EMBO/soda-nel huggingface dataset:
A plant-specific SWR1 chromatin remodeling complex couples histone H2A.Z deposition with nucleosome sliding. (2020)
Figure 5

Figure 5. Determination of the function of the SWR1 complex components in H2A.Z deposition.

(A) The effect of indicated mutations on H2A.Z deposition as determined by immunoblotting. Chromatin-associated proteins were extracted and subjected to immunoblotting by the anti-H2A.Z antibody. The histone H4 level was detected by the anti-H4 antibody and indicated as a loading control.

(B) Meta plots showing H2A.Z occupancy on H2A.Z target genes. TSS, transcription start site; TTS, transcription termination site. RPKM, reads per kilobase per million. The H2A.Z ChIP-seq results were generated from two independent biological replicates.

(C) Venn diagrams showing overlaps of genes with lower H2A.Z levels in the pie1, mbd9-1, and chr11/17 mutant than in the WT. The significance of the overlaps between each pair of samples was determined by hypergeometric test (P0).

(D) Heat maps showing H2A.Z enrichment in the WT, pie1, chr11/17, and mbd9-1 mutants over the PIE1-dependent H2A.Z target genes. The peaks were sorted by H2A.Z density in the WT.

(E) Shown are H2A.Z changes in the pie1, chr11/17, and mbd9-1 mutants relative to the WT. PIE1-dependent H2A.Z target genes were subjected to the analysis and sorted by H2A.Z density in the WT. The color bar shows log2(fold change).

(F) Meta plots showing CHR11 and PIE1 occupancy on PIE1-dependent H2A.Z target genes. TSS, transcription start site; TTS, transcription termination site. The CHR11-Flag and PIE1-Flag ChIP-seq data are from two independent biological replicates.

(G) Heat maps showing the enrichment of PIE1-Flag and CHR11-Flag over the PIE1-dependent H2A.Z target genes. The peaks were sorted by H2A.Z density in the WT.

(H) Box plot showing the enrichment of CHR11 and PIE1 over H2A.Z target genes and non-H2A.Z target genes. The log2(fold change) values of RPKM between the CHR11-Flag or PIE1-Flag signals and the WT signals are shown. Center lines and box edges represent medians and the interquartile range (IQR), respectively. Whiskers extend to minimum and maximum values within 1.5 times of the IQR. The data shown are from two independent biological replicates. The difference between the CHR11-Flag and PIE1-Flag ChIP-seq levels was determined by student's t test. ** P<0.01.
A plant-specific SWR1 chromatin remodeling complex couples histone H2A.Z deposition with nucleosome sliding. (2020)
Figure 6

Figure 6. Determination of the function of CHR11/17 and PIE1 in nucleosome sliding.

(A) Increasing amounts of CHR11 (left panel) or PIE1 (right panel) were incubated with end-positioned (lane 1-4 and 9-12) or center-positioned (lane 5-8 and 13-16) mono-nucleosomes in the presence of ATP. The sliding results were then visualized on native PAGE gels by EB staining.

(B) Distribution of nucleosomes over TSS of all protein-coding genes in the WT, chr11/17, pie1, and mbd9-1 mutants. The MNase-seq results were generated from two independent biological replicates.

(C) The proposed model for the function of SWR1-MBD9-CHR11/17 complex in nucleosome regulation. NFR, nucleosome free region.

Expanded View Figures
The HSP110/HSP70 disaggregation system generates spreading-competent toxic α-synuclein species (2020)
Figure 1

Figure 1. Tissue-specific KD of HSP-110 prevents resolubilization of HS-induced firefly luciferase aggregates

A Single plane confocal fluorescent microscopy images of 5-day-old animals harboring GFP-tagged endogenous HSP-110 (HSP-110::GFP). Muscle cells are outlined. M: muscle, H: hypodermis. In animals that co-express hairpin constructs targeting hsp-110 in muscle cells (HSP-110;HPI and HSP-110;HPII), GFP fluorescence is strongly reduced in muscle cells in comparison to control animals, indicating HSP-110::GFP depletion specifically in muscle cells. Scale bar: 10 µm.

B Experimental set-up: Age-synchronized 4-day-old animals were subjected to heat stress for 3 h at 33°C (+HS) then returned to 20°C or left at 20°C (-HS), respectively. Arrows indicate imaging time points. The -HS controls were imaged at the same time point as the +HS animals after 24h at 20°C. Maximum intensity projections of fluorescent microscopy z-stacks of animals expressing the indicated transgenes. HS-induced FLUCSM::GFP foci are cleared after a recovery for 24h at 20°C in the WT background, but persist in the presence of the hsp-110 hairpin construct. Scale bar: 20 µm.

C Quantification of FLUCSM::GFP foci disaggregation. % disaggregation is calculated as 100 - ratio of FLUCSM::GFP foci area relative to total muscle area at the +HS 24h time point to the +HS 2h time point. Data are displayed as mean ± SEM (in %) (N = 3). Statistical analysis was done using one-way ANOVA with Dunnett's adjustment for multiple comparisons. ** p ≤ 0.01.

Data information: All strains harbor the sid-1(pk3321) allele.
The HSP110/HSP70 disaggregation system generates spreading-competent toxic α-synuclein species (2020)
Figure 2

Figure 2. KD of HSP-110 reduces cytosolic α-Syn and Q35 foci formation and toxicity

A Maximum intensity projections of fluorescent microscopy z-stacks of 5-day-old nematodes expressing the indicated transgenes are shown. White dashed lines outline the borders of muscle cells. Scale bar: 10 µm. The co-expression of HPI and HPII reduces the amount of α-Syn::YFP inclusions.

B Quantification of α-Syn::YFP foci in 3- to 6-day-old animals. Product of mean foci fluorescence and foci area relative to muscle area is displayed (relFluoFoci). Data are shown as mean ± SEM (in %) (N = 3). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. ** = p ≤ 0.01, *** p ≤ 0.001.

C Motility Assay as a measure for transgene toxicity. Displayed is the mean number of body bends per 30 s ± SEM (N = 3). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. ** p ≤ 0.01, *** p ≤ 0.001.

D Maximum intensity projections of fluorescent microscopy z-stacks of 6-day-old animals expressing the indicated transgenes. Scale bar: 100 µm. The co-expression of HPI and HPII reduces the amount of Q35::YFP inclusions.

E Quantification of Q35::YFP foci in WT and hsp-110 hairpin background. Shown is the mean ± SEM (N = 3). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. * p ≤ 0.05, ** p ≤ 0.01.

F Motility Assay as a measure for transgene toxicity. Displayed is the mean number of body bends per 30 s ± SEM (N = 3). Statistical analysis was done using one-way ANOVA with Dunnett's multiple comparisons test. * p ≤ 0.05, ** p ≤ 0.01.

G Maximum intensity projections of fluorescent microscopy z-stacks of of 5-day-old nematodes expressing the indicated transgenes. White dashed lines outline the borders of muscle cells. Scale bar: 10 µm. M: muscle, H: hypodermis. Signal outside of muscle cells reveals spreading of α-Syn.

H Quantification of animals showing α-Syn transmission at indicated ages. Displayed is the mean ± SEM (in %) (N = 4). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. *** p ≤ 0.001.

Data information: All strains harbor the sid-1(pk3321) allele, except for the YFP only and Q24 controls.
The HSP110/HSP70 disaggregation system generates spreading-competent toxic α-synuclein species (2020)
Figure 3

Figure 3. WT but not NEF deficient yeast SSE1 can compensate the loss of C. elegans HSP-110

A Maximum intensity projections of confocal microscopy z-stacks of 5-day-old animals expressing the indicated transgenes. White dashed lines outline the borders of muscle cells. Scale bar: 10 µm. The expression of the yeast SSE1N575Y E575A mutant cannot reverse the reduction of α-Syn::YFP foci in the hsp-110 hairpin expressing animals.

B Quantification of motility as a measure for transgene toxicity. Displayed is the mean ± SEM (N = 3) amount of body bends/30s of animals expressing the indicated transgenes. Statistical analysis was done using two-way ANOVA and with Dunnett's multiple comparison test. n.s. = not significant, ** p ≤ 0.01, *** p ≤ 0.001. The presence of the yeast SSE1N575Y E575A mutant was not able to suppress the rescue of α-Syn::YFP toxicity in hsp-110 hairpin expressing animals.

C Maximum intensity projections of fluorescent microscopy z-stacks of 5-day-old nematodes expressing the indicated transgenes. White dashed lines outline the borders of muscle cells. M: muscle, H: hypodermis. Scale bars: 10 µm. Signal outside of muscle cells reveals spreading of α-Syn.

D Quantification of animals showing α-Syn transmission into the hypodermis at indicated ages. Displayed is the mean ± SEM (in %) (N = 3). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. n.s. = not significant, *** p ≤ 0.001.

E Experimental set-up: Age-synchronized 4-day-old animals were subjected to heat stress for 3 h at 33°C (+HS) then returned to 20°C or left at 20°C (-HS), respectively. Arrows indicate imaging time points. The -HS controls were imaged at the same time point as the +HS animals after 24h at 20°C. Maximum intensity projections of fluorescent microscopy z-stacks of animals expressing the indicated transgenes. Scale bar: 20 µm. The impaired clearance of HS-induced FLUCSM::GFP foci in the hsp-110 hairpin expressing cells is rescued by co-expression of WT but not by the HSP-70 binding deficient SSE1N575Y E575A mutant.

F Quantification of FLUCSM::GFP foci disaggregation. % disaggregation is calculated as 100 - ratio of FLUCSM::GFP foci area relative to total muscle area at the +HS 24h time point to the +HS 2h time point. Data are displayed as mean ± SEM (in %) (N = 3). Statistical analysis was done using one-way ANOVA with Dunnett's multiple comparison test. n.s. = not significant, ** p ≤ 0.01.

All strains harbor the sid-1(pk3321) allele.
The HSP110/HSP70 disaggregation system generates spreading-competent toxic α-synuclein species (2020)
Figure 4

Figure 4. HSP-110 HP expression impairs cellular protein folding homeostasis

A Maximum intensity projections of confocal z-stacks of 4-day-old animals expressing the indicated transgenes or harboring the indicated temperature-sensitive (ts) mutations. Muscle cells were stained with anti-myosin (green) and Alexa Flour 647-phalloidin (purple). Scale bar: 5 µm. Myosin misfolding reflects the exposure of the ts mutant phenotype of myosin(ts) [unc-54(e1301)] at the permissive temperate of 15°C by co-expression of the hsp-110 hairpins.

B Quantification of motility as a measure for toxicity. Displayed is the mean ± SEM (N = 4) of body bends/30s of animals expressing the indicated transgenes or harboring the indicated mutations. Statistical analysis was done using one-way ANOVA with Dunnett's multiple comparison test. *** p ≤ 0.001. Co-expression of the hsp-110 hairpins exposed the ts mutant phenotype at the permissive temperate of 15°C, resulting in a significant increase in movement defects. All strains contain the sid-1(pk3321) allele.

C Quantification of motility as a measure for transgene toxicity. Displayed is the mean ± SEM (N = 4) amount of body bends/30s of animals expressing the indicated transgenes or harboring the indicated mutations. Statistical analysis was done using two-way ANOVA with Tukey's multiple comparison test. * p ≤ 0.05. Expression of the hsp-110 hairpins caused movement defects with increasing age.

D Maximum intensity projections of confocal z-stacks of 12-day-old animals expressing the indicated transgenes. Scale bar: 10 µm. FLUCSM forms foci with increasing age, which is markedly accelerated upon hsp-110 KD.

E Quantification of animals with FLUCSM::GFP foci at indicated days of life. Data are displayed as mean ± SEM (in %) (N = 3). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. n.s. = not significant, ** p ≤ 0.01, *** p ≤ 0.001.

F Quantification of motility as a measure for transgene toxicity. Displayed is the mean ± SEM (N = 3) amount of body bends/30s of animals expressing the indicated transgenes or harboring the indicated mutations. Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. n.s. = not significant, *** p ≤ 0.001. Expression of the hsp-110 hairpins caused movement defects with increasing age. The beneficial effects of HSP-110 KD on amyloid toxicity is masked in older animals.

G Quantification of motility as a measure for transgene toxicity. Displayed is the mean ± SEM (N = 3) amount of body bends/30s of animals expressing the indicated transgenes or harboring the indicated mutations. Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. n.s. = not significant, , ** p ≤ 0.01, *** p ≤ 0.001. Expression of the hsp-110 hairpins caused movement defects with increasing age. The beneficial effects of HSP-110 KD on amyloid toxicity is masked in older animals.

Data information: All strains harbor the sid-1(pk3321) allele.
The PAX5-JAK2 translocation acts as dual-hit mutation that promotes aggressive B-cell leukemia via nuclear STAT5 activation (2022)
Figure 1

Figure 1. B-ALL development in Pax5Jak2/+ mice.

(A) Schematic diagram of the Pax5Jak2 allele. Human cDNA sequences, starting in PAX5 exon 4 and encoding the remaining PAX5-JAK2 protein, were inserted in frame into exon 4 of the mouse Pax5 locus to generate the Pax5Jak2 allele (Appendix Fig S1B). The C-terminal tag sequence (grey) contains an epitope for the V5 antibody, two cleavage sites for the TEV protease and a biotin acceptor sequence (Biotin). A black oval denotes the B cell-specific enhancer (En) in intron 5 (Decker et al., 2009). pA, polyadenylation site. Notably, the human and mouse Pax5 protein sequences encoded from exon 1 to exon 5 contain only one amino acid substitution (human Ser13 vs mouse Ile13 in exon 1) located upstream of the paired domain (Adams et al., 1992).

(B) Expression of the Pax5 protein in Pax5Jak2/+ pro-B cells. Nuclear extracts of short-term cultured pro-B cells from 3-week-old Pax5Jak2/+ and Pax5+/+ mice were analyzed by immunoblot analysis with an anti-Pax5 antibody recognizing the N-terminal paired domain. The size of marker proteins is indicated in kilodaltons (kDa) to the left.

(C) Flow-cytometric analysis of bone marrow cells from the two hindlegs of 3-week-old Pax5+/+ and Pax5Jak2/+ mice. The percentage of cells in each gate or quadrant is indicated.

(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). See Methods section for flow-cytometric definition of the different cell types. One of 3 independent experiments is shown.

(E) Flow-cytometric analysis of bone marrow cells and splenocytes from Pax5Jak2/+ mice at the age of 4 or 6 weeks. The newly emerging B220low B cells are highlighted by black boxes.

(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.

(G) Size comparison of the spleen and lymph nodes from a control Pax5+/+ mouse and a Pax5Jak2/+ tumor mouse.

(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.
The PAX5-JAK2 translocation acts as dual-hit mutation that promotes aggressive B-cell leukemia via nuclear STAT5 activation (2022)
Figure 2

Figure 2. Gene regulation in Pax5Jak2/+ pro-B cells and Pax5Jak2/+ B-ALL cells.

(A) Scatter plot of gene expression differences between Pax5Jak2/+ and Pax5+/+ pro-B cells, which were isolated from the bone marrow of 3-week-old mice prior to RNA-sequencing. The expression data of individual genes (indicated by dots) are plotted as mean normalized rlog (regularized logarithm) values. Genes with an expression difference of > 2-fold, an adjusted P value of < 0.05 and a TPM value of > 5 (in at least one cell type) are colored in blue and red, corresponding to upregulation or downregulation in Pax5Jak2/+ pro-B cells (Dataset EV1).

(B) Bar graph indicating how many of the up- or down-regulated genes in Pax5Jak2/+ pro-B cells correspond to repressed (orange) or activated (green) Pax5 target genes identified in pro-B cells (Appendix Fig S2A).

(C) Scatter plot of gene expression differences between Pax5Jak2/+ and Pax5+/- Cdkn2ab+/- B-ALL cells of tumors that were isolated from lymph nodes prior to RNA-sequencing. Genes with an expression difference of > 3-fold, an adjusted P value of < 0.05 and a TPM value of > 5 (in at least one tumor cell type) are colored in blue and red, corresponding to upregulation or downregulation in Pax5Jak2/+ B-ALL cells (Dataset EV3).

(D-G) GSEA analysis of 327 repressed (D) or 330 activated (F) Pax5 target genes identified in pro-B cells (Appendix Fig S2A), 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 (see Appendix Fig S2I).
The PAX5-JAK2 translocation acts as dual-hit mutation that promotes aggressive B-cell leukemia via nuclear STAT5 activation (2022)
Figure 4

Figure 4. The development of Pax5Jak2/+ tumors depends on the Jak2 kinase activity

(A) Schematic diagram of the kinase-dead (KD) Pax5-Jak2 fusion protein, which is encoded by the Pax5Jak2-KD allele and contains the K272E mutation known to abolish the Jak2 kinase function.

(B, C) 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. See Methods for flow-cytometric definition of the different cell types. The expression of CD19 and B220 on bone marrow B cells of the indicated genotypes is shown in (C).

(D, E) 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 in (D). The bioluminescence measurements of transplanted mice treated with ruxolitinib (black) or vehicle (grey) at the indicated days after cell transfer are shown as mean radiance values with SEM (E). n, number of mice analyzed. Statistical data are shown as mean value with SEM and were analyzed by the mixed-effect model REML with Geisser-Greenhouse's correction and Tukey's multiple comparison test: *P < 0.05, ***P < 0.001.

(F) Kaplan-Meier survival analysis of the transplanted mice evaluated in (D, E) 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.
The PAX5-JAK2 translocation acts as dual-hit mutation that promotes aggressive B-cell leukemia via nuclear STAT5 activation (2022)
Figure 6

Figure 6. Pax5-Jak2 does not function as an epigenetic regulator.

(A) Schematic diagram of histone H3 indicating the position of Y41. The pY41-peptide (amino acids 37-46, red) was used for immunizing mice and the subsequent generation of the hybridoma cell line 8B2-C1 (see Methods). The longer phosphorylated (pY41) and non-phosphorylated peptides (amino acids 29-53, black) were used for ELISA and immunoblot analysis.

(B) Specific detection of the pY41-peptide by the antibody produced by the hybridoma cell line 8B2-C1. The plate-bound pY41-peptide, in contrast to the corresponding non-phosphorylated peptide, is specifically detected by antibodies in the serially diluted supernatant of the hybridoma cells by ELISA assays, as measured by absorption at the wavelength of 450 nm. Mean values with SEM are shown for two independent experiments.

(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 (Appendix Fig S6B). 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.

(E) No evidence for an epigenetic function of Pax5-Jak2 in early B cell development. The genome-wide distribution of the histone marks H3K4me3, H3K27ac and H3K27me3 in in vitro cultured Pax5Jak2/- and Pax5Prd/- progenitor cells was determined by ChIP-seq analysis (Appendix Fig S6C). The average density of the three histone marks in these progenitor cells was determined for a region from -2.5 kb to +2.5 kb from the summit of the Pax5-Jak2 peaks identified in Pax5Jak2/+ B-ALL cells (Fig 5B). The results of two different ChIP-seq experiments per cell type are shown. The expression of the Pax5-Jak2 protein and paired domain (Prd) polypeptide in Pax5Jak2/- and Pax5Prd/- progenitor cells is shown in Appendix Fig S2D.