B . Anti - FLAG ChIP of wt - ESCs and F / H - BAZ2A - ESCs cultured in 2i or serum . Data were measured by qPCR and normalized to input and a control region that is not bound by BAZ2A . Average values of three independent experiments . Error bars represent s . d . and statistical significance ( P - values ) was calculated using the paired two - tailed t - test ( * < 0 . 05 , * * < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Anti-FLAG ChIP of wt-ESCs and F/H-BAZ2A-ESCs cultured in 2i or serum. Data were measured by qPCR and normalized to input and a control region that is not bound by BAZ2A. Average values of three independent experiments. Error bars represent s.d. and statistical significance (P-values) was calculated using the paired two-tailed t-test (* < 0.05, ** < 0.01).


C . Representative images showing the association of BAZ2A at regions enriched in H3K27ac and low in H3K27me3 . The grey rectangles highlighted some of BAZ2A associated regions .

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

C. Representative images showing the association of BAZ2A at regions enriched in H3K27ac and low in H3K27me3. The grey rectangles highlighted some of BAZ2A associated regions.


D . Pearson correlation heat map for the indicated ChIPseqs in ESC + 2i . BAZ2A ChIPseqs were performed with FLAG antibodies in F / H - BAZ2A - ESCs cultured in 2i or serum and with HA antibodies in F / H - BAZ2A - ESC + 2i . Data of H3K4me3 , H3K27ac and H3K27me3 are from this work . H3K36me3 , H3K9me3 and H3K4me1 values in ESC + 2i were taken from published data

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

D. Pearson correlation heat map for the indicated ChIPseqs in ESC+2i. BAZ2A ChIPseqs were performed with FLAG antibodies in F/H-BAZ2A-ESCs cultured in 2i or serum and with HA antibodies in F/H-BAZ2A-ESC+2i. Data of H3K4me3, H3K27ac and H3K27me3 are from this work. H3K36me3, H3K9me3 and H3K4me1 values in ESC+2i were taken from published data sets


E . Boxplot showing the mean of normalized BAZ2A occupancies in ESC + 2i and ESC + serum at BAZ2A - bound regions . Statistical significance ( P - values ) for the experiments was calculated using the unpaired two - tailed t - test ( * * * * < 0 . 0001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

E. Boxplot showing the mean of normalized BAZ2A occupancies in ESC+2i and ESC+serum at BAZ2A-bound regions. Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (**** < 0.0001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


F . Differential BAZ2A - binding in 2i and serum correlates with H3K27ac occupancies . Boxplot showing the mean of normalized H3K27ac and H3K27me3 read counts in ESC + 2i and ESC + serum at all BAZ2A - bound regions and BAZ2A - bound regions only in 2i were calculated and their log2 - fold changes are plotted . H3K27ac in ESC + serum Statistical significance ( P - values ) was calculated using the paired two - tailed t - test ( * * * < 0 . 001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

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

F. Differential BAZ2A-binding in 2i and serum correlates with H3K27ac occupancies. Boxplot showing the mean of normalized H3K27ac and H3K27me3 read counts in ESC+2i and ESC+serum at all BAZ2A-bound regions and BAZ2A-bound regions only in 2i were calculated and their log2-fold changes are plotted. H3K27ac in ESC+serum Statistical significance (P-values) was calculated using the paired two-tailed t-test (*** < 0.001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


G . Representative images showing 2i - specific BAZ2A - bound regions and H3K27ac and H3K27me3 in ESC + 2i and ESC + serum . The grey box highlights strong TIP5 - bound regions and corresponding histone modifications .

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

G. Representative images showing 2i-specific BAZ2A-bound regions and H3K27ac and H3K27me3 in ESC+2i and ESC+serum. The grey box highlights strong TIP5-bound regions and corresponding histone modifications.


H . Representative images showing that genes regulated by BAZ2A are enriched in H3K27me3 , depleted of H3K27ac and not bound by BAZ2A .

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

H. Representative images showing that genes regulated by BAZ2A are enriched in H3K27me3, depleted of H3K27ac and not bound by BAZ2A.


I . Heat map profiles of H3K27me3 and H3K27ac at ± 5 kb from the TSS of genes differentially regulated by BAZ2A in ESC + 2i and random genes . Differentially regulated genes are shown as upregulated or downregulated in ESC + 2i upon BAZ2A knockdown . Data were ranked by H3K27me3 levels . Transcription start site ( TSS ) .

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

I. Heat map profiles of H3K27me3 and H3K27ac at ±5 kb from the TSS of genes differentially regulated by BAZ2A in ESC+2i and random genes. Differentially regulated genes are shown as upregulated or downregulated in ESC+2i upon BAZ2A knockdown. Data were ranked by H3K27me3 levels. Transcription start site (TSS).


J . Boxplot showing the mean of normalized H3K27me3 and H3K27ac read counts over + / - 1kb of TSSs from upregulated , downregulated and random genes . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

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

J. Boxplot showing the mean of normalized H3K27me3 and H3K27ac read counts over +/- 1kb of TSSs from upregulated, downregulated and random genes. Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


A . Number of BAZ2A - bound regions in A and B compartments of ESC + serum . B . Hi - C contact matrices for a zoomed in region on chromosome 3 at 100 - kb resolution showing the presence of strong long - distance contacts corresponding to regions in A compartment bound by BAZ2A in ESC + serum . Low panels . HiC contacts of a zoomed region at 50 - kb resolution in ESC + serum , NPCs and CNs are shown . Data are from ( Bonev et al . , 2017 ) . HiC contacts were visualized using Juicebox C . BAZ2A - bound regions mark A subcompartments that are characterized by the strongest far - cis contacts in ESC + serum . Boxplots indicating observed / expected contact values between BAZ2A - bound regions and loci not bound by BAZ2A at near - cis and far - cis contacts in all genome or in A compartments . Statistical significance ( P - values ) was calculated using the unpaired two - tailed t - test ( * * * * < 0 . 0001 ) . D . Long - range contacts marked by BAZ2A binding are specific for ESCs . Boxplots indicating observed / expected contact values between BAZ2A - bound regions and loci not bound by BAZ2A at far - cis contacts in all genome or in A compartments of ESC + serum , NPC and CN . Statistical significance ( P - values ) was calculated using the unpaired two - tailed t - test ( * * * * < 0 . 0001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

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

A. Number of BAZ2A-bound regions in A and B compartments of ESC+serum. B. Hi-C contact matrices for a zoomed in region on chromosome 3 at 100-kb resolution showing the presence of strong long-distance contacts corresponding to regions in A compartment bound by BAZ2A in ESC+serum. Low panels. HiC contacts of a zoomed region at 50-kb resolution in ESC+serum, NPCs and CNs are shown. Data are from (Bonev et al., 2017). HiC contacts were visualized using Juicebox C. BAZ2A-bound regions mark A subcompartments that are characterized by the strongest far-cis contacts in ESC+serum. Boxplots indicating observed/expected contact values between BAZ2A-bound regions and loci not bound by BAZ2A at near-cis and far-cis contacts in all genome or in A compartments. Statistical significance (P-values) was calculated using the unpaired two-tailed t-test (****< 0.0001). D. Long-range contacts marked by BAZ2A binding are specific for ESCs. Boxplots indicating observed/expected contact values between BAZ2A-bound regions and loci not bound by BAZ2A at far-cis contacts in all genome or in A compartments of ESC+serum, NPC and CN. Statistical significance (P-values) was calculated using the unpaired two-tailed t-test (**** < 0.0001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


E . Number of BAZ2A - bound regions in A and B compartments in ESC + 2i . F . BAZ2A - bound regions mark A subcompartments that are characterized by the strongest far - cis contacts in ESC + 2i . Boxplots indicating observed / expected contact values between BAZ2A - bound regions and loci not bound by BAZ2A at near - cis and far - cis contacts in all genome or in A compartments of ESC + 2i . Statistical significance ( P - values ) was calculated using the unpaired two - tailed t - test ( * * < 0 . 001 , * * * * < 0 . 0001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

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

E. Number of BAZ2A-bound regions in A and B compartments in ESC+2i. F. BAZ2A-bound regions mark A subcompartments that are characterized by the strongest far-cis contacts in ESC+2i. Boxplots indicating observed/expected contact values between BAZ2A-bound regions and loci not bound by BAZ2A at near-cis and far-cis contacts in all genome or in A compartments of ESC+2i. Statistical significance (P-values) was calculated using the unpaired two-tailed t-test (** < 0.001, ****< 0.0001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


G . BAZ2A binding correlates the best with eigenvector values . Heat map showing Pearson correlation values of ChIPseq data sets with the eigenvector values from HiC data in ESC + serum and ESC + 2i , respectively .

G. BAZ2A binding correlates the best with eigenvector values. Heat map showing Pearson correlation values of ChIPseq data sets with the eigenvector values from HiC data in ESC+serum and ESC+2i, respectively.


H . Boxplots showing H3K4me1 and H3K27ac content of BAZ2A - bound regions and loci not bound by BAZ2A at far - cis contacts in ESC + 2i . Data from regions with lowest 10 % and top 10 % contact strength are shown . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

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

H. Boxplots showing H3K4me1 and H3K27ac content of BAZ2A-bound regions and loci not bound by BAZ2A at far-cis contacts in ESC+2i. Data from regions with lowest 10% and top 10% contact strength are shown. Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


A . Numbers of ATAC peaks called separately in ESC + 2i and ESC + serum including their localization according to compartmentalization . B . Numbers of significant changed ATAC peaks in ESC + 2i vs . ESC + serum and their localization according to compartmentalization . Increased chromatin accessibility sites ( IAS ) , decreased accessibility sites ( DAS ) . C . Average profile of normalized ATAC read counts over differential peaks in ESC + 2i and ESC + serum changed ATAC peaks . The signal is plotted over + / - 1kb on the center of the ATAC peak .

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

A. Numbers of ATAC peaks called separately in ESC+2i and ESC+serum including their localization according to compartmentalization. B. Numbers of significant changed ATAC peaks in ESC+2i vs. ESC+serum and their localization according to compartmentalization. Increased chromatin accessibility sites (IAS), decreased accessibility sites (DAS). C. Average profile of normalized ATAC read counts over differential peaks in ESC+2i and ESC+serum changed ATAC peaks. The signal is plotted over +/- 1kb on the center of the ATAC peak.


D . H3K27ac correlates with differential accessible sites between ESC + 2i and ESC + serum . Boxplot showing the mean values of normalized H3K27ac at ATAC peaks ( + / - 500bp ) that are increased and decreased in ESC + 2i compared to ESC + serum , respectively . ESCs were subjected to two consecutive treatments with siRNA - control and siRNA - Baz2a , each one lasting for 4 days . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( * * * < 0 . 001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

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

D. H3K27ac correlates with differential accessible sites between ESC+2i and ESC+serum. Boxplot showing the mean values of normalized H3K27ac at ATAC peaks (+/- 500bp) that are increased and decreased in ESC+2i compared to ESC+serum, respectively. ESCs were subjected to two consecutive treatments with siRNA-control and siRNA-Baz2a, each one lasting for 4 days. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (*** < 0.001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


E . BAZ2A depletion increases chromatin accessibility only in ESC + 2i . Numbers of significant changed ATAC peaks in ESC + 2i and ESC + serum upon BAZ2A knockdown and their localization according to compartmentalization . F . Average density plots of normalized ATAC read counts over differential ATAC peaks of ESC + 2i and ESC + serum treated with siRNA - Baz2a and siRNA - control over changed ATAC peaks . The signal is plotted over + / - 1kb centered on the ATAC peak .

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

E. BAZ2A depletion increases chromatin accessibility only in ESC+2i. Numbers of significant changed ATAC peaks in ESC+2i and ESC+serum upon BAZ2A knockdown and their localization according to compartmentalization. F. Average density plots of normalized ATAC read counts over differential ATAC peaks of ESC+2i and ESC+serum treated with siRNA-Baz2a and siRNA-control over changed ATAC peaks. The signal is plotted over +/- 1kb centered on the ATAC peak.


G . The distances of increased ( IAS ) and decreased ( DAS ) chromatin accessibility sites in BAZ2A - depleted ESC + 2i to the nearest gene promoter ( + / - 5kb of TSS ) were calculated . Distances in bp to the nearest promoter of genes upregulated ( UP ) , downregulated ( DOWN ) or not affected ( NOT ) upon BAZ2A knockdown in ESC + 2i are plotted . Statistical significance ( P - values ) for the experiments was calculated using the unpaired two - tailed t - test ( * * < 0 . 01 ) . Error bars represent s . d . The mean is represented by a horizontal line .

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

G. The distances of increased (IAS) and decreased (DAS) chromatin accessibility sites in BAZ2A-depleted ESC+2i to the nearest gene promoter (+/- 5kb of TSS) were calculated. Distances in bp to the nearest promoter of genes upregulated (UP), downregulated (DOWN) or not affected (NOT) upon BAZ2A knockdown in ESC+2i are plotted. Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (** < 0.01). Error bars represent s.d. The mean is represented by a horizontal line.


H . Genomic annotation of IAS and DAS in ESC + 2i depleted of BAZ2A perfomed with

H. Genomic annotation of IAS and DAS in ESC+2i depleted of BAZ2A perfomed with ChIPseeker


I . Fold - enrichment relative to expected of regions with increased chromatin accessibility ( IAS ) and upregulated genes upon BAZ2A KD at sub - regions within compartment A and B ( center , inside and boundaries ) . Compartments smaller than 500 kb were excluded from the analysis . Statistical significance ( P - values ) was calculated with permutation test ( ns : non significant , * < 0 . 05 , * * * < 0 . 001 ) .

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

I. Fold-enrichment relative to expected of regions with increased chromatin accessibility (IAS) and upregulated genes upon BAZ2A KD at sub-regions within compartment A and B (center, inside and boundaries). Compartments smaller than 500 kb were excluded from the analysis. Statistical significance (P-values) was calculated with permutation test (ns: non significant, * < 0.05, *** < 0.001).


J . BAZ2A - upregulated genes are enriched in the B compartment . Numbers of BAZ2A - regulated genes upregulated and downregulated upon BAZ2A knockdown in ESC + 2i including their localization according to compartmentalization .

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

J. BAZ2A-upregulated genes are enriched in the B compartment. Numbers of BAZ2A-regulated genes upregulated and downregulated upon BAZ2A knockdown in ESC+2i including their localization according to compartmentalization.


K . Active histone marks increase in the B compartment upon BAZ2A knockdown . Boxplots indicating the log2 fold - changes of normalized H3K27ac and H3K4me3 read counts in ESC + 2i treated with siRNA - Baz2a and siRNA - control at boundaries and centers of A and B compartments . ) . Statistical significance ( P - values ) for the experiments was calculated using the unpaired two - tailed t - test ( * * * < 0 . 001 , * < 0 . 05 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K. Active histone marks increase in the B compartment upon BAZ2A knockdown. Boxplots indicating the log2 fold-changes of normalized H3K27ac and H3K4me3 read counts in ESC+2i treated with siRNA-Baz2a and siRNA-control at boundaries and centers of A and B compartments. ). Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (*** < 0.001, * < 0.05). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


A . Hi - C contact matrices for chromosome 1 of ESC + serum , ESC + 2i / siRNA - control and ESC + 2i / siRNA - Baz2a showing the presence of strong long - distance contacts corresponding to regions in A compartment bound by BAZ2A in ESC + serum . Cells were subjected to two consecutive treatments with siRNA - control and siRNA - Baz2a , each one lasting for 4 days . Eigenvector values are shown . Increase in far - cis contacts in ESC + 2i upon BAZ2A - KD are highlighted with a rectangle . B . Relative contact frequencies over genomic distances in ESCs treated with siRNA - control and siRNA - BAZ2A . Statistical significance ( P - values ) was calculated using the Kolmogorov - Smirnov test . C . Boxplot indicating differences in eigenvector values at boundaries and centers of A and B compartments . Differences in eigenvector values were calculated from HiC data sets from ESC + 2i treated with siRNA - Baz2a and siRNA - control . Statistical significance ( P - values ) for the experiments was calculated using the unpaired two - tailed t - test ( * * * < 0 . 001 , ns : non significant ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

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

A. Hi-C contact matrices for chromosome 1 of ESC+serum, ESC+2i/siRNA-control and ESC+2i/siRNA-Baz2a showing the presence of strong long-distance contacts corresponding to regions in A compartment bound by BAZ2A in ESC+serum. Cells were subjected to two consecutive treatments with siRNA-control and siRNA-Baz2a, each one lasting for 4 days. Eigenvector values are shown. Increase in far-cis contacts in ESC+2i upon BAZ2A-KD are highlighted with a rectangle. B. Relative contact frequencies over genomic distances in ESCs treated with siRNA-control and siRNA-BAZ2A. Statistical significance (P-values) was calculated using the Kolmogorov-Smirnov test. C. Boxplot indicating differences in eigenvector values at boundaries and centers of A and B compartments. Differences in eigenvector values were calculated from HiC data sets from ESC+2i treated with siRNA-Baz2a and siRNA-control. Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (*** < 0.001, ns: non significant). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


A . Western blot showing total levels of H3K27me3 in ESC + 2i transfected with siRNA - control and siRNA - Baz2a . Histone H3 is shown as a protein loading control .

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

A. Western blot showing total levels of H3K27me3 in ESC+2i transfected with siRNA-control and siRNA-Baz2a. Histone H3 is shown as a protein loading control.


B . Scatterplot showing the changes in H3K27me3 upon BAZ2A depletion in ESC + 2i and ESC + serum . ESCs were subjected to two consecutive treatments with siRNA - control and siRNA - Baz2a , each one lasting for 4 days . Mean of normalized H3K27me3 read counts in ESC + 2i and ESC + serum treated with siRNA - control or siRNA - Baz2a over all TSSs ( + / - 1kb ) were calculated . Log2 - fold changes of ( siRNA - Baz2a / siRNA - control ) are plotted relative to the mean of normalized H3K27me3 occupancies . The red line represents the mean of H3K27me3 fold - changes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Scatterplot showing the changes in H3K27me3 upon BAZ2A depletion in ESC+2i and ESC+serum. ESCs were subjected to two consecutive treatments with siRNA-control and siRNA-Baz2a, each one lasting for 4 days. Mean of normalized H3K27me3 read counts in ESC+2i and ESC+serum treated with siRNA-control or siRNA-Baz2a over all TSSs (+/- 1kb) were calculated. Log2-fold changes of (siRNA-Baz2a/siRNA-control) are plotted relative to the mean of normalized H3K27me3 occupancies. The red line represents the mean of H3K27me3 fold-changes.


C . Representative images showing the H3K27me3 occupancy .

C. Representative images showing the H3K27me3 occupancy.


D . Average density plot of ChIPseq read counts of H3K27me3 at - / + 5 kb from the TSS of refseq genes in ESCs treated with siRNA - control or siRNA - Baz2a Q1 : first quartile corresponding to sequences with high H3K27me3 content . Q2 - 4 : quartiles 2 - 4 corresponding to sequences with low H3K27me3 content .

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

D. Average density plot of ChIPseq read counts of H3K27me3 at -/+ 5 kb from the TSS of refseq genes in ESCs treated with siRNA-control or siRNA-Baz2a Q1: first quartile corresponding to sequences with high H3K27me3 content. Q2-4: quartiles 2-4 corresponding to sequences with low H3K27me3 content.


E . ChIP - qPCR of H3K27me3 in ESC + 2i and ESC + serum treated with siRNA - control or siRNA - Baz2a . Data were measured by qPCR and normalized to input and to Tshz1 value in control cells . Control represents an intergenic sequence that does not contain H3K27me3 . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( ns : non significant , * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ) .

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E. ChIP-qPCR of H3K27me3 in ESC+2i and ESC+serum treated with siRNA-control or siRNA-Baz2a. Data were measured by qPCR and normalized to input and to Tshz1 value in control cells. Control represents an intergenic sequence that does not contain H3K27me3. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, * < 0.05, ** < 0.01, *** < 0.001).


F . Boxplot showing changes in H3K27me3 in ESC + 2i upon BAZ2A at BAZ2A - regulated and random genes . Mean of normalized H3K27me3 read counts in ESC + 2i treated with siRNA - control or siRNA - Baz2a over TSSs ( + / - 1kb ) of upregulated , downregulated and random genes were calculated . Log2 - fold changes of ( siRNA - Baz2a / siRNA - control ) are plotted . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( * * < 0 . 01 and * * * < 0 . 001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

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F. Boxplot showing changes in H3K27me3 in ESC+2i upon BAZ2A at BAZ2A-regulated and random genes. Mean of normalized H3K27me3 read counts in ESC+2i treated with siRNA-control or siRNA-Baz2a over TSSs (+/- 1kb) of upregulated, downregulated and random genes were calculated. Log2-fold changes of (siRNA-Baz2a/siRNA-control) are plotted. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (** < 0.01 and *** < 0.001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


G . Average density and heatmap profile of H3K27me3 transition state regions marked by CTCF sites in ESC + 2i or ESC + serum transfected with siRNA - control or siRNA - Baz2a . Data were ranked by H3K27me3 content in the corresponding siRNA - control ESCs . Cluster 1 represents high to low H327me3 transition whereas Cluster 2 shows low to high H3K27me3 levels .

G. Average density and heatmap profile of H3K27me3 transition state regions marked by CTCF sites in ESC+2i or ESC+serum transfected with siRNA-control or siRNA-Baz2a. Data were ranked by H3K27me3 content in the corresponding siRNA-control ESCs. Cluster 1 represents high to low H327me3 transition whereas Cluster 2 shows low to high H3K27me3 levels.


H . H3K27me3 ChIP - qPCR in ESC + 2i ( top panel ) or ESC + serum ( bottom panel ) transfected with siRNA - control or siRNA - Baz2a . Values show H3K27me3 occupancy at four sequences ( # 1 - # 4 ) with elevated H3K27me3 at CTCF boundary ( H3K27me3hi / CTCF ) . Control represents an intergenic sequence that does not contain H3K27me3 . Data were measured by qPCR and normalized to input and to sequence # 1 value in control cells . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( ns : non significant , * < 0 . 05 , * * < 0 . 01 ) .

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H. H3K27me3 ChIP-qPCR in ESC+2i (top panel) or ESC+serum (bottom panel) transfected with siRNA-control or siRNA-Baz2a. Values show H3K27me3 occupancy at four sequences (#1-#4) with elevated H3K27me3 at CTCF boundary (H3K27me3hi/CTCF). Control represents an intergenic sequence that does not contain H3K27me3. Data were measured by qPCR and normalized to input and to sequence #1 value in control cells. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, * < 0.05, ** < 0.01).


I . Hi - C contact matrices for a zoomed in region on chromosome 6 containing HoxA cluster ( 10 - kb resolution in upper panels , 5 kb resolution in lower panels ) . Blue arrows ( A , B , and C ) indicate gain or increase of contacts in ESC + siRNA - Baz2a compared to control cells ( siRNA - control ) . The orange arrow ( D ) indicates loss or decrease in contacts .

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I. Hi-C contact matrices for a zoomed in region on chromosome 6 containing HoxA cluster (10-kb resolution in upper panels, 5 kb resolution in lower panels). Blue arrows (A, B, and C) indicate gain or increase of contacts in ESC+siRNA-Baz2a compared to control cells (siRNA-control). The orange arrow (D) indicates loss or decrease in contacts.


B . BAZ2A - interacting proteins on chromatin . Mass spectrometry analysis of FLAG immunoprecipitates from wt and F / H - BAZ2A ESC + 2i . Values of peptide number are shown as the difference between peptides obtained in FLAG - IP of F / H - BAZ2A and wt ESC chromatin extracts . Data are from three independent experiments . The proteins found enriched in FLAG - IP of F / H - BAZ2A ESCs in all three experiments are shown . Further BAZ2A - interacting proteins identified in only two or one of the immunoprecipitation ( IP ) experiments Error bars represent s . d .

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B. BAZ2A-interacting proteins on chromatin. Mass spectrometry analysis of FLAG immunoprecipitates from wt and F/H-BAZ2A ESC+2i. Values of peptide number are shown as the difference between peptides obtained in FLAG-IP of F/H-BAZ2A and wt ESC chromatin extracts. Data are from three independent experiments. The proteins found enriched in FLAG-IP of F/H-BAZ2A ESCs in all three experiments are shown. Further BAZ2A-interacting proteins identified in only two or one of the immunoprecipitation (IP) experiments Error bars represent s.d.


C . Anti - HA IP of wt - and F / H - BAZ2A - ESC + 2i and ESC + serum . Western blot shows the interaction of BAZ2A with SNF2H , TOP2A and SMC1a .

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C. Anti-HA IP of wt- and F/H-BAZ2A-ESC+2i and ESC+serum. Western blot shows the interaction of BAZ2A with SNF2H, TOP2A and SMC1a.


D . TOP2A and BAZ2A regulate gene expression in ESC + 2i via a shared pathway . qRT - PCR of genes regulated by BAZ2A in ESC + 2i ( left panel ) and in ESC + serum ( right panel ) upon TOP2A or BAZ2A knockdown by siRNA . ESCs were treated with the corresponding RNAs for 4 days . Upregulated genes in ESC + 2i upon BAZ2A - KD are labeled in blue , downregulated genes are in red . Nanog , Rex1 and Actin B ( ActB ) are shown as genes not regulated by BAZ2A . mRNA levels were normalized to Rps12 mRNA and to ESCs transfected with siRNA - Control . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( ns : non significant , * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 , * * * * < 0 . 0001 ) .

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D. TOP2A and BAZ2A regulate gene expression in ESC+2i via a shared pathway. qRT-PCR of genes regulated by BAZ2A in ESC+2i (left panel) and in ESC+serum (right panel) upon TOP2A or BAZ2A knockdown by siRNA. ESCs were treated with the corresponding RNAs for 4 days. Upregulated genes in ESC+2i upon BAZ2A-KD are labeled in blue, downregulated genes are in red. Nanog, Rex1 and Actin B (ActB) are shown as genes not regulated by BAZ2A. mRNA levels were normalized to Rps12 mRNA and to ESCs transfected with siRNA-Control. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001).


E . Inhibition of type 2 topoisomerase activity phenocopies the alterations in gene expression of BAZ2A - regulated genes in ESC + 2i observed upon TOP2A or BAZ2A knockdown . ESCs were treated with the TOP2 inhibitor ICF - 193 for 24 hours . mRNA levels were normalized to Rps12 mRNA and to ESCs treated with DMSO . Upregulated genes in ESC + 2i upon BAZ2A - KD are labeled in blue , downregulated genes are in red . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( ns : non significant , * * < 0 . 01 , * * * < 0 . 001 , * * * * < 0 . 0001 ) .

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E. Inhibition of type 2 topoisomerase activity phenocopies the alterations in gene expression of BAZ2A-regulated genes in ESC+2i observed upon TOP2A or BAZ2A knockdown. ESCs were treated with the TOP2 inhibitor ICF-193 for 24 hours. mRNA levels were normalized to Rps12 mRNA and to ESCs treated with DMSO. Upregulated genes in ESC+2i upon BAZ2A-KD are labeled in blue, downregulated genes are in red. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, ** < 0.01, *** < 0.001, **** < 0.0001).


F . ChIP analysis showing that TOP2A regulates H3K27me3 occupancy in ESC + 2i but not in ESC + serum . Data were measured by qPCR and normalized to input and to Tshz1 value in cells transfected with siRNA - control . # 1 - # 4 indicated sequences with elevated H3K27me3 at CTCF boundary ( H3K27me3hi / CTCF ) . Control represents an intergenic region that does not contain H3K27me3 . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( ns : non significant , * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ) .

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F. ChIP analysis showing that TOP2A regulates H3K27me3 occupancy in ESC+2i but not in ESC+serum. Data were measured by qPCR and normalized to input and to Tshz1 value in cells transfected with siRNA-control. #1-#4 indicated sequences with elevated H3K27me3 at CTCF boundary (H3K27me3hi/CTCF). Control represents an intergenic region that does not contain H3K27me3. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, * < 0.05, ** < 0.01, *** < 0.001).


B . Southern blotting confirms successful targeting . Introduction of an additional EcoRI site results in a 4 . 1 kb restriction fragment detectable by Southern for targeted ES cells ( A174T / + ) but not for parental DNA ( + / + ) .

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B. Southern blotting confirms successful targeting. Introduction of an additional EcoRI site results in a 4.1 kb restriction fragment detectable by Southern for targeted ES cells (A174T/+) but not for parental DNA (+/+).


C . Capillary sequencing for Rnaseh2b + / + , Rnaseh2bA174T / + , and Rnaseh2bA174T / A174T DNA confirmed the presence of the introduced missense mutation .

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C. Capillary sequencing for Rnaseh2b+/+, Rnaseh2bA174T/+, and Rnaseh2bA174T/A174T DNA confirmed the presence of the introduced missense mutation.


D . Mouse genotyping by multiplex PCR . Top : A 221 bp PCR product is present in wild - type mice ( + / + ) ; the Rnaseh2bA174T allele ( also ) give a 460 bp product . Bottom : Position of forward ( x ) and reverse primers ( y , z ) .

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D. Mouse genotyping by multiplex PCR. Top: A 221 bp PCR product is present in wild-type mice (+/+); the Rnaseh2bA174T allele (also) give a 460 bp product. Bottom: Position of forward (x) and reverse primers (y, z).


E . Immunoblotting demonstrates depletion of all three RNase H2 protein subunits in Rnaseh2bA174T / A174T MEFs and RNASEH2BA177T / A177T LCLs . Representative of three independent experiments .

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E. Immunoblotting demonstrates depletion of all three RNase H2 protein subunits in Rnaseh2bA174T/A174T MEFs and RNASEH2BA177T/A177T LCLs. Representative of three independent experiments.


G , H . RNase H2 enzyme activity is reduced in mouse and patient cells . ( G ) Enzyme activity for Rnaseh2bA174T / A174T MEFs and passage - matched Rnaseh2b + / + controls , against RNase H substrate ( RNA : DNA heteroduplex ) and RNase H2 - specific substrate , double stranded DNA with a single embedded ribonucleotide ( DRD : DNA ) . Mean activity for three independent cell lines , error bars , SEM . Enzymatic activity expressed relative to the average value of control MEFs . * * * = p < 0 . 001 , two - tailed t - test ( n = 3 Rnaseh2bA174T / A174T and n = 3 Rnaseh2b + / + control MEF lines ) . ( H ) RNase H2 activity in LCLs from two independent healthy controls and an AGS patient homozygous for the RNASEH2B - A177T mutation . Enzyme activity normalised to average activity of control lines . Three independent experiments , error bars SEM . * * * = p < 0 . 001 versus either control , two - tailed t - test .

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G, H. RNase H2 enzyme activity is reduced in mouse and patient cells. (G) Enzyme activity for Rnaseh2bA174T/A174T MEFs and passage-matched Rnaseh2b+/+ controls, against RNase H substrate (RNA:DNA heteroduplex) and RNase H2-specific substrate, double stranded DNA with a single embedded ribonucleotide (DRD:DNA). Mean activity for three independent cell lines, error bars, SEM. Enzymatic activity expressed relative to the average value of control MEFs. *** = p<0.001, two-tailed t-test (n=3 Rnaseh2bA174T/A174T and n=3 Rnaseh2b+/+ control MEF lines). (H) RNase H2 activity in LCLs from two independent healthy controls and an AGS patient homozygous for the RNASEH2B-A177T mutation. Enzyme activity normalised to average activity of control lines. Three independent experiments, error bars SEM. *** = p<0.001 versus either control, two-tailed t-test.


Figure 2 . Increased ISG expression in tissues from Rnaseh2bA174T / A174T miceA . Transcript levels of multiple ISGs are significantly elevated in heart . B . Transcript levels of a subset of ISGs are significantly increased in kidney . C . No ISG upregulation is evident in the brain . ISG transcript levels determined by RT - qPCR normalised to transcript levels of the housekeeping gene HPRT ( Oas1a was undetectable in brain ) . Each data point represents the mean of technical replicates of tissue RNA from a single mouse . n = 9 nine - month old Rnaseh2bA174T / A174T mice , n = 4 age - matched control wildtype C57BL / 6Jmice . Horizontal line , mean ; error bars , SEM . * = p < 0 . 05 , * * = p < 0 . 01 , two - tailed t - test .

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Figure 2. Increased ISG expression in tissues from Rnaseh2bA174T/A174T miceA. Transcript levels of multiple ISGs are significantly elevated in heart.B. Transcript levels of a subset of ISGs are significantly increased in kidney.C. No ISG upregulation is evident in the brain. ISG transcript levels determined by RT-qPCR normalised to transcript levels of the housekeeping gene HPRT (Oas1a was undetectable in brain). Each data point represents the mean of technical replicates of tissue RNA from a single mouse. n=9 nine-month old Rnaseh2bA174T/A174T mice, n=4 age-matched control wildtype C57BL/6Jmice. Horizontal line, mean; error bars, SEM. * = p<0.05, ** = p<0.01, two-tailed t-test.


A . Validation of Rnaseh2b - / - MEF lines . RER activity ( DRD : DNA ) is undetectable in a Rnaseh2b - / - line consistent with complete inactivation of the Rnaseh2b gene ( Reijns et al , 2012 ) . Mean of three independent experiments ; error bars , SEM , * * * * = p < 0 . 0001 , two - tailed t - test . Rnaseh2b - / - and Rnaseh2b + / + control MEFs on a C57BL / 6 p53 - / - background .

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A. Validation of Rnaseh2b-/- MEF lines. RER activity (DRD:DNA) is undetectable in a Rnaseh2b-/- line consistent with complete inactivation of the Rnaseh2b gene (Reijns et al, 2012). Mean of three independent experiments; error bars, SEM, **** = p<0.0001, two-tailed t-test. Rnaseh2b-/- and Rnaseh2b+/+ control MEFs on a C57BL/6 p53-/- background.


B . ISG transcript levels are increased in Rnaseh2b - / - MEFs .

B. ISG transcript levels are increased in Rnaseh2b-/- MEFs.


C . BeadArray transcript analysis ( Illumina ) detected induction of multiple ISGs , including the cytokines CXCL10 and CCL5 , but not other cytokine transcripts in Rnaseh2b - / - MEFs . Plotted , average fold enrichment versus p - value of significantly upregulated transcripts ( p < 0 . 05 , after multiple testing correction ) comparing two Rnaseh2b - / - MEF lines versus 4 Rnaseh2b + / + MEF lines . 17 out of 29 transcripts are ISGs .

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C. BeadArray transcript analysis (Illumina) detected induction of multiple ISGs, including the cytokines CXCL10 and CCL5, but not other cytokine transcripts in Rnaseh2b-/- MEFs. Plotted, average fold enrichment versus p-value of significantly upregulated transcripts (p<0.05, after multiple testing correction) comparing two Rnaseh2b-/- MEF lines versus 4 Rnaseh2b+/+ MEF lines. 17 out of 29 transcripts are ISGs.


D . CXCL10 and CCL5 transcripts ( detected by RT - qPCR ) are significantly elevated .

D. CXCL10 and CCL5 transcripts (detected by RT-qPCR) are significantly elevated.


E . Increased CXCL10 and CCL5 protein ( detected by ELISA ) is secreted by Rnaseh2b - / - MEFs . ( B , D , E ) Mean data from three experiments for six independent Rnaseh2b - / - MEF lines versus four independent Rnaseh2b + / + MEF lines . Error bars , SEM . * = p < 0 . 05 , * * = p < 0 . 01 , Mann - Whitney U test .

E. Increased CXCL10 and CCL5 protein (detected by ELISA) is secreted by Rnaseh2b-/- MEFs. (B, D, E) Mean data from three experiments for six independent Rnaseh2b-/- MEF lines versus four independent Rnaseh2b+/+ MEF lines. Error bars, SEM. * = p<0.05, ** = p<0.01, Mann-Whitney U test.


A . ISG activation and cytokine secretion in Rnaseh2b - / - MEFs is markedly impaired by cGAS or STING siRNA depletion . Upper left and lower panels : RT - qPCR of IFIT1 , cGAS and STING transcripts , 48 h after siRNA targeting luciferase ( control ) , cGAS or STING . Upper right panel : CCL5 is significantly reduced in culture supernatants 48 h after cGAS or STING depletion . Concentration of CCL5 ( ELISA ) , normalised to luciferase siRNA control levels in each experiment . Mean from three independent experiments using one Rnaseh2b - / - MEF line ; error bars , SEM . * = p < 0 . 05 , * * = p < 0 . 01 , two - tailed t - test for RT - qPCR , one sample t - test for CCL5ELISA .

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A. ISG activation and cytokine secretion in Rnaseh2b-/- MEFs is markedly impaired by cGAS or STING siRNA depletion. Upper left and lower panels: RT-qPCR of IFIT1, cGAS and STING transcripts, 48 h after siRNA targeting luciferase (control), cGAS or STING. Upper right panel: CCL5 is significantly reduced in culture supernatants 48 h after cGAS or STING depletion. Concentration of CCL5 (ELISA), normalised to luciferase siRNA control levels in each experiment. Mean from three independent experiments using one Rnaseh2b-/- MEF line; error bars, SEM. * = p<0.05, ** = p<0.01, two-tailed t-test for RT-qPCR, one sample t-test for CCL5ELISA.


B - D . ISG induction and cytokine secretion is abolished in Rnaseh2b - / - cGAS - / - MEFs . cGAS was targeted by CRISPR / Cas9 genome editing of a Rnaseh2b - / - MEF line to inactivate cGAS / STING signalling . In addition to sequence validation , functional inactivation of cGAS was confirmed in Rnaseh2b - / - cGas - / - CRISPR lines by the absence of CCL5 secretion in response to dsDNA ( Appendix Fig S4 ) . CCL5 ( B ) and CXCL10 ( C ) production , as well as ISG expression ( D ) was abrogated in Rnaseh2b - / - cGAS - / - clones , assessed by ELISA and RT - qPCR respectively . Four independent experiments , n = 2 Rnaseh2b - / - cGAS - / - clones , n = 4 Rnaseh2b - / - cGAS + / + clones , error bars , SEM of each experiment * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , two - tailed t - test .

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B-D. ISG induction and cytokine secretion is abolished in Rnaseh2b-/- cGAS-/- MEFs. cGAS was targeted by CRISPR/Cas9 genome editing of a Rnaseh2b-/- MEF line to inactivate cGAS/STING signalling. In addition to sequence validation, functional inactivation of cGAS was confirmed in Rnaseh2b-/- cGas-/- CRISPR lines by the absence of CCL5 secretion in response to dsDNA (Appendix Fig S4). CCL5 (B) and CXCL10 (C) production, as well as ISG expression (D) was abrogated in Rnaseh2b-/- cGAS-/- clones, assessed by ELISA and RT-qPCR respectively. Four independent experiments, n=2 Rnaseh2b-/- cGAS-/- clones, n=4 Rnaseh2b-/- cGAS+/+ clones, error bars, SEM of each experiment * = p<0.05, ** = p<0.01, *** = p<0.001, two-tailed t-test.


E . ISG induction in Rnaseh2bA174T / A174T mice is STING dependent . RT - qPCR of RNA extracted from hearts from Sting + / + Rnaseh2bA174T / A174T ( n = 4 ) and Sting - / - Rnaseh2bA174T / A174T ( n = 6 ) three - month old mice .

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E. ISG induction in Rnaseh2bA174T/A174T mice is STING dependent. RT-qPCR of RNA extracted from hearts from Sting+/+ Rnaseh2bA174T/A174T (n=4) and Sting-/-Rnaseh2bA174T/A174T (n=6) three-month old mice.


F . Absence of STING does not significantly decrease basal ISG expression . RT - qPCR of RNA extracted from hearts from Sting + / + ( n = 3 ) and Sting - / - ( n = 5 ) three - month old mice . ( E , F ) Each data point represents the mean of technical triplicates from one mouse . Error bars , SEM . * = p < 0 . 05 , Mann - Whitney U test .

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

F. Absence of STING does not significantly decrease basal ISG expression. RT-qPCR of RNA extracted from hearts from Sting+/+ (n=3) and Sting-/- (n=5) three-month old mice. (E, F) Each data point represents the mean of technical triplicates from one mouse. Error bars, SEM. * = p<0.05, Mann-Whitney U test.


A . Overexpression of RNase H1 in Rnaseh2b - / - cells restores RNase H activity against RNA : DNA hybrids to 81 ± 10 % of wildtype levels , while overexpression of RNase H2B restores cellular enzyme activity for cleavage of both RNA : DNA and DRD : DNA substrates ( RER ) . Rnaseh2b - / - MEFs were complemented with Rnaseh1 ( + H1 ) , Rnaseh2b ( + H2B ) or EGFP by retroviral infection . Mean of n = 3 independent experiments ± SEM .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Overexpression of RNase H1 in Rnaseh2b-/- cells restores RNase H activity against RNA:DNA hybrids to 81 ± 10% of wildtype levels, while overexpression of RNase H2B restores cellular enzyme activity for cleavage of both RNA:DNA and DRD:DNA substrates (RER). Rnaseh2b-/- MEFs were complemented with Rnaseh1 (+H1), Rnaseh2b (+H2B) or EGFP by retroviral infection. Mean of n=3 independent experiments ± SEM.


B , C . DNA damage is reduced to wildtype levels by complementation with Rnaseh2b but not Rnaseh1 , measured by 53BP1 foci formation in detergent - extracted fixed cells . ( B ) Representative images ( scale bar , 10 µm ) . ( C ) At least 150 cells were counted for each cell line in three independent experiments . Mean ± SEM , * * * * = p < 0 . 0001 two - tailed t - test .

B, C. DNA damage is reduced to wildtype levels by complementation with Rnaseh2b but not Rnaseh1, measured by 53BP1 foci formation in detergent-extracted fixed cells. (B) Representative images (scale bar, 10 µm). (C) At least 150 cells were counted for each cell line in three independent experiments. Mean ± SEM, **** = p<0.0001 two-tailed t-test.


D - F . CCL5 ( D ) and CXCL10 production ( E ) , as well as ISG induction ( F ) in Rnaseh2b - / - MEFs are reduced close to wildtype levels ( Rnaseh2b + / + ) , by complementation with Rnaseh2b but not Rnaseh1 . Mean of n = 6 independent experiments ± SEM for complemented cells ; n = 3 independent experiments for Rnaseh2b - / - parental and Rnaseh2b + / + controls cells . * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 two - tailed t - test indicates significantly reduced expression compared to Rnaseh2b - / - parental cells .

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D-F. CCL5 (D) and CXCL10 production (E), as well as ISG induction (F) in Rnaseh2b-/- MEFs are reduced close to wildtype levels (Rnaseh2b+/+), by complementation with Rnaseh2b but not Rnaseh1. Mean of n=6 independent experiments ± SEM for complemented cells; n=3 independent experiments for Rnaseh2b-/- parental and Rnaseh2b+/+ controls cells. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001 two-tailed t-test indicates significantly reduced expression compared to Rnaseh2b-/- parental cells.


Representative confocal images of control ( CTL - 1 , 2 , 3 ) and DOA + patient fibroblasts carrying indicated monoallelic mutations imaged as described in A . Scale bar = 20μm . Passage number between P12 - 15 . Mitochondrial morphology quantification of B . Data represent mean ± SD of two independent experiments , ( 195 - 2496 cells per cell line ) , One - way ANOVA ; * * p < 0 . 01 , * * * * p < 0 . 0001 , ns ; not

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

Representative confocal images of control (CTL-1, 2, 3) and DOA+ patient fibroblasts carrying indicated monoallelic mutations imaged as described in A. Scale bar=20μm. Passage number between P12-15. Mitochondrial morphology quantification of B. Data represent mean ± SD of two independent experiments, (195-2496 cells per cell line), One-way ANOVA; ** p < 0.01, ****p < 0.0001, ns; not significant


Representative confocal images of control ( CTL - 1 ) and OPA1S545R patient fibroblasts treated with OPA1 , DNM1L , or non - targeting ( NT ) siRNAs for 72 hours and imaged as described in A . Scale bar = 20μm . Passage number between P12 - 14 . Mitochondrial morphology quantification of D . Data represent mean ± SD of three independent experiments , ( 3219 - 5857 cells per cell line ) , One - way ANOVA ; * * * * p < 0 . 0001 , ns ; not significant .

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Representative confocal images of control (CTL-1) and OPA1S545R patient fibroblasts treated with OPA1, DNM1L, or non-targeting (NT) siRNAs for 72 hours and imaged as described in A. Scale bar=20μm. Passage number between P12-14. Mitochondrial morphology quantification of D. Data represent mean ± SD of three independent experiments, (3219-5857 cells per cell line), One-way ANOVA; ****p < 0.0001, ns; not significant.


Representative confocal images of control ( CTL - 1 ) and OPA1S545R patient fibroblasts treated with 50μM cycloheximide ( CHX ) where indicated for 6 hours . Imaging as described in A . Scale bar = 20μm . Passage number between P14 - P15 . Mitochondrial morphology quantification of F . Data represent mean ± SD of two independent experiments , ( 879 - 4154 cells per cell line ) , One - way ANOVA ; * * * * p < 0 . 0001 , ns ; not significant .

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

Representative confocal images of control (CTL-1) and OPA1S545R patient fibroblasts treated with 50μM cycloheximide (CHX) where indicated for 6 hours. Imaging as described in A. Scale bar=20μm. Passage number between P14-P15. Mitochondrial morphology quantification of F. Data represent mean ± SD of two independent experiments, (879-4154 cells per cell line), One-way ANOVA; ****p < 0.0001, ns; not significant.


Representative confocal images of control ( CTL - 1 ) and OPA1S545R patient fibroblasts treated with OPA1 , DNM1L , PGS1 , and non - targeting ( NT ) siRNAs or indicated combinations for 72 hours . Mitochondria ( anti - TOM40 , green ) and nuclei ( DAPI , blue ) . Scale bar = 20μm . Passages number between P10 - 15 . Mitochondrial morphology quantification of ( A ) using control fibroblasts with fragmented ( OPA1 siRNA ) , normal ( non - targeting NT siRNA ) , and hypertubulated ( DNM1L siRNA ) mitochondria . Data represent mean ± SD of three independent experiments , One - way ANOVA ( 905 - 3695 cells per cell line ) , ( % fragmented ) ; ; * * * * p < 0 . 0001 , ns ; not significant . .

Representative confocal images of control (CTL-1) and OPA1S545R patient fibroblasts treated with OPA1, DNM1L, PGS1, and non-targeting (NT) siRNAs or indicated combinations for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=20μm. Passages number between P10-15. Mitochondrial morphology quantification of (A) using control fibroblasts with fragmented (OPA1 siRNA), normal (non-targeting NT siRNA), and hypertubulated (DNM1L siRNA) mitochondria. Data represent mean ± SD of three independent experiments, One-way ANOVA (905-3695 cells per cell line), (% fragmented); ; ****p < 0.0001, ns; not significant..


Representative confocal images of wild type ( WT ) and Opa1Crispr MEFs treated with NT or Pgs1 siRNA for 72 hours . Live imaging of mitochondria ( mitoYFP , green ) and nuclei ( NucBlue , blue ) . Scale bar = 10μm . Mitochondrial morphology quantification of ( C ) using WT MEFs treated with Opa1 siRNA ( fragmented ) , NT siRNA ( normal ) , or Dnm1l siRNA ( hypertubulated ) ground truth training sets . Data represent mean ± SD of three independent experiments , One - way ANOVA ( 6613 - 8758 cells per cell line ) , ( % fragmented ) ; * * * * p < 0 . 0001 , ns ; not significant .

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

Representative confocal images of wild type (WT) and Opa1Crispr MEFs treated with NT or Pgs1 siRNA for 72 hours. Live imaging of mitochondria (mitoYFP, green) and nuclei (NucBlue, blue). Scale bar=10μm. Mitochondrial morphology quantification of (C) using WT MEFs treated with Opa1 siRNA (fragmented), NT siRNA (normal), or Dnm1l siRNA (hypertubulated) ground truth training sets. Data represent mean ± SD of three independent experiments, One-way ANOVA (6613-8758 cells per cell line), (% fragmented) ; ****p < 0.0001, ns; not significant.


Representative confocal images of WT , Opa1Crispr MEFs complemented with pLenti - Opa1 , Opa1CrisprPgs1Crispr MEFs and Pgs1Crispr MEFs complemented with pLenti - Pgs1 by lentiviral delivery . Live imaging of mitochondria ( mitoYFP , green ) and nuclei ( NucBlue , blue ) . Scale bar = 10μm . Supervised ML mitochondrial morphology quantification of ( E ) using WT MEFs treated with Opa1 siRNA ( fragmented ) , NT siRNA ( normal ) , or Dnm1l siRNA ( hypertubulated ) training sets . Data represent mean ± SD of three independent experiments , One - way ANOVA ( 691 - 3990 cells per cell line ) , ( % fragmented ) ; * * * * p < 0 . 0001 , ns ; not significant . .

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

Representative confocal images of WT, Opa1Crispr MEFs complemented with pLenti-Opa1, Opa1CrisprPgs1Crispr MEFs and Pgs1Crispr MEFs complemented with pLenti-Pgs1 by lentiviral delivery. Live imaging of mitochondria (mitoYFP, green) and nuclei (NucBlue, blue). Scale bar=10μm. Supervised ML mitochondrial morphology quantification of (E) using WT MEFs treated with Opa1 siRNA (fragmented), NT siRNA (normal), or Dnm1l siRNA (hypertubulated) training sets. Data represent mean ± SD of three independent experiments, One-way ANOVA (691-3990 cells per cell line), (% fragmented) ; ****p < 0.0001, ns; not significant..


Representative confocal images of live cell imaging of MEFs of the indicated genotypes subjected fragmentation with 5μM carbonyl cyanide m - chlorophenyl hydrazine ( CCCP ) for the indicated time points . Images were captured every hour for 18 hours . Scale bar = 10μm . Supervised ML mitochondrial morphology quantification using WT MEFs treated with 5μM CCCP for 18h ( fragmented ) , untreated ( normal ) , or treated with 10μM CHX for 9h ( hypertubular ) training sets . Data represent mean ± SD of three independent experiments , ( 131 - 426 cells per cell line ) , One - way ANOVA ; * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , ns ; not significant .

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

Representative confocal images of live cell imaging of MEFs of the indicated genotypes subjected fragmentation with 5μM carbonyl cyanide m-chlorophenyl hydrazine (CCCP) for the indicated time points. Images were captured every hour for 18 hours. Scale bar=10μm. Supervised ML mitochondrial morphology quantification using WT MEFs treated with 5μM CCCP for 18h (fragmented), untreated (normal), or treated with 10μM CHX for 9h (hypertubular) training sets. Data represent mean ± SD of three independent experiments, (131-426 cells per cell line), One-way ANOVA; *p < 0.05, ** p < 0.01, ****p < 0.0001, ns; not significant.


Representative confocal images of live cell imaging of MEFs of the indicated genotypes subjected hyperfusion ( SiMH ) with 10μM cycloheximide ( CHX ) for the indicated time points . Images were captured every hour for 9 hours . Mitochondrial morphology quantification of using WT MEFs treated with 5μM CCCP for 18h ( fragmented ) , untreated ( normal ) , or treated with 10μM CHX for 9h ( hypertubular ) training sets . Data represent mean ± SD of four independent experiments , ( 155 - 745 cells per cell line ) , One - way ANOVA .

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

Representative confocal images of live cell imaging of MEFs of the indicated genotypes subjected hyperfusion (SiMH) with 10μM cycloheximide (CHX) for the indicated time points. Images were captured every hour for 9 hours. Mitochondrial morphology quantification of using WT MEFs treated with 5μM CCCP for 18h (fragmented), untreated (normal), or treated with 10μM CHX for 9h (hypertubular) training sets. Data represent mean ± SD of four independent experiments, (155-745 cells per cell line), One-way ANOVA.


Representative confocal micrographs of MEFs WT and Opa1Crispr MEFs treated with indicated siRNAs for 72 hours . Mitochondria ( anti - TOM40 , green ) and nuclei ( DAPI , blue ) . Scale bar = 10μm . Supervised ML mitochondrial morphology quantification of ( B ) using WT MEFs with fragmented ( Opa1 siRNA ) , normal ( non - targetting NT siRNA ) , and hypertubular ( Dnm1l siRNA ) mitochondria . Data represent mean ± SD of three independent experiments , One - way ANOVA ( 726 - 4236 cells per cell line ) , ( % fragmented ) ; * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns ; not significant .

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

Representative confocal micrographs of MEFs WT and Opa1Crispr MEFs treated with indicated siRNAs for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=10μm. Supervised ML mitochondrial morphology quantification of (B) using WT MEFs with fragmented (Opa1 siRNA), normal (non-targetting NT siRNA), and hypertubular (Dnm1l siRNA) mitochondria. Data represent mean ± SD of three independent experiments, One-way ANOVA (726-4236 cells per cell line), (% fragmented); *** p < 0.001, ****p < 0.0001, ns; not significant.


Representative confocal micrographs of MEFs WT , Pgs1Crispr and Dnm1lCrispr MEFs treated with indicated siRNAs for 72 hours . Mitochondria ( anti - TOM40 , green ) and nuclei ( DAPI , blue ) . Scale bar = 10μm . Supervised ML mitochondrial morphology quantification of ( G ) using WT MEFs with fragmented ( Opa1 siRNA ) , normal ( non - targetting NT siRNA ) , and hypertubulated ( Dnm1l siRNA ) mitochondria . Data represent mean ± SD of > 3 independent experiments , ( 3096 - 7238 cells per cell line ) , One - way ANOVA ( % fragmented ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , ns ; not significant .

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

Representative confocal micrographs of MEFs WT, Pgs1Crispr and Dnm1lCrispr MEFs treated with indicated siRNAs for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=10μm. Supervised ML mitochondrial morphology quantification of (G) using WT MEFs with fragmented (Opa1 siRNA), normal (non-targetting NT siRNA), and hypertubulated (Dnm1l siRNA) mitochondria. Data represent mean ± SD of >3 independent experiments, (3096-7238 cells per cell line), One-way ANOVA (% fragmented) ; *p < 0.05, ** p < 0.01, ****p < 0.0001, ns; not significant.


Representative transmission electron micrographs of MEFs of the indicated genotypes showing loss of lamellar cristae in Opa1Crispr and Opa1CrisprPgs1Crispr MEFs . Scale bar = 200nm . Quantification of ( C ) of mitochondrial ultrastructure ; outer membrane / inner membrane ration ( IMM / OMM ) and cristae number per mitochondrion . Violin plot of > 50 mitochondria per cell line , One - way ANOVA ; * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , ns ; not significant .

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Representative transmission electron micrographs of MEFs of the indicated genotypes showing loss of lamellar cristae in Opa1Crispr and Opa1CrisprPgs1Crispr MEFs. Scale bar=200nm. Quantification of (C) of mitochondrial ultrastructure; outer membrane/inner membrane ration (IMM/OMM) and cristae number per mitochondrion. Violin plot of >50 mitochondria per cell line, One-way ANOVA; *p < 0.05, ** p < 0.01, ****p < 0.0001, ns; not significant.


mtDNA content in MEFs from ( F ) was quantified by amplification of MTTL1 , 16S and MT - ND1 genes relative to the GAPDH nuclear gene in MEFs . Data represent mean ± SD of three independent experiments , One - way ANOVA ; * * * * p < 0 . 0001 , ns ; not significant . mtDNA content in WT and mutant MEFs treated with indicated siRNAs for 72 hours was quantified by amplification of MTTL1 , 16S and ND1 genes relative to the GAPDH nuclear gene in MEFs . Data represent mean ± SD of three independent experiments , One - way ANOVA ; * * p < 0 . 01 , * * * * p < 0 . 0001 , ns ; not significant .

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mtDNA content in MEFs from (F) was quantified by amplification of MTTL1, 16S and MT-ND1 genes relative to the GAPDH nuclear gene in MEFs. Data represent mean ± SD of three independent experiments, One-way ANOVA; ****p < 0.0001, ns; not significant. mtDNA content in WT and mutant MEFs treated with indicated siRNAs for 72 hours was quantified by amplification of MTTL1, 16S and ND1 genes relative to the GAPDH nuclear gene in MEFs. Data represent mean ± SD of three independent experiments, One-way ANOVA; **p < 0.01, ****p < 0.0001, ns; not significant.


( G ) Equal amounts of protein extracted from WT and mutant MEFs were separated by SDS - PAGE ( horizontal line denotes separate membranes ) , immunoblotted with indicated antibodies and quantified by densitometry ( H ) . Data represent mean ± SD of three independent experiments , One - way ANOVA ; * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns ; not significant .

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

(G) Equal amounts of protein extracted from WT and mutant MEFs were separated by SDS-PAGE (horizontal line denotes separate membranes), immunoblotted with indicated antibodies and quantified by densitometry (H). Data represent mean ± SD of three independent experiments, One-way ANOVA; **p < 0.01, ***p < 0.001, ****p < 0.0001, ns; not significant.


A ) Differential interference contrast ( DIC ) image of cortical neurons in culture . The arrowhead points to a neuron expressing Ub - R - GFP . B ) GFP fluorescence before and after exposure to lactacystin ( 10µM ) .

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

A) Differential interference contrast (DIC) image of cortical neurons in culture. The arrowhead points to a neuron expressing Ub-R-GFP. B) GFP fluorescence before and after exposure to lactacystin (10µM).


C ) A neuron coexpressing Ub - R - GFP and CFP before ( left ) and 10 hours after exposure to lactacystin ( right ) . Top row - GFP fluorescence ; bottom row - CFP fluorescence . Bars : 50µm ( A , B ) , 20 µm ( C ) . D ) Changes in GFP and CFP fluorescence following exposure to lactacystin at t = 1 hour ( vertical dashed line ) . Changes normalized to initial fluorescence in same cells . 9 neurons from 5 experiments ; averages and SEM . E ) Same as in D , except that here neurons were exposed only to carrier solution . 11 neurons from 5 experiments ; averages and SEM ( barely observable ) . F ) Comparison of GFP fluorescence accumulation rates . Linear fits shown as black lines ; fit parameters shown next to fits . Same data as in D , and E .

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

C) A neuron coexpressing Ub-R-GFP and CFP before (left) and 10 hours after exposure to lactacystin (right). Top row - GFP fluorescence; bottom row - CFP fluorescence. Bars: 50µm (A,B), 20 µm (C). D) Changes in GFP and CFP fluorescence following exposure to lactacystin at t=1 hour (vertical dashed line). Changes normalized to initial fluorescence in same cells. 9 neurons from 5 experiments; averages and SEM. E) Same as in D, except that here neurons were exposed only to carrier solution. 11 neurons from 5 experiments; averages and SEM (barely observable). F) Comparison of GFP fluorescence accumulation rates. Linear fits shown as black lines; fit parameters shown next to fits. Same data as in D, and E.


A ) Hippocampal neurons in primary culture immunolabeled against three synaptic proteins ( the postsynaptic scaffold molecule Dlg4 / PSD - 95 , the presynaptic active zone molecule RIM and the presynaptic vesicle protein SV2A ) following 10 hour incubations in carrier solution ( control , upper panels ) or Lactacystin ( 10 μM ; bottom panels ) . Scale bar ,

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

A) Hippocampal neurons in primary culture immunolabeled against three synaptic proteins (the postsynaptic scaffold molecule Dlg4/PSD-95, the presynaptic active zone molecule RIM and the presynaptic vesicle protein SV2A) following 10 hour incubations in carrier solution (control, upper panels) or Lactacystin (10 μM; bottom panels). Scale bar, 10μm


B ) Average synaptic immunofluorescence following 10 hour incubations in Lactacystin or carrier solution . Numbers within bars represent the number of fields of view analyzed for each condition ( ~ 170 synapses / field of view , on average ) . Error bars , SEM ; P values - two tailed unpaired t - test . Note that a statistically significant accumulation was observed only for RIM , whereas immunolabeling of all other proteins was either unchanged or reduced .

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B) Average synaptic immunofluorescence following 10 hour incubations in Lactacystin or carrier solution. Numbers within bars represent the number of fields of view analyzed for each condition (~170 synapses/field of view, on average). Error bars, SEM; P values - two tailed unpaired t-test. Note that a statistically significant accumulation was observed only for RIM, whereas immunolabeling of all other proteins was either unchanged or reduced.


C ) Hippocampal neurons in primary culture were grown in the presence of Lactacystin ( 10 μM ) , fixed at two hour intervals and immunolabeled against Shank3 / ProSAP2 . Two representative fields of view at t = 0 and t = 24h . Scale bar , 10μm . D ) Average synaptic Shank3 / ProSAP2 immunofluorescence at increasing times in the presence of Lactacystin . Fluorescence was normalized to mean fluorescence at t = 0 . Numbers under data points reflect the numbers of fields of view analyzed at each time point . Error bars , SEM .

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

C) Hippocampal neurons in primary culture were grown in the presence of Lactacystin (10 μM), fixed at two hour intervals and immunolabeled against Shank3/ProSAP2. Two representative fields of view at t=0 and t=24h. Scale bar, 10μm. D) Average synaptic Shank3/ProSAP2 immunofluorescence at increasing times in the presence of Lactacystin. Fluorescence was normalized to mean fluorescence at t=0. Numbers under data points reflect the numbers of fields of view analyzed at each time point. Error bars, SEM.


B ) Comparisons of H / L ratios obtained for 1409 proteins in lactacystin and untreated cells . H / L ratios are averages of 2 experiments .

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

B) Comparisons of H/L ratios obtained for 1409 proteins in lactacystin and untreated cells. H/L ratios are averages of 2 experiments.


A ) Fold changes in the H / L ratios ( ) measured for each protein in the experiments described in Fig . 3 ( expressed as log2 ( H / L ) Lactacystin - log2 ( H / L ) control ) . H / L ratios used here are the average of ratios obtained in two experiments for each protein and each condition . Proteins were sorted according to the fold change in H / L ratios - from the proteins whose ratios were most strongly reduced ( ~ 8 - fold reduction ) to proteins whose H / L ratios increased to the greatest extent ( > 8 - fold increase ) . Red circles indicate the location of synaptic proteins along this sorted list . The names of 34 synaptic proteins located at the left most region of the sorted list are provided in the order of their appearance . B ) The apparent fold - change in protein half - lives under certain assumptions described in Results . See Materials and Methods for further details on the calculation of the apparent fold change in half - lives .

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A) Fold changes in the H/L ratios ( ) measured for each protein in the experiments described in Fig. 3 (expressed as log2(H/L)Lactacystin - log2(H/L)control). H/L ratios used here are the average of ratios obtained in two experiments for each protein and each condition. Proteins were sorted according to the fold change in H/L ratios - from the proteins whose ratios were most strongly reduced (~8-fold reduction) to proteins whose H/L ratios increased to the greatest extent (>8-fold increase). Red circles indicate the location of synaptic proteins along this sorted list. The names of 34 synaptic proteins located at the left most region of the sorted list are provided in the order of their appearance. B) The apparent fold-change in protein half-lives under certain assumptions described in Results. See Materials and Methods for further details on the calculation of the apparent fold change in half-lives.


A ) Average log2 ( H / M ) ratios for 1 , 750 proteins at four time points in three separate experiments . Each time point shows the average and standard deviation of three mean log2 ( H / M ) values obtained in three separate experiments . Each mean , in turn , represents the mean log2 ( H / M ) value measured in the population of 1 , 750 proteins in that experiment and time point . B ) Distribution of log2 ( H / M ) values for 1 , 750 proteins at t = 0 . The log2 ( H / M ) value of each protein represents the average value for that protein in the three experiments . In principle , log2 ( H / M ) for all proteins at t = 0 should be 0 , but in practice they are distributed around 0 as shown here . The H / M ratios for 3 standard deviation limits are shown beneath the X axis . C , D ) The same as in A and B but for two experiments in which Epoxomicin was used ( 1 , 823 proteins ) . E , F ) The same as in A and B but for the combined data from all experiments ( 3 experiments in which lactacystin was used and 2 experiments in which epoxomicin was used ; 1 , 469 proteins ) .

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A) Average log2(H/M) ratios for 1,750 proteins at four time points in three separate experiments. Each time point shows the average and standard deviation of three mean log2(H/M) values obtained in three separate experiments. Each mean, in turn, represents the mean log2(H/M) value measured in the population of 1,750 proteins in that experiment and time point. B) Distribution of log2(H/M) values for 1,750 proteins at t=0. The log2(H/M) value of each protein represents the average value for that protein in the three experiments. In principle, log2(H/M) for all proteins at t=0 should be 0, but in practice they are distributed around 0 as shown here. The H/M ratios for 3 standard deviation limits are shown beneath the X axis. C, D) The same as in A and B but for two experiments in which Epoxomicin was used (1,823 proteins). E, F) The same as in A and B but for the combined data from all experiments (3 experiments in which lactacystin was used and 2 experiments in which epoxomicin was used; 1,469 proteins).


A ) log2 ( H / M ) values for 5 synaptic proteins for which degradation rates were significantly slowed down in the presence of lactacystin . Each point represents average log2 ( H / M ) from 3 experiments . B ) log2 ( H / M ) values for the same 5 synaptic proteins in experiments in which epoxomicin was used . Each point represents average log2 ( H / M ) from 2 experiments . C ) Average ( ± SEM ) log2 ( H / M ) ratios for the same 5 synaptic proteins , based on all five experiments ( 3 lactacystin , 2 epoxomicin ) . P values - two - tailed Welch ' s t - test , comparing the five log2 ( H / M ) measurements at t = 24 to those measured for the same proteins at t = 0 .

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

A) log2(H/M) values for 5 synaptic proteins for which degradation rates were significantly slowed down in the presence of lactacystin. Each point represents average log2(H/M) from 3 experiments. B) log2(H/M) values for the same 5 synaptic proteins in experiments in which epoxomicin was used. Each point represents average log2(H/M) from 2 experiments. C) Average (± SEM) log2(H/M) ratios for the same 5 synaptic proteins, based on all five experiments (3 lactacystin, 2 epoxomicin). P values - two-tailed Welch's t-test, comparing the five log2(H/M) measurements at t=24 to those measured for the same proteins at t=0.


D , E , F ) Average ( ± SEM ) Log2 ( H / M ) values for three groups of synaptic proteins ( synaptic vesicle , presynaptic , postsynaptic ) , based on all five experiments . None of the changes observed for these proteins were statistically significant . The temporal changes common to all proteins and all groups most likely represent slight imperfections in the H / M normalization process ( Compare with Fig . 6E ; see Results for further details ) .

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D,E,F) Average (± SEM) Log2(H/M) values for three groups of synaptic proteins (synaptic vesicle, presynaptic, postsynaptic), based on all five experiments. None of the changes observed for these proteins were statistically significant. The temporal changes common to all proteins and all groups most likely represent slight imperfections in the H/M normalization process (Compare with Fig. 6E; see Results for further details).


G ) log2 ( H / M ) values at t = 24 hours for a group of well - studied synaptic proteins , sorted from highest to lowest . P values ( two - tailed Welch ' s t - test ) of comparisons with log2 ( H / M ) values for the same proteins at t = 0 are shown in parentheses . Proteins that exhibited statistically significant changes ( P ≤ 0 . 05 ) at t = 24h are shown in red . Proteins for which ( 0 . 05 < P ≤ 0 . 10 ) at t = 24h are shown in blue / red . The gray region depicts three standard deviations of log2 ( H / M ) values at t = 0 ( Fig . 6F ) , to provide a measure of the method ' s sensitivity .

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G) log2(H/M) values at t=24 hours for a group of well-studied synaptic proteins, sorted from highest to lowest. P values (two-tailed Welch's t-test) of comparisons with log2(H/M) values for the same proteins at t=0 are shown in parentheses. Proteins that exhibited statistically significant changes (P≤0.05) at t=24h are shown in red. Proteins for which (0.05 < P ≤ 0.10) at t=24h are shown in blue/red. The gray region depicts three standard deviations of log2(H/M) values at t=0 (Fig. 6F), to provide a measure of the method's sensitivity.


Measured log2 ( H / M ) values at four time points as a function of their half - lives estimated here and elsewhere ( Cohen et al , 2013 ) . All log2 ( H / M ) values shown here are averages of values obtained in 5 experiments ( 3 using lactacystin , 2 using epoxomicin ) . Proteins for which statistically significant differences at t = 24 hours were observed ( P < 0 . 05 , two - tailed Welch ' s t - test ) are shown in red . Expected log2 ( H / M ) values based on equation 4 are plotted as light blue lines . To minimize the masking of potential dependencies by the slight imperfections in the H / M normalization process ( which introduces small offsets along the Y scale ) , average population log2 ( H / M ) values of each time point ( Fig . 6E ) were subtracted from all measured Log2 ( H / M ) values . Data for 1 , 416 proteins for which half - life estimates were available .

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

Measured log2(H/M) values at four time points as a function of their half-lives estimated here and elsewhere (Cohen et al, 2013). All log2(H/M) values shown here are averages of values obtained in 5 experiments (3 using lactacystin, 2 using epoxomicin). Proteins for which statistically significant differences at t=24 hours were observed (P<0.05, two-tailed Welch's t-test) are shown in red. Expected log2(H/M) values based on equation 4 are plotted as light blue lines. To minimize the masking of potential dependencies by the slight imperfections in the H/M normalization process (which introduces small offsets along the Y scale), average population log2(H/M) values of each time point (Fig. 6E) were subtracted from all measured Log2(H/M) values. Data for 1,416 proteins for which half-life estimates were available.


Measured Log2 ( H / M ) values at four time points as a function of their half - lives estimated here and elsewhere ( Cohen et al , 2013 ) . All log2 ( H / M ) values shown here are averages of values obtained in 5 experiments ( 3 using lactacystin , 2 using epoxomicin ) . Proteins for which statistically significant differences at t = 24 hours were observed ( P < 0 . 05 , two - tailed Welch ' s t - test ) are shown in red . Expected log2 ( H / M ) values based on equation 4 are plotted as light blue lines . To minimize the masking of potential dependencies by the slight imperfections in the H / M normalization process ( which introduces small offsets along the Y scale ) , average population log2 ( H / M ) values of each time point ( Fig . 6E ) were subtracted from all measured Log2 ( H / M ) values . Data for 174 synaptic proteins for which half - life estimates were available .

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

Measured Log2(H/M) values at four time points as a function of their half-lives estimated here and elsewhere (Cohen et al, 2013). All log2(H/M) values shown here are averages of values obtained in 5 experiments (3 using lactacystin, 2 using epoxomicin). Proteins for which statistically significant differences at t=24 hours were observed (P<0.05, two-tailed Welch's t-test) are shown in red. Expected log2(H/M) values based on equation 4 are plotted as light blue lines. To minimize the masking of potential dependencies by the slight imperfections in the H/M normalization process (which introduces small offsets along the Y scale), average population log2(H/M) values of each time point (Fig. 6E) were subtracted from all measured Log2(H/M) values. Data for 174 synaptic proteins for which half-life estimates were available.


B ) Kaplan - Meier curve showing % of mice surviving S . pneumoniae infection . Data was analyzed using Kaplan - Meier statistical analysis . N = 5 - 10 mice / group . Data information : Data presented show mean ± SEM of biological replicates .

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B) Kaplan-Meier curve showing % of mice surviving S. pneumoniae infection. Data was analyzed using Kaplan-Meier statistical analysis. N = 5 - 10 mice/group. Data information: Data presented show mean ± SEM of biological replicates.


F ) Bar graphs comparing bacterial burden ( CFU ) in the lung and spleen at 48h , 72h and 7d post - S . pneumoniae infection in unvaccinated and BCG vaccinated mice . N = 3 - 5 mice / group . Data information : Data presented in F ) show mean ± SEM of biological replicates . Statistical analysis for F ) were two - way ANOVA with Sidak ' s multiple comparisons test * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 .

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F) Bar graphs comparing bacterial burden (CFU) in the lung and spleen at 48h, 72h and 7d post-S. pneumoniae infection in unvaccinated and BCG vaccinated mice. N = 3 - 5 mice/group. Data information: Data presented in F) show mean ± SEM of biological replicates. Statistical analysis for F) were two-way ANOVA with Sidak's multiple comparisons test *p < 0.05; **p < 0.01; ****p < 0.0001.


B ) Dot plots of gated mononuclear cells from the BAL of control and BCG vaccinated mice . Analysis was performed utilizing default FlowJo V . 10 software settings . C ) Line graphs comparing absolute neutrophil cell counts in the airways at 18 and 36h post - S . pneumoniae infection in BCG vaccinated and unvaccinated hosts . N = 3 - 4 mice / group . Data information : Data presented represent mean ± SEM of biological replicates . Statistical analysis for ( C , were two - way ANOVA with Sidak ' s multiple comparisons test . * p < 0 . 05 ; * * * p < 0 . 001 .

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B) Dot plots of gated mononuclear cells from the BAL of control and BCG vaccinated mice. Analysis was performed utilizing default FlowJo V.10 software settings. C) Line graphs comparing absolute neutrophil cell counts in the airways at 18 and 36h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis for (C, were two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05; ***p < 0.001.


D ) Dot plots of gated mononuclear cells from the airways of control and BCG vaccinated hosts . Analysis was performed utilizing default FlowJo V . 10 software settings . Line graphs comparing absolute AM cell counts in the airways at 18 and 36h post - S . pneumoniae infection in BCG vaccinated and unvaccinated hosts . N = 3 - 4 mice / group . Data information : Data presented represent mean ± SEM of biological replicates . Statistical analysis were two - way ANOVA with Sidak ' s multiple comparisons test . * p < 0 . 05 ; * * * p < 0 . 001 .

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D) Dot plots of gated mononuclear cells from the airways of control and BCG vaccinated hosts. Analysis was performed utilizing default FlowJo V.10 software settings. Line graphs comparing absolute AM cell counts in the airways at 18 and 36h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis were two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05; ***p < 0.001.


G ) Line graphs comparing the levels of neutrophilic chemokines in the BAL fluid at 18 and 36h post - S . pneumoniae infection in BCG vaccinated and unvaccinated hosts . N = 3 - 4 mice / group . Data information : Data presented represent mean ± SEM of biological replicates . Statistical analysis were two - way ANOVA with Sidak ' s multiple comparisons test . * p < 0 . 05 ; * * * p < 0 . 001 .

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G) Line graphs comparing the levels of neutrophilic chemokines in the BAL fluid at 18 and 36h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis were two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05; ***p < 0.001.


A ) Dot plots of gated mononuclear cells from the lung of control and BCG vaccinated hosts . Analysis was performed utilizing default FlowJo V . 10 software settings . B ) Line graphs comparing absolute neutrophil cell counts in the lung at 18 and 36h post - S . pneumoniae infection in BCG vaccinated and unvaccinated hosts . N = 3 - 4 mice / group . Data information : Data presented represent mean ± SEM of biological replicates . Statistical analysis for ( B , were two - way ANOVA with Sidak ' s multiple comparisons test .

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

A) Dot plots of gated mononuclear cells from the lung of control and BCG vaccinated hosts. Analysis was performed utilizing default FlowJo V.10 software settings. B) Line graphs comparing absolute neutrophil cell counts in the lung at 18 and 36h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis for (B, were two-way ANOVA with Sidak's multiple comparisons test.


C ) Dot plots of gated mononuclear cells from the lung of control and BCG vaccinated hosts . Analysis was performed utilizing default FlowJo V . 10 software settings . D - Line graphs comparing absolute AM , cell counts in the lung at 18 and 36h post - S . pneumoniae infection in BCG vaccinated and unvaccinated hosts . N = 3 - 4 mice / group . Data information : Data presented represent mean ± SEM of biological replicates . Statistical analysis were two - way ANOVA with Sidak ' s multiple comparisons test .

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C) Dot plots of gated mononuclear cells from the lung of control and BCG vaccinated hosts. Analysis was performed utilizing default FlowJo V.10 software settings. D - Line graphs comparing absolute AM, cell counts in the lung at 18 and 36h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis were two-way ANOVA with Sidak's multiple comparisons test.