Figure 2 ( A ) Comparison of the dynamic behavior of the GFP - tagged Chs5 and Bch1 with the GFP - tagged Sec28 , Cop1 , Clc1 and Apm1 . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 . ( B ) Binding kinetics of the GFP - tagged Chs5 and ChAPs at the TGN is different . Data processing was carried out as in ( A ) . ( C ) Quantification of the relative expression levels of the exomer components in GFP - tagged strains . A representative immunoblot of yeast lysates is shown ; Pgk1 serves as loading control . The asterisks depict the analyzed bands . The plot shows average and SD of three independent biological experiments . The relative expression levels are normalized to Chs6 ( depicted by the red line ) .

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 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 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 2(A) Comparison of the dynamic behavior of the GFP-tagged Chs5 and Bch1 with the GFP-tagged Sec28, Cop1, Clc1 and Apm1. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.(B) Binding kinetics of the GFP-tagged Chs5 and ChAPs at the TGN is different. Data processing was carried out as in (A).(C) Quantification of the relative expression levels of the exomer components in GFP-tagged strains. A representative immunoblot of yeast lysates is shown; Pgk1 serves as loading control. The asterisks depict the analyzed bands. The plot shows average and SD of three independent biological experiments. The relative expression levels are normalized to Chs6 (depicted by the red line).


Figure 3 ( A ) Chs5 is necessary for recruitment and stable binding of ChAPs at the TGN . Differential centrifugation of cell lysates obtained from Chs6 - GFP WT and Δchs5 strain . TCL , total cell lysate ; S10 , 10 , 000 g supernatant ; P10 , 10 , 000 g pellet ; S100 , 100 , 000 g supernatant ; P100 , 100 , 000 g pellet . Anp1 serves as the Golgi marker and Pgk1 as the cytoplasm marker . A representative immunoblot of three independent biological experiments is shown . ( B ) Diffusional behavior of GFP - tagged ChAPs in the cytoplasm ( open triangles , dashed line ) and at the TGN ( closed circles , full line ) . The mean of 10 - 20 FRAP measurements from different cells is shown . Calculated diffusion coefficients are shown in Expanded View Table 1 . ( C ) Chs5 - GFP displays reduced membrane association in the absence of ChAPs . Differential centrifugation of cell lysates obtained from Chs5 GFP WT and ΔΔΔΔ strains . TCL , total cell lysate ; S10 , 10 , 000 g supernatant ; P10 , 10 , 000 g pellet ; S100 , 100 , 000 g supernatant ; P100 , 100 , 000 g pellet . Anp1 serves as the Golgi marker and Pgk1 as the cytoplasm marker . A representative immunoblot of three independent biological experiments is shown . ( D ) Fluorescence images of cells expressing Chs5 - GFP and TGN marker Sec7 - DsRed in WT , ChAP and strains . Merged images depict the differences in Chs5 - GFP localization to the TGN , cytoplasm and distinct foci ( arrows ) in these strains . The brightness and contrast were adjusted differently in the merged images . Bar , 5 µm . ( E ) Co - localization analysis of Chs5 - GFP and TGN marker Sec7 - DsRed in WT , ChAP and strains . Mander 1 depicts the relative amount of the GFP signal co - localizing with the overall DsRed signal , and Mander 2 vice versa . Manders coefficients were obtained using the JACoP plug - in for ImageJ after background subtraction ( N = 20 ) and are given in Expanded View Table 2 . ( F ) Binding kinetics of Chs5 - GFP at the TGN in WT , ChAP and strains . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 . The schematic cartoons depict the predicted majority of the analyzed complexes . The violet ovals represent Chs5 dimer and circles represent ChAPs : green : Bch1 , red : Bud7 , dark blue : Bch2 , light blue : Chs6 .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 3(A) Chs5 is necessary for recruitment and stable binding of ChAPs at the TGN. Differential centrifugation of cell lysates obtained from Chs6-GFP WT and Δchs5 strain. TCL, total cell lysate; S10, 10,000 g supernatant; P10, 10,000 g pellet; S100, 100,000 g supernatant; P100, 100,000 g pellet. Anp1 serves as the Golgi marker and Pgk1 as the cytoplasm marker. A representative immunoblot of three independent biological experiments is shown.(B) Diffusional behavior of GFP-tagged ChAPs in the cytoplasm (open triangles, dashed line) and at the TGN (closed circles, full line). The mean of 10-20 FRAP measurements from different cells is shown. Calculated diffusion coefficients are shown in Expanded View Table 1.(C) Chs5-GFP displays reduced membrane association in the absence of ChAPs. Differential centrifugation of cell lysates obtained from Chs5 GFP WT and ΔΔΔΔ strains. TCL, total cell lysate; S10, 10,000 g supernatant; P10, 10,000 g pellet; S100, 100,000 g supernatant; P100, 100,000 g pellet. Anp1 serves as the Golgi marker and Pgk1 as the cytoplasm marker. A representative immunoblot of three independent biological experiments is shown.(D) Fluorescence images of cells expressing Chs5-GFP and TGN marker Sec7-DsRed in WT, ChAP and strains. Merged images depict the differences in Chs5-GFP localization to the TGN, cytoplasm and distinct foci (arrows) in these strains. The brightness and contrast were adjusted differently in the merged images. Bar, 5 µm.(E) Co-localization analysis of Chs5-GFP and TGN marker Sec7-DsRed in WT, ChAP and strains. Mander 1 depicts the relative amount of the GFP signal co-localizing with the overall DsRed signal, and Mander 2 vice versa. Manders coefficients were obtained using the JACoP plug-in for ImageJ after background subtraction (N = 20) and are given in Expanded View Table 2.(F) Binding kinetics of Chs5-GFP at the TGN in WT, ChAP and strains. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2. The schematic cartoons depict the predicted majority of the analyzed complexes. The violet ovals represent Chs5 dimer and circles represent ChAPs: green:Bch1, red:Bud7, dark blue:Bch2, light blue:Chs6.


Figure 4 ( A - B ) Overexpression of Bch2 in absence of other ChAPs results in more efficient Chs5 recruitment and stabilization at the TGN . Fluorescence images of cells expressing Chs5 - GFP ( A ) and binding kinetics of Chs5 - GFP at the TGN ( B ) in WT , BCH2 and GPD - BCH2 strains . Bar , 5 µm . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 . The schematic cartoons depict the predicted majority of the analyzed complexes ; Chs5 dimer : violet ovals , Bch2 : dark blue circles . ( C - D ) Overexpression of Chs6 in absence of other ChAPs results in more efficient Chs5 recruitment and stabilization at the TGN . ( C ) Fluorescence images of cells expressing Chs5 - GFP in WT , CHS6 and GPD - CHS6 strains . Bar , 5 µm . ( D ) Binding kinetics of Chs5 - GFP at the TGN in WT , , CHS6 and GPD - CHS6 strains . Data processing was carried out as in ( A - B ) . The schematic cartoons depict the predicted majority of the analyzed complexes ; Chs5 dimer : violet ovals , Chs6 : light blue circles . ( E - G ) Overexpression of Chs6 rescues Chs3 export in absence of other ChAPs . ( E ) Fluorescence images of cells expressing Chs3 - GFP in WT , CHS6 and GPD - CHS6 strains . Arrows point to Chs3 - GFP signals at the bud neck . Bar , 5 µm . The inset represents a twofold magnification . ( F ) Quantification of phenotypes in ( E ) ; 100 small budded and 100 large budded cells were quantified per experiment . Average and SD of three independent biological experiments is shown . ( G ) Chs3 GFP GPD - CHS6 strain was sensitive to calcofluor to a similar extent as the WT strain . Plates were incubated at 30 ° C for 2 - 3 days . A representative drop test of three independent biological experiments is shown .

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

Figure 4(A-B) Overexpression of Bch2 in absence of other ChAPs results in more efficient Chs5 recruitment and stabilization at the TGN. Fluorescence images of cells expressing Chs5-GFP (A) and binding kinetics of Chs5-GFP at the TGN (B) in WT, BCH2 and GPD-BCH2 strains. Bar, 5 µm. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2. The schematic cartoons depict the predicted majority of the analyzed complexes; Chs5 dimer:violet ovals, Bch2:dark blue circles.(C-D) Overexpression of Chs6 in absence of other ChAPs results in more efficient Chs5 recruitment and stabilization at the TGN. (C) Fluorescence images of cells expressing Chs5-GFP in WT, CHS6 and GPD-CHS6 strains. Bar, 5 µm. (D) Binding kinetics of Chs5-GFP at the TGN in WT, , CHS6 and GPD-CHS6 strains. Data processing was carried out as in (A-B). The schematic cartoons depict the predicted majority of the analyzed complexes; Chs5 dimer: violet ovals, Chs6: light blue circles.(E-G) Overexpression of Chs6 rescues Chs3 export in absence of other ChAPs. (E) Fluorescence images of cells expressing Chs3-GFP in WT, CHS6 and GPD-CHS6 strains. Arrows point to Chs3-GFP signals at the bud neck. Bar, 5 µm. The inset represents a twofold magnification.(F) Quantification of phenotypes in (E); 100 small budded and 100 large budded cells were quantified per experiment. Average and SD of three independent biological experiments is shown.(G) Chs3 GFP GPD-CHS6 strain was sensitive to calcofluor to a similar extent as the WT strain. Plates were incubated at 30°C for 2-3 days. A representative drop test of three independent biological experiments is shown.


Figure 5Fluorescence images of cells expressing Chs5 - GFP and TGN marker Sec7DsRed ( A ) and binding kinetics of Chs5 - GFP at the TGN ( B ) in WT , CHS6 and CHS6BUD7 ( A , B ) The brightness and contrast were adjusted differently in the merged images . Bar , 5 µm . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 . Fluorescence images of cells expressing Chs5 - GFP and TGN marker Sec7DsRed ( C ) and binding kinetics of Chs5 - GFP at the TGN ( D ) in WT , or CHS6BCH2 ( C , D ) strains . The brightness and contrast were adjusted differently in the merged images . Bar , 5 µm . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 .

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 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

Figure 5Fluorescence images of cells expressing Chs5-GFP and TGN marker Sec7DsRed (A) and binding kinetics of Chs5-GFP at the TGN (B) in WT, CHS6 and CHS6BUD7 (A, B) The brightness and contrast were adjusted differently in the merged images. Bar, 5 µm. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.Fluorescence images of cells expressing Chs5-GFP and TGN marker Sec7DsRed (C) and binding kinetics of Chs5-GFP at the TGN (D) in WT, or CHS6BCH2 (C, D) strains. The brightness and contrast were adjusted differently in the merged images. Bar, 5 µm. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.


Figure 6 ( A - D ) ChAPs are differently dependent on each other in terms of binding at the TGN . Binding kinetics of GFP - tagged Chs6 ( A ) , Bud7 ( B ) , Bch2 ( C ) and Bch1 ( D ) at the TGN in the presence ( WT ) or absence of the other ChAPs ( ) . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 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 O O O O O O O O O O O O O O O O O O O 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(A-D) ChAPs are differently dependent on each other in terms of binding at the TGN. Binding kinetics of GFP-tagged Chs6 (A), Bud7 (B), Bch2 (C) and Bch1 (D) at the TGN in the presence (WT) or absence of the other ChAPs (). The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.


Figure 7 ( A - B ) Overexpression of Bch1 displaces Chs6 from the TGN and impairs plasma membrane localization of Chs6 - dependent cargo Chs3 . Fluorescence images of Chs6 - GFP ( A ) and Chs3 - GFP ( B ) in WT and GPD - BCH1 strains . Bar , 5 µm . Arrows point to Chs3 - GFP signals at the bud neck . The inset represents a twofold magnification . ( C ) Quantification of the phenotypes in ( B ) ; 100 small budded and 100 large budded cells were quantified per experiment . Average and SD of three independent biological experiments is shown . ( D ) Chitin production is reduced upon Bch1 overexpression . Fluorescence images of chitin stained with calcofluor in Chs3 - GFP WT , chs5 and GPD - BCH1 strains . ( E ) Overexpression of Bch1 does not impair plasma membrane localization of the Bch1 - dependent cargo Pin2 . Fluorescence images of Pin2 - GFP in WT and GPD - BCH1 strains . ( F - I ) Overexpression of Chs6 displaces Bch1 and Chs5 from the TGN . Fluorescence images ( F - G ) and binding kinetics at the TGN ( H - I ) of cells expressing Bch1 - GFP ( F , H ) and Chs5 - GFP ( G , I ) upon overexpression of Chs6 under GPD promoter from 2μ plasmid . The insets in ( F , G ) represent two - fold magnifications . The arrows in ( F ) point to residual Bch1 staining at the TGN . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 . Size bars represent 5 µm .

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 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 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 O O O O O O O O O O O O O O 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 7(A-B) Overexpression of Bch1 displaces Chs6 from the TGN and impairs plasma membrane localization of Chs6-dependent cargo Chs3. Fluorescence images of Chs6-GFP (A) and Chs3-GFP (B) in WT and GPD-BCH1 strains. Bar, 5 µm. Arrows point to Chs3-GFP signals at the bud neck. The inset represents a twofold magnification.(C) Quantification of the phenotypes in (B); 100 small budded and 100 large budded cells were quantified per experiment. Average and SD of three independent biological experiments is shown.(D) Chitin production is reduced upon Bch1 overexpression. Fluorescence images of chitin stained with calcofluor in Chs3-GFP WT, chs5 and GPD-BCH1 strains.(E) Overexpression of Bch1 does not impair plasma membrane localization of the Bch1-dependent cargo Pin2. Fluorescence images of Pin2-GFP in WT and GPD-BCH1 strains.(F-I) Overexpression of Chs6 displaces Bch1 and Chs5 from the TGN. Fluorescence images (F-G) and binding kinetics at the TGN (H-I) of cells expressing Bch1-GFP (F, H) and Chs5-GFP (G, I) upon overexpression of Chs6 under GPD promoter from 2μ plasmid. The insets in (F, G) represent two-fold magnifications. The arrows in (F) point to residual Bch1 staining at the TGN. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2. Size bars represent 5 µm.


Figure 8 ( A ) Overexpression of Pin2 enhances Chs5 stabilization at the TGN . Binding kinetics of Chs5 GFP in WT , bud7bch1 and bud7bch1 strain expressing Bch2 - dependent cargo Pin2 from a centromeric or a 2μ plasmid . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 . ( B ) Comparison of the expression levels of the endogenous Pin2 with the levels of Pin2 expressed from a centromeric or a 2μ plasmid . Pgk1 serves as loading control . A representative immunoblot of three independent biological experiments is shown .

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 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

Figure 8(A) Overexpression of Pin2 enhances Chs5 stabilization at the TGN. Binding kinetics of Chs5 GFP in WT, bud7bch1 and bud7bch1 strain expressing Bch2-dependent cargo Pin2 from a centromeric or a 2μ plasmid. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.(B) Comparison of the expression levels of the endogenous Pin2 with the levels of Pin2 expressed from a centromeric or a 2μ plasmid. Pgk1 serves as loading control. A representative immunoblot of three independent biological experiments is shown.


Figure 1 ( a ) ( top ) Experimental design . Cultured cortical neurons are stimulated with BDNF , KCl or no treatment ( Control ) and activated neurons are prepared in three specific time points for RNA - seq and ATAC - seq at early ( 1h ) and late time points ( 6 and 10h ) ; ( bottom ) Blot for pMAPK in neurons treated with KCl 55 mM and several concentrations of BDNF ( Con = no stimulation ) . 44 and 42 KDa bands indicated with lines . Nucleolin shown as internal control . ( b ) Hierarchical clustering for all differentially - expressed genes ( DE - genes , adjusted P - value < 0 . 1 , using Benjamini Hochberg ' s correction ) using correlation of log2 - fold changes when compared to unstimulated control samples at each time point . ( d ) Partitioning around medoids clustering of expression dynamics using genewise - scaled Z - scores of top 5 , 000 DE - genes from ( c ) with lowest adjusted p - value ( k = 10 clusters ) . Numbers indicate clusters . Left line plots indicate expression levels for signature genes selected for each cluster . 4 , 907 DE - genes are also selected with FDR = 1 % and more than 100 counts across samples . ( e ) Gene Ontology enrichment analysis for clusters shown in ( d ) . Each ribbon shows clusters with their respective significant gene groups using topGO ( Alexa et al , 2006 ) . Up to five significant terms per cluster are shown ( P - values adjusted with BH ) . Circle sizes indicate enrichment of ontology genes in each cluster versus all other clusters . Names on the right y - axis indicate ontology common names .

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 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

Figure 1(a) (top) Experimental design. Cultured cortical neurons are stimulated with BDNF, KCl or no treatment (Control) and activated neurons are prepared in three specific time points for RNA-seq and ATAC-seq at early (1h) and late time points (6 and 10h); (bottom) Blot for pMAPK in neurons treated with KCl 55 mM and several concentrations of BDNF (Con = no stimulation). 44 and 42 KDa bands indicated with lines. Nucleolin shown as internal control.(b) Hierarchical clustering for all differentially-expressed genes (DE-genes, adjusted P-value < 0.1, using Benjamini Hochberg's correction) using correlation of log2-fold changes when compared to unstimulated control samples at each time point.(d) Partitioning around medoids clustering of expression dynamics using genewise-scaled Z-scores of top 5,000 DE-genes from (c) with lowest adjusted p-value (k = 10 clusters). Numbers indicate clusters. Left line plots indicate expression levels for signature genes selected for each cluster. 4,907 DE-genes are also selected with FDR=1% and more than 100 counts across samples. (e) Gene Ontology enrichment analysis for clusters shown in (d). Each ribbon shows clusters with their respective significant gene groups using topGO(Alexa et al, 2006). Up to five significant terms per cluster are shown (P-values adjusted with BH). Circle sizes indicate enrichment of ontology genes in each cluster versus all other clusters. Names on the right y-axis indicate ontology common names.


Figure 2 ( a ) Hierarchical clustering for all differentially accessible peaks ( DA - peaks , adjusted P < 0 . 1 , using Benjamini Hochberg ' s correction ) using correlation of log2 - fold changes when compared versus matched control samples . ( d ) ( top ) Association between distal regulatory elements ( DREs ) and gene expression at the BDNF 1h time - point . Each point indicates the log2 - fold change of an ATAC - seq peak ( x - axis ) and the gene expression of a closest gene ( y - axis ) with distance between 2 - 20 Kbp . Colors indicate whether none ( gray ) , only the peak ( orange ) , or both peak and gene ( red ) show significant changes versus control neurons . ( bottom ) Enrichment for paired DA - peak and DE - gene in the four quadrants are summarized for BDNF and KCl . Asterisks indicate P values as corrected by a Benjamini Hochberg procedure ( * = P < 0 . 05 ; * * = P < 0 . 01 ) .

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 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

Figure 2(a) Hierarchical clustering for all differentially accessible peaks (DA-peaks, adjusted P < 0.1, using Benjamini Hochberg's correction) using correlation of log2-fold changes when compared versus matched control samples.(d) (top) Association between distal regulatory elements (DREs) and gene expression at the BDNF 1h time-point. Each point indicates the log2-fold change of an ATAC-seq peak (x-axis) and the gene expression of a closest gene (y-axis) with distance between 2-20 Kbp. Colors indicate whether none (gray), only the peak (orange), or both peak and gene (red) show significant changes versus control neurons. (bottom) Enrichment for paired DA-peak and DE-gene in the four quadrants are summarized for BDNF and KCl. Asterisks indicate P values as corrected by a Benjamini Hochberg procedure (* = P < 0.05; ** = P < 0.01).


Figure 3 ( c ) RNA expression changes for TFs related to 8 - mer groups bZIP , CTCF , HIC1 , and EGR . Significant changes relative to control samples are shown with asterisks ( Wilcoxon rank sums test , two - sided ) .

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Figure 3(c) RNA expression changes for TFs related to 8-mer groups bZIP, CTCF, HIC1, and EGR. Significant changes relative to control samples are shown with asterisks (Wilcoxon rank sums test, two-sided).


Figure 4 ( c ) Chromosome 15 genome tracks neighboring Arc , displaying ATAC - seq read counts per million ( CPM ) ; RNA - seq CPM ; H3K27ac normalized signal upon KCl stimulation , H3K4me1 ( Malik et al , 2014 ) ; CTCF ( Sams et al , 2016 ) ; and Cortical neurons Hi - C data ( Bonev et al , 2017 ) . Red bars in ATAC - seq tracks indicate gained DA - peaks in BDNF , and red bars in RNA - seq tracks indicate Arc differential expression in BDNF and KCl 1h . Green blocks in Hi - C tracks indicate anchor points for the calculation of contact scores , using shaman ( Cohen et al , 2017 ) . Line and Spearman ' s rho value indicate counts rank - based correlation between highlighted peaks across all samples ( Appendix Figure S4c ) . TF module names indicate the presence of 8 - mers in those peaks .

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

Figure 4(c) Chromosome 15 genome tracks neighboring Arc, displaying ATAC-seq read counts per million (CPM); RNA-seq CPM; H3K27ac normalized signal upon KCl stimulation, H3K4me1(Malik et al, 2014); CTCF(Sams et al, 2016); and Cortical neurons Hi-C data(Bonev et al, 2017). Red bars in ATAC-seq tracks indicate gained DA-peaks in BDNF, and red bars in RNA-seq tracks indicate Arc differential expression in BDNF and KCl 1h. Green blocks in Hi-C tracks indicate anchor points for the calculation of contact scores, using shaman(Cohen et al, 2017). Line and Spearman's rho value indicate counts rank-based correlation between highlighted peaks across all samples (Appendix Figure S4c). TF module names indicate the presence of 8-mers in those peaks.


Figure 6 ( a ) Associations between chromatin accessibility DA - peaks and GWAS summary statistics . All DA - peaks ( DA ) , gained DA - peaks ( gained ) , closing DA - peaks , and consensus peaks ( all ) are fitted to summary statistics data of multiple GWAS studies ( y - axis ) . Circle color indicates LDSC regression coefficient ( effect size ) , and circle size indicates association significance . Shadings indicate treatments . Time points are shown ( orange = 1h , brown = 6h ) . Red lines indicate LD score regression coefficient adjusted P < 0 . 1 after BH correction . Orange lines indicate P < 0 . 1 . ( BD = Bipolar Disorder ; SZ = Schizophrenia ; BD + SZ : BP and SZ samples combined ; MDD = Major Depressive Disorder ; ADHD = Attention deficit hyperactivity disorder ; AD = Alzheimer ' s Disease ) . ( b ) Correlations between differentially accessible elements conserved mouse and human regions across conditions ( N = 16 , 580 ) . Asterisks indicate significance of Pearson ' s R correlation after correction with BH - procedure .

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 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

Figure 6(a) Associations between chromatin accessibility DA-peaks and GWAS summary statistics. All DA-peaks (DA), gained DA-peaks (gained), closing DA-peaks, and consensus peaks (all) are fitted to summary statistics data of multiple GWAS studies (y-axis). Circle color indicates LDSC regression coefficient (effect size), and circle size indicates association significance. Shadings indicate treatments. Time points are shown (orange = 1h, brown = 6h). Red lines indicate LD score regression coefficient adjusted P < 0.1 after BH correction. Orange lines indicate P < 0.1. (BD = Bipolar Disorder; SZ = Schizophrenia; BD+SZ: BP and SZ samples combined; MDD = Major Depressive Disorder; ADHD = Attention deficit hyperactivity disorder; AD = Alzheimer's Disease).(b) Correlations between differentially accessible elements conserved mouse and human regions across conditions (N=16,580). Asterisks indicate significance of Pearson's R correlation after correction with BH-procedure.


Figure 1 ( A ) DNA damage induces H2AX ADP - ribosylation . 293T cells were treated with H2O2 ( 2 mM in PBS , 5 minutes ) , MMS ( 1 mM in medium , 30 minutes ) or mock ( PBS , 30 minutes ) . ADP - ribosylated proteins were IPed with anti - ADP - ribose ( anti - ADPR ) antibody . ADP - ribosylated H2AX was examined by Western blotting using anti - H2AX antibody . ( B ) H2AX is ADP - ribosylated at glutamate 141 ( E141 ) . shPARG HCT116 cells were treated with H2O2 ( 2 mM in PBS , 5 minutes ) and ADP - ribosylated residues were tagged by a hydroxamic acid derivative with an addition of 15 . 0109 Da , an increment that can be readily distinguished by mass spectrometry . Fragmentation of the NH2OH - derivatized peptides yielded typical b - and y - ion series , allowing easy localization of ADP - ribosylation sites . Glutamate 141 ( E141 ) is indicated by an asterisk . ( C ) The E141A mutation abolishes the ADP - ribosylation of H2AX . Empty vector ( EV ) , wild type ( WT ) or the E141A mutant ( E141A ) of H2AX was expressed in U2OS H2AX knockout cells that were treated with H2O2 . ADP - ribosylated proteins were IPed with anti - ADPR antibody . ADP - ribosylated H2AX was examined by Western blotting using anti - H2AX antibody . ( D ) ADP - ribosylation on H2AX is suppressed by olaparib treatment ( 1 µM , 1 hour ) . 0 . 1 % DMSO in medium for 1 hour as mock . The samples were examined by IP and Western blotting . ( E ) PARP1 , but not PARP2 , mediates the ADP - ribosylation on H2AX . Wild type ( WT ) cells , PARP1 - null cells or PARP2 - null cells were treated with H2O2 .

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 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 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 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

Figure 1(A) DNA damage induces H2AX ADP-ribosylation. 293T cells were treated with H2O2 (2 mM in PBS, 5 minutes), MMS (1 mM in medium, 30 minutes) or mock (PBS, 30 minutes). ADP-ribosylated proteins were IPed with anti-ADP-ribose (anti-ADPR) antibody. ADP-ribosylated H2AX was examined by Western blotting using anti-H2AX antibody.(B) H2AX is ADP-ribosylated at glutamate 141 (E141). shPARG HCT116 cells were treated with H2O2 (2 mM in PBS, 5 minutes) and ADP-ribosylated residues were tagged by a hydroxamic acid derivative with an addition of 15.0109 Da, an increment that can be readily distinguished by mass spectrometry. Fragmentation of the NH2OH-derivatized peptides yielded typical b- and y-ion series, allowing easy localization of ADP-ribosylation sites. Glutamate 141 (E141) is indicated by an asterisk.(C) The E141A mutation abolishes the ADP-ribosylation of H2AX. Empty vector (EV), wild type (WT) or the E141A mutant (E141A) of H2AX was expressed in U2OS H2AX knockout cells that were treated with H2O2. ADP-ribosylated proteins were IPed with anti-ADPR antibody. ADP-ribosylated H2AX was examined by Western blotting using anti-H2AX antibody.(D) ADP-ribosylation on H2AX is suppressed by olaparib treatment (1 µM, 1 hour). 0.1 % DMSO in medium for 1 hour as mock. The samples were examined by IP and Western blotting.(E) PARP1, but not PARP2, mediates the ADP-ribosylation on H2AX. Wild type (WT) cells, PARP1-null cells or PARP2-null cells were treated with H2O2.


Figure 2 ( A ) U2OS cells ( WT ) and U2OS H2AX knockout cells reconstituted with empty vector ( H2AX KO + EV ) , wild type H2AX ( H2AX KO + WT ) , or the E141A mutant H2AX ( E141A ) were treated with H2O2 or MMS . Cell viability was measured after 24 hours using MTT assay . Values are mean ± SD of three assays . ( B ) U2OS cells ( WT ) and U2OS H2AX knockout cells reconstituted with empty vector ( H2AX KO + EV ) , wild type H2AX ( H2AX KO + WT ) , or the E141A mutant ( E141A ) H2AX were treated with H2O2 ( 2 mM in PBS , 5 minutes ) or MMS ( 1 mM in medium , 30 minutes ) , subsequently ; the cells were cultured in fresh DMEM medium for 2 weeks . The numbers of colony formation were counted . Values are mean ± SD of three assays . ( C ) Damaged bases repair are suppressed in U2OS cells expressing the E141A mutant ( E141A ) . U2OS cells ( WT ) and U2OS H2AX knockout cells reconstituted with wild type H2AX ( H2AX KO + WT ) , or the E141A mutant H2AX ( E141A ) were treated with H2O2 ( 2 mM in PBS , 5 minutes ) or MMS ( 1 mM in medium , 30 minutes ) and harvested at the indicated recovery time points . Then FPG - modified comet assay was performed . NT : no treatment . Representative comet tails were shown . The olive tail moments ( OTM ) were summarized from at least 50 cells in each experiment . Values are mean ± SD of three assays . ( D ) DNA replication in U2OS cells expressing the E141A mutant ( E141A ) is suppressed . Cells were treated with H2O2 ( 2 mM in PBS , 5 minutes ) , MMS ( 1 mM in medium , 30 minutes ) or mock ( PBS , 30 minutes ) , followed by BrdU incorporation ( 30 μM in medium , 30 minutes ) . Representative images show BrdU - positive cells in cells expressing WT H2AX or E141A following genotoxic stress . Nuclei were stained with DAPI ( blue ) . The bottom panel histogram shows the percentage of BrdU incorporation in each sample , Values are mean ± SD of three assays . ( E ) Generation of AP sites is suppressed in cells expressing E141A . Cells were treated with 2 mM H2O2 for 5 minutes or 1 mM MMS for 30 minutes . AP sites were calculated according to the standard curve . Values are mean ± SD of three assays .

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

Figure 2(A) U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with empty vector (H2AX KO+EV), wild type H2AX (H2AX KO+WT), or the E141A mutant H2AX (E141A) were treated with H2O2 or MMS. Cell viability was measured after 24 hours using MTT assay. Values are mean ± SD of three assays.(B) U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with empty vector (H2AX KO+EV), wild type H2AX (H2AX KO+WT), or the E141A mutant (E141A) H2AX were treated with H2O2 (2 mM in PBS, 5 minutes) or MMS (1 mM in medium, 30 minutes), subsequently; the cells were cultured in fresh DMEM medium for 2 weeks. The numbers of colony formation were counted. Values are mean ± SD of three assays.(C) Damaged bases repair are suppressed in U2OS cells expressing the E141A mutant (E141A). U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with wild type H2AX (H2AX KO+WT), or the E141A mutant H2AX (E141A) were treated with H2O2 (2 mM in PBS, 5 minutes) or MMS (1 mM in medium, 30 minutes) and harvested at the indicated recovery time points. Then FPG-modified comet assay was performed. NT: no treatment. Representative comet tails were shown. The olive tail moments (OTM) were summarized from at least 50 cells in each experiment. Values are mean ± SD of three assays.(D) DNA replication in U2OS cells expressing the E141A mutant (E141A) is suppressed. Cells were treated with H2O2 (2 mM in PBS, 5 minutes), MMS (1 mM in medium, 30 minutes) or mock (PBS, 30 minutes), followed by BrdU incorporation (30 μM in medium, 30 minutes). Representative images show BrdU-positive cells in cells expressing WT H2AX or E141A following genotoxic stress. Nuclei were stained with DAPI (blue). The bottom panel histogram shows the percentage of BrdU incorporation in each sample, Values are mean ± SD of three assays.(E) Generation of AP sites is suppressed in cells expressing E141A. Cells were treated with 2 mM H2O2 for 5 minutes or 1 mM MMS for 30 minutes. AP sites were calculated according to the standard curve. Values are mean ± SD of three assays.


Figure 3 ( A ) ADP - ribosylation mediates the recruitment of Neil3 . GFP - Neil3 was expressed in U2OS cells , and cells were treated with olaparib ( 1 µM , 1 hour ) or mock ( 0 . 1 % DMSO in medium , 1 hour ) . Following laser microirradiation treatment , the recruitment of Neil3 was examined with live - cell imaging at the indicated time points ( left panel ) . The laser stripe is indicated with yellow arrowheads . The relocation kinetics is shown in the right panel . Data represents mean ± SD from three biologically independent experiments ( right panel ) . At least 20 cells were included in each experiment . ( B ) The GRFs of Neil3 alone is sufficient to be recruited to the sites of DNA damage . Truncated mutants of Neil3 were generated and fused with a GFP tag . Following laser microirradiation treatment , the recruitment of truncated mutants Neil3 were examined with live - cell imaging at the indicated time points ( left panel ) . The laser stripe is indicated with yellow arrowheads . The relocation kinetics is shown in the right panel . Data represents mean ± SD from three biologically independent experiments ( right panel ) . At least 20 cells were included in each experiment . ( C ) PARP inhibitor treatment ( olaparib : 1 µM , 1 hour ) or lack of PARP1 suppresses the relocation of the GRFs of Neil3 to the DNA lesions . The laser stripe is indicated with yellow arrowheads . The relocation kinetics is shown in the right panel . Data represents mean ± SD from three biologically independent experiments ( right panel ) . At least 20 cells were included in each experiment . ( D ) The recruitment of Neil3 to the oxidative lesions is inhibited by olaparib treatment ( 1 µM , 1 hour ) . 0 . 1 % DMSO in medium for 1 hour as mock . Oxidative damage was induced by KillerRed ( KR ) system with the exposure to a 15 - W SYLVANIA cool white fluorescent bulb for 10 minutes . Scale bar , 2 μm . The foci were indicated with white arrowheads . Right panel : the percentage of Neil3 foci co - localized with KR was quantified .

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 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 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 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

Figure 3(A) ADP-ribosylation mediates the recruitment of Neil3. GFP-Neil3 was expressed in U2OS cells, and cells were treated with olaparib (1 µM, 1 hour) or mock (0.1 % DMSO in medium, 1 hour). Following laser microirradiation treatment, the recruitment of Neil3 was examined with live-cell imaging at the indicated time points (left panel). The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represents mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment.(B) The GRFs of Neil3 alone is sufficient to be recruited to the sites of DNA damage. Truncated mutants of Neil3 were generated and fused with a GFP tag. Following laser microirradiation treatment, the recruitment of truncated mutants Neil3 were examined with live-cell imaging at the indicated time points (left panel). The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represents mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment.(C) PARP inhibitor treatment (olaparib: 1 µM, 1 hour) or lack of PARP1 suppresses the relocation of the GRFs of Neil3 to the DNA lesions. The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represents mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment.(D) The recruitment of Neil3 to the oxidative lesions is inhibited by olaparib treatment (1 µM, 1 hour). 0.1 % DMSO in medium for 1 hour as mock. Oxidative damage was induced by KillerRed (KR) system with the exposure to a 15-W SYLVANIA cool white fluorescent bulb for 10 minutes. Scale bar, 2 μm. The foci were indicated with white arrowheads. Right panel: the percentage of Neil3 foci co-localized with KR was quantified.


Figure 4 ( A ) The GRFs of Neil3 recognizes PAR . The recombinant GST fusion proteins were incubated with PAR and the protein - PAR complex was pulled down by glutathione agarose beads . Samples were serially diluted three - fold , spotted onto the nitrocellulose membrane , and subjected to dot blotting using anti - PAR antibody ( top panel ) . FL represents full length Neil3 , N - ter represents N - terminal Neil3 ( 1 - 300 aa ) , Mid represents middle Neil3 ( 300 - 404 aa ) , and GRFs represents C - terminal Neil3 GRFs domain ( 480 - 606 aa ) . Recombinant GST protein was used as the negative control ( NC ) . The GST fusion proteins were also examined by the SDS - PAGE followed with Coomassie blue staining ( middle panel ) . The relative PAR - binding in the pull down assay was summarized in the graph ( bottom panel ) . Values are mean ± SD of three assays . ( B ) Tandem co - immunoprecipitation and Western blotting assays demonstrate that Neil3 interacts with ADP - ribosylated H2AX but not the E141A mutant H2AX . ( C , D ) The GRFs of Neil3 is sufficient and required for the interaction with ADP - ribosylated H2AX . Tandem co - immunoprecipitation and Western blotting assays were used ( E , F ) The relocation kinetic of full - length Neil3 or the GRFs of Neil3 to DNA lesions was analyzed . GFP - tagged full length Neil3 or the GRFs of Neil3 was expressed in the indicated cells , and relocation kinetics was monitored in a time course following laser microirradiation ( left panel ) . The laser stripe is indicated with yellow arrowheads . The relocation kinetics is shown in the right panel . Data represent mean ± SD from three biologically independent experiments ( right panel ) . At least 20 cells were included in each experiment . ( G , H ) The recruitment of full - length Neil3 or the GRFs of Neil3 to the KR - induced oxidative lesions is mediated by H2AX ADP - ribosylation ( left panel ) . Scale bars , 2 μm . The foci were indicated with white arrowheads . Scale bars , 0 . 5 μm . Right panel : the percentage of Neil3 or GRFs Neil3 foci co - localized with KR was quantified . Mean value with SD is from 20 cells .

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

Figure 4(A) The GRFs of Neil3 recognizes PAR. The recombinant GST fusion proteins were incubated with PAR and the protein-PAR complex was pulled down by glutathione agarose beads. Samples were serially diluted three-fold, spotted onto the nitrocellulose membrane, and subjected to dot blotting using anti-PAR antibody (top panel). FL represents full length Neil3, N-ter represents N-terminal Neil3 (1-300 aa), Mid represents middle Neil3 (300-404 aa), and GRFs represents C-terminal Neil3 GRFs domain (480-606 aa). Recombinant GST protein was used as the negative control (NC). The GST fusion proteins were also examined by the SDS-PAGE followed with Coomassie blue staining (middle panel). The relative PAR-binding in the pull down assay was summarized in the graph (bottom panel). Values are mean ± SD of three assays.(B) Tandem co-immunoprecipitation and Western blotting assays demonstrate that Neil3 interacts with ADP-ribosylated H2AX but not the E141A mutant H2AX.(C,D) The GRFs of Neil3 is sufficient and required for the interaction with ADP-ribosylated H2AX. Tandem co-immunoprecipitation and Western blotting assays were used(E,F) The relocation kinetic of full-length Neil3 or the GRFs of Neil3 to DNA lesions was analyzed. GFP-tagged full length Neil3 or the GRFs of Neil3 was expressed in the indicated cells, and relocation kinetics was monitored in a time course following laser microirradiation (left panel). The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represent mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment.(G,H) The recruitment of full-length Neil3 or the GRFs of Neil3 to the KR-induced oxidative lesions is mediated by H2AX ADP-ribosylation (left panel). Scale bars, 2 μm. The foci were indicated with white arrowheads. Scale bars, 0.5 μm. Right panel: the percentage of Neil3 or GRFs Neil3 foci co-localized with KR was quantified. Mean value with SD is from 20 cells.


Figure 5 ( A ) PARP inhibition facilitates γH2AX following oxidative stress . The cells were pre - treated with olaparib ( 1 µM , 1 hour ) prior to H2O2 stimulation . 0 . 1 % DMSO in medium for 1 hour as mock . γH2AX or H2AX was detected using Western blotting and immunostaining assays . The intensity of γH2AX levels were examined by Western blotting normalized by β - actin and averaged . Values are mean ± SD of three assays . P - values were calculated using Student ' s t - test . * * P < 0 . 01 ( left panel ) . The number of γH2AX foci examined in immunostaining assay was counted by Image J software and graphed by GraphPad Prism 8 . ( B ) The effect of H2AX E141 ADP - ribosylation on γH2AX following oxidative stress . γH2AX or H2AX was detected using Western blotting and immunostaining assays . The intensity of γH2AX levels were examined by Western blotting normalized by β - actin and averaged . Values are mean ± SD of three assays . P - values were calculated using Student ' s t - test . * * P < 0 . 01 ( left panel ) . The number of γH2AX foci examined in immunostaining assay was counted by Image J software and graphed by GraphPad Prism 8 . ( C ) The intensity of γH2AX is increased in cell expressing the E141A mutant following KillerRed - induced oxidative DNA lesions ( left panel ) . Scale bar , 2 μm . The foci were indicated with white arrowheads . Scale bar , 0 . 5 μm . Right panel : the relative intensity of γH2AX at the site of tetR ( intensity of γH2AX at the site of tetR / intensity of γH2AX area away from tetR ) was quantified . Mean value with SD is the relative intensity of γH2AX in 20 cells . ( D ) The level of MDC1 at KillerRed - induced oxidative DNA lesions is increased in the cells expressing the E141A mutant ( left panel ) . Scale bar , 2 μm . The foci were indicated with white arrowheads . Right panel : the percentage of MDC1 foci co - localized with KR was quantified . Mean value with SD is from 20 cells . ( E ) The recruitment of Neil3 / MDC1 chimera but not wild type Neil3 to the KR - induced oxidative lesions in cells expressing the E141A mutant ( left panel ) . Neil3 / MDC1 chimera : replaced the GRFs of Neil3 with the tandem BRCT domains of MDC1 . Scale bar , 2 μm . The foci were indicated with white arrowheads . Right panel : The percentage of detected protein foci co - localized with KR was quantified . Mean value with SD is from 20 cells . ( F ) AP sites were measured in cells expressing the E141A mutant or the Neil3 / MDC1 chimera , according to the standard curve . Values are mean ± SD of three assays . ( G ) DNA replication of U2OS cells expression with individual wild type ( WT ) , the H2AX KO + WT , the E141A mutant ( E141A ) or the Neil3 / MDC1 Chimera ( Chimera ) were analyzed by BrdU incorporation following treatment with H2O2 , MMS or mock ( PBS ) . Represented images are shown in the left panel . The graph shows percentages of BrdU - positive cells expressing WT , E141A or Chimera ( right panel ) .

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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O 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) PARP inhibition facilitates γH2AX following oxidative stress. The cells were pre-treated with olaparib (1 µM, 1 hour) prior to H2O2 stimulation. 0.1 % DMSO in medium for 1 hour as mock. γH2AX or H2AX was detected using Western blotting and immunostaining assays. The intensity of γH2AX levels were examined by Western blotting normalized by β-actin and averaged. Values are mean ± SD of three assays. P-values were calculated using Student's t-test. **P < 0.01 (left panel). The number of γH2AX foci examined in immunostaining assay was counted by Image J software and graphed by GraphPad Prism 8.(B) The effect of H2AX E141 ADP-ribosylation on γH2AX following oxidative stress. γH2AX or H2AX was detected using Western blotting and immunostaining assays. The intensity of γH2AX levels were examined by Western blotting normalized by β-actin and averaged. Values are mean ± SD of three assays. P-values were calculated using Student's t-test. **P < 0.01 (left panel). The number of γH2AX foci examined in immunostaining assay was counted by Image J software and graphed by GraphPad Prism 8.(C) The intensity of γH2AX is increased in cell expressing the E141A mutant following KillerRed-induced oxidative DNA lesions (left panel). Scale bar, 2 μm. The foci were indicated with white arrowheads. Scale bar, 0.5 μm. Right panel: the relative intensity of γH2AX at the site of tetR (intensity of γH2AX at the site of tetR/intensity of γH2AX area away from tetR) was quantified. Mean value with SD is the relative intensity of γH2AX in 20 cells.(D) The level of MDC1 at KillerRed-induced oxidative DNA lesions is increased in the cells expressing the E141A mutant (left panel). Scale bar, 2 μm. The foci were indicated with white arrowheads. Right panel: the percentage of MDC1 foci co-localized with KR was quantified. Mean value with SD is from 20 cells.(E) The recruitment of Neil3/MDC1 chimera but not wild type Neil3 to the KR-induced oxidative lesions in cells expressing the E141A mutant (left panel). Neil3/MDC1 chimera: replaced the GRFs of Neil3 with the tandem BRCT domains of MDC1. Scale bar, 2 μm. The foci were indicated with white arrowheads. Right panel: The percentage of detected protein foci co-localized with KR was quantified. Mean value with SD is from 20 cells.(F) AP sites were measured in cells expressing the E141A mutant or the Neil3/MDC1 chimera, according to the standard curve. Values are mean ± SD of three assays.(G) DNA replication of U2OS cells expression with individual wild type (WT), the H2AX KO + WT, the E141A mutant (E141A) or the Neil3/MDC1 Chimera (Chimera) were analyzed by BrdU incorporation following treatment with H2O2, MMS or mock (PBS). Represented images are shown in the left panel. The graph shows percentages of BrdU-positive cells expressing WT, E141A or Chimera (right panel).


Figure 1A . Representative immunofluorescence images and quantifications showing that 3 months old Hnf1apKO mice have increased number of KI67 + ( red ) acinar cell nuclei co - staining with DAPI ( blue ) and Amylase ( green ) . Arrows point to KI67 + acinar cells in Hnf1apKO mouse . Scale bar is 200 μm . Acinar proliferation is represented as the average of the KI67 + / Amylase + cell ratio . Quantifications were performed on 3 random fields from 3 Pdx1Cre and 3 Hnf1apKO mice . P - values are from two - tailed Student ' s t test . Representative H & E stainings of pancreata from KrasG12D and Hnf1apKO ; KrasG12D mice . B - D . KrasG12D and Hnf1apKO ; KrasG12D mice have normal morphology at 7 days . E - J . At 21 days Hnf1apKO ; KrasG12D mice show acinar - to - ductal metaplasia ( dashed encircled areas ) and regions with desmoplastic reaction ( asterisk ) , which are not observed in KrasG12D mice ( E , H ) . K - P . At 8 weeks KrasG12D pancreas show occasional abnormal ductal structures ( dashed encircled areas in N which is a magnification of squared dotted box in K ) and Hnf1apKO ; KrasG12D mice ( L , M , O , P ) present mucinous tubular complexes ( black arrows ) , and more advanced PanINs with luminal budding ( open arrows ) including foci of spindle cell proliferation ( asterisks ) and incipient infiltrative growth ( black dashed box area in O ) . Data information : Black dashed boxes in ( E , F , K , L and O ) indicate magnified areas in ( H , G , N , M and P ) respectively . Scale bars indicate 100 µm ( C , E , F , K , L ) , 50 µm ( O ) and 20 µm ( B , D , G , H - J , M , N , P ) .

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 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 O O O O O O O

Figure 1A. Representative immunofluorescence images and quantifications showing that 3 months old Hnf1apKO mice have increased number of KI67+ (red) acinar cell nuclei co-staining with DAPI (blue) and Amylase (green). Arrows point to KI67+ acinar cells in Hnf1apKO mouse. Scale bar is 200 μm. Acinar proliferation is represented as the average of the KI67+/Amylase+ cell ratio. Quantifications were performed on 3 random fields from 3 Pdx1Cre and 3 Hnf1apKO mice. P-values are from two-tailed Student's t test.Representative H&E stainings of pancreata from KrasG12D and Hnf1apKO;KrasG12D mice. B-D. KrasG12D and Hnf1apKO;KrasG12D mice have normal morphology at 7 days. E-J. At 21 days Hnf1apKO;KrasG12D mice show acinar-to-ductal metaplasia (dashed encircled areas) and regions with desmoplastic reaction (asterisk), which are not observed in KrasG12D mice (E, H). K-P. At 8 weeks KrasG12D pancreas show occasional abnormal ductal structures (dashed encircled areas in N which is a magnification of squared dotted box in K) and Hnf1apKO;KrasG12D mice (L,M,O,P) present mucinous tubular complexes (black arrows), and more advanced PanINs with luminal budding (open arrows) including foci of spindle cell proliferation (asterisks) and incipient infiltrative growth (black dashed box area in O). Data information: Black dashed boxes in (E,F, K, L and O) indicate magnified areas in (H, G, N, M and P) respectively. Scale bars indicate 100 µm (C,E,F,K,L), 50 µm (O) and 20 µm (B,D,G,H-J,M,N,P).


Figure 2A . Fold change ( FC ) in transcripts in Hnf1aaKO vs . control pancreas , plotted against significance ( - Log10 q ; genes significant at q < 0 . 05 are shown as colored dots above the horizontal line ) . B . GSEA showing that genes specific to differentiated acinar cells were downregulated in Hnf1aaKO pancreas , but not genes specific to islets or duct cells . Upregulated genes were enriched in genes specific to mesenchymal cells . Lineage - enriched genes were obtained from Muraro et al . ( Muraro et al . , 2016 ) . D . GSEA revealed that Hnf1aaKO pancreas showed increased transcripts involved in oncogenic pathways such as EMT , MAPK , KRAS , PI3K - AKT . E . HNF1A promotes transcriptional activation of direct target genes . Left : HNF1A bound - genes were enriched among genes that showed downregulation in Hnf1a mutants , but not among upregulated genes . P - values and odds ratios ( O . R . ) calculated by Fisher ' s exact test . Right : Venn diagrams showing overlap of HNF1A - bound genes with genes that were downregulated and upregulated in Hnf1a mutant pancreas .

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 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 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

Figure 2A. Fold change (FC) in transcripts in Hnf1aaKO vs. control pancreas, plotted against significance (-Log10 q; genes significant at q<0.05 are shown as colored dots above the horizontal line).B. GSEA showing that genes specific to differentiated acinar cells were downregulated in Hnf1aaKO pancreas, but not genes specific to islets or duct cells. Upregulated genes were enriched in genes specific to mesenchymal cells. Lineage-enriched genes were obtained from Muraro et al. (Muraro et al., 2016).D. GSEA revealed that Hnf1aaKO pancreas showed increased transcripts involved in oncogenic pathways such as EMT, MAPK, KRAS, PI3K-AKT.E. HNF1A promotes transcriptional activation of direct target genes. Left: HNF1A bound-genes were enriched among genes that showed downregulation in Hnf1a mutants, but not among upregulated genes. P-values and odds ratios (O.R.) calculated by Fisher's exact test. Right: Venn diagrams showing overlap of HNF1A-bound genes with genes that were downregulated and upregulated in Hnf1a mutant pancreas.


Figure 3A . Human orthologs of genes that were up - and down - regulated in Hnf1aaKO pancreas were also up - and down - regulated in human pancreas with low vs . high HNF1A expression ( lowest vs . highest expression deciles , respectively ) . A random list of 717 genes controlled for similar expression levels was used for comparison . Violin plots include median and interquartile ranges . Dots are average values for each gene . Kruskall Wallis P < 0 . 0001 . D . HNF1A mRNA levels differed in HNF1A LoF and control groups ( Kruskal - Wallis , P < 0 . 01 ) , despite considerable variability and overlap between groups . E . KDM6A mRNA levels were downregulated in HNF1A LoF tumors ( Kruskal - Wallis , P < 0 . 001 ) . Data information : Box plots in ( D and E ) show HNF1A and KDM6A expression in HNF1A LoF tumors ( n = 26 ) and Control 1 ( n = 39 ) and Control 2 ( n = 57 ) tumors . The horizontal central line marks the median . Box limits indicate the first and third quartiles and whiskers extend to highest and lowest data points within 1 . 5 x interquartile range outside box limits .

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 O O O O O O O O O O O O O O O O O O O O O O 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 3A. Human orthologs of genes that were up- and down-regulated in Hnf1aaKO pancreas were also up- and down-regulated in human pancreas with low vs. high HNF1A expression (lowest vs. highest expression deciles, respectively). A random list of 717 genes controlled for similar expression levels was used for comparison. Violin plots include median and interquartile ranges. Dots are average values for each gene. Kruskall Wallis P<0.0001.D. HNF1A mRNA levels differed in HNF1A LoF and control groups (Kruskal-Wallis, P<0.01), despite considerable variability and overlap between groups. E. KDM6A mRNA levels were downregulated in HNF1A LoF tumors (Kruskal-Wallis, P<0.001).Data information: Box plots in (D and E) show HNF1A and KDM6A expression in HNF1A LoF tumors (n=26) and Control 1 (n=39) and Control 2 (n=57) tumors. The horizontal central line marks the median. Box limits indicate the first and third quartiles and whiskers extend to highest and lowest data points within 1.5 x interquartile range outside box limits.


Figure 4H & E staining of pancreata from Kdm6apKO ; KrasG12D and KrasG12D mice . A - C . 7 day - old KrasG12D and Kdm6apKO ; KrasG12D mice showed normal morphology . D - I . At 21 days , KrasG12D mice showed normal pancreas morphology ( dotted area at in D is shown at large magnification in G ) whereas Kdm6apKO ; KrasG12D mice already show acinar - to - ductal metaplasia ( dashed encircled area in F ) , spindle cell proliferation ( asterisks in E and H ) , sarcomatoid architecture ( I ) and desmoplastic reaction ( black arrowhead in H ) . J - O . KrasG12D mice present occasional acinar - to - ductal metaplasia and low grade PanINs at 8 weeks ( M is a magnification of squared dotted box in J and see Fig 1N ) whereas at the same age pancreas from Kdm6apKO ; KrasG12D mice show massive remodeling ( K ) , extensive acinar - to - ductal metaplasia ( L and dashed encircled area in N ) , cancer with prominent spindle / mesenchymal proliferation , infiltrative growth ( black arrows and asterisk respectively in O ) and abundant stroma ( black arrowheads in N ) . Data information : Scale bars : 100 µm ( B , D , E , J , K ) or 20 µm ( A , C , F - I , L - N ) .

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 O O O O O O O O O

Figure 4H&E staining of pancreata from Kdm6apKO;KrasG12D and KrasG12D mice. A-C. 7 day-old KrasG12D and Kdm6apKO;KrasG12D mice showed normal morphology. D-I. At 21 days, KrasG12D mice showed normal pancreas morphology (dotted area at in D is shown at large magnification in G) whereas Kdm6apKO;KrasG12D mice already show acinar-to-ductal metaplasia (dashed encircled area in F), spindle cell proliferation (asterisks in E and H), sarcomatoid architecture (I) and desmoplastic reaction (black arrowhead in H). J-O. KrasG12D mice present occasional acinar-to-ductal metaplasia and low grade PanINs at 8 weeks (M is a magnification of squared dotted box in J and see Fig 1N) whereas at the same age pancreas from Kdm6apKO;KrasG12D mice show massive remodeling (K), extensive acinar-to-ductal metaplasia (L and dashed encircled area in N), cancer with prominent spindle/mesenchymal proliferation, infiltrative growth (black arrows and asterisk respectively in O) and abundant stroma (black arrowheads in N). Data information: Scale bars: 100 µm (B,D,E,J,K) or 20 µm (A,C,F-I, L-N).


Figure 5A . Fold change ( FC ) and significance for transcripts in Kdm6apKO vs . control pancreas . Genes significant at q < 0 . 05 are shown as colored dots above the horizontal line . B . GSEA showing that genes specific to differentiated acinar cells were downregulated in KDM6A - deficient pancreas , but not genes specific to islets or duct cells . Upregulated genes were enriched in genes specific to mesenchymal cells . Lineage - enriched genes were obtained from ( Muraro et al . , 2016 ) . D . GSEA showed increased expression of indicated oncogenic pathway genes in Kdm6apKO pancreas . E . KDM6A functions as a transcriptional activator of direct target genes in pancreatic cells . Left : Downregulated , but not upregulated , genes in Kdm6apKO pancreas were enriched for KDM6A binding . P - values and odds ratios ( O . R . ) were calculated by Fisher ' s exact test . Right : Venn diagrams showing overlap of down and upregulated genes with KDM6A binding . F . KDM6A - bound enhancers and promoters showed increased H3K27me3 and decreased H3K27ac in Kdm6apKO pancreas . G . Genes that were downregulated in Kdm6apKO pancreas ( KDM6A - dependent genes ) showed greatest changes in histone marks . Box plots show fold - changes of H3K27me3 ( left ) and H3K27ac ( right ) signals in Kdm6apKO compared to control . Signals were analyzed in KDM6A bound regions in promoters and enhancers of genes that were KDM6A dependent ( grey ; n = 420 regions ) and independent ( white ; n = 8035 regions ) . The signals are average values from ChIP - seq experiments in two biological replicates . The horizontal central line marks the median . Box limits indicate the first and third quartiles and whiskers extend to highest and lowest data points within 1 . 5 x IQR outside box limits . P - values were determined by two - tailed Mann - Whitney U test . H . Changes in KDM6A , H3K27me3 and H3K27ac ChIP - seq profiles in the Ppp4r4 - Serpina10 locus in Kdm6apKO pancreas . I . qPCR of H3K27me3 , H3K27ac and H3K4me1 changes in promoter and enhancer regions highlighted in ( H ) ( R1 , R2 , R3 ) , and Serpin10 mRNA . Error bars show + / - SD and P - values are from two - tailed Student ' s t test , n = 3 . J . KDM6A - bound genes that showed decreased expression in Kdm6apKO pancreas ( functional KDM6A binding sites ) showed simultaneous gain of H3K27me3 and loss and of H3K27ac .

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 B-PANEL_START 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 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 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 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

Figure 5A. Fold change (FC) and significance for transcripts in Kdm6apKO vs. control pancreas. Genes significant at q<0.05 are shown as colored dots above the horizontal line.B. GSEA showing that genes specific to differentiated acinar cells were downregulated in KDM6A-deficient pancreas, but not genes specific to islets or duct cells. Upregulated genes were enriched in genes specific to mesenchymal cells. Lineage-enriched genes were obtained from (Muraro et al., 2016).D. GSEA showed increased expression of indicated oncogenic pathway genes in Kdm6apKO pancreas.E. KDM6A functions as a transcriptional activator of direct target genes in pancreatic cells. Left: Downregulated, but not upregulated, genes in Kdm6apKO pancreas were enriched for KDM6A binding. P-values and odds ratios (O.R.) were calculated by Fisher's exact test. Right: Venn diagrams showing overlap of down and upregulated genes with KDM6A binding.F. KDM6A-bound enhancers and promoters showed increased H3K27me3 and decreased H3K27ac in Kdm6apKO pancreas.G. Genes that were downregulated in Kdm6apKO pancreas (KDM6A-dependent genes) showed greatest changes in histone marks. Box plots show fold-changes of H3K27me3 (left) and H3K27ac (right) signals in Kdm6apKO compared to control. Signals were analyzed in KDM6A bound regions in promoters and enhancers of genes that were KDM6A dependent (grey; n=420 regions) and independent (white; n=8035 regions). The signals are average values from ChIP-seq experiments in two biological replicates. The horizontal central line marks the median. Box limits indicate the first and third quartiles and whiskers extend to highest and lowest data points within 1.5 x IQR outside box limits. P-values were determined by two-tailed Mann-Whitney U test.H. Changes in KDM6A, H3K27me3 and H3K27ac ChIP-seq profiles in the Ppp4r4-Serpina10 locus in Kdm6apKO pancreas.I. qPCR of H3K27me3, H3K27ac and H3K4me1 changes in promoter and enhancer regions highlighted in (H) (R1, R2, R3), and Serpin10 mRNA. Error bars show +/-SD and P-values are from two-tailed Student's t test, n=3.J. KDM6A-bound genes that showed decreased expression in Kdm6apKO pancreas (functional KDM6A binding sites) showed simultaneous gain of H3K27me3 and loss and of H3K27ac.


Figure 6B . Co - binding analysis in functional KDM6A - bound enhancer and promoter regions revealed that HNF1A was the most enriched co - bound TF amongst three other acinar cell TFs . Binding regions of TAL1 in a non - pancreatic cell type and random binding sites were used as negative controls . P - values was determined by Fisher ' s exact test for peak comparisons using all enhancer and promoter regions as background . C . The most downregulated genes in Kdm6apKO pancreas are shown ranked by q - value , and are almost invariably bound by HNF1A and down regulated in Hnf1aaKO pancreas , or known to be direct HNF1A - dependent target genes from other studies ( red and purple , respectively ) . D , E . GSEA analysis on the Hnf1aaKO and Kdm6apKO ranked - ordered gene lists vs . their reciprocal up - or downregulated gene sets , demonstrated that KDM6A and HNF1A regulate similar genes . F . Expression changes in Hnf1aaKO and Kdm6apKO pancreas , showing that genes bound by KDM6A and downregulated in Kdm6apKO pancreas ( red dots ) were generally downregulated in Hnf1aaKO pancreas . G . HNF1A and KDM6A co - occupy the same regions in Pah , which is downregulated in Hnf1a and Kdm6a knock - out pancreas . H . Genes that were co - bound by KDM6A and HNF1A showed greatest downregulation in Kdm6apKO pancreas , compared with KDM6A - bound genes that were not bound by HNF1A . Box plots show median and IQR of Log2 TPM fold - changes and whiskers extend to highest and lowest data points within 1 . 5 x IQR outside box limits . P - values were determined by two - tailed Student ' s t tests and n = 4 replicates per condition .

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Figure 6B. Co-binding analysis in functional KDM6A-bound enhancer and promoter regions revealed that HNF1A was the most enriched co-bound TF amongst three other acinar cell TFs. Binding regions of TAL1 in a non-pancreatic cell type and random binding sites were used as negative controls. P-values was determined by Fisher's exact test for peak comparisons using all enhancer and promoter regions as background.C. The most downregulated genes in Kdm6apKO pancreas are shown ranked by q-value, and are almost invariably bound by HNF1A and down regulated in Hnf1aaKO pancreas, or known to be direct HNF1A-dependent target genes from other studies (red and purple, respectively).D,E. GSEA analysis on the Hnf1aaKO and Kdm6apKO ranked-ordered gene lists vs. their reciprocal up- or downregulated gene sets, demonstrated that KDM6A and HNF1A regulate similar genes.F. Expression changes in Hnf1aaKO and Kdm6apKO pancreas, showing that genes bound by KDM6A and downregulated in Kdm6apKO pancreas (red dots) were generally downregulated in Hnf1aaKO pancreas.G. HNF1A and KDM6A co-occupy the same regions in Pah, which is downregulated in Hnf1a and Kdm6a knock-out pancreas.H. Genes that were co-bound by KDM6A and HNF1A showed greatest downregulation in Kdm6apKO pancreas, compared with KDM6A-bound genes that were not bound by HNF1A. Box plots show median and IQR of Log2 TPM fold-changes and whiskers extend to highest and lowest data points within 1.5 x IQR outside box limits. P-values were determined by two-tailed Student's t tests and n=4 replicates per condition.


Figure 7A . Co - immunoprecipitation of endogenous HNF1A and KDM6A followed by western blot demonstrated that HNF1A is in the same complex as KDM6A . B . Western blot showing loss of HNF1A and unchanged KDM6A in Hnf1a - / - pancreas . C . Differential binding analysis of KDM6A in Hnf1a - / - vs . wild type pancreas . Pink dots below zero ( 1873 sites ) show regions with reduced KDM6A binding and pink dots above zero ( 118 sites ) are regions with increased binding at FDR < 0 . 05 . Regions that show reduced KDM6A binding in Hnf1a - / - chromatin are strongly bound by HNF1A and are highly enriched in HNF1 motifs . P - values in ( D ) were calculated with two - tailed Mann - Whitney U testRegions that show reduced KDM6A binding in Hnf1a - / - chromatin are strongly bound by HNF1A and are highly enriched in HNF1 motifs . P - values in ( E ) with Fisher ' s exact test . F . KDM6A binding is markedly reduced in HNF1A and KDM6A co - bound regions in Hnf1a - / - pancreas , but not in other KDM6A bound regions . G . Genes that loose KDM6A binding in Hnf1a - mutant pancreas are predominantly downregulated in Hnf1aaKO pancreas and are direct HNF1A target genes ( red dots ) .

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 B-PANEL_START 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 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 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 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

Figure 7A. Co-immunoprecipitation of endogenous HNF1A and KDM6A followed by western blot demonstrated that HNF1A is in the same complex as KDM6A.B. Western blot showing loss of HNF1A and unchanged KDM6A in Hnf1a-/- pancreas.C. Differential binding analysis of KDM6A in Hnf1a-/- vs. wild type pancreas. Pink dots below zero (1873 sites) show regions with reduced KDM6A binding and pink dots above zero (118 sites) are regions with increased binding at FDR<0.05.Regions that show reduced KDM6A binding in Hnf1a-/- chromatin are strongly bound by HNF1A and are highly enriched in HNF1 motifs. P-values in (D) were calculated with two-tailed Mann-Whitney U testRegions that show reduced KDM6A binding in Hnf1a-/- chromatin are strongly bound by HNF1A and are highly enriched in HNF1 motifs. P-values in (E) with Fisher's exact test.F. KDM6A binding is markedly reduced in HNF1A and KDM6A co-bound regions in Hnf1a-/- pancreas, but not in other KDM6A bound regions.G. Genes that loose KDM6A binding in Hnf1a-mutant pancreas are predominantly downregulated in Hnf1aaKO pancreas and are direct HNF1A target genes (red dots).


Figure 1B . The ER marker ER - tdTomato ( magenta ) is co - localized with ectopically expressed Wrb - EGFP ( green ) in inner saccular HCs of 5 - dpf zebrafishlarvae ( ' control ' refers to either wild - type or + / pwi fish that were picked in a phenotypic screen and did not exert any abnormal behavioral phenotype ) . While the left HC solely expressed ER - tdTomato , the neighboring HC expressed both , ER - tdTomato and Wrb - GFP . Colocalization between both proteins occurs in areas exhibiting white pixels . Scale bars : 5 µmC . Projection of confocal sections of control inner ear HCs immunolabelled for otoferlin ( magenta ) and expressing an EGFP - tagged truncated Wrb fragment containing only the cytosolic coiled - coiled domains ( Wrbcc - EGFP , green ) . Wrbcc - EGFP distribution was diffuse and found throughout the HC . D . Projection of confocal sections of inner ear HCs of 5 - dpf control ( D ) and Wrb - deficient pwi mutant fish ( wrb pwi / pwi ; D ' ) , showing strongly reduced otoferlin immunofluorescence in the mutant HCs . The color look - up table used represents higher pixel intensities with warmer colors . E . A representative transgenically - rescued pwi - mutant HC ( white arrow ) , expressing Wrb - GFP , exhibits a strongly increased otoferlin signal ( magenta ) in direct comparison to the neighboring non - rescued mutant HCs . ( E ' ) Same image as in E but intensity - coded for otoferlin fluorescence . F . A representative control HC expressing Wrbcc - EGFP ( white arrow ) , immunolabelled for otoferlin ( magenta ) . The Wrbcc - EGFP - expressing HC shows significantly less otoferlin ( F ' ) , suggesting a dominant negative effect of Wrbcc in otoferlin biogenesis . The transfected HC is encircled with a dashed line . G . Quantification otoferlin downregulation of Wrbcc - EGFP shown in F . Otoferlin immunofluorescence intensity of Wrbcc - EGFP transfected inner ear HCs n = 22 , from three 5 - dpf control larva ) showed significant decrease of ~ 43 % compared to the adjacent Wrbcc - EGFP negative wild - type HCs ( n = 22 , from the same larva ) . Fluorescent intensity values were normalized with the average value of Wrbcc - EGFP negative HCs . * * p < 0 . 01H . Quantification of otoferlin immunohistochemistry data shown in ( D ) from hair cells of control ( n = 88 HCs from three 5 - dpf larva ) , wrb pwi / pwi ( n = 80 HCs from 3 sibling larva ) normalized against the mean intensity value of the control group and Wrb - GFP transfected wrb pwi / pwi zebrafish inner ears . Mutant HCs showed ~ 82 % reduction in otoferlin fluorescence intensity when compared with control HCs imaged under the same conditions . Otoferlin fluorescent intensity of Wrb - EGFP transfected mutant HCs ( n = 24 , from three , 5 - dpf larva ) showed significant increases compared to adjacent Wrb - EGFP negative mutant HCs ( n = 24 from the same larva ) . Fluorescent intensity values were normalized with the average value of Wrb - EGFP negative HCs . * * * p < 0 . 001I - I ' . Acoustic startle reflex measurements of ( I ) live , intact , 5 - dpf control ( n = 20 ) and wrb pwi / pwi ( n = 18 ) zebrafishlarva , where almost 100 % of the larva responded to five successive acoustic stimuli at 3 seconds intervals . In wrb pwi / pwi animals , only 39 % of larva responded to the first stimulus , thereafter , the number of responding larva decreased rapidly to less than 6 % . ( I ' ) Acoustic startle reflexes of 5 - dpf zebrafish larva after injection with either EGFP capped mRNA ( control , n = 16 , or wrb pwi / pwi n = 17 ) , Wrb - EGFP capped mRNA ( control , n = 20 , or wrb pwi / pwi n = 20 ) or two different concentrations of otoferlin mRNA ( 1x : control , n = 13 ; wrb pwi / pwi n = 11 ; 2x : control , n = 16 ; wrb pwi / pwi n = 6 ) . In contrast to EGFP mRNA injection , Wrb - EGFP as well as otoferlin mRNAs could partially rescue the acoustic startle reflex in the mutants , while not displaying detrimental effects on overall zebrafish morphology and development . Please note the dose - dependent effect of otoferlin mRNA injection . Similar results as in 1x EGFP mRNA were also obtained from injection of 2X EGFP mRNA as control ( data not shown ) . Measurements from two ( 2x otoferlin ) or three ( all other ) different experiments were compiled in the graph .

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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O 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 1B. The ER marker ER-tdTomato (magenta) is co-localized with ectopically expressed Wrb-EGFP (green) in inner saccular HCs of 5-dpf zebrafishlarvae ('control' refers to either wild-type or +/pwi fish that were picked in a phenotypic screen and did not exert any abnormal behavioral phenotype). While the left HC solely expressed ER-tdTomato, the neighboring HC expressed both, ER-tdTomato and Wrb-GFP. Colocalization between both proteins occurs in areas exhibiting white pixels. Scale bars: 5 µmC. Projection of confocal sections of control inner ear HCs immunolabelled for otoferlin (magenta) and expressing an EGFP-tagged truncated Wrb fragment containing only the cytosolic coiled-coiled domains (Wrbcc-EGFP, green). Wrbcc-EGFP distribution was diffuse and found throughout the HC.D. Projection of confocal sections of inner ear HCs of 5-dpf control (D) and Wrb-deficient pwi mutant fish (wrb pwi/pwi; D'), showing strongly reduced otoferlin immunofluorescence in the mutant HCs. The color look-up table used represents higher pixel intensities with warmer colors.E. A representative transgenically-rescued pwi-mutant HC (white arrow), expressing Wrb-GFP, exhibits a strongly increased otoferlin signal (magenta) in direct comparison to the neighboring non-rescued mutant HCs. (E') Same image as in E but intensity-coded for otoferlin fluorescence.F. A representative control HC expressing Wrbcc-EGFP (white arrow), immunolabelled for otoferlin (magenta). The Wrbcc-EGFP-expressing HC shows significantly less otoferlin (F'), suggesting a dominant negative effect of Wrbcc in otoferlin biogenesis. The transfected HC is encircled with a dashed line.G. Quantification otoferlin downregulation of Wrbcc-EGFP shown in F. Otoferlin immunofluorescence intensity of Wrbcc-EGFP transfected inner ear HCs n = 22, from three 5-dpf control larva) showed significant decrease of ~43% compared to the adjacent Wrbcc-EGFP negative wild-type HCs (n = 22, from the same larva). Fluorescent intensity values were normalized with the average value of Wrbcc-EGFP negative HCs. **p < 0.01H. Quantification of otoferlin immunohistochemistry data shown in (D) from hair cells of control (n = 88 HCs from three 5-dpf larva), wrb pwi/pwi (n = 80 HCs from 3 sibling larva) normalized against the mean intensity value of the control group and Wrb-GFP transfected wrb pwi/pwi zebrafish inner ears. Mutant HCs showed ~82% reduction in otoferlin fluorescence intensity when compared with control HCs imaged under the same conditions. Otoferlin fluorescent intensity of Wrb-EGFP transfected mutant HCs (n = 24, from three, 5-dpf larva) showed significant increases compared to adjacent Wrb-EGFP negative mutant HCs (n = 24 from the same larva). Fluorescent intensity values were normalized with the average value of Wrb-EGFP negative HCs. ***p<0.001I-I'. Acoustic startle reflex measurements of (I) live, intact, 5-dpf control (n = 20) and wrb pwi/pwi (n = 18) zebrafishlarva, where almost 100% of the larva responded to five successive acoustic stimuli at 3 seconds intervals. In wrb pwi/pwi animals, only 39% of larva responded to the first stimulus, thereafter, the number of responding larva decreased rapidly to less than 6%. (I') Acoustic startle reflexes of 5-dpf zebrafish larva after injection with either EGFP capped mRNA (control, n = 16, or wrb pwi/pwi n = 17), Wrb-EGFP capped mRNA (control, n = 20, or wrb pwi/pwi n = 20) or two different concentrations of otoferlin mRNA (1x: control, n = 13; wrb pwi/pwi n = 11; 2x: control, n = 16; wrb pwi/pwi n = 6). In contrast to EGFP mRNA injection, Wrb-EGFP as well as otoferlin mRNAs could partially rescue the acoustic startle reflex in the mutants, while not displaying detrimental effects on overall zebrafish morphology and development. Please note the dose-dependent effect of otoferlin mRNA injection. Similar results as in 1x EGFP mRNA were also obtained from injection of 2X EGFP mRNA as control (data not shown). Measurements from two (2x otoferlin) or three (all other) different experiments were compiled in the graph.


Figure 2A . HZZ - OTOFop , carrying a C - terminal glycosylation site ( opsin tag ) was purified alone or in complex with wild - type or an ATPase - deficient mutant version of TRC40 and incubated in the absence or presence of ER - derived rough microsomes ( RM ) . Membrane integration ( glycosylation ) was monitored by SDS - PAGE and immunoblot using an anti - opsin antibody . Where indicated , EndoH was used to remove N - linked oligosaccharides . B . HZZ - OTOFop in complex with wild - type or mutant TRC40 was incubated with RM in the presence or absence of ATP and membrane insertion was monitored by opsin - specific immunoblot . C . HZZ - OTOFop in complex with wild - type TRC40 was incubated in the presence of RM , trypsin - treated rough microsomes ( TRM ) or in the presence of WRBcc or CAMLcyt . Membrane insertion was monitored by opsin - specific immunoblot . D . Quantification of relative protein glycosylation shown in B ( n = 3 ) . Data are represented as mean ± SEM . TRC40gr , mutated version of TRC40 ; * * p < 0 . 01 ; * * * p < 0 . 001 ( Student ' s two - sample t - test ) . E . Quantification of relative protein glycosylation of the data shown in C ( n = 3 ) . Data are represented as mean ± SEM . * * * p < 0 . 001 ( Student ' s two - sample t - test )

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 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 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 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

Figure 2A. HZZ-OTOFop, carrying a C-terminal glycosylation site (opsin tag) was purified alone or in complex with wild-type or an ATPase-deficient mutant version of TRC40 and incubated in the absence or presence of ER-derived rough microsomes (RM). Membrane integration (glycosylation) was monitored by SDS-PAGE and immunoblot using an anti-opsin antibody. Where indicated, EndoH was used to remove N-linked oligosaccharides.B. HZZ-OTOFop in complex with wild-type or mutant TRC40 was incubated with RM in the presence or absence of ATP and membrane insertion was monitored by opsin-specific immunoblot.C. HZZ-OTOFop in complex with wild-type TRC40 was incubated in the presence of RM, trypsin-treated rough microsomes (TRM) or in the presence of WRBcc or CAMLcyt. Membrane insertion was monitored by opsin-specific immunoblot.D. Quantification of relative protein glycosylation shown in B (n = 3). Data are represented as mean ± SEM. TRC40gr, mutated version of TRC40; **p<0.01; ***p<0.001 (Student's two-sample t-test).E. Quantification of relative protein glycosylation of the data shown in C (n = 3). Data are represented as mean ± SEM. ***p<0.001 (Student's two-sample t-test)


Figure 3A . Grand averages of auditory brainstem responses ( ABR ) from Wrbfl / fl : CreA ( red traces ; SEM pink ) and Wrb + / + : CreA ( black traces ; SEM grey ) mice . ABRs were recorded for three separate age groups ( A - A ' ' , as indicated in the graph ) using click stimulation at 80 dB ( peak equivalent ) and 20 Hz stimulation rate . There was an age - progressive reduction of ABR amplitude in Wrbfl / fl : CreA mice . B . ABRs from Wrbfl / fl : CreA mice showed a progressive threshold increase , Wrb + / + : CreA mice and Wrbfl / fl mice lacking Cre recombinase had normal thresholds . Statistical comparison of the threshold at 12 kHz was done by Kruskal - Wallis test , p values are from post - hoc Dunn ' s multiple comparison test . Measurements in which no ABR was observed at the maximal available tone burst level ( 90 dB ) or click level ( 120 dB ) scored as 100 dB threshold . Data are represented as mean ± SEM .

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

Figure 3A. Grand averages of auditory brainstem responses (ABR) from Wrbfl/fl:CreA (red traces; SEM pink) and Wrb+/+:CreA (black traces; SEM grey) mice. ABRs were recorded for three separate age groups (A-A'', as indicated in the graph) using click stimulation at 80 dB (peak equivalent) and 20 Hz stimulation rate. There was an age- progressive reduction of ABR amplitude in Wrbfl/fl:CreA mice.B. ABRs from Wrbfl/fl:CreA mice showed a progressive threshold increase, Wrb+/+:CreA mice and Wrbfl/fl mice lacking Cre recombinase had normal thresholds. Statistical comparison of the threshold at 12 kHz was done by Kruskal-Wallis test, p values are from post-hoc Dunn's multiple comparison test. Measurements in which no ABR was observed at the maximal available tone burst level (90 dB) or click level (120 dB) scored as 100 dB threshold. Data are represented as mean ± SEM.