EMBO/SourceData huggingface dataset:
Figure 1B. Analysis of cell cycle arrest (in G2) in response to DSB induction assayed by flow cytometry.C. Rad53 phosphorylation (Rad53-P) in response to DSB assayed by western blotting.D. Cell survival upon DSB induction. Average ± SD (n=4) is shown for each genotype.
Figure 2B. Srs2 requirement in de novo telomere addition in cells with and without functional HR. All strains are pif1-m2. Strains used: NK1264; NK2375, NK2376; NK2014, NK2015; NK2451, NK2452; NK2012, NK2013; NK2457, NK2458; NK2363, NK2364; NK2469; 2369, NK2370; NK2473-2475.D. Dynamics of (TG1-3)n addition monitored by qPCR through a time-course experiment (asynchronous populations). The Y axis shows a fold increase in de novo telomere-specific PCR product relative to the background levels at 0 h and normalised against an internal control (ARO1 locus). Average ± SD (n=3) is shown for each time-point of each genotype. Strains used: NK3292, NK3293; NK4670, NK4671; NK4112, NK4113; NK4114, NK4115; NK3292 est2∆, NK3293 est2∆; NK4232, NK4233.F. Comparative analysis of ssDNA/dsDNA at de novo telomere addition loci in SRS2 and srs2Δ during a time-course experiment (synchronised populations). The Y axis shows a fold increase in de novo telomere-specific PCR product relative to the background levels at 0 h and normalised against an internal control (ARO1 locus). Average ± SD (n=3) is shown for each time-point of each genotype. Top set of error bars represents SD in relative increase of the de novo telomere-specific PCR product (as in panel D) while the lower set of error bars corresponds to quantifications of ss/dsDNA fractions. Strains used: NK3292, NK3293; NK4670, NK4671.
Figure 3B. Southern blot analysis of re-synthesis of resected DNA during BIR in SRS2 and srs2Δ corresponding to the data quantifications in C. DNA was digested with EcoRI and BamHI, resolved on 0.7% agarose gels, transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes (three RS probes and a reference probe, REF, hybridizing to an ARS522-containing fragment on chr. V which is not involved in the repair). A representative image of one of the three repeats is shown.C. Re-synthesis of resected DNA on chr.VIIL in SRS2 and srs2Δ cells (solid and dashed lines, respectively) at the distance of 15.2 (RS15.2), 6.8 (RS6.8) and 2.6 (RS2.6) kb away from the homology region. Average ± SD (n=3) is shown for each time point.D. Southern blot analysis of BIR-dependent DNA synthesis in SRS2 and srs2Δ corresponding to the data quantifications in D. DNA was digested with EcoRI and BamHI, resolved on 0.7% agarose gels, transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes (three BIR probes and a reference probe, REF, hybridizing to an ARS522-containing fragment on chr. V which is not involved in the repair). A representative image of one of the three repeats is shown. C - control strain NK3980.E. BIR-dependent DNA synthesis in SRS2 and srs2Δ cells (solid and dashed lines, respectively) at the distance of 6 (BIR6), 36 (BIR36) and 77 (BIR77) kb away from the homology region. Average ± SD (n=3) is shown for each time point.
Figure 4B. Frequency of DSB repair via SSA in SRS2 and srs2Δ cells with and without functional HR in the assay based on the system shown in panel A. Average ± SD (n=4) is shown for each genotype. Strains used in B and D: NK4691-4693; NK4805-4808; NK5081-5084; NK5085-5091.D. Fragment L formation in SRS2 and srs2Δ cells in the presence and absence of Rad51. Also see Figure EV3 for blot images. Average ± SD (n=4) is shown for each time point.
Figure 1(D) Absolute cell numbers of the indicated cell types were determined by flow-cytometric analysis of the bone marrow from 3-week-old Pax5+/+ and Pax5Jak2/+ mice. Average cell numbers are shown with SEM and were statistically analyzed by multiple t-tests (unpaired two-tailed with Holm-Šídák's correction); ns, not significant (P > 0.05). One of 3 independent experiments is shown.(F) Kaplan-Meier survival analysis of Pax5Jak2/+ (black) and control Pax5+/+ (grey) mice. n, number of mice analyzed. A P value of < 0.0001 was determined for the survival curves by statistical analysis with the log-rank (Mantel-Cox) test.(H) Eosin-hematoxylin-stained sections of the lung and liver of a Pax5Jak2/+ tumor mouse. Infiltrating and blasting tumor cells are indicated by an arrow.(I) Flow-cytometric analysis of lymph node cells from a control Pax5+/+ mouse and a 10-week-old Pax5Jak2/+ tumor mouse. (J) Flow-cytometric analysis of B220lowCD19+ B cells from the bone marrow of a 4-week-old Pax5Jak2/+ mouse.
Figure 2(D-G) GSEA analysis of 327 repressed (D) or 330 activated (F) Pax5 target genes identified in pro-B cells as compared with the ranked log2-fold gene expression changes in Pax5Jak2/+ (PJ) B-ALLs versus control (Ctrl) Pax5+/- Cdkn2ab+/- B-ALLs (left). NES, normalized enrichment score. The expression of five genes that are upregulated in Pax5Jak2/+ B-ALL cells (upper row) and Pax5-/- (KO) pro-B cells (lower row) is shown in (E). Likewise, the expression of five genes that are upregulated in control B-ALL cells (upper row) and Pax5+/+ (WT) pro-B cells (lower row) is shown in (G). TPM, transcripts per million. Mean TPM values with SEM are shown for the following RNA-seq experiments: 3 (WT pro-B), 2 (KO pro-B), 2 (Ctrl B-ALL), and 4 (PJ B-ALL).(H) Expression of the Ptprc (CD45) gene from exon 3 to exon 8, as determined by RNA-seq of Pax5Jak2/+ and Pax5+/- Cdkn2ab+/- B-ALL cells. The alternatively spliced exons 4 (A), 5 (B) and 6 (C) are indicated in orange in the respective exon-intron structure of the Ptprc gene. Ptprc transcripts of B220+ Pax5+/- Cdkn2ab+/- B-ALL cells contain all three exons, thus giving rise to expression of the CD45 isoform RABC (known as B220). In contrast, reads at exon 4 are barely detectable and reads at exon 6 are strongly reduced in the mRNA of Pax5Jak2/+ B-ALL cells, which likely gives rise to the CD45 isoforms RBC and RB
Figure 3(D) Flow-cytometric analysis of bone marrow cells from 4-week-old Pax5ihCd2/+ and Pax5Jak2/ihCd2 mice. The expression of human (h) CD2 from the Pax5ihCd2 allele is shown for CD19+B220+ (grey) and CD19+B220low (black) B cells.(G) Kaplan-Meier survival analysis of Cd79a-Cre Ikzf1neo/+ Pax5LSL-Jak2/+ (black) and Cd79a-Cre Pax5LSL-Jak2/+ (grey) mice. Statistical analysis of the survival curves was performed with the log-rank (Mantel-Cox) test; ****P < 0.0001. n, number of mice analyzed.(H) Flow-cytometric analysis of B220 and CD19 expression in B-ALL tumor cells from the lymph node of a Cd79a-Cre Ikzf1neo/+ Pax5LSL-Jak2/+ mouse (black; left). Pax5 expression in these B-ALL tumor cells (black line) and control Pax5+/+ lymph node B cells (grey filled) was determined by intracellular Pax5 staining (right).
Figure 4(B, Flow-cytometric analysis of bone marrow and spleen from Pax5Jak2-KD/+ (black; n = 11) and control Pax5+/+ (grey; n = 7) mice at the age of 6-8 weeks. The frequencies of the indicated B cell types are shown for each organ (B). The data are presented as mean percentages with SEM and were statistically analyzed by multiple t-tests (unpaired and two-tailed with Holm-Šídák's correction); ns (P > 0.5), *P < 0.05, **P < 0.01.(D, Tumor progression in mice, transplanted with Pax5Jak2-Luc/+ tumor cells, in the presence or absence of the JAK1/2 inhibitor ruxolitinib. At day 14 after cell transfer, the transplanted mice were treated twice daily with ruxolitinib or vehicle, and the tumor mass was monitored by bioluminescence measurements. Images of three representative mice with or without ruxolitinib treatment are shown(F) Kaplan-Meier survival analysis of the transplanted mice upon prolonged treatment with ruxolitinib (black) and vehicle (grey). Statistical analysis of the survival curves (F) was performed with the log-rank (Mantel-Cox) test; **P < 0.01.
Figure 5(B) Genome-wide binding of Pax5-Jak2 in in vitro cultured pro-B cells from Pax5Jak2/+ Rosa26BirA/+ mice at the age of 3 weeks (expressing Pax5) and ex vivo Pax5Jak2/+ Rosa26BirA/+ B-ALL tumors (lacking Pax5), as determined by Bio-ChIP-seq analysis The DNA-binding pattern of full-length Pax5 was determined by Bio-ChIP-seq analysis of ex vivo sorted Pax5Bio/Bio pro-B cells, which carried a C‐terminal biotin acceptor sequence together with an IRES‐BirA gene insertion in the 3' untranslated region of Pax5 Two independent Bio-ChIP-seq experiments were performed for each cell type. Representative binding patterns of Pax5 and Pax5-Jak2 in the three B cell types are shown for a selected genomic region, with horizontal bars indicating Pax5 or Pax5-Jak2 peaks that were identified by MACS peak calling (left). The number of Pax5 (white) and Pax5-Jak2 (grey or black) peaks, which were defined by stringent MACS peak calling with a P value of < 10-10 in the three B cell types, are shown to the right.(F, G) Flow-cytometric analysis of the bone marrow and spleen from Pax5Prd*-Jak2 /+ (black; n = 6) and control Pax5+/+ (grey; n = 5) mice at the age of 6-7 weeks. The frequency of the indicated B cell types in each organ is indicated in (F), and the flow-cytometric analysis of CD19 and B220 expression on bone marrow B cells is shown in (G). Statistical data are shown as mean percentages with SEM and were analyzed by multiple t-tests (unpaired and two-tailed with Holm-Šídák's correction): ns > 0.05; *P < 0.05; **P < 0.01. Each dot corresponds to one mouse.(H) Kaplan-Meier survival analysis of Pax5Prd*-Jak2 /+ (black) and Pax5+/+ (grey) mice. A P value of < 0.0001 was determined for the survival curves by statistical analysis with the log-rank (Mantel-Cox) test. n, number of mice analyzed.
Figure 6(C) Specificity of the anti-H3Y41ph antibody, as shown by immunoblot analysis. One phosphorylated (pY41) or non-phosphorylated peptide was coupled to ubiquitin (Ubi, 8.5 kDa), followed by separation of the protein conjugates on SDS-PAGE and immunoblot detection with the anti-H3Y41ph antibody present in the 8B2-C1 cell supernatant. Following treatment with calf intestinal alkaline phosphatase (CIP), the dephosphorylated Ubi-pY41 peptide conjugate could no longer be detected with the anti-H3Y41ph antibody.(D) Immunoblot analysis of whole-cell extracts prepared from Pax5Jak2/+ and control Pax5Etv6/+ Cdkn2ab+/- B-ALL cells as well as from the human HEL, TMD8 and K1106 cell lines. Phosphorylated (p) STAT5 was detected with an anti-STAT5 (pY694) antibody and the pY41-peptide with the purified anti H3Y41ph antibody One to five pY41-peptides were coupled to ubiquitin, which was added in the range of 50 ng per well. The Gapdh and histone H3 proteins were analyzed as loading control. An unspecific protein is denoted by an asterisk. One representative of 5 immunoblot experiments is shown.
Figure 7(A, Tumor progression in Il7+/+ (grey), Il7+/- (dashed) and Il7-/- (black) mice transplanted with Pax5Jak2-Luc/+ tumor cells, as determined by bioluminescence measurements at the indicated days after cell transfer. Images of three transplanted mice for each Il7 genotype are shown at the indicated days after cell transfer(C) Kaplan-Meier survival analysis of Il7+/+, Il7+/- and Il7-/- mice transplanted with Pax5Jak2-Luc/+ tumors cells. Statistical analysis of the survival curves was performed with the log-rank (Mantel-Cox) test; ns P > 0.05, *P < 0.05, **P < 0.01.(E) Quantification of the median fluorescence intensity (MFI) values of the p-STAT5 levels determined by intracellular staining of splenic FO B cells from Pax5+/+ (grey), Pax5Jak2/+ (black) and Pax5Jak2-KD/+ (white) mice at the age of 3-10 weeks Statistical data are presented as mean values with SEM and were analyzed by the two-tailed unpaired Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot corresponds to one mouse.(F) Flow cytometric analysis of p-STAT5 levels in pro-B cells (B220+CD19+Kit+IgM-) and leukemic B220low B cells of Pax5Jak2/+ mice (black) as well as in control pro-B cells of Pax5+/+ (WT) mice (grey) at the age of 4-5 weeks. A representative flow cytometric analysis of the bone marrow of a Pax5Jak2/+ mouse (left) and representative intracellular p-STAT5 stainings (middle) are shown. The p-STAT5 levels in pro-B and leukemic B220low B cells (right) are quantified as MFI values relative to that of Pax5+/+ pro-B cells. Statistical data are presented as mean values with SEM and were analyzed by the two-tailed unpaired Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot corresponds to one mouse.(I) Upregulation of the genes Cxcr5, Syndig1l and Sema4a in Pax5Jak2/+ (PJ) B-ALL cells. Mean TPM values with SEM are shown for the following RNA-seq experiments: 2 (Ctrl B-ALL), 4 (PJ B-ALL)K) Identification of Cxcr5, Syndig1l and Sema4a as activated STAT5 target genes. ChIP-seq analysis identified STAT5-binding regions at all three loci (K). Horizontal bars below the ChIP-seq track indicate STAT5-binding regions identified by MACS peak calling. RPM, reads per million.
Figure 1MPK4‐HA is detected in PAT1‐Myc immunoprecipitates from double transgenic Arabidopsis plants. Immunoblots of input and anti‐Myc IPs probed with anti‐HA and anti‐Myc antibodies. Left panel, input; right panel, anti‐Myc IP.Yeast pat1Δ mutants are unable to grow at 37°C while wild‐type (B4742) strain grows at 30°C (left panel) and 37°C (right). Growth at 37°C is restored in pat1Δ expressing AtPAT1. Serial dilutions of each yeast strain were plated on YPAD agar plates and grown at the indicated temperatures.Co‐IP between PAT1‐HA and LSM1‐GFP. Proteins were transiently co‐expressed in N. benthamiana and tissue harvested 3 days post‐infiltration. Immunoblots of GFP IPs (top panel) and inputs (20 μg each, bottom panel). Immunoblots were cut in half and probed with anti‐HA antibodies and anti‐GFP antibodies.Agarose gel electrophoresis of 5′ RACE PCR to detect capped transcripts of EIF4A1, UGT87A2 and EXPL1 in 14‐day‐old Col‐0 and pat1‐1.
Figure 2MPK4 and MPK6 were immunoprecipitated from extracts of Col‐0 seedlings treated with 200 nM flg22 for 10 min. For the negative control (−), extracts were incubated with agarose beads without antibodies. IPs were incubated with His6‐PAT1, His6‐PAT1 S208A or MBP for 60 min at 37°C before boiling and SDS‐PAGE. Autoradiogram (top panel), Coomassie‐stained gel for loading control (bottom).
Figure 3pat1 mutants have a serrated leaf, semi‐dwarf phenotypePlants photographed at 4 weeks of growth in short day conditions, genotypes as indicated. Col‐0 and eds1‐2 plants and pat1‐1 and pat1‐1/eds1‐2, respectively, were placed in the middle of the same pots before the pictures were taken.
Figure 4PR gene expression is elevated in mpk4‐2 and pat1‐1 mutants. Four‐week‐old plants were used for RNA extraction, followed by qRT‐PCR. Fold‐change in PR1 (gray bars) and PR2 (black) expression is relative to Col‐0, normalized to UBQ10. Standard error of the mean is indicated by errors bars (n = 3). Statistical significance between the mean values was determined by ANOVA followed by Fisher's LSD test, P‐values are only shown for mean values significantly different from Col‐0.pat1 is more resistant to colonization by P. syringae pv. tomato DC3000. Bacteria were syringe‐infiltrated and samples taken 3 days post‐infiltration. Data are shown as log10‐transformed colony‐forming units/cm2 leaf tissue (cfu/cm2). Standard error of the mean is indicated by errors bars (n = 4). Statistical significance between the mean values was determined by ANOVA followed by Fisher′s LSD test.
Figure 5Confocalmicroscopy with root elongation zones. Five‐day‐old Col‐0/PAT1‐GFP seedlings were treated with 1 μM flg22 on glass slides for 20 min (second panel) followed by treatment with 100 μg/ml cycloheximide for 60 min (bottom panel). The scale bar corresponds to 10 μm.Confocalmicroscopy with 5‐day‐old VCS‐GFP seedlings treated as in (A). The scale bar corresponds to 10 μm.PAT1 protein is induced by PAMP treatment. Immunoblot detection of equal amounts of protein from Col‐0 seedlings at times in minutes as indicated following vacuum infiltration with 1 μM flg22 or water (180 min after infiltration). Immunoblots were probed with anti‐PAT1 antibodies. Negative control pat1‐1 was loaded for comparison. Coomassie brilliant blue protein loading control is indicated by CBB.Anti‐GFP immunoblotting of proteins extracted from Col‐0/PAT1‐GFP seedlings treated over a time course with flg22 (top panel) and Coomassie brilliant blue (CBB)‐stained PVDF loading control is shown (bottom panel). No PAT1‐GFP was detected in negative control Col‐0.
Figure 6Elevated PR gene expression in pat1 mutants is suppressed by summ2‐8. Four‐week‐old plants were used for RNA extraction, followed by qRT‐PCR. Fold‐change in PR1 (gray bars) and PR2 (black bars) expression is relative to Col‐0, normalized to UBQ10. Standard error of the mean is indicated by errors bars (n = 3).pat1 resistance to P. syringae pv. tomato DC3000 is suppressed by summ2‐8. Bacteria were syringe‐infiltrated and samples taken 3 days post‐infiltration. Data are log10‐transformed colony‐forming units/cm2 leaf tissue (cfu/cm2). Standard error of the mean is indicated by errors bars (n = 4).14‐day‐old seedlings grown on ms plates were used for RNA extraction. Next, 1 μg of RNA from each sample was treated with XRN1 (NEB) or mock treated before RT‐qPCR. Data were normalized to ACT2, and transcript levels were compared between XRN1 and mock‐treated RNA for each genotype. Standard error of the mean is indicated by error bars (n = 4). Statistical significance between the mean values was determined by ANOVA followed by Fisher's LSD test.Phosphorylation of the PAT1 peptide SSFVSYPPPGSISPDQR, which include Ser208, in Col‐0 and mkk1/1‐summ2 before and after flg22 treatment. The phosphorylation stoichiometry is illustrated relative to Col PAT1 without flg22 treatment. The ratios were obtained using quantitative iTRAQ mass spectrometry.
Figure 7Proteins were transiently co‐expressed (PAT1‐GFP + MPK4‐HA or SUMM2‐HA) in N. benthamiana and tissue harvested 2 days post‐infiltration. Anti‐HA immunoblots of inputs (20 μg each) are shown in the bottom panel. Immunoblots of GFP IPs were probed with anti‐HA antibodies, then stripped and probed with anti‐GFP antibodies. Molecular weights are shown in kDa on the right.Confocalimages of yellow fluorescent protein complementation. PAT1‐Nc was transiently co‐expressed with either SUMM2‐Nn or At4g12010 in N. benthamiana. Confocalmicroscopy on leaf disks was conducted 2 days post‐infiltration. Merged pictures show overlay of brightfield, chlorophyll and eYFP. The scale bar corresponds to 25 μm.
Figure 2(a-b) Plots as in Figure 1, showing changes in Lamin B1 DamID signals upon CRISPRa activation of NLGN1 (a) and SOX6 (b). Control cells were treated either without sgRNA or with one of various sgRNAs targeting promoters on different chromosomes. Vertical green dotted lines mark the position of the sgRNA target sequence.(c) Domainograms showing regions with reduced NL interactions around 12 genes individually activated by CRISPRa. Genomic regions are aligned by the respective sgRNA target positions, and oriented so that the activated genes are all transcribed from left to right.
Figure 6(a) DamID domainograms of NL interactions after activation of CCSER1, GRID2, or both. All data are from human RPE-1 cells.(b) DamID domainograms after activation of ADAM22, ABCB1, or the genes ABCB4, ABCB1, ADAM22 and RUNC3B simultaneously. All data are from human RPE-1 cells. DamID data of activation of ADAM22 and ABCB1 alone are same as in Figure 4a, b.
Figure 7(a) DamID profiles of the 129Sv allele of the Morc1 locus with deletions of the promoters of Morc, Morc1 and DppA2 (deletions marked by yellow vertical boxes), and in control cells. (b) Same as (a), but for the non-mutated Cast alleles. (c-d) Same design as (a-b), but with only a single mono-allelic deletion of the Morc promoter (vertical yellow box).(e-f) Effect of PAS insertion on NL interactions of Cobl gene locus. Same design as (a-b) but with insertion of a PAS (located at red triangle and vertical dotted line) that truncates the 129Sv allele of the Cobl transcription unit. 129Sv allele is shown in (e), non-mutated Cast allele in (f). Clones with Morc1 locus mutations (each assayed in four independent biological replicates) served collectively as control in (e-f)
Figure 8(c) Average DamID profiles across 17 transgene integration sites inside LADs. Blue curve shows alleles without integrations, red curve shows the corresponding alleles with integrations. Shading between the lines shows which curve has the highest value.(d) Relative expression levels of individual barcoded transgenes in LADs and iLADs.(e) Estimated expression levels of integrated transgenes in LADs (vertical orange lines; median value indicated by arrowhead), compared to the distribution of expression levels of all active endogenous genes (black curve).
Figure 1A The family pedigree, where circles denote female members, squares male members, solid symbols affected members, and white symbols asymptomatic members with normal physical exam; the dots indicate heterozygous carriers and double line denotes a consanguineous marriage. The pictures show scapular winging which is a consistent clinical sign in affected individuals.B Hematoxylin and eosin staining (H&amp;amp;E) of skeletal muscle from patient II.1 shows histological features of moderate to severe dystrophic pattern. (Scale bar, 50 μm).C T1-weighted MRI axial images at thigh and calf levels show that the fatty degeneration is more prominent in thigh muscles, equally affecting posterior and anterior compartment, with relative sparing of the rectus femoris, sartorius and gracilismuscles until late stages (4, 10 and 11, respectively). Strikingly, the fatty tissue is located in the internal parts of almost all the affected muscles in thigh (1, 2, 3, 5-9), while the external regions are spared. At calf level, only the gastrocnemius medialis muscle (12) shows this pattern, while the soleus (13) is diffusely involved. Patient II.2 (PII.2) shows late-stage thigh muscles with an unusual involvement of the tibialis posteriormuscle (14) in the lower leg.
Figure 2A Muscle sections show variable labeling using an antibody against glycosylated α-dystroglycan (αDG-IIH6), whereas labeling using antibodies against α-dystroglycan core protein (αDG-Core), β-dystroglycan (βDG) and laminin-2 is similar to control (Scale bar, 100 μm).B Western blots and ligand-overlay (O/L) of wheat-germ agglutinin-enriched muscle and fibroblasts lysates from PII.2, PII.5, the healthy sibling (HII.3) and healthy controls (Ctr, Ctr1, and Ctr2) (Muscle: 250μg protein/lane, Fibroblasts: 800μg/lane). In muscle, expression of αDG-IIH6 is reduced but the αDG-Core shows similar expression as controls, with a slight reduction in molecular weight; laminin overlay assay detected some binding activity but was diminished compared to controls, whereas agrin-binding activity showed no difference with controls. In fibroblasts, both αDG-IIH6 and αDG-Core expression and laminin-binding activity were normal, suggesting that unlike other muscular dystrophies, α-dystroglycan glycosylation defect is muscle-specific in POGLUT1 patients.C No ultrastructural alterations are observed in muscle by electron microscopy (6-7 fields were captured from PII.1, PII.3, and PII.5). Note normal basement membrane compactation (arrowheads). Original magnification: 26,700.