Journal: Scientific Reports

Article Title: Gradual chromosomal lagging drive programmed genome elimination in hemiclonal fishes from the genus Hypseleotris

doi: 10.1038/s41598-024-78278-6

Figure Lengend Snippet: Analysis of epigenetic histone modifications during mitosis and micronuclei formation. Whole-mount immunofluorescent staining of gonads from sexual species ( a , b ) and hybrids (all rest) with antibodies against various epigenetic modification stained in red: H3S10P ( a , c , d ), H3S28P ( b , e ), H3K9me3 ( f , g ) and H3T11P ( h ). Accumulation of all analyzed chromatin modifications indicate normal chromosome condensation during prophase and metaphase. In hybrids, misaligned chromosomes (indicated by white arrows) have similar distribution of the H3S10P ( c ), H3S28P ( e ), H3K9me3 ( f ) and H3T11P ( h ) chromatin modifications as the rest of the chromosomes (showed by white arrowheads) suggesting that misaligned chromosomes condense properly during prophase and metaphase. Micronuclei present during metaphases (indicated by thin white arrows) do not accumulate epigenetic histone modification such as H3S10P ( c ), H3K9me3 ( f ). ( d ) Accumulation of H3S10P epigenetic modification in some micronuclei (indicated by thin red arrows) suggest their formation by chromosomal lagging. Some micronuclei do not show accumulation of H3S10P epigenetic modification (indicated by thin white arrows). ( g ) In contrast to the chromatin in the interphase nucleus of the gonial cell, chromatin in micronuclei accumulated H3K9me3 epigenetic modifications indicating its inactivation and heterochromatinization (micronuclei indicated with thin red arrows). Tubulin (stained in green) visualize cytoskeleton components of the spindle ( a , f ). Chromatin is visualized with DAPI in blue. All pictures represent a single gonadal section of 0.4 μm in thickness after 3D immunofluorescent staining. Scale bars = 10 μm.

Article Snippet: We used the following primary antibodies: rabbit polyclonal antibody against DDX4 (concentration 1:50; C1C3, GeneTex) to detect Vasa protein; mouse monoclonal antibodies against alpha tubulin (concentration 1:100; ab7291, Abcam); rabbit polyclonal antibody against H3S10P (concentration 1:100; GTX128116, GeneTex); rabbit polyclonal antibody against H3S28P (concentration 1:100; #9713, Cell Signaling); rabbit polyclonal antibody against H3T11P (concentration 1:100; #9764, Cell Signaling); rabbit polyclonal antibody against H3K9me3 (concentration 1:100; ab8898, Abcam).

Techniques: Staining, Modification

Journal: Scientific Reports

Article Title: Gradual chromosomal lagging drive programmed genome elimination in hemiclonal fishes from the genus Hypseleotris

doi: 10.1038/s41598-024-78278-6

Figure Lengend Snippet: Analysis of epigenetic histone modifications during mitosis and micronuclei formation. Whole-mount immunofluorescent staining of gonads from sexual species ( a , b ) and hybrids (all rest) with antibodies against various epigenetic modification stained in red: H3S10P ( a , c , d ), H3S28P ( b , e ), H3K9me3 ( f , g ) and H3T11P ( h ). Accumulation of all analyzed chromatin modifications indicate normal chromosome condensation during prophase and metaphase. In hybrids, misaligned chromosomes (indicated by white arrows) have similar distribution of the H3S10P ( c ), H3S28P ( e ), H3K9me3 ( f ) and H3T11P ( h ) chromatin modifications as the rest of the chromosomes (showed by white arrowheads) suggesting that misaligned chromosomes condense properly during prophase and metaphase. Micronuclei present during metaphases (indicated by thin white arrows) do not accumulate epigenetic histone modification such as H3S10P ( c ), H3K9me3 ( f ). ( d ) Accumulation of H3S10P epigenetic modification in some micronuclei (indicated by thin red arrows) suggest their formation by chromosomal lagging. Some micronuclei do not show accumulation of H3S10P epigenetic modification (indicated by thin white arrows). ( g ) In contrast to the chromatin in the interphase nucleus of the gonial cell, chromatin in micronuclei accumulated H3K9me3 epigenetic modifications indicating its inactivation and heterochromatinization (micronuclei indicated with thin red arrows). Tubulin (stained in green) visualize cytoskeleton components of the spindle ( a , f ). Chromatin is visualized with DAPI in blue. All pictures represent a single gonadal section of 0.4 μm in thickness after 3D immunofluorescent staining. Scale bars = 10 μm.

Article Snippet: We used the following primary antibodies: rabbit polyclonal antibody against DDX4 (concentration 1:50; C1C3, GeneTex) to detect Vasa protein; mouse monoclonal antibodies against alpha tubulin (concentration 1:100; ab7291, Abcam); rabbit polyclonal antibody against H3S10P (concentration 1:100; GTX128116, GeneTex); rabbit polyclonal antibody against H3S28P (concentration 1:100; #9713, Cell Signaling); rabbit polyclonal antibody against H3T11P (concentration 1:100; #9764, Cell Signaling); rabbit polyclonal antibody against H3K9me3 (concentration 1:100; ab8898, Abcam).

Techniques: Staining, Modification

Journal: Scientific Reports

Article Title: Gradual chromosomal lagging drive programmed genome elimination in hemiclonal fishes from the genus Hypseleotris

doi: 10.1038/s41598-024-78278-6

Figure Lengend Snippet: Schematic overview of selective genome elimination in carp gudgeons. ( a ) Scheme of the reproduction of hybrid individuals and individuals of sexual species, H. bucephala . During gametogenesis in hybrids, H. bucephala genome (red) is eliminated, while H. gymnocephala genome (green) is transmitted to gametes. After fertilization by co-occurring sexual species H. bucephala , hybrid chromosomal composition is restored. ( b ) Suggested simplified scheme of gradual chromosome elimination via micronucleus formation in hybrids. ( c ) During eliminating mitosis all chromosomes accumulate epigenetic chromatin marks H3S10P, H3S28P and H3K9me3. After lagging and inclusion of individual chromosomes in micronuclei, we detected H3S10P modification in at least some micronuclei. In the micronuclei, chromatin accumulates heterochromatin marks.

Article Snippet: We used the following primary antibodies: rabbit polyclonal antibody against DDX4 (concentration 1:50; C1C3, GeneTex) to detect Vasa protein; mouse monoclonal antibodies against alpha tubulin (concentration 1:100; ab7291, Abcam); rabbit polyclonal antibody against H3S10P (concentration 1:100; GTX128116, GeneTex); rabbit polyclonal antibody against H3S28P (concentration 1:100; #9713, Cell Signaling); rabbit polyclonal antibody against H3T11P (concentration 1:100; #9764, Cell Signaling); rabbit polyclonal antibody against H3K9me3 (concentration 1:100; ab8898, Abcam).

Techniques: Modification

Journal: Scientific reports

Article Title: Gradual chromosomal lagging drive programmed genome elimination in hemiclonal fishes from the genus Hypseleotris.

doi: 10.1038/s41598-024-78278-6

Figure Lengend Snippet: Fig. 2. Analysis of epigenetic histone modifications during mitosis and micronuclei formation. Whole-mount immunofluorescent staining of gonads from sexual species (a, b) and hybrids (all rest) with antibodies against various epigenetic modification stained in red: H3S10P (a, c, d), H3S28P (b, e), H3K9me3 (f, g) and H3T11P (h). Accumulation of all analyzed chromatin modifications indicate normal chromosome condensation during prophase and metaphase. In hybrids, misaligned chromosomes (indicated by white arrows) have similar distribution of the H3S10P (c), H3S28P (e), H3K9me3 (f) and H3T11P (h) chromatin modifications as the rest of the chromosomes (showed by white arrowheads) suggesting that misaligned chromosomes condense properly during prophase and metaphase. Micronuclei present during metaphases (indicated by thin white arrows) do not accumulate epigenetic histone modification such as H3S10P (c), H3K9me3 (f). (d) Accumulation of H3S10P epigenetic modification in some micronuclei (indicated by thin red arrows) suggest their formation by chromosomal lagging. Some micronuclei do not show accumulation of H3S10P epigenetic modification (indicated by thin white arrows). (g) In contrast to the chromatin in the interphase nucleus of the gonial cell, chromatin in micronuclei accumulated H3K9me3 epigenetic modifications indicating its inactivation and heterochromatinization (micronuclei indicated with thin red arrows). Tubulin (stained in green) visualize cytoskeleton components of the spindle (a, f). Chromatin is visualized with DAPI in blue. All pictures represent a single gonadal section of 0.4 μm in thickness after 3D immunofluorescent staining. Scale bars = 10 μm.

Article Snippet: We used the following primary antibodies: rabbit polyclonal antibody against DDX4 (concentration 1:50; C1C3, GeneTex) to detect Vasa protein; mouse monoclonal antibodies against alpha tubulin (concentration 1:100; ab7291, Abcam); rabbit polyclonal antibody against H3S10P (concentration 1:100; GTX128116, GeneTex); rabbit polyclonal antibody against H3S28P (concentration 1:100; #9713, Cell Signaling); rabbit polyclonal antibody against H3T11P (concentration 1:100; #9764, Cell Signaling); rabbit polyclonal antibody against H3K9me3 (concentration 1:100; ab8898, Abcam).

Techniques: Staining, Modification

Journal: Scientific reports

Article Title: Gradual chromosomal lagging drive programmed genome elimination in hemiclonal fishes from the genus Hypseleotris.

doi: 10.1038/s41598-024-78278-6

Figure Lengend Snippet: Fig. 5. Schematic overview of selective genome elimination in carp gudgeons. (a) Scheme of the reproduction of hybrid individuals and individuals of sexual species, H. bucephala. During gametogenesis in hybrids, H. bucephala genome (red) is eliminated, while H. gymnocephala genome (green) is transmitted to gametes. After fertilization by co-occurring sexual species H. bucephala, hybrid chromosomal composition is restored. (b) Suggested simplified scheme of gradual chromosome elimination via micronucleus formation in hybrids. (c) During eliminating mitosis all chromosomes accumulate epigenetic chromatin marks H3S10P, H3S28P and H3K9me3. After lagging and inclusion of individual chromosomes in micronuclei, we detected H3S10P modification in at least some micronuclei. In the micronuclei, chromatin accumulates heterochromatin marks.

Article Snippet: We used the following primary antibodies: rabbit polyclonal antibody against DDX4 (concentration 1:50; C1C3, GeneTex) to detect Vasa protein; mouse monoclonal antibodies against alpha tubulin (concentration 1:100; ab7291, Abcam); rabbit polyclonal antibody against H3S10P (concentration 1:100; GTX128116, GeneTex); rabbit polyclonal antibody against H3S28P (concentration 1:100; #9713, Cell Signaling); rabbit polyclonal antibody against H3T11P (concentration 1:100; #9764, Cell Signaling); rabbit polyclonal antibody against H3K9me3 (concentration 1:100; ab8898, Abcam).

Techniques: Modification

Journal: Molecular Oncology

Article Title: DAPK3 participates in the mRNA processing of immediate early genes in chronic lymphocytic leukaemia

doi: 10.1002/1878-0261.12692

Figure Lengend Snippet: H3T6 and H3T11 phosphorylation at IEGs is catalysed by DAPK3 and is similarly abrogated by both BTK and DAPK3 inhibition. (A) qPCR data analysis of EGR1 and DUSP2 gene expression at 30 ( n = 6 patients) and 60 min ( n = 4 patients) post‐anti‐IgM stimulation in U‐CLL cells. CLL cells were pretreated with 1 µ m ibrutinib or 25 µ m DAPKi for 1 h as indicated below the graphs before anti‐IgM stimulation. Expression changes were quantified using the ∆ C t method with TBP and PPP6C as control genes. Bars represent the grand mean of the samples. Significant differences calculated using two‐way ANOVA followed by Dunnett's multiple comparison test with anti‐IgM as control. EGR1 (30 m) P values = 0.0009, 0.0015 and 0.0009 for anti‐IgM vs Unstim, Ibr and DAPKi respectively. DUSP2 (30 m) P values = 0.0013, 0.0023 and 0.0011 for the same comparisons. EGR1 (60 m) P values = 0.0039, 0.0052 and 0.0052 for the same comparisons. DUSP2 (60 m) P values = 0.0204, 0.0269 and 0.0205 for the same comparisons. (B) ChIP‐qPCR data assessing levels of H3T6‐P and H3T11‐P at the EGR1 and DUSP2 gene loci. CLL cells pretreated with either 1 µ m ibrutinib or 25 µ m DAPKi for 1 h before stimulation with anti‐IgM for 30 min. Values on the x axis refer to specific gene regions relative to the TSS in kilobases (kb) as indicated on the gene schematics below (not to scale). Error bars represent the SD of three independent experiments. CTCF1/3 (C1/C3) were used as negative control regions which are not indicated on the gene schematics. (C) co‐IP of CLL cells stimulated with anti‐IgM for 1 h as indicated. Immunoprecipitates from histone H3 pulldown were analysed by SDS/PAGE followed by western blot probing for DAPK3‐T265P. Untreated, crude cell lysate was used as positive control (input), and IgG beads were used for negative control (Ctrl IgG). Blot is representative of three independent co‐IP experiments. Western blot is cropped due to different exposure times for input and co‐IP lysates. (D) Kinase assay quantified by dot‐blot with recombinant histone H3 and recombinant DAPK3 to measure DAPK3 kinase activity over time (5–60 min) with or without (negative control) ATP and in the presence of 1 µ m ibrutinib and 25 µ m DAPK inhibitor. (E) Kinase assay displaying histone H3 phosphorylation quantified relative to untreated assay with error bars representing the SD of three independent experiments. Significant differences calculated using two‐way ANOVA followed by Dunnett's multiple comparison test with the untreated sample as control for each time point. H3T6‐P P values = 0.3075 and 0.0139 (5 m), 0.4157 and 0.0021 (20 m), 0.1583 and 0.0001 (60 m) for untreated vs. Ibr and DAPKi, respectively. H3T11‐P P values = 0.6679 and 0.6844 (5 m), 0.0672 and 0.0095 (20 m), 0.0043 and 0.0001 (60 m) for the same comparisons. Ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: Reaction samples were spotted onto nitrocellulose membranes and air dried before blocking in 5% BSA at room temperature for 1 h. Membranes were incubated at 4 °C overnight with anti‐histone H3T11‐P (Abcam; ab5168) and anti‐histone H3T6‐P (Diagenode; C15410282).

Techniques: Inhibition, Expressing, Negative Control, Co-Immunoprecipitation Assay, SDS Page, Western Blot, Positive Control, Kinase Assay, Dot Blot, Recombinant, Activity Assay

Journal: Molecular Oncology

Article Title: DAPK3 participates in the mRNA processing of immediate early genes in chronic lymphocytic leukaemia

doi: 10.1002/1878-0261.12692

Figure Lengend Snippet: DAPK3‐mediated histone H3 threonine phosphorylation is required for faithful co‐transcriptional splicing. (A) EGR1/DUSP2 gene schematics showing introns and exons. Primer locations for qPCR and RT‐PCR indicated by vertical arrows and blue boxes. Numbers after gene names correspond to the position of the primers in and around the gene relative to the TSS, where + denotes 3′ of the TSS and − denotes 5′ of the TSS. Reverse transcription PCR (RT‐PCR) analysis comparing levels of processed EGR1/DUSP2 mRNA (top left) with EGR1/DUSP2 primary transcript levels (bottom left, top right, bottom right). CLL cells were pretreated with either 1 µ m ibrutinib or 25 µ m DAPKi for 1 h as indicated and then stimulated with anti‐IgM and sCD40L to activate IEG expression. Extracted RNA was reverse transcribed with either random primer (RP) or oligo‐dT, subjected to RT‐PCR and analysed by agarose gel electrophoresis. (B) qPCR analysis of EGR1/DUSP2 primary transcript (shades of blue) vs processed mRNA (red) from HBL1 cells pretreated with either 1 µ m ibrutinib or 25 µ m DAPKi for 1 h and then stimulated with or without anti‐IgM and CD40L for 1 h as indicated. Error bars represent the SD of n = 3 independent experiments. (C) Western blots of HBL1 cells transfected with siRNAs against DAPK3 (#1 and #2) and with a nonspecific control siRNA (siCtrl). On day 5 post‐transfection, cells were harvested and lysates probed with antibodies against DAPK3, DAPK3‐T265‐P, histone H3T6‐P and histone H3T11‐P. β‐actin was used as a loading control and for normalisation to siCtrl using image lab software (right). Blots are representative of three independent transfections. Significant differences calculated using two‐way ANOVA followed by Dunnett's multiple comparison test. P values for DAPK3 expression normalised to siCtrl = 0.0036 and 0.0004 for siCtrl vs. siDAPK3 #1 and siDAPK3 #2, respectively. (D) qPCR analysis of HBL1 cells transfected with siRNAs against DAPK3 (#1 and #2) and with a nonspecific control siRNA (siCtrl). On day 5 post‐transfection, HBL1 cells were stimulated with anti‐IgM for 1 h and then harvested for analysis. Expression changes were quantified using the ∆ C t method with TBP, GAPDH and PPP6C as control genes. Error bars represent the SD of three independent transfections. Significant differences calculated using two‐way ANOVA followed by Dunnett's multiple comparison test with anti‐IgM stimulated siCtrl as control. P values for EGR1, EGR1 +0.55 kb and EGR1 +1.0 kb = 0.0082 and 0.0009, 0.1163 and 0.2759, 0.0047 and 0.0270 for siCtrl vs. siDAPK3 #1 and siDAPK3 #2, respectively. Ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: Reaction samples were spotted onto nitrocellulose membranes and air dried before blocking in 5% BSA at room temperature for 1 h. Membranes were incubated at 4 °C overnight with anti‐histone H3T11‐P (Abcam; ab5168) and anti‐histone H3T6‐P (Diagenode; C15410282).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Western Blot, Transfection, Software