the anti - FOXA2 antibody . The red dotted areas on the left represent poorly differentiated ( G3 ) tumor regions .

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Colocalization of HNF1β and FOXA2 in human PDAC areas. PDAC sections from three different patients were stained with the anti-HNF1β antibody, stripped and re-stained with the anti-FOXA2 antibody. The red dotted areas on the left represent poorly differentiated (G3) tumor regions.


FOXA2 peaks in CFPAC1 cells were divided into 5 clusters based on their association with HNF1β and the effects of HNF1β loss on FOXA2 binding and chromatin accessibility ( determined by ATAC - seq ) . Clusters 1 - 4 were HNF1β - positive while cluster 5 was HNF1β - negative . The heat - maps on the left show FOXA2 , HNF1β , ATAC - seq and H3K27Ac profiles at FOXA2 - bound genomic regions . The three heat - maps on the right show JunB ChIP - seq data and ATAC - seq data in wild type or Jun / JunB double knockout ( DKO ) CFPAC1 cells . Data are shown in + / - 3 kb regions centered on the summit of FOXA2 peaks .

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FOXA2 peaks in CFPAC1 cells were divided into 5 clusters based on their association with HNF1β and the effects of HNF1β loss on FOXA2 binding and chromatin accessibility (determined by ATAC-seq). Clusters 1-4 were HNF1β- positive while cluster 5 was HNF1β-negative. The heat-maps on the left show FOXA2, HNF1β, ATAC-seq and H3K27Ac profiles at FOXA2-bound genomic regions. The three heat-maps on the right show JunB ChIP-seq data and ATAC-seq data in wild type or Jun/JunB double knockout (DKO) CFPAC1 cells. Data are shown in +/- 3 kb regions centered on the summit of FOXA2 peaks.


Characterization of clusters 1 - 3 . Left : quantification of the indicated signals at FOXA2 bound regions in wild - type ( WT ) and HNF1β knock - out ( KO ) cells . Central values represent the median , the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers . Middle : distribution of FOXA and HNF1β motifs relative to the summit of FOXA2 peaks . Right : over - represented motifs in clusters 1 - 3 and inferred binding models .

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Characterization of clusters 1-3. Left: quantification of the indicated signals at FOXA2 bound regions in wild-type (WT) and HNF1β knock-out (KO) cells. Central values represent the median, the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers. Middle: distribution of FOXA and HNF1β motifs relative to the summit of FOXA2 peaks. Right: over-represented motifs in clusters 1-3 and inferred binding models.


Quantification of JunB ChIP - seq signals in clusters 1 to 5 . ( Right ) Quantification of ATAC - seq signals in clusters 1 to 5 in wild type and Jun / JunB double KO ( DKO ) CFPAC1 cells . Central values represent the median , the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers .

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Quantification of JunB ChIP-seq signals in clusters 1 to 5. (Right) Quantification of ATAC-seq signals in clusters 1 to 5 in wild type and Jun/JunB double KO (DKO) CFPAC1 cells. Central values represent the median, the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers.


Representative snapshot showing changes in FOXA2 occupancy , chromatin accessibility and histone acetylation in HNF1β knock - out CFPAC1 cells . The FOXA2 ChIP - seq track in PANC1 cells is also shown for comparison .

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Representative snapshot showing changes in FOXA2 occupancy, chromatin accessibility and histone acetylation in HNF1β knock-out CFPAC1 cells. The FOXA2 ChIP-seq track in PANC1 cells is also shown for comparison.


Western blot showing HNF1β over - expression after lentiviral transduction of PANC1 cells . Expression of FOXA2 in wild type and over - expressing ( O . E . ) cells is shown . Vinculin : loading control .

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Western blot showing HNF1β over-expression after lentiviral transduction of PANC1 cells. Expression of FOXA2 in wild type and over-expressing (O.E.) cells is shown. Vinculin: loading control.


Scatter plot showing the genomic distribution of FOXA2 in PANC1 cells transduced with the HNF1β - expressing lentivirus ( HNF1B O . E . ) and in their matched mock - infected control . The fraction of gained , unchanged and lost FOXA2 peaks overlapping HNF1β ChIP - seq peaks in transduced PANC1 cells is shown in the pie - charts on the right .

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Scatter plot showing the genomic distribution of FOXA2 in PANC1 cells transduced with the HNF1β-expressing lentivirus (HNF1B O.E.) and in their matched mock-infected control. The fraction of gained, unchanged and lost FOXA2 peaks overlapping HNF1β ChIP-seq peaks in transduced PANC1 cells is shown in the pie-charts on the right.


TF motif over - represented in gained or lost FOXA2 - bound genomic regions .

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TF motif over-represented in gained or lost FOXA2-bound genomic regions.


Representative snapshot from PANC1 cells transduced with the HNF1β - expressing lentivirus . HNF1B O . E . : HNF1B over - expression .

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Representative snapshot from PANC1 cells transduced with the HNF1β-expressing lentivirus. HNF1B O.E.: HNF1B over-expression.


Percentage of different sets of FOXA2 peaks overlapping HOXB8 ChIP - seq peaks .

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Percentage of different sets of FOXA2 peaks overlapping HOXB8 ChIP-seq peaks.


Representative snapshots showing the overlap between HOXB8 and FOXA2 ChIP - seq peaks in PANC1 cells . The FOXA2 ChIP - seq track in CFPAC1 cells is also shown for comparison .

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Representative snapshots showing the overlap between HOXB8 and FOXA2 ChIP-seq peaks in PANC1 cells. The FOXA2 ChIP-seq track in CFPAC1 cells is also shown for comparison.


FOXA2 ChIP - seq in CFPAC1 cells transduced with a HOXB8 - expressing lentivirus and in their matched mock - infected control . Gained ( brown ) and lost ( yellow ) FOXA2 peaks are shown . Pie - charts on the right show the fraction of gained , unchanged and lost peaks overlapping HOXB8 ChIP - seq peaks in transduced CFPAC1 cells .

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FOXA2 ChIP-seq in CFPAC1 cells transduced with a HOXB8-expressing lentivirus and in their matched mock-infected control. Gained (brown) and lost (yellow) FOXA2 peaks are shown. Pie-charts on the right show the fraction of gained, unchanged and lost peaks overlapping HOXB8 ChIP-seq peaks in transduced CFPAC1 cells.


Expression of HNF1β protein in wild - type and HOXB8 - expressing PANC1 cells .

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Expression of HNF1β protein in wild-type and HOXB8-expressing PANC1 cells.


Representative snapshots from CFPAC1 cells transduced with the HOXB8 - expressing lentivirus .

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Representative snapshots from CFPAC1 cells transduced with the HOXB8-expressing lentivirus.


( A ) Co - localization of c - terminally GFP - or mCherry - tagged Apl5 , Sec7 and Vps41 in a vam3 temperature sensitive ( ts ) background strain . Cells were grown over night at 23 ° C and where indicated shifted to 37 ° C for 3 h prior to imaging . White arrowheads indicate co - localization events . Scale bar , 5 µm . Scale bar in inset , 2 µm .

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(A) Co-localization of c-terminally GFP- or mCherry-tagged Apl5, Sec7 and Vps41 in a vam3 temperature sensitive (ts) background strain. Cells were grown over night at 23°C and where indicated shifted to 37°C for 3 h prior to imaging. White arrowheads indicate co-localization events. Scale bar, 5 µm. Scale bar in inset, 2 µm.


( B ) Quantification of co - localization events Single events were counted and the percentage of co - localization normalized to the number of Apl5 - punctae was plotted ( n ≥ 187 cells ) . Bars show the mean percentage of co - localization ± standard deviation .

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(B) Quantification of co-localization events Single events were counted and the percentage of co-localization normalized to the number of Apl5-punctae was plotted (n ≥ 187 cells). Bars show the mean percentage of co-localization ± standard deviation.


( C ) Re - localization of Apl5 upon Vps41 re - localization . Vps41 was tagged with a C - terminal FRBGFP tag in a strain , where Apl5 carries a C - terminal mCherry and Pma1 a FKBP - tag Cells were grown in either the absence ( control ) or presence of 10 μM rapamycin , and localization of Vps41 and Apl5 was analysed by fluorescence microscopy . Scale bar , 5 μm .

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(C) Re-localization of Apl5 upon Vps41 re-localization. Vps41 was tagged with a C-terminal FRBGFP tag in a strain, where Apl5 carries a C-terminal mCherry and Pma1 a FKBP-tag Cells were grown in either the absence (control) or presence of 10 μM rapamycin, and localization of Vps41 and Apl5 was analysed by fluorescence microscopy. Scale bar, 5 μm.


( D ) Effect of clathrin deletion on the AP - 3 pathway . GFP - tagged Nyv1 with a C - terminal Snc1 transmembrane domain ( GNS ) was localized relative to FM4 - 64 stained vacuoles in the indicated strains . Labeling of the cell surface by GFP indicates a defect in the AP - 3 pathway ( E ) Quantification of AP - 3 defect from ( D ) . Linear intensity plots were laid over the cells , the AP - 3 defect was calculated by dividing GFP - intensities on the vacuolar membrane by the sum of the intensity of vacuolar , and plasma membrane signal ( n ≥ 30 cells ) . Bars show the mean vacuolar signal over total signal ± standard deviation .

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(D) Effect of clathrin deletion on the AP-3 pathway. GFP-tagged Nyv1 with a C-terminal Snc1 transmembrane domain (GNS) was localized relative to FM4-64 stained vacuoles in the indicated strains. Labeling of the cell surface by GFP indicates a defect in the AP-3 pathway (E) Quantification of AP-3 defect from (D). Linear intensity plots were laid over the cells, the AP-3 defect was calculated by dividing GFP-intensities on the vacuolar membrane by the sum of the intensity of vacuolar, and plasma membrane signal (n ≥ 30 cells). Bars show the mean vacuolar signal over total signal ± standard deviation.


( C ) Tethering of AP - 3 vesicles to the mitochondrial surface . Cells were grown at 30 ° C in YPG and kept in logarithmic phase . Just prior to imaging , the mitochondrial DNA was stained with DAPI Localization of Apl6 and Vps41 was analysed by fluorescence microscopy . First column shows DIC ( difference interference contrast ) image of cells . Scale bar , 5 µm . The inset shows the indicated magnification . Scale bar in inset , 2 µm .

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(C) Tethering of AP-3 vesicles to the mitochondrial surface. Cells were grown at 30°C in YPG and kept in logarithmic phase. Just prior to imaging, the mitochondrial DNA was stained with DAPI Localization of Apl6 and Vps41 was analysed by fluorescence microscopy. First column shows DIC (difference interference contrast) image of cells. Scale bar, 5 µm. The inset shows the indicated magnification. Scale bar in inset, 2 µm.


Apl5 - dots co - localizing with GFP signal were counted and plotted in percent of the total number of Apl5 - dots . Bars show the mean percentage of co - localization ± standard deviation ( n ≥ 60 cells ) .

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Apl5-dots co-localizing with GFP signal were counted and plotted in percent of the total number of Apl5-dots. Bars show the mean percentage of co-localization ± standard deviation (n ≥ 60 cells).


( E , F ) Purification of mitochondria from selected strains . Mitochondria were purified from the indicated strains as described in the methods . The amount corresponding to 1 % of the total volume was loaded of the ' load ' ' sample , 5 % were used for ' crude ' and ' pure ' mitochondria fractions . Samples were loaded on a 10 % SDS PAGE gel and analysed by Western Blot . White asterisks indicate unspecific bands . ( G ) Quantification of relative Nyv1 signal intensity from ( F ) . Signal intensity was determined using ImageJ and a ratio of pure over crude signal intensity normalized to Tom40 signal intensity was plotted . Bars show the relative Nvy1 signal intensity ± standard deviation from n = 3 independent experiments .

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(E, F) Purification of mitochondria from selected strains. Mitochondria were purified from the indicated strains as described in the methods. The amount corresponding to 1 % of the total volume was loaded of the 'load'' sample, 5% were used for 'crude' and 'pure' mitochondria fractions. Samples were loaded on a 10% SDS PAGE gel and analysed by Western Blot. White asterisks indicate unspecific bands. (G) Quantification of relative Nyv1 signal intensity from (F). Signal intensity was determined using ImageJ and a ratio of pure over crude signal intensity normalized to Tom40 signal intensity was plotted. Bars show the relative Nvy1 signal intensity ± standard deviation from n = 3 independent experiments.


( A - H ) Indicated strains were grown in YPG for expression of Vps41 - GFP - Fis constructs . Two representative images are shown for each strain . Mitochondria ( M ) are highlighted in red and vesicular structures in teal ( unlabeled for VPS41 - GFP - Fis ) or green ( unlabeled for VPS41 - GFP - Fis ) . Cell wall ( CW ) , plasma membrane ( PM ) , and vacuoles ( V ) are indicated . Scale bars , 250 nm .

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(A-H) Indicated strains were grown in YPG for expression of Vps41-GFP-Fis constructs. Two representative images are shown for each strain. Mitochondria (M) are highlighted in red and vesicular structures in teal (unlabeled for VPS41-GFP-Fis) or green (unlabeled for VPS41-GFP-Fis). Cell wall (CW), plasma membrane (PM), and vacuoles (V) are indicated. Scale bars, 250 nm.


( A ) GNS was localized by fluorescence microscopy in the indicated strains grown in medium containing glucose or galactose Images were inverted to visualize possible defects . First column shows DIC image of cells . Scale bar , 5 µm . ( B ) Quantification of the AP - 3 defect from ( A ) . Linear intensity plots were laid over cells grown in medium containing glucose ( D ) or galactose ( G ) , the AP - 3 defect was calculated by dividing GFP - intensities on the vacuolar membrane by the sum of the intensity of vacuolar , and plasma membrane signal ( n ≥ 15 cells ) . Bars show the mean vacuolar signal over total signal ± standard deviation .

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(A) GNS was localized by fluorescence microscopy in the indicated strains grown in medium containing glucose or galactose Images were inverted to visualize possible defects. First column shows DIC image of cells. Scale bar, 5 µm. (B) Quantification of the AP-3 defect from (A). Linear intensity plots were laid over cells grown in medium containing glucose (D) or galactose (G), the AP-3 defect was calculated by dividing GFP-intensities on the vacuolar membrane by the sum of the intensity of vacuolar, and plasma membrane signal (n ≥ 15 cells). Bars show the mean vacuolar signal over total signal ± standard deviation.


( C ) Effect of different truncations and deletions on AP - 3 targeting to mitochondria . Indicated strains were grown at 30 ° C in YPG and kept in logarithmic phase . Localization of Apl5 or Apl6 and Vps41 was analysed by fluorescence microscopy . First column shows DIC image of cells . Scale bar , 5 µm . Scale bar in inset , 2 µm . ( D ) Quantification of ( C ) . ( - ) Control corresponds to Vps41 ( ΔPEST ) - GFP - Fis1 Apl6 - mCherry ( CUY12558 ) and ( + ) corresponds to Vps41 ( S - D ) - GFP - Fis1 Apl6 - mCherry ( CUY12560 ) . Apl5 - / Apl6 - dots co - localizing with GFP signal were counted and plotted in percent of the total number of Apl5 - / Apl6 - dots ( n ≥ 63 ) . Bars show the mean percentage of co - localization ± standard deviation .

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(C) Effect of different truncations and deletions on AP-3 targeting to mitochondria. Indicated strains were grown at 30°C in YPG and kept in logarithmic phase. Localization of Apl5 or Apl6 and Vps41 was analysed by fluorescence microscopy. First column shows DIC image of cells. Scale bar, 5 µm. Scale bar in inset, 2 µm. (D) Quantification of (C). (-) Control corresponds to Vps41(ΔPEST)-GFP-Fis1 Apl6-mCherry (CUY12558) and (+) corresponds to Vps41(S-D)-GFP-Fis1 Apl6-mCherry (CUY12560). Apl5- /Apl6-dots co-localizing with GFP signal were counted and plotted in percent of the total number of Apl5- /Apl6-dots (n ≥ 63). Bars show the mean percentage of co-localization ± standard deviation.


( C ) Volcano plots of proteins in close proximity to Apl5 - TurboID versus control cells , from a label - free proteomics analysis of streptavidin - biotin pulldowns . The logarithmic ratios of protein intensities are plotted against negative logarithmic P values of two tailed Student ' s t - test , equal variance , performed from n = 3 independent experiments . The red dashed line ( significance , 0 . 05 ) separates specifically identified proteins ( top right portion of plot ) from background . Selected top hits are indicated with black dots ,

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(C) Volcano plots of proteins in close proximity to Apl5-TurboID versus control cells, from a label-free proteomics analysis of streptavidin-biotin pulldowns. The logarithmic ratios of protein intensities are plotted against negative logarithmic P values of two tailed Student's t-test, equal variance, performed from n = 3 independent experiments. The red dashed line (significance, 0.05) separates specifically identified proteins (top right portion of plot) from background. Selected top hits are indicated with black dots,


( E ) Volcano plots of proteins in close proximity to Apl5 - TurboID versus control cells in a GAL1pr - Vps41 - GFP - Fis1 background , from a label - free proteomics analysis of streptavidin - biotin pulldowns with cells grown in YPG . The logarithmic ratios of protein intensities are plotted against negative logarithmic P values of two tailed Student ' s t - test , equal variance , performed from n = 3 independent experiments . The red dashed line ( significance , 0 . 001 ) separates specifically identified proteins ( top right portion of plot ) from background . Selected top hits are indicated with black

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(E) Volcano plots of proteins in close proximity to Apl5-TurboID versus control cells in a GAL1pr-Vps41-GFP-Fis1 background, from a label-free proteomics analysis of streptavidin-biotin pulldowns with cells grown in YPG. The logarithmic ratios of protein intensities are plotted against negative logarithmic P values of two tailed Student's t-test, equal variance, performed from n = 3 independent experiments. The red dashed line (significance, 0.001) separates specifically identified proteins (top right portion of plot) from background. Selected top hits are indicated with black dots


( B ) AP - 3 sorting defects in GAP deletions . The AP - 3 reporter was localized relative to FM4 - 64 stained vacuoles in the indicated strains . First column shows the DIC image of cells . Scale bar , 5 µm .

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(B) AP-3 sorting defects in GAP deletions. The AP-3 reporter was localized relative to FM4-64 stained vacuoles in the indicated strains. First column shows the DIC image of cells. Scale bar, 5 µm.


( D ) Localization of AP - 3 in ArfGAP deletion strains . Localization of C - terminally GFP - tagged Apl5 relative to FM4 - 64 stained vacuoles in the indicated strains . First column shows DIC image of cells . Scale bar , 5 µm . ( E ) Quantification of Apl5 - dots in ( D ) . Apl5 - positve dots were counted and averaged to the number of dots per cell . Box boundaries indicate 25 % and 75 % of the dataset . The middle line indicates the median and the small square the mean .

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(D) Localization of AP-3 in ArfGAP deletion strains. Localization of C-terminally GFP-tagged Apl5 relative to FM4-64 stained vacuoles in the indicated strains. First column shows DIC image of cells. Scale bar, 5 µm. (E) Quantification of Apl5-dots in (D). Apl5-positve dots were counted and averaged to the number of dots per cell. Box boundaries indicate 25% and 75% of the dataset. The middle line indicates the median and the small square the mean.


( F ) Co - localization of C - terminally GFP - tagged Apl5 and C - terminally mCherry - tagged Sec7 in wt and age2Δ background . Cells were grown at 30 ° C and kept in logarithmic phase . Scale bar , 5 µm . Scale bar in inset , 2 µm . ( G ) Quantification of co - localization from ( F ) normalized per cell . Number of co - localizing dots was determined and normalized to the number of co - localization events per cell . Bars show the mean number of co - localizing dots per cell ± standard deviation ( n ≥ 60 ) .

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(F) Co-localization of C-terminally GFP-tagged Apl5 and C-terminally mCherry-tagged Sec7 in wt and age2Δ background. Cells were grown at 30°C and kept in logarithmic phase. Scale bar, 5 µm. Scale bar in inset, 2 µm. (G) Quantification of co-localization from (F) normalized per cell. Number of co-localizing dots was determined and normalized to the number of co-localization events per cell. Bars show the mean number of co-localizing dots per cell ± standard deviation (n ≥ 60).


( A , B ) AP - 3 sorting defects in strains overexpressing Gcs1 ( A ) or Age2 ( B ) . The AP - 3 reporter GNS was localized relative to FM4 - 64 stained vacuoles in the indicated strains . Scale bar , 5 µm .

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(A, B) AP-3 sorting defects in strains overexpressing Gcs1 (A) or Age2 (B). The AP-3 reporter GNS was localized relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm.


( C ) Localization of AP - 3 in ArfGAP overexpression strains . Localization of C - terminally GFP - tagged Apl5 relative to FM4 - 64 stained vacuoles in the indicated strains . Scale bar , 5 µm . ( D ) Quantification of Apl5 dots in ( C ) . Apl5 - positve dots were counted and averaged to the number of dots per cell . Box boundaries indicate 25 % and 75 % of the dataset . The middle line indicates the median and the small square the mean .

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(C) Localization of AP-3 in ArfGAP overexpression strains. Localization of C-terminally GFP-tagged Apl5 relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm. (D) Quantification of Apl5 dots in (C). Apl5-positve dots were counted and averaged to the number of dots per cell. Box boundaries indicate 25% and 75% of the dataset. The middle line indicates the median and the small square the mean.


( E , F ) AP - 3 sorting defects in GAP - dead mutants of Gcs1 ( E ) and Age2 ( F ) . The AP - 3 reporter GNS was localized relative to FM4 - 64 stained vacuoles in the indicated strains . Scale bar , 5 µm .

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(E, F) AP-3 sorting defects in GAP-dead mutants of Gcs1 (E) and Age2 (F). The AP-3 reporter GNS was localized relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm.


( G ) Localization of AP - 3 in GAP - dead mutants of Gcs1 and Age2 . Localization of C - terminally GFP - tagged Apl5 relative to FM4 - 64 stained vacuoles in the indicated strains . Scale bar , 5 µm . ( H ) Quantification of Apl5 dots in ( G ) . Apl5 - positve dots were counted and averaged to the number of dots per cell . Box boundaries indicate 25 % and 75 % of the dataset . The middle line indicates the median and the small square the mean .

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(G) Localization of AP-3 in GAP-dead mutants of Gcs1 and Age2. Localization of C-terminally GFP-tagged Apl5 relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm. (H) Quantification of Apl5 dots in (G). Apl5-positve dots were counted and averaged to the number of dots per cell. Box boundaries indicate 25% and 75% of the dataset. The middle line indicates the median and the small square the mean.


B . Analysis of cell cycle arrest ( in G2 ) in response to DSB induction assayed by flow cytometry .

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B. Analysis of cell cycle arrest (in G2) in response to DSB induction assayed by flow cytometry.


C . Rad53 phosphorylation ( Rad53 - P ) in response to DSB assayed by western blotting .

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C. Rad53 phosphorylation (Rad53-P) in response to DSB assayed by western blotting.


D . Cell survival upon DSB induction . Average ± SD ( n = 4 ) is shown for each genotype .

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D. Cell survival upon DSB induction. Average ± SD (n=4) is shown for each genotype.


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B. Srs2 requirement in de novo telomere addition in cells with and without functional HR. All strains are pif1-m2. Strains used: NK1264; NK2375, NK2376; NK2014, NK2015; NK2451, NK2452; NK2012, NK2013; NK2457, NK2458; NK2363, NK2364; NK2469; 2369, NK2370; NK2473-2475.


D . Dynamics of ( TG1 - 3 ) n addition monitored by qPCR through a time - course experiment ( asynchronous populations ) . The Y axis shows a fold increase in de novo telomere - specific PCR product relative to the background levels at 0 h and normalised against an internal control ( ARO1 locus ) . Average ± SD ( n = 3 ) is shown for each time - point of each genotype . Strains used : NK3292 , NK3293 ; NK4670 , NK4671 ; NK4112 , NK4113 ; NK4114 , NK4115 ; NK3292 est2 ∆ , NK3293 est2 ∆ ; NK4232 , NK4233 .

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

D. Dynamics of (TG1-3)n addition monitored by qPCR through a time-course experiment (asynchronous populations). The Y axis shows a fold increase in de novo telomere-specific PCR product relative to the background levels at 0 h and normalised against an internal control (ARO1 locus). Average ± SD (n=3) is shown for each time-point of each genotype. Strains used: NK3292, NK3293; NK4670, NK4671; NK4112, NK4113; NK4114, NK4115; NK3292 est2∆, NK3293 est2∆; NK4232, NK4233.


F . Comparative analysis of ssDNA / dsDNA at de novo telomere addition loci in SRS2 and srs2Δ during a time - course experiment ( synchronised populations ) . The Y axis shows a fold increase in de novo telomere - specific PCR product relative to the background levels at 0 h and normalised against an internal control ( ARO1 locus ) . Average ± SD ( n = 3 ) is shown for each time - point of each genotype . Top set of error bars represents SD in relative increase of the de novo telomere - specific PCR product ( as in panel D ) while the lower set of error bars corresponds to quantifications of ss / dsDNA fractions . Strains used : NK3292 , NK3293 ; NK4670 , NK4671 .

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F. Comparative analysis of ssDNA/dsDNA at de novo telomere addition loci in SRS2 and srs2Δ during a time-course experiment (synchronised populations). The Y axis shows a fold increase in de novo telomere-specific PCR product relative to the background levels at 0 h and normalised against an internal control (ARO1 locus). Average ± SD (n=3) is shown for each time-point of each genotype. Top set of error bars represents SD in relative increase of the de novo telomere-specific PCR product (as in panel D) while the lower set of error bars corresponds to quantifications of ss/dsDNA fractions. Strains used: NK3292, NK3293; NK4670, NK4671.


B . Southern blot analysis of re - synthesis of resected DNA during BIR in SRS2 and srs2Δ corresponding to the data quantifications in C . DNA was digested with EcoRI and BamHI , resolved on 0 . 7 % agarose gels , transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes ( three RS probes and a reference probe , REF , hybridizing to an ARS522 - containing fragment on chr . V which is not involved in the repair ) . A representative image of one of the three repeats is shown . C . Re - synthesis of resected DNA on chr . VIIL in SRS2 and srs2Δ cells ( solid and dashed lines , respectively ) at the distance of 15 . 2 ( RS15 . 2 ) , 6 . 8 ( RS6 . 8 ) and 2 . 6 ( RS2 . 6 ) kb away from the homology region . Average ± SD ( n = 3 ) is shown for each time point .

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B. Southern blot analysis of re-synthesis of resected DNA during BIR in SRS2 and srs2Δ corresponding to the data quantifications in C. DNA was digested with EcoRI and BamHI, resolved on 0.7% agarose gels, transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes (three RS probes and a reference probe, REF, hybridizing to an ARS522-containing fragment on chr. V which is not involved in the repair). A representative image of one of the three repeats is shown.C. Re-synthesis of resected DNA on chr.VIIL in SRS2 and srs2Δ cells (solid and dashed lines, respectively) at the distance of 15.2 (RS15.2), 6.8 (RS6.8) and 2.6 (RS2.6) kb away from the homology region. Average ± SD (n=3) is shown for each time point.


D . Southern blot analysis of BIR - dependent DNA synthesis in SRS2 and srs2Δ corresponding to the data quantifications in D . DNA was digested with EcoRI and BamHI , resolved on 0 . 7 % agarose gels , transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes ( three BIR probes and a reference probe , REF , hybridizing to an ARS522 - containing fragment on chr . V which is not involved in the repair ) . A representative image of one of the three repeats is shown . C - control strain NK3980 . E . BIR - dependent DNA synthesis in SRS2 and srs2Δ cells ( solid and dashed lines , respectively ) at the distance of 6 ( BIR6 ) , 36 ( BIR36 ) and 77 ( BIR77 ) kb away from the homology region . Average ± SD ( n = 3 ) is shown for each time point .

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

D. Southern blot analysis of BIR-dependent DNA synthesis in SRS2 and srs2Δ corresponding to the data quantifications in D. DNA was digested with EcoRI and BamHI, resolved on 0.7% agarose gels, transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes (three BIR probes and a reference probe, REF, hybridizing to an ARS522-containing fragment on chr. V which is not involved in the repair). A representative image of one of the three repeats is shown. C - control strain NK3980.E. BIR-dependent DNA synthesis in SRS2 and srs2Δ cells (solid and dashed lines, respectively) at the distance of 6 (BIR6), 36 (BIR36) and 77 (BIR77) kb away from the homology region. Average ± SD (n=3) is shown for each time point.


B . Frequency of DSB repair via SSA in SRS2 and srs2Δ cells with and without functional HR in the assay based on the system shown in panel A . Average ± SD ( n = 4 ) is shown for each genotype . Strains used in B and D : NK4691 - 4693 ; NK4805 - 4808 ; NK5081 - 5084 ; NK5085 - 5091 .

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B. Frequency of DSB repair via SSA in SRS2 and srs2Δ cells with and without functional HR in the assay based on the system shown in panel A. Average ± SD (n=4) is shown for each genotype. Strains used in B and D: NK4691-4693; NK4805-4808; NK5081-5084; NK5085-5091.


D . Fragment L formation in SRS2 and srs2Δ cells in the presence and absence of Rad51 . Also see Figure EV3 for blot images . Average ± SD ( n = 4 ) is shown for each time point .

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D. Fragment L formation in SRS2 and srs2Δ cells in the presence and absence of Rad51. Also see Figure EV3 for blot images. Average ± SD (n=4) is shown for each time point.


( D ) Absolute cell numbers of the indicated cell types were determined by flow - cytometric analysis of the bone marrow from 3 - week - old Pax5 + / + and Pax5Jak2 / + mice . Average cell numbers are shown with SEM and were statistically analyzed by multiple t - tests ( unpaired two - tailed with Holm - Šídák ' s correction ) ; ns , not significant ( P > 0 . 05 ) . One of 3 independent experiments is shown .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE I-TISSUE O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Absolute cell numbers of the indicated cell types were determined by flow-cytometric analysis of the bone marrow from 3-week-old Pax5+/+ and Pax5Jak2/+ mice. Average cell numbers are shown with SEM and were statistically analyzed by multiple t-tests (unpaired two-tailed with Holm-Šídák's correction); ns, not significant (P > 0.05). One of 3 independent experiments is shown.


( F ) Kaplan - Meier survival analysis of Pax5Jak2 / + ( black ) and control Pax5 + / + ( grey ) mice . n , number of mice analyzed . A P value of < 0 . 0001 was determined for the survival curves by statistical analysis with the log - rank ( Mantel - Cox ) test .

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(F) Kaplan-Meier survival analysis of Pax5Jak2/+ (black) and control Pax5+/+ (grey) mice. n, number of mice analyzed. A P value of < 0.0001 was determined for the survival curves by statistical analysis with the log-rank (Mantel-Cox) test.


( H ) Eosin - hematoxylin - stained sections of the lung and liver of a Pax5Jak2 / + tumor mouse . Infiltrating and blasting tumor cells are indicated by an arrow .

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(H) Eosin-hematoxylin-stained sections of the lung and liver of a Pax5Jak2/+ tumor mouse. Infiltrating and blasting tumor cells are indicated by an arrow.


( I ) Flow - cytometric analysis of lymph node cells from a control Pax5 + / + mouse and a 10 - week - old Pax5Jak2 / + tumor mouse . ( J ) Flow - cytometric analysis of B220lowCD19 + B cells from the bone marrow of a 4 - week - old Pax5Jak2 / + mouse .

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(I) Flow-cytometric analysis of lymph node cells from a control Pax5+/+ mouse and a 10-week-old Pax5Jak2/+ tumor mouse. (J) Flow-cytometric analysis of B220lowCD19+ B cells from the bone marrow of a 4-week-old Pax5Jak2/+ mouse.


( D - G ) GSEA analysis of 327 repressed ( D ) or 330 activated ( F ) Pax5 target genes identified in pro - B cells as compared with the ranked log2 - fold gene expression changes in Pax5Jak2 / + ( PJ ) B - ALLs versus control ( Ctrl ) Pax5 + / - Cdkn2ab + / - B - ALLs ( left ) . NES , normalized enrichment score . The expression of five genes that are upregulated in Pax5Jak2 / + B - ALL cells ( upper row ) and Pax5 - / - ( KO ) pro - B cells ( lower row ) is shown in ( E ) . Likewise , the expression of five genes that are upregulated in control B - ALL cells ( upper row ) and Pax5 + / + ( WT ) pro - B cells ( lower row ) is shown in ( G ) . TPM , transcripts per million . Mean TPM values with SEM are shown for the following RNA - seq experiments : 3 ( WT pro - B ) , 2 ( KO pro - B ) , 2 ( Ctrl B - ALL ) , and 4 ( PJ B - ALL ) .

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

(D-G) GSEA analysis of 327 repressed (D) or 330 activated (F) Pax5 target genes identified in pro-B cells as compared with the ranked log2-fold gene expression changes in Pax5Jak2/+ (PJ) B-ALLs versus control (Ctrl) Pax5+/- Cdkn2ab+/- B-ALLs (left). NES, normalized enrichment score. The expression of five genes that are upregulated in Pax5Jak2/+ B-ALL cells (upper row) and Pax5-/- (KO) pro-B cells (lower row) is shown in (E). Likewise, the expression of five genes that are upregulated in control B-ALL cells (upper row) and Pax5+/+ (WT) pro-B cells (lower row) is shown in (G). TPM, transcripts per million. Mean TPM values with SEM are shown for the following RNA-seq experiments: 3 (WT pro-B), 2 (KO pro-B), 2 (Ctrl B-ALL), and 4 (PJ B-ALL).


( H ) Expression of the Ptprc ( CD45 ) gene from exon 3 to exon 8 , as determined by RNA - seq of Pax5Jak2 / + and Pax5 + / - Cdkn2ab + / - B - ALL cells . The alternatively spliced exons 4 ( A ) , 5 ( B ) and 6 ( C ) are indicated in orange in the respective exon - intron structure of the Ptprc gene . Ptprc transcripts of B220 + Pax5 + / - Cdkn2ab + / - B - ALL cells contain all three exons , thus giving rise to expression of the CD45 isoform RABC ( known as B220 ) . In contrast , reads at exon 4 are barely detectable and reads at exon 6 are strongly reduced in the mRNA of Pax5Jak2 / + B - ALL cells , which likely gives rise to the CD45 isoforms RBC and

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(H) Expression of the Ptprc (CD45) gene from exon 3 to exon 8, as determined by RNA-seq of Pax5Jak2/+ and Pax5+/- Cdkn2ab+/- B-ALL cells. The alternatively spliced exons 4 (A), 5 (B) and 6 (C) are indicated in orange in the respective exon-intron structure of the Ptprc gene. Ptprc transcripts of B220+ Pax5+/- Cdkn2ab+/- B-ALL cells contain all three exons, thus giving rise to expression of the CD45 isoform RABC (known as B220). In contrast, reads at exon 4 are barely detectable and reads at exon 6 are strongly reduced in the mRNA of Pax5Jak2/+ B-ALL cells, which likely gives rise to the CD45 isoforms RBC and RB


( D ) Flow - cytometric analysis of bone marrow cells from 4 - week - old Pax5ihCd2 / + and Pax5Jak2 / ihCd2 mice . The expression of human ( h ) CD2 from the Pax5ihCd2 allele is shown for CD19 + B220 + ( grey ) and CD19 + B220low ( black ) B cells .

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(D) Flow-cytometric analysis of bone marrow cells from 4-week-old Pax5ihCd2/+ and Pax5Jak2/ihCd2 mice. The expression of human (h) CD2 from the Pax5ihCd2 allele is shown for CD19+B220+ (grey) and CD19+B220low (black) B cells.


( G ) Kaplan - Meier survival analysis of Cd79a - Cre Ikzf1neo / + Pax5LSL - Jak2 / + ( black ) and Cd79a - Cre Pax5LSL - Jak2 / + ( grey ) mice . Statistical analysis of the survival curves was performed with the log - rank ( Mantel - Cox ) test ; * * * * P < 0 . 0001 . n , number of mice analyzed .

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(G) Kaplan-Meier survival analysis of Cd79a-Cre Ikzf1neo/+ Pax5LSL-Jak2/+ (black) and Cd79a-Cre Pax5LSL-Jak2/+ (grey) mice. Statistical analysis of the survival curves was performed with the log-rank (Mantel-Cox) test; ****P < 0.0001. n, number of mice analyzed.


( H ) Flow - cytometric analysis of B220 and CD19 expression in B - ALL tumor cells from the lymph node of a Cd79a - Cre Ikzf1neo / + Pax5LSL - Jak2 / + mouse ( black ; left ) . Pax5 expression in these B - ALL tumor cells ( black line ) and control Pax5 + / + lymph node B cells ( grey filled ) was determined by intracellular Pax5 staining ( right ) .

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(H) Flow-cytometric analysis of B220 and CD19 expression in B-ALL tumor cells from the lymph node of a Cd79a-Cre Ikzf1neo/+ Pax5LSL-Jak2/+ mouse (black; left). Pax5 expression in these B-ALL tumor cells (black line) and control Pax5+/+ lymph node B cells (grey filled) was determined by intracellular Pax5 staining (right).


( B , Flow - cytometric analysis of bone marrow and spleen from Pax5Jak2 - KD / + ( black ; n = 11 ) and control Pax5 + / + ( grey ; n = 7 ) mice at the age of 6 - 8 weeks . The frequencies of the indicated B cell types are shown for each organ ( B ) . The data are presented as mean percentages with SEM and were statistically analyzed by multiple t - tests ( unpaired and two - tailed with Holm - Šídák ' s correction ) ; ns ( P > 0 . 5 ) , * P < 0 . 05 , * * P < 0 . 01 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE O B-TISSUE O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Flow-cytometric analysis of bone marrow and spleen from Pax5Jak2-KD/+ (black; n = 11) and control Pax5+/+ (grey; n = 7) mice at the age of 6-8 weeks. The frequencies of the indicated B cell types are shown for each organ (B). The data are presented as mean percentages with SEM and were statistically analyzed by multiple t-tests (unpaired and two-tailed with Holm-Šídák's correction); ns (P > 0.5), *P < 0.05, **P < 0.01.


( D , Tumor progression in mice , transplanted with Pax5Jak2 - Luc / + tumor cells , in the presence or absence of the JAK1 / 2 inhibitor ruxolitinib . At day 14 after cell transfer , the transplanted mice were treated twice daily with ruxolitinib or vehicle , and the tumor mass was monitored by bioluminescence measurements . Images of three representative mice with or without ruxolitinib treatment are

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

(D, Tumor progression in mice, transplanted with Pax5Jak2-Luc/+ tumor cells, in the presence or absence of the JAK1/2 inhibitor ruxolitinib. At day 14 after cell transfer, the transplanted mice were treated twice daily with ruxolitinib or vehicle, and the tumor mass was monitored by bioluminescence measurements. Images of three representative mice with or without ruxolitinib treatment are shown


( F ) Kaplan - Meier survival analysis of the transplanted mice upon prolonged treatment with ruxolitinib ( black ) and vehicle ( grey ) . Statistical analysis of the survival curves ( F ) was performed with the log - rank ( Mantel - Cox ) test ; * * P < 0 . 01 .

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

(F) Kaplan-Meier survival analysis of the transplanted mice upon prolonged treatment with ruxolitinib (black) and vehicle (grey). Statistical analysis of the survival curves (F) was performed with the log-rank (Mantel-Cox) test; **P < 0.01.


( B ) Genome - wide binding of Pax5 - Jak2 in in vitro cultured pro - B cells from Pax5Jak2 / + Rosa26BirA / + mice at the age of 3 weeks ( expressing Pax5 ) and ex vivo Pax5Jak2 / + Rosa26BirA / + B - ALL tumors ( lacking Pax5 ) , as determined by Bio - ChIP - seq analysis The DNA - binding pattern of full - length Pax5 was determined by Bio - ChIP - seq analysis of ex vivo sorted Pax5Bio / Bio pro - B cells , which carried a C ‐ terminal biotin acceptor sequence together with an IRES ‐ BirA gene insertion in the 3 ' untranslated region of Pax5 Two independent Bio - ChIP - seq experiments were performed for each cell type . Representative binding patterns of Pax5 and Pax5 - Jak2 in the three B cell types are shown for a selected genomic region , with horizontal bars indicating Pax5 or Pax5 - Jak2 peaks that were identified by MACS peak calling ( left ) . The number of Pax5 ( white ) and Pax5 - Jak2 ( grey or black ) peaks , which were defined by stringent MACS peak calling with a P value of < 10 - 10 in the three B cell types , are shown to the right .

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

(B) Genome-wide binding of Pax5-Jak2 in in vitro cultured pro-B cells from Pax5Jak2/+ Rosa26BirA/+ mice at the age of 3 weeks (expressing Pax5) and ex vivo Pax5Jak2/+ Rosa26BirA/+ B-ALL tumors (lacking Pax5), as determined by Bio-ChIP-seq analysis The DNA-binding pattern of full-length Pax5 was determined by Bio-ChIP-seq analysis of ex vivo sorted Pax5Bio/Bio pro-B cells, which carried a C‐terminal biotin acceptor sequence together with an IRES‐BirA gene insertion in the 3' untranslated region of Pax5 Two independent Bio-ChIP-seq experiments were performed for each cell type. Representative binding patterns of Pax5 and Pax5-Jak2 in the three B cell types are shown for a selected genomic region, with horizontal bars indicating Pax5 or Pax5-Jak2 peaks that were identified by MACS peak calling (left). The number of Pax5 (white) and Pax5-Jak2 (grey or black) peaks, which were defined by stringent MACS peak calling with a P value of < 10-10 in the three B cell types, are shown to the right.


( F , G ) Flow - cytometric analysis of the bone marrow and spleen from Pax5Prd * - Jak2 / + ( black ; n = 6 ) and control Pax5 + / + ( grey ; n = 5 ) mice at the age of 6 - 7 weeks . The frequency of the indicated B cell types in each organ is indicated in ( F ) , and the flow - cytometric analysis of CD19 and B220 expression on bone marrow B cells is shown in ( G ) . Statistical data are shown as mean percentages with SEM and were analyzed by multiple t - tests ( unpaired and two - tailed with Holm - Šídák ' s correction ) : ns > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 . Each dot corresponds to one mouse .

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

(F, G) Flow-cytometric analysis of the bone marrow and spleen from Pax5Prd*-Jak2 /+ (black; n = 6) and control Pax5+/+ (grey; n = 5) mice at the age of 6-7 weeks. The frequency of the indicated B cell types in each organ is indicated in (F), and the flow-cytometric analysis of CD19 and B220 expression on bone marrow B cells is shown in (G). Statistical data are shown as mean percentages with SEM and were analyzed by multiple t-tests (unpaired and two-tailed with Holm-Šídák's correction): ns > 0.05; *P < 0.05; **P < 0.01. Each dot corresponds to one mouse.


( H ) Kaplan - Meier survival analysis of Pax5Prd * - Jak2 / + ( black ) and Pax5 + / + ( grey ) mice . A P value of < 0 . 0001 was determined for the survival curves by statistical analysis with the log - rank ( Mantel - Cox ) test . n , number of mice analyzed .

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(H) Kaplan-Meier survival analysis of Pax5Prd*-Jak2 /+ (black) and Pax5+/+ (grey) mice. A P value of < 0.0001 was determined for the survival curves by statistical analysis with the log-rank (Mantel-Cox) test. n, number of mice analyzed.


( C ) Specificity of the anti - H3Y41ph antibody , as shown by immunoblot analysis . One phosphorylated ( pY41 ) or non - phosphorylated peptide was coupled to ubiquitin ( Ubi , 8 . 5 kDa ) , followed by separation of the protein conjugates on SDS - PAGE and immunoblot detection with the anti - H3Y41ph antibody present in the 8B2 - C1 cell supernatant . Following treatment with calf intestinal alkaline phosphatase ( CIP ) , the dephosphorylated Ubi - pY41 peptide conjugate could no longer be detected with the anti - H3Y41ph antibody .

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(C) Specificity of the anti-H3Y41ph antibody, as shown by immunoblot analysis. One phosphorylated (pY41) or non-phosphorylated peptide was coupled to ubiquitin (Ubi, 8.5 kDa), followed by separation of the protein conjugates on SDS-PAGE and immunoblot detection with the anti-H3Y41ph antibody present in the 8B2-C1 cell supernatant. Following treatment with calf intestinal alkaline phosphatase (CIP), the dephosphorylated Ubi-pY41 peptide conjugate could no longer be detected with the anti-H3Y41ph antibody.


( D ) Immunoblot analysis of whole - cell extracts prepared from Pax5Jak2 / + and control Pax5Etv6 / + Cdkn2ab + / - B - ALL cells as well as from the human HEL , TMD8 and K1106 cell lines . Phosphorylated ( p ) STAT5 was detected with an anti - STAT5 ( pY694 ) antibody and the pY41 - peptide with the purified anti H3Y41ph antibody One to five pY41 - peptides were coupled to ubiquitin , which was added in the range of 50 ng per well . The Gapdh and histone H3 proteins were analyzed as loading control . An unspecific protein is denoted by an asterisk . One representative of 5 immunoblot experiments is shown .

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(D) Immunoblot analysis of whole-cell extracts prepared from Pax5Jak2/+ and control Pax5Etv6/+ Cdkn2ab+/- B-ALL cells as well as from the human HEL, TMD8 and K1106 cell lines. Phosphorylated (p) STAT5 was detected with an anti-STAT5 (pY694) antibody and the pY41-peptide with the purified anti H3Y41ph antibody One to five pY41-peptides were coupled to ubiquitin, which was added in the range of 50 ng per well. The Gapdh and histone H3 proteins were analyzed as loading control. An unspecific protein is denoted by an asterisk. One representative of 5 immunoblot experiments is shown.


( A , Tumor progression in Il7 + / + ( grey ) , Il7 + / - ( dashed ) and Il7 - / - ( black ) mice transplanted with Pax5Jak2 - Luc / + tumor cells , as determined by bioluminescence measurements at the indicated days after cell transfer . Images of three transplanted mice for each Il7 genotype are shown at the indicated days after cell

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(A, Tumor progression in Il7+/+ (grey), Il7+/- (dashed) and Il7-/- (black) mice transplanted with Pax5Jak2-Luc/+ tumor cells, as determined by bioluminescence measurements at the indicated days after cell transfer. Images of three transplanted mice for each Il7 genotype are shown at the indicated days after cell transfer


( C ) Kaplan - Meier survival analysis of Il7 + / + , Il7 + / - and Il7 - / - mice transplanted with Pax5Jak2 - Luc / + tumors cells . Statistical analysis of the survival curves was performed with the log - rank ( Mantel - Cox ) test ; ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 .

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(C) Kaplan-Meier survival analysis of Il7+/+, Il7+/- and Il7-/- mice transplanted with Pax5Jak2-Luc/+ tumors cells. Statistical analysis of the survival curves was performed with the log-rank (Mantel-Cox) test; ns P > 0.05, *P < 0.05, **P < 0.01.


( E ) Quantification of the median fluorescence intensity ( MFI ) values of the p - STAT5 levels determined by intracellular staining of splenic FO B cells from Pax5 + / + ( grey ) , Pax5Jak2 / + ( black ) and Pax5Jak2 - KD / + ( white ) mice at the age of 3 - 10 weeks Statistical data are presented as mean values with SEM and were analyzed by the two - tailed unpaired Student ' s t - test : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Each dot corresponds to one mouse .

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(E) Quantification of the median fluorescence intensity (MFI) values of the p-STAT5 levels determined by intracellular staining of splenic FO B cells from Pax5+/+ (grey), Pax5Jak2/+ (black) and Pax5Jak2-KD/+ (white) mice at the age of 3-10 weeks Statistical data are presented as mean values with SEM and were analyzed by the two-tailed unpaired Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot corresponds to one mouse.


( F ) Flow cytometric analysis of p - STAT5 levels in pro - B cells ( B220 + CD19 + Kit + IgM - ) and leukemic B220low B cells of Pax5Jak2 / + mice ( black ) as well as in control pro - B cells of Pax5 + / + ( WT ) mice ( grey ) at the age of 4 - 5 weeks . A representative flow cytometric analysis of the bone marrow of a Pax5Jak2 / + mouse ( left ) and representative intracellular p - STAT5 stainings ( middle ) are shown . The p - STAT5 levels in pro - B and leukemic B220low B cells ( right ) are quantified as MFI values relative to that of Pax5 + / + pro - B cells . Statistical data are presented as mean values with SEM and were analyzed by the two - tailed unpaired Student ' s t - test : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Each dot corresponds to one mouse .

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(F) Flow cytometric analysis of p-STAT5 levels in pro-B cells (B220+CD19+Kit+IgM-) and leukemic B220low B cells of Pax5Jak2/+ mice (black) as well as in control pro-B cells of Pax5+/+ (WT) mice (grey) at the age of 4-5 weeks. A representative flow cytometric analysis of the bone marrow of a Pax5Jak2/+ mouse (left) and representative intracellular p-STAT5 stainings (middle) are shown. The p-STAT5 levels in pro-B and leukemic B220low B cells (right) are quantified as MFI values relative to that of Pax5+/+ pro-B cells. Statistical data are presented as mean values with SEM and were analyzed by the two-tailed unpaired Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot corresponds to one mouse.


( I ) Upregulation of the genes Cxcr5 , Syndig1l and Sema4a in Pax5Jak2 / + ( PJ ) B - ALL cells . Mean TPM values with SEM are shown for the following RNA - seq experiments : 2 ( Ctrl B - ALL ) , 4 ( PJ B - ALL )

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(I) Upregulation of the genes Cxcr5, Syndig1l and Sema4a in Pax5Jak2/+ (PJ) B-ALL cells. Mean TPM values with SEM are shown for the following RNA-seq experiments: 2 (Ctrl B-ALL), 4 (PJ B-ALL)


K ) Identification of Cxcr5 , Syndig1l and Sema4a as activated STAT5 target genes . ChIP - seq analysis identified STAT5 - binding regions at all three loci ( K ) . Horizontal bars below the ChIP - seq track indicate STAT5 - binding regions identified by MACS peak calling . RPM , reads per million .

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K) Identification of Cxcr5, Syndig1l and Sema4a as activated STAT5 target genes. ChIP-seq analysis identified STAT5-binding regions at all three loci (K). Horizontal bars below the ChIP-seq track indicate STAT5-binding regions identified by MACS peak calling. RPM, reads per million.


MPK4 ‐ HA is detected in PAT1 ‐ Myc immunoprecipitates from double transgenic Arabidopsis plants . Immunoblots of input and anti ‐ Myc IPs probed with anti ‐ HA and anti ‐ Myc antibodies . Left panel , input ; right panel , anti ‐ Myc IP .

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MPK4‐HA is detected in PAT1‐Myc immunoprecipitates from double transgenic Arabidopsis plants. Immunoblots of input and anti‐Myc IPs probed with anti‐HA and anti‐Myc antibodies. Left panel, input; right panel, anti‐Myc IP.


Yeast pat1Δ mutants are unable to grow at 37 ° C while wild ‐ type ( B4742 ) strain grows at 30 ° C ( left panel ) and 37 ° C ( right ) . Growth at 37 ° C is restored in pat1Δ expressing AtPAT1 . Serial dilutions of each yeast strain were plated on YPAD agar plates and grown at the indicated temperatures .

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Yeast pat1Δ mutants are unable to grow at 37°C while wild‐type (B4742) strain grows at 30°C (left panel) and 37°C (right). Growth at 37°C is restored in pat1Δ expressing AtPAT1. Serial dilutions of each yeast strain were plated on YPAD agar plates and grown at the indicated temperatures.


Co ‐ IP between PAT1 ‐ HA and LSM1 ‐ GFP . Proteins were transiently co ‐ expressed in N . benthamiana and tissue harvested 3 days post ‐ infiltration . Immunoblots of GFP IPs ( top panel ) and inputs ( 20 μg each , bottom panel ) . Immunoblots were cut in half and probed with anti ‐ HA antibodies and anti ‐ GFP antibodies .

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Co‐IP between PAT1‐HA and LSM1‐GFP. Proteins were transiently co‐expressed in N. benthamiana and tissue harvested 3 days post‐infiltration. Immunoblots of GFP IPs (top panel) and inputs (20 μg each, bottom panel). Immunoblots were cut in half and probed with anti‐HA antibodies and anti‐GFP antibodies.


Agarose gel electrophoresis of 5 ′ RACE PCR to detect capped transcripts of EIF4A1 , UGT87A2 and EXPL1 in 14 ‐ day ‐ old Col ‐ 0 and pat1 ‐ 1 .

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Agarose gel electrophoresis of 5′ RACE PCR to detect capped transcripts of EIF4A1, UGT87A2 and EXPL1 in 14‐day‐old Col‐0 and pat1‐1.


MPK4 and MPK6 were immunoprecipitated from extracts of Col ‐ 0 seedlings treated with 200 nM flg22 for 10 min . For the negative control ( − ) , extracts were incubated with agarose beads without antibodies . IPs were incubated with His6 ‐ PAT1 , His6 ‐ PAT1 S208A or MBP for 60 min at 37 ° C before boiling and SDS ‐ PAGE . Autoradiogram ( top panel ) , Coomassie ‐ stained gel for loading control ( bottom ) .

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MPK4 and MPK6 were immunoprecipitated from extracts of Col‐0 seedlings treated with 200 nM flg22 for 10 min. For the negative control (−), extracts were incubated with agarose beads without antibodies. IPs were incubated with His6‐PAT1, His6‐PAT1 S208A or MBP for 60 min at 37°C before boiling and SDS‐PAGE. Autoradiogram (top panel), Coomassie‐stained gel for loading control (bottom).


pat1 mutants have a serrated leaf , semi ‐ dwarf phenotypePlants photographed at 4 weeks of growth in short day conditions , genotypes as indicated . Col ‐ 0 and eds1 ‐ 2 plants and pat1 ‐ 1 and pat1 ‐ 1 / eds1 ‐ 2 , respectively , were placed in the middle of the same pots before the pictures were taken .

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pat1 mutants have a serrated leaf, semi‐dwarf phenotypePlants photographed at 4 weeks of growth in short day conditions, genotypes as indicated. Col‐0 and eds1‐2 plants and pat1‐1 and pat1‐1/eds1‐2, respectively, were placed in the middle of the same pots before the pictures were taken.


PR gene expression is elevated in mpk4 ‐ 2 and pat1 ‐ 1 mutants . Four ‐ week ‐ old plants were used for RNA extraction , followed by qRT ‐ PCR . Fold ‐ change in PR1 ( gray bars ) and PR2 ( black ) expression is relative to Col ‐ 0 , normalized to UBQ10 . Standard error of the mean is indicated by errors bars ( n = 3 ) . Statistical significance between the mean values was determined by ANOVA followed by Fisher ' s LSD test , P ‐ values are only shown for mean values significantly different from Col ‐ 0 .

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PR gene expression is elevated in mpk4‐2 and pat1‐1 mutants. Four‐week‐old plants were used for RNA extraction, followed by qRT‐PCR. Fold‐change in PR1 (gray bars) and PR2 (black) expression is relative to Col‐0, normalized to UBQ10. Standard error of the mean is indicated by errors bars (n = 3). Statistical significance between the mean values was determined by ANOVA followed by Fisher's LSD test, P‐values are only shown for mean values significantly different from Col‐0.


pat1 is more resistant to colonization by P . syringae pv . tomato DC3000 . Bacteria were syringe ‐ infiltrated and samples taken 3 days post ‐ infiltration . Data are shown as log10 ‐ transformed colony ‐ forming units / cm2 leaf tissue ( cfu / cm2 ) . Standard error of the mean is indicated by errors bars ( n = 4 ) . Statistical significance between the mean values was determined by ANOVA followed by Fisher ′ s LSD test .

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pat1 is more resistant to colonization by P. syringae pv. tomato DC3000. Bacteria were syringe‐infiltrated and samples taken 3 days post‐infiltration. Data are shown as log10‐transformed colony‐forming units/cm2 leaf tissue (cfu/cm2). Standard error of the mean is indicated by errors bars (n = 4). Statistical significance between the mean values was determined by ANOVA followed by Fisher′s LSD test.


Confocalmicroscopy with root elongation zones . Five ‐ day ‐ old Col ‐ 0 / PAT1 ‐ GFP seedlings were treated with 1 μM flg22 on glass slides for 20 min ( second panel ) followed by treatment with 100 μg / ml cycloheximide for 60 min ( bottom panel ) . The scale bar corresponds to 10 μm .

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Confocalmicroscopy with root elongation zones. Five‐day‐old Col‐0/PAT1‐GFP seedlings were treated with 1 μM flg22 on glass slides for 20 min (second panel) followed by treatment with 100 μg/ml cycloheximide for 60 min (bottom panel). The scale bar corresponds to 10 μm.


Confocalmicroscopy with 5 ‐ day ‐ old VCS ‐ GFP seedlings treated as in ( A ) . The scale bar corresponds to 10 μm .

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Confocalmicroscopy with 5‐day‐old VCS‐GFP seedlings treated as in (A). The scale bar corresponds to 10 μm.


PAT1 protein is induced by PAMP treatment . Immunoblot detection of equal amounts of protein from Col ‐ 0 seedlings at times in minutes as indicated following vacuum infiltration with 1 μM flg22 or water ( 180 min after infiltration ) . Immunoblots were probed with anti ‐ PAT1 antibodies . Negative control pat1 ‐ 1 was loaded for comparison . Coomassie brilliant blue protein loading control is indicated by CBB .

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PAT1 protein is induced by PAMP treatment. Immunoblot detection of equal amounts of protein from Col‐0 seedlings at times in minutes as indicated following vacuum infiltration with 1 μM flg22 or water (180 min after infiltration). Immunoblots were probed with anti‐PAT1 antibodies. Negative control pat1‐1 was loaded for comparison. Coomassie brilliant blue protein loading control is indicated by CBB.


Anti ‐ GFP immunoblotting of proteins extracted from Col ‐ 0 / PAT1 ‐ GFP seedlings treated over a time course with flg22 ( top panel ) and Coomassie brilliant blue ( CBB ) ‐ stained PVDF loading control is shown ( bottom panel ) . No PAT1 ‐ GFP was detected in negative control Col ‐ 0 .

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Anti‐GFP immunoblotting of proteins extracted from Col‐0/PAT1‐GFP seedlings treated over a time course with flg22 (top panel) and Coomassie brilliant blue (CBB)‐stained PVDF loading control is shown (bottom panel). No PAT1‐GFP was detected in negative control Col‐0.


Elevated PR gene expression in pat1 mutants is suppressed by summ2 ‐ 8 . Four ‐ week ‐ old plants were used for RNA extraction , followed by qRT ‐ PCR . Fold ‐ change in PR1 ( gray bars ) and PR2 ( black bars ) expression is relative to Col ‐ 0 , normalized to UBQ10 . Standard error of the mean is indicated by errors bars ( n = 3 ) .

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Elevated PR gene expression in pat1 mutants is suppressed by summ2‐8. Four‐week‐old plants were used for RNA extraction, followed by qRT‐PCR. Fold‐change in PR1 (gray bars) and PR2 (black bars) expression is relative to Col‐0, normalized to UBQ10. Standard error of the mean is indicated by errors bars (n = 3).


pat1 resistance to P . syringae pv . tomato DC3000 is suppressed by summ2 ‐ 8 . Bacteria were syringe ‐ infiltrated and samples taken 3 days post ‐ infiltration . Data are log10 ‐ transformed colony ‐ forming units / cm2 leaf tissue ( cfu / cm2 ) . Standard error of the mean is indicated by errors bars ( n = 4 ) .

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pat1 resistance to P. syringae pv. tomato DC3000 is suppressed by summ2‐8. Bacteria were syringe‐infiltrated and samples taken 3 days post‐infiltration. Data are log10‐transformed colony‐forming units/cm2 leaf tissue (cfu/cm2). Standard error of the mean is indicated by errors bars (n = 4).


14 ‐ day ‐ old seedlings grown on ms plates were used for RNA extraction . Next , 1 μg of RNA from each sample was treated with XRN1 ( NEB ) or mock treated before RT ‐ qPCR . Data were normalized to ACT2 , and transcript levels were compared between XRN1 and mock ‐ treated RNA for each genotype . Standard error of the mean is indicated by error bars ( n = 4 ) . Statistical significance between the mean values was determined by ANOVA followed by Fisher ' s LSD test .

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14‐day‐old seedlings grown on ms plates were used for RNA extraction. Next, 1 μg of RNA from each sample was treated with XRN1 (NEB) or mock treated before RT‐qPCR. Data were normalized to ACT2, and transcript levels were compared between XRN1 and mock‐treated RNA for each genotype. Standard error of the mean is indicated by error bars (n = 4). Statistical significance between the mean values was determined by ANOVA followed by Fisher's LSD test.


Phosphorylation of the PAT1 peptide SSFVSYPPPGSISPDQR , which include Ser208 , in Col ‐ 0 and mkk1 / 1 ‐ summ2 before and after flg22 treatment . The phosphorylation stoichiometry is illustrated relative to Col PAT1 without flg22 treatment . The ratios were obtained using quantitative iTRAQ mass spectrometry .

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Phosphorylation of the PAT1 peptide SSFVSYPPPGSISPDQR, which include Ser208, in Col‐0 and mkk1/1‐summ2 before and after flg22 treatment. The phosphorylation stoichiometry is illustrated relative to Col PAT1 without flg22 treatment. The ratios were obtained using quantitative iTRAQ mass spectrometry.


Proteins were transiently co ‐ expressed ( PAT1 ‐ GFP + MPK4 ‐ HA or SUMM2 ‐ HA ) in N . benthamiana and tissue harvested 2 days post ‐ infiltration . Anti ‐ HA immunoblots of inputs ( 20 μg each ) are shown in the bottom panel . Immunoblots of GFP IPs were probed with anti ‐ HA antibodies , then stripped and probed with anti ‐ GFP antibodies . Molecular weights are shown in kDa on the right .

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Proteins were transiently co‐expressed (PAT1‐GFP + MPK4‐HA or SUMM2‐HA) in N. benthamiana and tissue harvested 2 days post‐infiltration. Anti‐HA immunoblots of inputs (20 μg each) are shown in the bottom panel. Immunoblots of GFP IPs were probed with anti‐HA antibodies, then stripped and probed with anti‐GFP antibodies. Molecular weights are shown in kDa on the right.


Confocalimages of yellow fluorescent protein complementation . PAT1 ‐ Nc was transiently co ‐ expressed with either SUMM2 ‐ Nn or At4g12010 in N . benthamiana . Confocalmicroscopy on leaf disks was conducted 2 days post ‐ infiltration . Merged pictures show overlay of brightfield , chlorophyll and eYFP . The scale bar corresponds to 25 μm .

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Confocalimages of yellow fluorescent protein complementation. PAT1‐Nc was transiently co‐expressed with either SUMM2‐Nn or At4g12010 in N. benthamiana. Confocalmicroscopy on leaf disks was conducted 2 days post‐infiltration. Merged pictures show overlay of brightfield, chlorophyll and eYFP. The scale bar corresponds to 25 μm.


A , B Bottom panels : gene annotation track ( mm10 ) ; the activated gene is marked in green and the location of the TALE - VP64 target sequence is shown by the vertical dashed green line . Middle panels : DamID tracks of NL interactions in control cells ( " control " , blue line ) and cells expressing TALE - VP64 ( " experimental " , red line ) . n indicates the number of independent biological replicates that were combined . Noise was suppressed by a running mean filter of indicated window size . Shading between the lines corresponds to the color of the sample with the highest value . Arrowheads on the right - hand side mark the 5th and 95th percentiles of genome - wide DamID values . Top panels : domainograms ; for every window of indicated size ( vertical axis ) and centered on a genomic position ( horizontal axis ) the pixel shade indicates the ranking of the change in DamID score ( experimental minus control ) in this window compared to the genome - wide changes in DamID scores across all possible windows of the same size . Blue : DamID score is highest in control samples ; red : DamID score is highest in experimental samples ( color key on the right of panel ( a ) ) . In ( a ) activation of Sox6 was the experimental perturbation , activation of Nrp1 ( which is located on a different chromosome ) served as control ; in ( b ) activation of Nrp1 was the experimental condition and activation of Sox6 served as control .

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

A, B Bottom panels: gene annotation track (mm10); the activated gene is marked in green and the location of the TALE-VP64 target sequence is shown by the vertical dashed green line. Middle panels: DamID tracks of NL interactions in control cells ("control", blue line) and cells expressing TALE-VP64 ("experimental", red line). n indicates the number of independent biological replicates that were combined. Noise was suppressed by a running mean filter of indicated window size. Shading between the lines corresponds to the color of the sample with the highest value. Arrowheads on the right-hand side mark the 5th and 95th percentiles of genome-wide DamID values. Top panels: domainograms; for every window of indicated size (vertical axis) and centered on a genomic position (horizontal axis) the pixel shade indicates the ranking of the change in DamID score (experimental minus control) in this window compared to the genome-wide changes in DamID scores across all possible windows of the same size. Blue: DamID score is highest in control samples; red: DamID score is highest in experimental samples (color key on the right of panel (a)). In (a) activation of Sox6 was the experimental perturbation, activation of Nrp1 (which is located on a different chromosome) served as control; in (b) activation of Nrp1 was the experimental condition and activation of Sox6 served as control.


( a - b ) Plots as in Figure 1 , showing changes in Lamin B1 DamID signals upon CRISPRa activation of NLGN1 ( a ) and SOX6 ( b ) . Control cells were treated either without sgRNA or with one of various sgRNAs targeting promoters on different chromosomes . Vertical green dotted lines mark the position of the sgRNA target sequence .

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

(a-b) Plots as in Figure 1, showing changes in Lamin B1 DamID signals upon CRISPRa activation of NLGN1 (a) and SOX6 (b). Control cells were treated either without sgRNA or with one of various sgRNAs targeting promoters on different chromosomes. Vertical green dotted lines mark the position of the sgRNA target sequence.


( c ) Domainograms showing regions with reduced NL interactions around 12 genes individually activated by CRISPRa . Genomic regions are aligned by the respective sgRNA target positions , and oriented so that the activated genes are all transcribed from left to right .

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

(c) Domainograms showing regions with reduced NL interactions around 12 genes individually activated by CRISPRa. Genomic regions are aligned by the respective sgRNA target positions, and oriented so that the activated genes are all transcribed from left to right.


Average DamID values plotted against average expression levels of eight genes activated by CRISPRa ( red ; n = 2 ) or in control cells that were treated either without sgRNA or with one of various sgRNAs targeting promoters on different chromosomes ( blue ; n ranging from 19 to 27 ) .

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

Average DamID values plotted against average expression levels of eight genes activated by CRISPRa (red; n = 2) or in control cells that were treated either without sgRNA or with one of various sgRNAs targeting promoters on different chromosomes (blue; n ranging from 19 to 27).


A - C CRISPRa activation of ADAM22 ( a ) , ABCB1 ( b ) and PTN ( c ) in human RPE - 1 cells . Top panels visualize DamID data similar to Figure 2a - b . Middle panels show maps of replication timing at the same resolution and in the same plotting style as panels , except that different colors are used as indicated . Bottom panels show gene track , with activated gene highlighted in green .

O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-ORGANISM B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-C CRISPRa activation of ADAM22 (a), ABCB1 (b) and PTN (c) in human RPE-1 cells. Top panels visualize DamID data similar to Figure 2a-b. Middle panels show maps of replication timing at the same resolution and in the same plotting style as panels, except that different colors are used as indicated. Bottom panels show gene track, with activated gene highlighted in green.