( c ) MALDI MS analysis of the ε - cleavage sites of wt and mutant TREM2 C - terminal fragments . Mass spectrometry reveals ε - cleavage C - terminal of A192 for the wt CTF . MS determination of the ε - cleavage site of TREM2 CTF K186A reveals major and minor ε - cleavage sites C - terminal of A195 and L193 , respectively . The exact same cleavage pattern is observed for the K186L mutant . MS analysis of the cleavage pattern of the K186P mutant reveals major and minor ε - cleavage sites C - terminal of L193 and S191 . Minor peaks that could not be assigned are indicated by an asterisk .

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(c) MALDI MS analysis of the ε-cleavage sites of wt and mutant TREM2 C-terminal fragments. Mass spectrometry reveals ε-cleavage C-terminal of A192 for the wt CTF. MS determination of the ε-cleavage site of TREM2 CTF K186A reveals major and minor ε-cleavage sites C-terminal of A195 and L193, respectively. The exact same cleavage pattern is observed for the K186L mutant. MS analysis of the cleavage pattern of the K186P mutant reveals major and minor ε-cleavage sites C-terminal of L193 and S191. Minor peaks that could not be assigned are indicated by an asterisk.


( d , e ) Chemically induced unfolding of TREM2 - TMH wild - type ( d ) and the K186A variant ( e ) demonstrates that the less dynamic variant is more stable than the wild - type protein .

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(d, e) Chemically induced unfolding of TREM2-TMH wild-type (d) and the K186A variant (e) demonstrates that the less dynamic variant is more stable than the wild-type protein.


( f ) Water exchange peak as observed in a 3D - 15N - edited - [ 1H , 1H ] - NOESY experiment which is only absent if the amide moiety is present in a stable hydrogen bond .

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(f) Water exchange peak as observed in a 3D-15N-edited-[1H,1H]-NOESY experiment which is only absent if the amide moiety is present in a stable hydrogen bond.


Representative images of the size of Mex3a mutant mice and control littermates at postnatal day ( P ) 15 . Scale bar , 1 cm . Genotypes were confirmed by Mex3a mRNA ISH in intestinal tissue ( right panels ) . Scale bars , 50 μm . The offspring number ( n ) and observed genotype frequencies ( % ) resulting from heterozygous crosses are indicated below .

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Representative images of the size of Mex3a mutant mice and control littermates at postnatal day (P)15. Scale bar, 1 cm. Genotypes were confirmed by Mex3a mRNA ISH in intestinal tissue (right panels). Scale bars, 50 μm. The offspring number (n) and observed genotype frequencies (%) resulting from heterozygous crosses are indicated below.


Absolute weight of Mex3a KO mice and control littermates at different ages . Data is represented in a box and whiskers plot as mean ( middle line ) with the minimum and maximum distribution values . Each point depicts one animal ( WT : P1 , n = 15 ; P8 , n = 36 ; P15 , n = 43 ; P22 , n = 14 ; Adult , n = 14 ; Mex3a + / - : P1 , n = 24 ; P8 , n = 90 ; P15 , n = 90 ; P22 , n = 37 ; Adult , n = 21 ; Mex3a - / - : P1 , n = 9 ; P8 , n = 24 ; P15 , n = 26 ; P22 , n = 14 ; Adult , n = 8 ) * * * P = 0 . 0003 , * * * * P < 0 . 0001 , two - way ANOVA test .

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Absolute weight of Mex3a KO mice and control littermates at different ages. Data is represented in a box and whiskers plot as mean (middle line) with the minimum and maximum distribution values. Each point depicts one animal (WT: P1, n = 15; P8, n = 36; P15, n = 43; P22, n = 14; Adult, n = 14; Mex3a+/-: P1, n = 24; P8, n = 90; P15, n = 90; P22, n = 37; Adult, n = 21; Mex3a-/-: P1, n = 9; P8, n = 24; P15, n = 26; P22, n = 14; Adult, n = 8) ***P = 0.0003, ****P < 0.0001, two-way ANOVA test.


Kaplan - Meier survival curves for the different Mex3a genotypes ( n = 80 for each genotype ) * * * * P < 0 . 0001 , Log - rank ( Mantel - Cox ) test .

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Kaplan-Meier survival curves for the different Mex3a genotypes (n = 80 for each genotype) ****P < 0.0001, Log-rank (Mantel-Cox) test.


Macroscopic assessment of the gastrointestinal tract of Mex3a KO and WT mice . These images are representative of the phenotype observed in mutant animals euthanized at different postnatal days . Boxed areas depict the distal small intestinal section ( ileum ) used for subsequent immunohistochemical analyses .

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Macroscopic assessment of the gastrointestinal tract of Mex3a KO and WT mice. These images are representative of the phenotype observed in mutant animals euthanized at different postnatal days. Boxed areas depict the distal small intestinal section (ileum) used for subsequent immunohistochemical analyses.


H & E staining of a Mex3a KO and WT littermate at P19 . Inserts depict high magnification of the boxed areas .

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H&E staining of a Mex3a KO and WT littermate at P19. Inserts depict high magnification of the boxed areas.


Average crypt depth ( C ) of WT animals ( n = 3 , > 40 crypts counted per animal ) and Mex3a mutants ( n = 6 , > 20 crypts counted per animal ) . Also shown is the average villi height ( D ) of WT animals ( n = 3 , > 25 villi counted per animal ) and Mex3a mutants ( n = 6 , > 15 villi counted per animal ) . Data is represented as mean ± standard error between P16 and P18 . * P = 0 . 03 , * * * P = 0 . 0006 , Student ` s t test .

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Average crypt depth (C) of WT animals (n = 3, > 40 crypts counted per animal) and Mex3a mutants (n = 6, > 20 crypts counted per animal). Also shown is the average villi height (D) of WT animals (n = 3, > 25 villi counted per animal) and Mex3a mutants (n = 6, > 15 villi counted per animal). Data is represented as mean ± standard error between P16 and P18. *P = 0.03, ***P = 0.0006, Student`s t test.


Immunohistochemistry staining for ( E ) the tuft cell marker DCLK1 ( black arrowheads indicate positive cells ) , ( F ) the Paneth cell marker

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Immunohistochemistry staining for (E) the tuft cell marker DCLK1 (black arrowheads indicate positive cells), (F) the Paneth cell marker LYZ1


Immunohistochemistry staining for the proliferation marker KI67 .

Immunohistochemistry staining for the proliferation marker KI67.


mRNA ISH staining for the stem cell markers ( H ) Lgr5 , ( I ) Olfm4 , ( J ) Lrig1 , and ( K ) Hopx in ileal sections of Mex3a mutants and littermate controls at P19 . Inserts depict high magnification of the boxed areas .

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mRNA ISH staining for the stem cell markers (H) Lgr5, (I) Olfm4, (J) Lrig1, and (K) Hopx in ileal sections of Mex3a mutants and littermate controls at P19. Inserts depict high magnification of the boxed areas.


qPCR analysis of the expression level of the indicated stem cell markers in freshly isolated crypt fractions from Mex3a KO and WT animals ( n = 3 of each genotype ) . Data is represented as mean fold - change plus standard error in Mex3a KO mice relatively to WT animals ( dashed line ) . * P = 0 . 0208 , * * P < 0 . 01 , * * * P = 0 . 0007 , Student ` s t test .

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qPCR analysis of the expression level of the indicated stem cell markers in freshly isolated crypt fractions from Mex3a KO and WT animals (n = 3 of each genotype). Data is represented as mean fold-change plus standard error in Mex3a KO mice relatively to WT animals (dashed line). *P = 0.0208, **P < 0.01, ***P = 0.0007, Student`s t test.


Quantification of the size ( diameter length ) of Mex3a KO and WT organoid - initiating structures 2 days after crypt isolation ( n = 3 for each genotype , > 150 structures counted in total ) . Data is represented in a box and whiskers plot as mean ( middle line ) with the minimum and maximum distribution values . Each point depicts one organoid - initiating structure . * * * * P < 0 . 0001 , Student ` s t test . Phase contrast microscopy images of intestinal organoids generated by Mex3a - / - and WT crypts 6 days after crypt isolation . Scale bars , 100 μm .

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Quantification of the size (diameter length) of Mex3a KO and WT organoid-initiating structures 2 days after crypt isolation (n = 3 for each genotype, > 150 structures counted in total). Data is represented in a box and whiskers plot as mean (middle line) with the minimum and maximum distribution values. Each point depicts one organoid-initiating structure. ****P < 0.0001, Student`s t test. Phase contrast microscopy images of intestinal organoids generated by Mex3a-/- and WT crypts 6 days after crypt isolation. Scale bars, 100 μm.


Representative phase contrast microscopy images of Mex3a KO and WT intestinal organoids at days 2 and 4 of culture after 10 weekly passages . Scale bars , 100 μm .

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Representative phase contrast microscopy images of Mex3a KO and WT intestinal organoids at days 2 and 4 of culture after 10 weekly passages. Scale bars, 100 μm.


qPCR analysis of crypt lineage marker genes in organoid cultures at day 2 . Data is presented as the mean fold - change plus standard error ( n = 4 for each genotype ) for target gene expression in Mex3a KO intestinal organoids relative to WT ( dashed line ) . * P < 0 . 05 , Student ` s t test .

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qPCR analysis of crypt lineage marker genes in organoid cultures at day 2. Data is presented as the mean fold-change plus standard error (n = 4 for each genotype) for target gene expression in Mex3a KO intestinal organoids relative to WT (dashed line). *P < 0.05, Student`s t test.


Immunohistochemistry staining for the Paneth cell marker LYZ1 in Mex3a KO and WT intestinal organoid sections at day 2 of culture . Scale bars , 50 μm .

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Immunohistochemistry staining for the Paneth cell marker LYZ1 in Mex3a KO and WT intestinal organoid sections at day 2 of culture. Scale bars, 50 μm.


qPCR analysis of crypt lineage marker genes in organoid cultures at day 4 . Data is presented as the mean fold - change plus standard error ( n = 4 for each genotype ) for target gene expression in Mex3a KO intestinal organoids relative to WT ( dashed line ) .

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qPCR analysis of crypt lineage marker genes in organoid cultures at day 4. Data is presented as the mean fold-change plus standard error (n = 4 for each genotype) for target gene expression in Mex3a KO intestinal organoids relative to WT (dashed line).


mRNA ISH staining for the stem cell markers Mex3a , Lgr5 , Hopx and Lrig1 in Mex3a KO and WT intestinal organoid sections at day 2 of culture . Scale bars , 50 μm .

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mRNA ISH staining for the stem cell markers Mex3a, Lgr5, Hopx and Lrig1 in Mex3a KO and WT intestinal organoid sections at day 2 of culture. Scale bars, 50 μm.


Representative images of immunofluorescence staining for the proliferation marker KI67 and BrdU incorporation in Mex3a KO and WT animals . Mice were injected at P17 and sacrificed at P20 ( n = 3 for each genotype ) . Scale bars , 50 μm .

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Representative images of immunofluorescence staining for the proliferation marker KI67 and BrdU incorporation in Mex3a KO and WT animals. Mice were injected at P17 and sacrificed at P20 (n = 3 for each genotype). Scale bars, 50 μm.


Hierarchical clustering of samples ( n = 3 for each genotype ) and heatmap of differentially expressed genes between the Mex3a - / - and WT mice . The red colour represents over - expression , while the blue colour under - expression .

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Hierarchical clustering of samples (n = 3 for each genotype) and heatmap of differentially expressed genes between the Mex3a-/- and WT mice. The red colour represents over-expression, while the blue colour under-expression.


GSEA pre - ranked analysis comparing the Mex3a - / - transcriptional profile against ( E ) the

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GSEA pre-ranked analysis comparing the Mex3a-/- transcriptional profile against (E) the Mex3ahigh


GSEA pre - ranked analysis comparing the Mex3a - / - transcriptional profile against the Lgr5high and ( G ) the Wnt signalling pathway gene signatures .

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GSEA pre-ranked analysis comparing the Mex3a-/- transcriptional profile against the Lgr5high and (G) the Wnt signalling pathway gene signatures.


qPCR analysis of selected genes in a cohort of independent mice . Data is presented as the mean fold - change of target gene expression in 2 biological replicates ( Mex3a - / - Vs WT # 1 and Mex3a - / - Vs WT # 2 ) of Mex3a KO intestinal crypts relative to the respective WT controls ( dashed line ) .

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qPCR analysis of selected genes in a cohort of independent mice. Data is presented as the mean fold-change of target gene expression in 2 biological replicates (Mex3a-/- Vs WT #1 and Mex3a-/- Vs WT #2) of Mex3a KO intestinal crypts relative to the respective WT controls (dashed line).


Immunohistochemistry staining for PPARγ in Mex3a KO and WT mice ileum . Inserts depict high magnification of the boxed areas . Scale bars , 50 μm .

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Immunohistochemistry staining for PPARγ in Mex3a KO and WT mice ileum. Inserts depict high magnification of the boxed areas. Scale bars, 50 μm.


Pparg mRNA ISH in ileal sections of Mex3a mutants and littermate controls at P18 . Inserts depict high magnification of the boxed areas . Scale bars , 50 μm .

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Pparg mRNA ISH in ileal sections of Mex3a mutants and littermate controls at P18. Inserts depict high magnification of the boxed areas. Scale bars, 50 μm.


qPCR analysis of PPARγ transcriptional targets in organoid cultures at day 4 . Data is presented as the mean fold - change plus standard error ( n = 3 for each genotype ) for target gene expression in Mex3a KO intestinal organoids relative to WT ( dashed line ) .

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qPCR analysis of PPARγ transcriptional targets in organoid cultures at day 4. Data is presented as the mean fold-change plus standard error (n = 3 for each genotype) for target gene expression in Mex3a KO intestinal organoids relative to WT (dashed line).


Representative phase contrast microscopy images of Mex3a KO and WT intestinal organoids after 5 days of treatment with 50 μM Pioglitazone or vehicle ( DMSO ) . Scale bars , 100 μm .

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Representative phase contrast microscopy images of Mex3a KO and WT intestinal organoids after 5 days of treatment with 50 μM Pioglitazone or vehicle (DMSO). Scale bars, 100 μm.


qPCR analysis of Lgr5 , Pcna and Angptl4 expression in organoid cultures after 5 days of treatment with 50 μM Pioglitazone . Data is presented as the mean fold - change plus standard error ( n = 4 for each genotype ) for target gene expression in Pioglitazone - treated organoids relative to the DMSO vehicle ( dashed line ) . * P = 0 . 02 , Student ` s t test .

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qPCR analysis of Lgr5, Pcna and Angptl4 expression in organoid cultures after 5 days of treatment with 50 μM Pioglitazone. Data is presented as the mean fold-change plus standard error (n = 4 for each genotype) for target gene expression in Pioglitazone-treated organoids relative to the DMSO vehicle (dashed line). * P = 0.02, Student`s t test.


Representative phase contrast microscopy images of Mex3a KO and WT intestinal organoids at day 1 after passaging Pioglitazone - or vehicle ( DMSO ) - treated organoids . Scale bars , 100 μm .

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Representative phase contrast microscopy images of Mex3a KO and WT intestinal organoids at day 1 after passaging Pioglitazone- or vehicle (DMSO)-treated organoids. Scale bars, 100 μm.


C . Coomassie staining of SDS - PAGE - separated pulldown samples with / without cell extracts ( C . E ) using GST - tagged FET - NTDs as baits . Sepharose with bound GST was included as control . Background from recombinant protein baits ( shown by white arrowheads ) as well as smaller partial recombinant products is visualized in the left panel ( C . E - ) . Several high - molecular weight proteins are retained by the FET - NTDs ( C . E + ) . Black arrowheads indicate protein bands and gel parts analyzed by mass

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C. Coomassie staining of SDS-PAGE-separated pulldown samples with/without cell extracts (C.E) using GST-tagged FET-NTDs as baits. Sepharose with bound GST was included as control. Background from recombinant protein baits (shown by white arrowheads) as well as smaller partial recombinant products is visualized in the left panel (C.E -). Several high-molecular weight proteins are retained by the FET-NTDs (C.E +). Black arrowheads indicate protein bands and gel parts analyzed by mass spectrometry


D . Immunoblot analysis ( IB ) of pulldown samples with FET - NTD baits with / without cell extracts ( C . E ) . Antibodies against ARID1A , BRG1 and BAF60A were used for detection of SWI / SNF components . Input samples diluted 1 : 5 were included as a control .

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D. Immunoblot analysis (IB) of pulldown samples with FET-NTD baits with/without cell extracts (C.E). Antibodies against ARID1A, BRG1 and BAF60A were used for detection of SWI/SNF components. Input samples diluted 1:5 were included as a control.


E . In situ proximity ligation assays ( PLA ) using antibodies against BRG1 , ARID1A , DDIT3 and C - terminal parts of normal FUS show protein complexes containing FUS / BRG1 , FUS / ARID1A , FUS - DDIT3 / BRG1 and FUS - DDIT3 / ARID1A as red fluorescent spots in nuclei of MLS cell lines 2645 - 94 and 402 - 91 . C - terminal parts of FUS are not present in the FUS - DDIT3 fusion protein and normal DDIT3 is not expressed in these cell lines . Merged images also include DAPI nuclear counterstain in blue . Combinations of primary antibodies are used to detect interactions ( left panel ) . In control experiments ( right panel ) , one primary antibody is omitted to evaluate the background fluorescent signals . Scale bars = 10 μm .

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E. In situ proximity ligation assays (PLA) using antibodies against BRG1, ARID1A, DDIT3 and C-terminal parts of normal FUS show protein complexes containing FUS/BRG1, FUS/ARID1A, FUS-DDIT3/BRG1 and FUS-DDIT3/ARID1A as red fluorescent spots in nuclei of MLS cell lines 2645-94 and 402-91. C-terminal parts of FUS are not present in the FUS-DDIT3 fusion protein and normal DDIT3 is not expressed in these cell lines. Merged images also include DAPI nuclear counterstain in blue. Combinations of primary antibodies are used to detect interactions (left panel). In control experiments (right panel), one primary antibody is omitted to evaluate the background fluorescent signals. Scale bars = 10 μm.


B . Immunoblot analysis ( IB ) of GST - pulldown samples with deletion mutants of FUS ‑ NTD ‑ GST as baits with / without cell extract ( C . E ) . ARID1A and BRG1 were used as tracers of SWI / SNF binding . Coomassie staining of recombinant proteins are shown in the lower panel . Amino acid numbers are indicated

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

B. Immunoblot analysis (IB) of GST-pulldown samples with deletion mutants of FUS‑NTD‑GST as baits with/without cell extract (C.E). ARID1A and BRG1 were used as tracers of SWI/SNF binding. Coomassie staining of recombinant proteins are shown in the lower panel. Amino acid numbers are indicated


C . Immunoblot analysis ( IB ) of FUS - NTD ‑ GST pulldown samples of cell extracts ( C . E . ) after treatment with RNase or DNase , using antibodies against ARID1A and BRG1

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

C. Immunoblot analysis (IB) of FUS-NTD‑GST pulldown samples of cell extracts (C.E.) after treatment with RNase or DNase, using antibodies against ARID1A and BRG1


D . Immunoblot analysis ( IB ) of SWI / SNF components ( ARID1A , BRG1 , BAF170 , BAF155 , BAF60A , SS18 and BAF47 ) and full - length FET proteins ( EWSR1 and FUS ) remaining on sepharose - bound FUS - NTD ‑ GST pulldown baits after increasing stringency washes with 100 , 150 , 250 , 500 or 1000 mM NaCl . Note loss of BRG1 at NaCl concentration > 250mM

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

D. Immunoblot analysis (IB) of SWI/SNF components (ARID1A, BRG1, BAF170, BAF155, BAF60A, SS18 and BAF47) and full-length FET proteins (EWSR1 and FUS) remaining on sepharose-bound FUS-NTD‑GST pulldown baits after increasing stringency washes with 100, 150, 250, 500 or 1000 mM NaCl. Note loss of BRG1 at NaCl concentration > 250mM


A . Immunoblot analysis ( IB ) of proteins co - immunoprecipitated with nuclear extracted SWI / SNF from MLS cell line 402 ‑ 91 and EWS cell line TC - 71 . Antibodies against BRG1 , DDIT3 ( FUS - DDIT3 ) , the C - terminal parts of normal FUS and EWSR1 , and FLI1 ( EWSR1 - FLI1 ) were used for detection . In order to directly quantify the fraction of bound and non - bound protein , relative amounts of protein for each IP ‑ sample were loaded on the gel , with consideration taken for dilutions during the immunoprecipitation procedure . I : input of nuclear extract , B : bound proteins , NB : proteins not bound . One representative immunoblot is shown . Immunoblots from all replicates are shown in the source data . B . Graphs showing the percentage of bound to total ( bound + non ‑ bound ) signal intensities from immunoblots for FUS ‑ DDIT3 , EWSR1 and FUS in MLS 402 - 91 cell line and EWSR1 - FLI1 , EWSR1 and FUS in EWS TC - 71 cell line . Mean ± SEM is shown with individual replicates indicated by circles ( n = 3 ) . Student ' s t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Original data for all quantifications , including p - values , are shown as source data .

O O B-EXP_ASSAY O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_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

A. Immunoblot analysis (IB) of proteins co-immunoprecipitated with nuclear extracted SWI/SNF from MLS cell line 402‑91 and EWS cell line TC-71. Antibodies against BRG1, DDIT3 (FUS-DDIT3), the C-terminal parts of normal FUS and EWSR1, and FLI1 (EWSR1-FLI1) were used for detection. In order to directly quantify the fraction of bound and non-bound protein, relative amounts of protein for each IP‑sample were loaded on the gel, with consideration taken for dilutions during the immunoprecipitation procedure. I: input of nuclear extract, B: bound proteins, NB: proteins not bound. One representative immunoblot is shown. Immunoblots from all replicates are shown in the source data. B. Graphs showing the percentage of bound to total (bound + non‑bound) signal intensities from immunoblots for FUS‑DDIT3, EWSR1 and FUS in MLS 402-91 cell line and EWSR1-FLI1, EWSR1 and FUS in EWS TC-71 cell line. Mean ± SEM is shown with individual replicates indicated by circles (n=3). Student's t-test, *p<0.05, **p<0.01, ***p<0.001. Original data for all quantifications, including p-values, are shown as source data.


C . Immunoblot analysis of proteins co - immunoprecipitated with nuclear extracted SWI / SNF from the model cell line HT1080 with overexpressed FUS - DDIT3 - dsRED , EWSR1 - FLI1 - EGFP or dsRED are shown as representative immunoblots . Antibodies against BRG1 , DDIT3 ( FUS - DDIT3 - dsRED ) , the C ‑ terminal parts of normal FUS and EWSR1 , EZH2 and FLI1 ( EWSR1 - FLI1 - EGFP ) were used for detection . In order to directly quantify the fraction of bound and non - bound protein , relative amounts of protein for each IP ‑ sample were loaded on the gel , with consideration taken for dilutions during the immunoprecipitation procedure . I : input of nuclear extract , B : bound proteins , NB : proteins not bound . Immunoblots from all replicates are shown in the source data ( with either dsRED - or EGFP ‑ tagged proteins expressed ) .

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

C. Immunoblot analysis of proteins co-immunoprecipitated with nuclear extracted SWI/SNF from the model cell line HT1080 with overexpressed FUS-DDIT3-dsRED, EWSR1-FLI1-EGFP or dsRED are shown as representative immunoblots. Antibodies against BRG1, DDIT3 (FUS-DDIT3-dsRED), the C‑terminal parts of normal FUS and EWSR1, EZH2 and FLI1 (EWSR1-FLI1-EGFP) were used for detection. In order to directly quantify the fraction of bound and non-bound protein, relative amounts of protein for each IP‑sample were loaded on the gel, with consideration taken for dilutions during the immunoprecipitation procedure. I: input of nuclear extract, B: bound proteins, NB: proteins not bound. Immunoblots from all replicates are shown in the source data (with either dsRED- or EGFP‑tagged proteins expressed).


D . Graphs showing the percentage of bound to total ( bound + non ‑ bound ) signal intensities from immunoblots for the fusion protein ( FUS ‑ DDIT3 or EWSR1 - FLI1 ) , EWSR1 , FUS and EZH2 in the model cell line HT1080 with overexpressed FUS - DDIT3 - dsRED / - EGFP ( n = 4 , circles ) , EWSR1 - FLI1 - EGFP ( n = 3 , squares ) or dsRED / EGFP ( n = 4 , triangles ) . Mean ± SEM is shown with individual replicates indicated . Student ' s t - test , no significant changes , p > 0 . 05 . Original data for all quantifications , including p - values , are shown as source data .

O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Graphs showing the percentage of bound to total (bound + non‑bound) signal intensities from immunoblots for the fusion protein (FUS‑DDIT3 or EWSR1-FLI1), EWSR1, FUS and EZH2 in the model cell line HT1080 with overexpressed FUS-DDIT3-dsRED/-EGFP (n=4, circles), EWSR1-FLI1-EGFP (n=3, squares) or dsRED/EGFP (n=4, triangles). Mean ± SEM is shown with individual replicates indicated. Student's t-test, no significant changes, p>0.05. Original data for all quantifications, including p-values, are shown as source data.


A . Immunoblot analysis ( IB ) of histone modifications in stably transfected HT1080 cell lines expressing EGFP , FUS ‑ DDIT3 ‑ EGFP or EWSR1 ‑ FLI1 ‑ EGFP . Antibodies against H3K27Ac , H3K27me3 , H3K4me3 and histone loading control H4 for detection of histone modifications and antibodies against EZH2 and loading control GAPDH to evaluate catalytic PRC2 amount . Immunoblot analysis with GFP antibody is shown to verify expression of FET fusion oncoproteins and EGFP . One representative immunoblot is shown . Graphs showing amount of protein ( H3K27me3 , H3K4me3 , ratio H3K27me3 / H3K4me3 , H3K27Ac , and EZH2 ) quantified from immunoblots relative each corresponding loading control H4 or GAPDH , normalized to parental HT1080 . Mean ± SEM is shown with individual replicates indicated by circles from two experiments with stable transfection and by squares from two experiments with transient transfection ( 24h and 48h , n = 4 . Student ' s t - test , ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Original data for all quantifications , including p - values , are shown as source data .

O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-SUBCELLULAR O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_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

A. Immunoblot analysis (IB) of histone modifications in stably transfected HT1080 cell lines expressing EGFP, FUS‑DDIT3‑EGFP or EWSR1‑FLI1‑EGFP. Antibodies against H3K27Ac, H3K27me3, H3K4me3 and histone loading control H4 for detection of histone modifications and antibodies against EZH2 and loading control GAPDH to evaluate catalytic PRC2 amount. Immunoblot analysis with GFP antibody is shown to verify expression of FET fusion oncoproteins and EGFP. One representative immunoblot is shown. Graphs showing amount of protein (H3K27me3, H3K4me3, ratio H3K27me3/H3K4me3, H3K27Ac, and EZH2) quantified from immunoblots relative each corresponding loading control H4 or GAPDH, normalized to parental HT1080. Mean ± SEM is shown with individual replicates indicated by circles from two experiments with stable transfection and by squares from two experiments with transient transfection (24h and 48h , n=4. Student's t-test, ns= not significant, *p<0.05, **p<0.01, ***p<0.001. Original data for all quantifications, including p-values, are shown as source data.


( A , B ) t - SNE visualization of 20 , 009 human retinal cells colored by ( A ) annotation of 18 transcriptionally distinct clusters ( C0 - C17 ) and ( B ) their distribution in 3 donor retina samples .

O O O O O O O O O O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O

(A, B) t-SNE visualization of 20,009 human retinal cells colored by (A) annotation of 18 transcriptionally distinct clusters (C0-C17) and (B) their distribution in 3 donor retina samples.


C ) Feature expression heatmap showing expression patterns of major retinal class markers across 16 retinal cell clusters . The size of each circle depicts the percentage of cells expressing the marker within the cluster . Brown colour indicates ≥ 10 nTrans ( number of transcripts ) .

O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O B-TISSUE O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

C) Feature expression heatmap showing expression patterns of major retinal class markers across 16 retinal cell clusters. The size of each circle depicts the percentage of cells expressing the marker within the cluster. Brown colour indicates ≥10 nTrans (number of transcripts).


D ) t - SNE plots showing expression of a set of selected marker genes for major retinal classes .

O O O O O O O B-EXP_ASSAY O O O O O O O O O B-TISSUE O O

D) t-SNE plots showing expression of a set of selected marker genes for major retinal classes.


E ) Correlation matrix for the identified 18 clusters . The upper triangle depicts the z - value for correlation and the lower triangle depicts the correlation coefficient for gene expression in clusters .

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

E) Correlation matrix for the identified 18 clusters. The upper triangle depicts the z-value for correlation and the lower triangle depicts the correlation coefficient for gene expression in clusters.


F ) Heatmap of differentially expressed genes used to classify cell types for each cluster compared to all other clusters for the 18 retinal cell clusters . The rows correspond to top 10 genes most selectively upregulated in individual clusters ( p < 0 . 01 , Benjamini - Hochberg correction ) and the columns show individual cells ordered by cluster ( C0 - C17 ) .

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

F) Heatmap of differentially expressed genes used to classify cell types for each cluster compared to all other clusters for the 18 retinal cell clusters. The rows correspond to top 10 genes most selectively upregulated in individual clusters (p< 0.01, Benjamini-Hochberg correction) and the columns show individual cells ordered by cluster (C0-C17).


A ) Feature expression heatmap of a panel of known marker genes for rod photoreceptors across the identified 16 retinal cell clusters . Brown colour indicates ≥ 100 nTrans ( number of transcripts ) .

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

A) Feature expression heatmap of a panel of known marker genes for rod photoreceptors across the identified 16 retinal cell clusters. Brown colour indicates ≥100 nTrans (number of transcripts).


B ) Representation of the three donor retina samples in the six rod photoreceptor clusters .

O O O O O O O B-TISSUE O O O O B-CELL_TYPE I-CELL_TYPE O O

B) Representation of the three donor retina samples in the six rod photoreceptor clusters.


C ) Violin plot showing high or low expression levels of MALAT1 in rod photoreceptor clusters .

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

C) Violin plot showing high or low expression levels of MALAT1 in rod photoreceptor clusters.


D ) Distribution of rod photoreceptor populations with high MALAT1 expression ( MALAT1 - hi ) or low MALAT1 expression ( MALAT1 - lo ) in three donor retina samples .

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

D) Distribution of rod photoreceptor populations with high MALAT1 expression (MALAT1-hi) or low MALAT1 expression (MALAT1-lo) in three donor retina samples.


E ) Fluorescent in situ hybridization analysis of human peripheral retina showing heterogeneous levels of MALAT1 expression in the rod photoreceptors located in the outer nuclear layer ( ONL ) . INL : inner nuclear layer ; OPL : outer plexiform layer . Scale bar = 20µm .

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

E) Fluorescent in situ hybridization analysis of human peripheral retina showing heterogeneous levels of MALAT1 expression in the rod photoreceptors located in the outer nuclear layer (ONL). INL: inner nuclear layer; OPL: outer plexiform layer. Scale bar = 20µm.


A , B ) ( A ) t - SNE visualization of human retinal cells colored by annotation of 13 transcriptionally distinct clusters ( CCA0 - CCA12 ) and ( B ) their distribution in 3 donor retina samples .

O O O O O O O O O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O

A, B) (A) t-SNE visualization of human retinal cells colored by annotation of 13 transcriptionally distinct clusters (CCA0-CCA12) and (B) their distribution in 3 donor retina samples.


C ) Feature expression heatmap showing expression patterns of 7 rod photoreceptor markers across 12 retinal cell clusters . The size of each circle depicts the percentage of cells expressing the marker within the cluster . Brown colour indicates ≥ 50 nTrans ( number of transcripts ) .

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

C) Feature expression heatmap showing expression patterns of 7 rod photoreceptor markers across 12 retinal cell clusters. The size of each circle depicts the percentage of cells expressing the marker within the cluster. Brown colour indicates ≥50 nTrans (number of transcripts).


D ) t - SNE plots showing expression of MALAT1 .

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

D) t-SNE plots showing expression of MALAT1.


E ) Expression pattern of MALAT1 in the rod photoreceptor showing MALAT1 - hi ( CCA1 , CCA10 ) and MALAT1 - lo ( CCA0 ) subpopulations . x - axis depicts normalized transcript levels .

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

E) Expression pattern of MALAT1 in the rod photoreceptor showing MALAT1-hi (CCA1, CCA10) and MALAT1-lo (CCA0) subpopulations. x-axis depicts normalized transcript levels.


F ) t - SNE plots showing expression of major ribosomal genes .

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

F) t-SNE plots showing expression of major ribosomal genes.


G ) Heatmap of differentially expressed genes between the two MALAT1 - hi clusters CCA1 and CCA10 . The rows correspond to top 10 genes most selectively upregulated in individual clusters ( p < 0 . 01 , Benjamini - Hochberg correction ) and the columns show individual cells ordered in CCA1 and CCA10 .

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

G) Heatmap of differentially expressed genes between the two MALAT1-hi clusters CCA1 and CCA10. The rows correspond to top 10 genes most selectively upregulated in individual clusters (p< 0.01, Benjamini-Hochberg correction) and the columns show individual cells ordered in CCA1 and CCA10.


Fluorescent in situ hybridization analysis of the same donor human peripheral retina at different time points post - mortem ( 7 and 13 hours , Retina 7 ) , showing decreases in MALAT1 - hi rod subpopulations in the outer nuclear layer ( ONL ) at later time point . INL : inner nuclear layer ; OPL : outer plexiform layer . Scale bar = 20µm . White arrows indicated MALAT1 - hi rod photoreceptors .

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

Fluorescent in situ hybridization analysis of the same donor human peripheral retina at different time points post-mortem (7 and 13 hours, Retina 7), showing decreases in MALAT1-hi rod subpopulations in the outer nuclear layer (ONL) at later time point. INL: inner nuclear layer; OPL: outer plexiform layer. Scale bar = 20µm. White arrows indicated MALAT1-hi rod photoreceptors.


A ) Feature expression heatmap showing the expression of 11 known cone photoreceptor markers across 16 retinal cell clusters . Brown colour indicates ≥ 10 nTrans ( number of transcripts ) .

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

A) Feature expression heatmap showing the expression of 11 known cone photoreceptor markers across 16 retinal cell clusters. Brown colour indicates ≥10 nTrans (number of transcripts).


C ) Scatter plots showing expression of OPN1LW / OPN1MW or OPN1SW in individual cone photoreceptors for population C10 . The colour depicts expression level for OPN1LW / OPN1MW in individual cells .

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

C) Scatter plots showing expression of OPN1LW/OPN1MW or OPN1SW in individual cone photoreceptors for population C10. The colour depicts expression level for OPN1LW/OPN1MW in individual cells.


D ) Heatmap of top 20 differentially expressed genes between L / M cones and S cones . The colour depicts normalised gene expression ( z - score capped at 2 . 5 ) .

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

D) Heatmap of top 20 differentially expressed genes between L/M cones and S cones. The colour depicts normalised gene expression (z-score capped at 2.5).


E ) Expression pattern of glial markers in Muller glia ( C9 ) and retinal astrocytes ( C16 ) . x - axis depicts normalized transcript levels .

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

E) Expression pattern of glial markers in Muller glia (C9) and retinal astrocytes (C16). x-axis depicts normalized transcript levels.


C ) t - SNE analysis of the human Müller glia cell line MIO - M1 with the retinal cell types identified in this human neural retina atlas .

O O O O O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-CELL_TYPE I-CELL_TYPE O O O O B-ORGANISM B-TISSUE I-TISSUE O O

C) t-SNE analysis of the human Müller glia cell line MIO-M1 with the retinal cell types identified in this human neural retina atlas.


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 ( + / + ) .

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

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 .

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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 .

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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 .

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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.


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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 .

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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 .

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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 .

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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 .

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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.


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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 .

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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.


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( b ) Chromatin immunoprecipitation using α - FLAG antibody was performed to confirm the pull - down of Sα chromatin from the Sα - engineered ( FNLDD - Sα - LexA ) CH12F3 - 2A cells . The a , b , and c bars indicate the primers used for ChIP - qPCR as shown in Fig . 1a ( n = 3 ; mean + sd ; two tailed unpaired Student ' s t - test ) .

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(b) Chromatin immunoprecipitation using α-FLAG antibody was performed to confirm the pull-down of Sα chromatin from the Sα-engineered (FNLDD-Sα-LexA) CH12F3-2A cells. The a, b, and c bars indicate the primers used for ChIP-qPCR as shown in Fig. 1a (n=3; mean + sd; two tailed unpaired Student's t-test).


( e ) Bar graph showing the proteins with ≥ 2 - fold enrichment as calculated by the heavy to light SILAC ratio .

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(e) Bar graph showing the proteins with ≥2-fold enrichment as calculated by the heavy to light SILAC ratio.


( f ) Effect of the siRNA - mediated KD of histone chaperones and chromatin remodelers on IgM to IgA switching in CH12F3 - 2A cells . Data are presented as the % IgA switching relative to the IgA switching in cells transfected with control siRNA ( siCONT ) ( n = 2 ; mean + sd ) .

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(f) Effect of the siRNA-mediated KD of histone chaperones and chromatin remodelers on IgM to IgA switching in CH12F3-2A cells. Data are presented as the % IgA switching relative to the IgA switching in cells transfected with control siRNA (siCONT) (n=2; mean + sd).


( a ) Top : Scheme of the IgA - switching assay in CH12F3 - 2A cells . After electroporation of siRNAs , cells were cultured for 24 h , and then stimulated by CIT , cultured for another 24 h , and subjected to FACS analysis . Bottom : Confirmation of the siRNA - mediated KD of SAMHD1 in CH12F3 - 2A cells .

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(a) Top: Scheme of the IgA-switching assay in CH12F3-2A cells. After electroporation of siRNAs, cells were cultured for 24 h, and then stimulated by CIT, cultured for another 24 h, and subjected to FACS analysis. Bottom: Confirmation of the siRNA-mediated KD of SAMHD1 in CH12F3-2A cells.


( b ) FACS profile of the percentage of CH12F3 - 2A cells undergoing IgA switching after transfection of the indicated siRNAs into CH12F3 - 2A cells cultured with ( + ) or without ( - ) CIT stimulation for 24 h . The number in the FACS plot represents the percentage of cells expressing IgA on their surface .

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(b) FACS profile of the percentage of CH12F3-2A cells undergoing IgA switching after transfection of the indicated siRNAs into CH12F3-2A cells cultured with (+) or without (-) CIT stimulation for 24 h. The number in the FACS plot represents the percentage of cells expressing IgA on their surface.


( c ) Summary of the FACS data obtained from three independent experiments . Data are shown as the % IgA switching relative to the IgA switching in cells transfected with control siRNA ( siCONT ) ( n = 3 ; mean + sd ; two tailed unpaired Student ' s t - test ) .

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(c) Summary of the FACS data obtained from three independent experiments. Data are shown as the % IgA switching relative to the IgA switching in cells transfected with control siRNA (siCONT) (n=3; mean + sd; two tailed unpaired Student's t-test).


( d ) Quantitative RT - PCR analysis of the μGLT , αGLT , Aid , and Samhd1 expression in the indicated samples . The data were normalized to Gapdh , and represents as the mean of the two independent experiments with standard deviations . Statistical significance as determined by two - tailed unpaired Student ' s t - test is shown . ns = not significant .

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(d) Quantitative RT-PCR analysis of the μGLT, αGLT, Aid, and Samhd1 expression in the indicated samples. The data were normalized to Gapdh, and represents as the mean of the two independent experiments with standard deviations. Statistical significance as determined by two-tailed unpaired Student's t-test is shown. ns = not significant.


( e ) Confirmation of the CRISPR / Cas9 mediated KO of Samhd1 in CH12F3 - 2A cells . Beta - actin was used as a loading control .

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(e) Confirmation of the CRISPR/Cas9 mediated KO of Samhd1 in CH12F3-2A cells. Beta-actin was used as a loading control.


( f ) CSR time course analysis of WT and Samhd1 KO clones . The data are shown as percentage of IgA expressing cells ( n = 2 ; mean ± sd ; two tailed unpaired Student ' s t - test ; * * , p < 0 . 01 ) .

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(f) CSR time course analysis of WT and Samhd1 KO clones. The data are shown as percentage of IgA expressing cells (n=2; mean ± sd; two tailed unpaired Student's t-test; **, p<0.01).


( g ) Proliferation of WT and Samhd1 KO clones after stimulation with CIT ( n = 2 ; mean ± sd ; two tailed unpaired Student ' s t - test ; ns = not significant ) .

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(g) Proliferation of WT and Samhd1 KO clones after stimulation with CIT (n = 2; mean ± sd; two tailed unpaired Student's t-test; ns = not significant).


( h ) Analysis of the cell proliferation of WT or Samhd1 KO clones of CH12F3 - 2A . Histograms show the CFSE dye dilution derived from the indicated samples . Aphidicolin ( 2 μg / mL ) was also used as a positive control .

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(h) Analysis of the cell proliferation of WT or Samhd1 KO clones of CH12F3-2A. Histograms show the CFSE dye dilution derived from the indicated samples. Aphidicolin (2 μg/mL) was also used as a positive control.


( i ) Schematic representation of the IgG1 or IgG3 switching assay in spleen B cells . Bottom : Western blot analysis of the SAMHD1 expression in spleen B cells derived from WT or Samhd1 KO mice . Tubulin was used as a loading control .

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(i) Schematic representation of the IgG1 or IgG3 switching assay in spleen B cells. Bottom: Western blot analysis of the SAMHD1 expression in spleen B cells derived from WT or Samhd1 KO mice. Tubulin was used as a loading control.


( j ) Bar graphs show the compilation of IgG1 and IgG3 CSR assays from five independent experiments . The data are shown as percentage of IgG1 ( left ) or IgG3 ( right ) expressing cells ( n = 5 ; mean ± sd ; two - tailed unpaired Student ' s t - test ) .

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(j) Bar graphs show the compilation of IgG1 and IgG3 CSR assays from five independent experiments. The data are shown as percentage of IgG1 (left) or IgG3 (right) expressing cells (n=5; mean ± sd; two-tailed unpaired Student's t-test).


( b ) Southern blot analysis of PCR - amplified fragments with a Myc - specific probe from two independent experiments . CH12F3 - 2A cells were transfected with the indicated siRNAs . ( c ) Frequency of IgH / c - Myc chromosomal translocations derived from two independent experiments ( mean ± sd ; Fisher ' s exact test ) .

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(b) Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments. CH12F3-2A cells were transfected with the indicated siRNAs. (c) Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test).


Western blot analysis of the KD of SAMHD1 or TOP1 by the transfection of CH12F3 - 2A cells by SAMHD1 siRNA or by tetracycline ( 50 nM ) treatment , respectively . Expression of Tubulin is shown as a loading control ( e : left ) . Relative expression of TOP1 and SAMHD1 as determined by densitometric quantification of western blot images in Fig . 3e ( e : right ) .

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Western blot analysis of the KD of SAMHD1 or TOP1 by the transfection of CH12F3-2A cells by SAMHD1 siRNA or by tetracycline (50 nM) treatment, respectively. Expression of Tubulin is shown as a loading control (e: left). Relative expression of TOP1 and SAMHD1 as determined by densitometric quantification of western blot images in Fig. 3e (e: right).


Southern blot analysis of PCR - amplified fragments with a Myc - specific probe from two independent experiments ( f ) . Frequency of IgH / c - Myc chromosomal translocations derived from two independent experiments ( mean ± sd ; Fisher ' s exact test ) ( g ) .

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Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments (f). Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test) (g).


( h - j ) Analysis of the IgH / c - Myc translocation frequency in WT and Samhd1 KO CH12F3 - 2A cells . ( h ) Schematic illustration of the experimental design for the translocation assay . ( i ) Southern blot analysis of PCR - amplified fragments with a Myc - specific probe from two independent experiments . ( j ) Frequency of IgH / c - Myc chromosomal translocations derived from two independent experiments ( mean ± sd ; Fisher ' s exact test ) .

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(h-j) Analysis of the IgH/c-Myc translocation frequency in WT and Samhd1 KO CH12F3-2A cells. (h) Schematic illustration of the experimental design for the translocation assay. (i) Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments. (j) Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test).


( k - m ) Analysis of the IgH / c - Myc translocation frequency in spleen B cell derived from WT ( Samhd1 + / + ) or Samhd1 KO ( Samhd1 - / - ) mice . ( k ) Schematic illustration of the experimental design for the translocation assay . ( l ) Southern blot analysis of PCR - amplified fragments with a Myc - specific probe from two independent experiments . ( m ) Frequency of IgH / c - Myc chromosomal translocations derived from two independent experiments ( mean ± sd ; Fisher ' s exact test ) .

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(k-m) Analysis of the IgH/c-Myc translocation frequency in spleen B cell derived from WT (Samhd1+/+) or Samhd1 KO (Samhd1-/-) mice. (k) Schematic illustration of the experimental design for the translocation assay. (l) Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments. (m) Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test).


( b ) Mutation frequency in the 5 ′ Sµ region of WT or Samhd1 KO CH12F3 - 2A cells . Statistical significance as evaluated by Fisher ' s exact test is shown . ns = not significant .

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(b) Mutation frequency in the 5′ Sµ region of WT or Samhd1 KO CH12F3-2A cells. Statistical significance as evaluated by Fisher's exact test is shown. ns = not significant.


( d ) LM - PCR analysis of the AID - induced DNA double - strand breaks in the Sµ region of WT or Samhd1 KO CH12F3 - 2A cells . Wedges indicate a three - fold increase in the DNA amount . Amplification of Gapdh was used as an internal loading control .

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(d) LM-PCR analysis of the AID-induced DNA double-strand breaks in the Sµ region of WT or Samhd1 KO CH12F3-2A cells. Wedges indicate a three-fold increase in the DNA amount. Amplification of Gapdh was used as an internal loading control.


( e ) Gamma - H2AX - ChIP signal in the Sµ region is shown as the fraction of immunoprecipitated DNA ( % IP ) normalized to the total amount of DNA used for the immunoprecipitation ( n = 3 ; mean + sd ; two - tailed unpaired Student ' s t - test ; ns = not significant ) .

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(e) Gamma-H2AX-ChIP signal in the Sµ region is shown as the fraction of immunoprecipitated DNA (% IP) normalized to the total amount of DNA used for the immunoprecipitation (n=3; mean + sd; two-tailed unpaired Student's t-test; ns = not significant).


( f ) Left : Scheme of long - range interactions between Sμ - Sα elements in the IgH locus before and after AID activation . Right : Representative gel picture of the 3C assay detecting the Sμ - Sα interaction in WT or Samhd1 KO CH12F3 - 2A cells with or without CIT stimulation for 24 h . Gapdh was amplified as a loading control .

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(f) Left: Scheme of long-range interactions between Sμ-Sα elements in the IgH locus before and after AID activation. Right: Representative gel picture of the 3C assay detecting the Sμ-Sα interaction in WT or Samhd1 KO CH12F3-2A cells with or without CIT stimulation for 24 h. Gapdh was amplified as a loading control.


( a - d ) Analysis of Sμ - Sα recombination junctions from WT or CH12F3 - 2A cells after transfection with control ( siCONT ) or Ligase 4 ( siLIG4 ) siRNA . The genomic DNA isolated from WT or Samhd1 KO cells from three independent experiments were pooled prior to junction analysis . A total of 62 ( siCONT ) and 73 ( siLIG4 ) junctions from WT , and 60 ( siCONT ) and 60 ( siLIG4 ) junctions from Samhd1 KO cells were sequenced and analyzed . Statistical significance as evaluated by Fisher ' s exact test ( b ) or two - tailed unpaired Student ' s t - test ( d ) or is shown . Schematic representation of the Sμ - Sα junction analysis ( a ) . Summary of the characteristics of Sμ - Sα recombination junctions ( b ) . Sequences at Sμ - Sα recombination junctions with insertions ( c ) . Summary of the average length of insertions at Sμ - Sα recombination junctions ( d ) .

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

(a-d) Analysis of Sμ-Sα recombination junctions from WT or CH12F3-2A cells after transfection with control (siCONT) or Ligase 4 (siLIG4) siRNA. The genomic DNA isolated from WT or Samhd1 KO cells from three independent experiments were pooled prior to junction analysis. A total of 62 (siCONT) and 73 (siLIG4) junctions from WT, and 60 (siCONT) and 60 (siLIG4) junctions from Samhd1 KO cells were sequenced and analyzed. Statistical significance as evaluated by Fisher's exact test (b) or two-tailed unpaired Student's t-test (d) or is shown. Schematic representation of the Sμ-Sα junction analysis (a). Summary of the characteristics of Sμ-Sα recombination junctions (b). Sequences at Sμ-Sα recombination junctions with insertions (c). Summary of the average length of insertions at Sμ-Sα recombination junctions (d).


( e - i ) Analysis of the CRISPR / Cas9 induced chromosomal translocations between the Rosa26 ( Chr6 ) and H3f3b ( Chr 11 ) genes . The genomic DNA isolated from WT or Samhd1 KO cells from three independent experiments were pooled prior to chromosomal translocation or junction analysis . Schematic representation of the chromosomal translocations ( e ) . Representative images of agarose gels after PCR - amplification of the Rosa26 / H3f3b translocation from WT or Samhd1 KO CH12F3 - 2A cells ( f ) . Relative frequency of Rosa26 / H3b3f chromosomal translocations ( g ) . Frequency of junctions containing insertions ( h ) and average length of insertions ( i ) . Statistical significance as evaluated by two - tailed unpaired Student ' s t - test ( g , i ) or Fisher ' s exact test ( h ) is shown .

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(e-i) Analysis of the CRISPR/Cas9 induced chromosomal translocations between the Rosa26 (Chr6) and H3f3b (Chr 11) genes. The genomic DNA isolated from WT or Samhd1 KO cells from three independent experiments were pooled prior to chromosomal translocation or junction analysis. Schematic representation of the chromosomal translocations (e). Representative images of agarose gels after PCR-amplification of the Rosa26/H3f3b translocation from WT or Samhd1 KO CH12F3-2A cells (f). Relative frequency of Rosa26/H3b3f chromosomal translocations (g). Frequency of junctions containing insertions (h) and average length of insertions (i). Statistical significance as evaluated by two-tailed unpaired Student's t-test (g, i) or Fisher's exact test (h) is shown.


( b ) Confirmation of the expression of GFP - tagged human SAMHD1 ( WT , and DN , DA , TA , and RE mutants ) . Histone H3 expression is shown as a loading control .

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(b) Confirmation of the expression of GFP-tagged human SAMHD1 (WT, and DN, DA, TA, and RE mutants). Histone H3 expression is shown as a loading control.


( c ) CSR complementation assay in Samhd1 KO cells . Data are shown as the % IgA switching relative to the IgA switching in WT cells transfected with empty vector , and are derived from three independent experiments ( mean ± sd ; two - tailed unpaired Student ' s t - test ) .

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(c) CSR complementation assay in Samhd1 KO cells. Data are shown as the % IgA switching relative to the IgA switching in WT cells transfected with empty vector, and are derived from three independent experiments (mean ± sd; two-tailed unpaired Student's t-test).


( e ) Effect of deoxyribonucleoside supplementation individually or in combination on the rate of IgM to IgA switching in CH12F3 - 2A cells . Data are presented as % IgA switching relative to the IgA switching in cells treated with an equivalent volume of the solvent in which the deoxyribonucleosides were dissolved ( n = 3 ; mean ± sd ; two - tailed unpaired Student ' s t - test ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 ) .

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(e) Effect of deoxyribonucleoside supplementation individually or in combination on the rate of IgM to IgA switching in CH12F3-2A cells. Data are presented as % IgA switching relative to the IgA switching in cells treated with an equivalent volume of the solvent in which the deoxyribonucleosides were dissolved (n=3; mean ± sd; two-tailed unpaired Student's t-test; ** = p ≤ 0.01; *** = p ≤ 0.001).


( f ) Effect of deoxyribonucleoside supplementation individually or in combination on the frequency of IgH / c - Myc translocations in CH12F3 - 2A cells ( n = 2 ; mean ± sd ; Fisher ' s exact test ; ns = not significant ; * * * = p ≤ 0 . 001 ) . The concentration dNTPs used for supplementation either individually or in combination were as follows : 2 ′ - deoxyguanosine ( dG ) , 0 . 15 mM ; 2 ′ - deoxyadenosine ( dA ) , 1 . 0 mM ; 2 ′ - deoxycytidine ( dC ) , 12 mM ; and 2 ′ - deoxythymidine ( dT ) , 1 . 6 mM .

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(f) Effect of deoxyribonucleoside supplementation individually or in combination on the frequency of IgH/c-Myc translocations in CH12F3-2A cells (n = 2; mean ± sd; Fisher's exact test; ns = not significant; *** = p ≤ 0.001). The concentration dNTPs used for supplementation either individually or in combination were as follows: 2′-deoxyguanosine (dG), 0.15 mM; 2′-deoxyadenosine (dA), 1.0 mM; 2′-deoxycytidine (dC), 12 mM; and 2′-deoxythymidine (dT), 1.6 mM.


( b ) FACS profile of the percentage of WT and Samhd1 KO CH12F3 - 2A cells undergoing IgA switching following transfection with the indicated plasmids . The number in the FACS plot represents the percentage of cells expressing IgA on their surface . ( c ) Summary of the FACS data obtained from two independent experiments . The data are shown as percentage of IgA expressing cells upon CSR induced by blunt ( left ) or staggered ( right ) DSBs ( n = 2 ; mean + sd ; two - tailed unpaired Student ' s t - test ) ; ns = not significant .

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(b) FACS profile of the percentage of WT and Samhd1 KO CH12F3-2A cells undergoing IgA switching following transfection with the indicated plasmids. The number in the FACS plot represents the percentage of cells expressing IgA on their surface. (c) Summary of the FACS data obtained from two independent experiments. The data are shown as percentage of IgA expressing cells upon CSR induced by blunt (left) or staggered (right) DSBs (n = 2; mean + sd; two-tailed unpaired Student's t-test); ns = not significant.


( d ) Length of resected DNA at Sμ - Sα junctions obtained from the joining of DSBs with blunt ends . Statistical significance as evaluated by two - tailed unpaired Student ' s t - test is shown . ( e ) Summary of the characteristics of Sμ - Sα recombination junctions resulting from the joining of DSBs with blunt ends .

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

(d) Length of resected DNA at Sμ-Sα junctions obtained from the joining of DSBs with blunt ends. Statistical significance as evaluated by two-tailed unpaired Student's t-test is shown. (e) Summary of the characteristics of Sμ-Sα recombination junctions resulting from the joining of DSBs with blunt ends.


( f ) Length of resected DNA at Sμ - Sα junctions obtained from the joining of staggered DSBs . Left : Length of Sμ and Sα overhangs were 35 nt and 98 nt , respectively . Right : Length of Sμ and Sα overhangs were 35 nt and 187 nt , respectively . ( g ) Summary of the characteristics of Sμ - Sα recombination junctions resulting from the joining of staggered DSBs . Statistical significance as evaluated by Fisher ' s exact test is shown .

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

(f) Length of resected DNA at Sμ-Sα junctions obtained from the joining of staggered DSBs. Left: Length of Sμ and Sα overhangs were 35 nt and 98 nt, respectively. Right: Length of Sμ and Sα overhangs were 35 nt and 187 nt, respectively. (g) Summary of the characteristics of Sμ-Sα recombination junctions resulting from the joining of staggered DSBs. Statistical significance as evaluated by Fisher's exact test is shown.


( h ) Summary of the average length of insertions at Sμ - Sα recombination junctions . Statistical significance as evaluated by two - tailed unpaired Student ' s t - test is shown .

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

(h) Summary of the average length of insertions at Sμ-Sα recombination junctions. Statistical significance as evaluated by two-tailed unpaired Student's t-test is shown.


Analysis of the effect of TdT expression on the frequency of CSR as well as characteristics of Sμ - Sα recombination junctions resulting from the joining of DSBs with blunt ends . ( j ) Top : Schematic representation of the experimental design to analyze the effect of TdT expression on CSR in WT and Samhd1 KO cells . Bottom : Western blot analysis of the expression of TdT in WT and Samhd1 KO cells . Tubulin was used as a loading control .

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

Analysis of the effect of TdT expression on the frequency of CSR as well as characteristics of Sμ-Sα recombination junctions resulting from the joining of DSBs with blunt ends. (j) Top: Schematic representation of the experimental design to analyze the effect of TdT expression on CSR in WT and Samhd1 KO cells. Bottom: Western blot analysis of the expression of TdT in WT and Samhd1 KO cells. Tubulin was used as a loading control.


( k ) FACS profile of the percentage IgA switching in WT or Samhd1 KO CH12F3 - 2A cells with or without TdT expression . ( l ) Summary of the FACS data obtained from two independent experiments ( n = 2 ; mean + sd ; two - tailed unpaired Student ' s t - test ; * = p ≤ 0 . 05 ) .

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

(k) FACS profile of the percentage IgA switching in WT or Samhd1 KO CH12F3-2A cells with or without TdT expression. (l) Summary of the FACS data obtained from two independent experiments (n = 2; mean + sd; two-tailed unpaired Student's t-test; * = p ≤ 0.05).


( m ) Summary of the characteristics of Sμ - Sα recombination junctions . Statistical significance as evaluated by Fisher ' s exact test is shown . 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

(m) Summary of the characteristics of Sμ-Sα recombination junctions. Statistical significance as evaluated by Fisher's exact test is shown. ns = not significant.