EMBO/SourceData huggingface dataset:
Figure 2(B) U2OS WT or PIF1-KO cells were labeled with CldU for 30 min followed by incubation with 2 mM HU for 2 hr and then IdU for another 30 min. Labeled cells were processed for DNA fiber analysis. Representative images of stalled or restarted forks and forks with new origin firing were shown (left). The percentage of restarted forks was quantified by analyzing of 110-130 fibers for each experiment (right). Experiments were repeated four times for each sample. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01, ***P < 0.001.(D) U2OS (EGFP-BIR-5085) cell lines carrying Dox-inducible Cas9/sgRNA-5085 (Dox-Cas9) or Cas9n/sgRNA-5085 (Dox-Cas9n) were incubated with or without Dox (5 µg/ml) and assayed by FACS analysis 2 days later (left). U2OS (EGFP-BIR-5085, Dox-Cas9 or Dox-Cas9n) cells expressing shRNAs RAD51, POLD3 and PIF1 or shRNA vector (Ctrl) were incubated with 5 µg/ml Dox, and FACS analysis was performed after 2 days (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01, ***P < 0.001.
Figure 3(E) U2OS (EGFP-Flex1-BIR-5086) cells expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA vector (Ctrl) were infected by retroviruses expressing RAS, and the percentage of EGFP positive cells was quantified by FACS analysis 4 days after retrovirus infection (top). Expression level of RAD51 and POLD3 is shown by Western blot analysis (bottom). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01.(F) U2OS WT or PIF1-KO cells were infected by retrovirus expressing RAS or empty vector. Cell profiling was plotted by counting cell numbers every 24 hr, and normalized to the cell number on the first day. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01.
Figure 4(C) U2OS (EGFP-STGC-1731) cell lines carrying Dox-inducible Cas9/sgRNA-1731 (Dox-Cas9) or Cas9n/sgRNA (Dox-Cas9n) were incubated with or without Dox (5 µg/ml), and the percentage of EGFP positive cells was quantified by FACS analysis 2 days later (top). U2OS (EGFP-STGC-1731, Dox-Cas9 or Dox-Cas9n) cells expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA control (Ctrl) were incubated with Dox (5 µg/ml), and the percentage of EGFP positive cells was quantified by FACS analysis after 2 days (bottom). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.(E) U2OS (EGFP-Flex1-STGC-1541) cells were treated with or without 2 mM HU for 24 hr, and the percentage of EGFP positive cells by HU induction was quantified by FACS analysis 4 days after removal of HU (left). U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA vector (Ctrl) were treated with 2 mM HU for 24 hr, and the percentage of EGFP positive cells was quantified by FACS analysis 4 days later (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.(F) U2OS (EGFP-Flex1-STGC-1541) cells were infected by retroviruses expressing RAS or empty vector, and the percentage of EGFP positive cells was assayed by FACS 5 days following infection (left). U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RAD51, POLD3 and PIF1 shRNAs or shRNA vector were infected by retroviruses expressing RAS, and the percentage of EGFP positive cells was assayed by FACS 5 days after infection (middle). Expression level of RAD51 and POLD3 is shown by Western blot analysis (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.
Figure 5(D) U2OS (EGFP/STGC-mCherry/LTGC-5034, Dox-Cas9 (left) or Dox-Cas9n (right)) cells expressing shRNAs for POLD3 and PIF1 or shRNA vector (Ctrl) were incubated with Dox (5 µg/ml). The percentage of EGFP or mCherry positive cells after induction was quantified by FACS analysis 2 days later to determine the percentage of EGFP or mCherry positive cells. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.(E) U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RFC1 and PCNA or vector (Ctrl) were treated with 2 mM HU for 24 hr. The percentage of EGFP positive cells after HU treatment was quantified by FACS analysis 3 days after HU removal (left). U2OS (EGFP-STGC-1731) cells expressing shRNAs for RFC1 and PCNA or vector (Ctrl) were infected by lentivirus expressing I-SceI. The percentage of EGFP positive cells by I-SceI induction was quantified by FACS analysis 4 days later (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.(F) U2OS (EGFP/STGC-mCherry/LTGC-5034, Dox-Cas9 (left) or Dox-Cas9n (right)) cells expressing shRNAs for RFC1 and PCNA or vector (Ctrl) were incubated with Dox (5 µg/ml). The percentage of EGFP or mCherry positive cells after induction was quantified by FACS analysis 2 days later. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.(G) FLAG-PIF1 was stably expressed in U2OS (EGFP-Flex1-STGC-1541) cells by lentiviral infection. Enrichment of FLAG-PIF1 at Flex1 site or GAPDH site with or without HU (2 mM, 10 hr) treatment was calculated by anti-FLAG ChIP (left). When PCNA was depleted by shRNA using vector as a control (Ctrl), enrichment of FLAG-PIF1 at Flex1 site was calculated by anti-FLAG ChIP (right). ChIP value in cells without HU treatment is set up as 1 for normalization. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.
Figure 6(D) Growth curves of WT or FANCM-KO HCT116 cells were plotted after expressing POLD3 (left) or PIF1 (right) shRNA or shRNA vector (Ctrl). Cell number was normalized to that on day one. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01.
Figure 7(C) Anti-γH2AX ChIP analysis at FRA3B, FRA16D and GAPDH genomic loci was performed in U2OS WT and PIF1-KO cells before and after APH treatment (0.4 μM, 24 hr). Enrichment of γH2AX at FRA3B, FRA16D and GAPDH loci in WT and PIF1 KO cells was calculated using ChIP value in WT cells as 1 for normalization. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05.(E) U2OS (EGFP-BIR-5085) cells expressing PIF1 WT or L319P mutant with endogenous PIF1 silenced by shRNA were infected by lentiviruses to express I-SceI. The percentage of EGFP positive cells was quantified by FACS analysis 5 days after I-SceI infection. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05.(I) Anti-γH2AX ChIP analysis at FRA3B, FRA16D and GAPDH genomic loci was performed in U2OS WT and PIF1-KO cells, or PIF1-KO cells expressing PIF1-WT, and E307Q and L319P mutants before and after APH treatment (0.4 μM, 24 hr). Enrichment of γH2AX at FRA3B, FRA16D and GAPDH loci was calculated using ChIP value in WT cells as 1 for normalization. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05.
Figure 1Vector mediated eGFP expression after intravitreal injection of novel capsid variants compared to controls in 2-month-old mice. (A) In vivo cSLO examinations of the mouse retina at 1- and 2-weeks post injection (WPI) of 2E9 total vg (in 1 µl) of sc-CMV-eGFP packaged with parental serotype AAV2 , state-of-art AAV2.7m8, novel AAV2.GL and AAV2.NN capsid variants.(B) Confocal scans of retinal cross sections immunolabelled for eGFP and DAPI. Acquisition settings were kept constant for all samples. Scale bar: 100 μm. ONL, outer nuclear layer; INL, inner nuclear layer.(C) Confocal tile-scans of retinal cross sections immunolabelled for eGFP and Hoechst. Scale bar: 500 μm(D-E) Immunolabelled cross sections of mouse retina infected with AAV2.GL and AAV2.NN respectively, with focus on the ONL and INL, stained for eGFP, recoverin and DAPI. Scale bar: 50 μm ONL, outer nuclear layer; INL, inner nuclear layer.(F) qRT-PCR data comparing fold change in eGFP transcript levels from retinal samples infected with capsid variants AAV2.7m8, AAV2.GL and AAV2.NN compared to AAV2.
Figure 2Vector mediated eGFP expression after intravitreal injection of novel capsid variants compared to controls in 2-month-old mice. (A) In vivo cSLO examinations of the mouse retina at 1-, 2- and 3- weeks post injection (WPI) of 8E9 total vg of ss-hRho-eGFP packaged with parental serotype AAV2, state-of-art AAV2.7m8, novel AAV2.GL and AAV2.NN capsid variants. (B) qRT-PCR data comparing fold change in eGFP transcript levels relative to ITR2 amplicons from retinal samples infected with all four capsid variants.(C) Confocal scans of retinal cross sections, with focus on the ONL, immunolabelled for DAPI, eGFP and recoverin. The upper panel shows only the DAPI and eGFP signal in the ONL and the lower panel shows the same images (noted with asterisk *) with merged DAPI, eGFP and recoverin signal. Acquisition settings were kept constant for all samples. Scale bar: 50 μm. ONL, outer nuclear layer.
Figure 3Vector mediated eGFP expression after intravitreal injection in 10-month-old dog eyes. (A) In vivo cSLO examinations of the dog retina at four, five and six weeks post intravitreal injection of 2E11 total vg (in 200 µl) of sc-CMV-eGFP packaged with wildtype AAV2 (right eye), AAV2.GL (right eye) and AAV2.NN (left eye) capsid variants.(B-C) Confocal scans of immunolabelled retinal cross sections (14 μm) stained for GFP and DAPI at 6 WPI time point. : DAPI was used a nuclear marker. GFP=eGFP; RPE, retinal pigmented epithelium; OS, outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.(D-E) Confocal scans of immunolabelled retinal cross sections (14 μm) at 6 WPI time point, focusing on the photoreceptors, using antibodies against eGFP, Rhodopsin (Rho) and M-opsin (MO). OS, outer segments ONL, outer nuclear layer
Figure 4Vector mediated expression after intravitreal injection in ~3-year old cynomolgus macaque eyes. (A-B) In vivo cSLO examination images collaged to provide NHP fundus overview at 8 WPI of E12 total vg (in 38.9-100 µl) of sc-CMV-eGFP packaged with AAV2.GL and AAV2.NN capsid variants, respectively. (C-D) Confocal scans of immunolabelled retinal cross sections (14 μm) of the fovea centralis from AAV2.GL and AAV2.NN treated animals, respectively.(E) Percentage quantification of eGFP+ photoreceptors in the ONL of foveal cross sections. AAV2.GL technical repeats n=4, AAV2.NN technical repeats n=3.(F-G) Confocal scans of immunolabelled retinal cross sections (14 μm) of the periphery from AAV2.GL (F) and AAV2.NN (G) treated retinae. (Η-Ι) Higher magnification confocal scans of immunolabelled peripheral retina. ONL, outer nuclear layer; INL, inner nuclear layer
Figure 5Vector mediated eGFP expression in human retinal explants. (B) EVOS epifluorescence images at 2x magnification of the human retinal explants at three, six and nine days in vitro (DIV) after transduction with 1E11 total vg of sc-CMV-eGFP packaged with AAV2.GL and AAV2.NN capsid variants. White signal indicates eGFP.(C) Confocal scans of day 10 tissue sections immunolabelled for eGFP and Hoechst. Scale bar: 50 μm.
Figure 6(A) Representative photopic ERG flicker series at 1, 5 and 10 Hz with 3 cd.s/m2 illumination at 10 WPI. Arrowheads indicate light stimulus.(B) Representative photopic single flash ERG signal with 1, 3 and 10 cd.s/m2 illumination. The vertical dotted line indicates light stimulus. (C) Quantification of photopic B-wave amplitude from single flash responses to 1, 3 and 10 cd.s/m2 illumination of wildtype C57BL6/J x 129/sv eyes (WT, n = 3) compared to treated eyes (IVT/GL, n ≥ 3) and untreated eyes (UN, n ≥ 3) at 10 weeks post injection. (E-F) Confocal images of sections proximal to the injection site in Q2 (E) and distal to the injection site in Q4 (F), showing Cnga3 expression in PNA-positive cone outer segments. OS, outer segment layer; ONL, outer nuclear layer; INL, inner nuclear layer.(G-I) Confocal images of Cnga3-/- treated (G), wildtype (H) and Cnga3-/- untreated (I) retinal sections stained for Cnga3. Note some non-specific signal detected in INL and adjacent synaptic layer. Scale bar: 50 μm. OS, outer segment layer; ONL, outer nuclear layer; INL, inner nuclear layer.
Figure 1The refined models of four crystal structures of the ribosome bound to aminoacyl- or peptidyl-tRNA analogs are displayed in their respective unbiased electron density Fo-Fc maps (contoured at 2.5σ). The maps were calculated using phases produced by rigid body refinement of the ligand-free test structure put into the Fo data set.A, B. A-site bound substrates ACC-Puromycin (A) and ACCA-Pro (B).The refined models of four crystal structures of the ribosome bound to aminoacyl- or peptidyl-tRNA analogs are displayed in their respective unbiased electron density Fo-Fc maps (contoured at 2.5σ). The maps were calculated using phases produced by rigid body refinement of the ligand-free test structure put into the Fo data set.C, D. P-site bound substrates ACCA-Leu-Phe (C) and ACCA-Pro-Pro (D).
Figure 2This figure compares conformation of aminoacyl residues of the two A-site substrates:A. ACC-PuromycinB. ACCA-Pro.To show position of the A-site substrates relative to the P-site, the model P-site substrate tRNA-Phe is shown as in the pre-attack complex of the ribosome (pdb id 1vy4). In both illustrations, the A site is viewed as through the peptide exit tunnel. In addition to S. cerevisiae residues numbering, E. coli numbering is shown in parenthesis. Comparison of the two structures shows that the side chain of the methyl-tyrosine is bound to the conserved hydrophobic A-site cleft, whereas the side chain of the proline residue penetrates the actual site of peptide bond formation where it may interfere with alignment of the reacting groups and proper positioning of the attacking electron pair of the α-amine.
Figure 3This figure compares conformation of two dipeptides in the ribosome nascent peptide tunnel:A. Phe-Leu peptideB. Pro-Pro peptide.
Figure 1Mapping of open chromatin as well as active and repressive histone modifications at upstream regulatory regions of the Prdm1 locus in pro-B cells and plasmablasts. Open chromatin was determined by ATAC-seq (Buenrostro et al., 2013), and active (H3K4me2, H3K4me3, H3K9ac) and repressive (H3K27me3) histone marks were mapped by ChIP-seq analysis in ex vivo sorted pro-B cells (ATAC-seq and H3K27me3, this study), short-term in vitro cultured pro-B cells (H3K4me2, H3K4me3, H3K9ac) (Revilla-i-Domingo et al., 2012) and in vitro LPS-induced plasmablasts (Minnich et al., 2016) (Table EV4). The indicated upstream regions were previously shown by 3C-analysis to interact with the Prdm1 promoter (Wöhner et al., 2016). The mm9 genomic coordinates of mouse chromosome 10 and the respective positions of two human SNPs (rs658431 and rs548234) are indicated. RPM, reads per million mapped sequence readsMapping of nascent transcripts or mature mRNA at the Prdm1 and Atg5 loci by GRO-seq or RNA-seq analyses of ex vivo sorted pro-B and FO B cells, respectively (Table EV4). Strand-specific GRO-seq reads are indicated in blue and red, respectively Quantification of nascent transcript and mRNA levels. The nascent transcription or mRNA expression of the Atg5 and Prdm1 genes is shown as mean expression value (TPM) with SEM based on two different GRO-seq or RNA-seq experiments for each B cell type, respectively. TPM, transcripts per million (Wagner et al., 2012)
Figure 2Expression of Prdm1 and Atg5 mRNA in ex vivo sorted pro-B and pre-B cells from the bone marrow of Prdm1ihCd2/+ or wild-type (WT) mice. mRNA expression is shown as mean expression value (TPM) with SEM based on two independent RNA-seq experiments for each cell type and genotypeRT-qPCR analysis of nascent Prdm1 and Atg5 transcripts in ex vivo sorted pre-B cells and in in vitro differentiated plasmablasts (PB) of the indicated genotypes. Plasmablasts were generated by stimulation of splenic FO B cells for 4 days with LPS. The data were normalized to those of the ubiquitously expressed control gene Tbp and are presented relative to those of the wild-type pre-B cells (set as 1). Nascent transcripts were PCR-amplified with primers located in the indicated introns (Table EV3). Statistical data are shown as mean value with SEM and were analyzed by the Student"s t-test; *P < 0.05, **P < 0.01. Each dot corresponds to oneFlow cytometric analysis of hCD2 cell surface expression and intracellular Blimp1 expression of the indicated cell types. Bone marrow of Prdm1ihCd2/+ (red) or wild-type (WT, gray) mice was used to analyz ALPs, BLPs, pro-B, pre-B, immature B cells whereas the spleen was used for flow cytometric analysis o FO B, MZ B and plasma cell of both genotypes The histograms show hCD2 (top row) and Blimp1 (bottom row) expression for the different cell types Wild-type FO B cells (dashed line) were used as negative control for the Blimp1 staining in plasma cells. The difference in mean fluorescence intensity (ΔMFI) between the two genotypes is shown for each cell typeFlow cytometric analysis of hCD2 cell surface expression and intracellular Blimp1 expression of the indicated cell types. Bone marrow of Prdm1ihCd2/+ (red) or wild-type (WT, gray) mice was used to analyz NK cells and granulocytes, whereas the spleen was used for flow cytometric analysis o naïve CD4 T and naïve CD8 T cells of both genotypes The histograms show hCD2 (top row) and Blimp1 (bottom row) expression for the different cell types The difference in mean fluorescence intensity (ΔMFI) between the two genotypes is shown for each cell typeImmunoblot analysis of Blimp1 and the TATA-binding protein (TBP) in nuclear extracts prepared from short-term cultured wild-type or Prdm1ihCd2/+ pro-B cells. Marker proteins of the indicated size (in kilodaltons) are shown to the right
Figure 3Flow cytometric analysis of total B cells (A) as well as pro-B, pre-B, immature (imm) B and recirculating (recirc) B cells (B) from the bone marrow of wild-type (WT, gray), Prdm1ihCd2/+ (red) and Prdm1ihCd2/ihCd2 (white) mice at the age of 6-8 weeks Flow cytometric analysis of total B, FO B and MZ B cells from the spleens of wild-type, Prdm1ihCd2/+ and Prdm1ihCd2/ihCd2 miceDetermination of cell death of ex vivo pro-B and pre-B cells from the bone marrow of Prdm1ihCd2/+ (red) and wild-type (gray) mice by flow cytometric analysis of the loss of plasma membrane asymmetry (F2N12S ratiometric dye staining) and the loss of cell membrane integrity (SYTOX staining). Representative flow cytometry plots (left) and the quantification of dead and apoptotic (apop) cells (right) are shownBrdU labeling of immature (imm) and FO B cells in the spleen of 3-month-old Prdm1ihCd2/+ mice (red dots) and control wild-type littermates (gray dots). The percentage of BrdU+ B cells was determined for each B cell type by flow cytometric analysis after 10 days of continuous BrdU labeling (day 10, white bar) or after a subsequent 15-day chase period (day 25; hatched bar) without BrdU in the drinking wateBrdU labeling of immature (imm) and FO B cells in the spleen of 3-month-old Prdm1ihCd2/+ mice (red dots) and control wild-type littermates (gray dots) A diagram (below) indicates the design of the BrdU labeling and chase experiments. Flow cytometric data obtained with FO B cells from one mouse of each genotype are shown in (F) The percentage of BrdU+ FO B cells is shown above the indicated gates (F)