Journal: bioRxiv

Article Title: A time-resolved atlas of histone modifications during mitotic entry

doi: 10.1101/2025.09.29.679131

Figure Lengend Snippet: A – A Pearson correlation heatmap of ChIP-sequencing of H3T3ph, H3K4me1, H3K4me2 and H3K4me3 throughout mitotic entry. B – Numbers of enriched peaks at different timepoints. Bars represent mean ± standard deviation. n=2. C – A UCSC genome browser view showing the normalized (log 2 (IP/Input)) ChIP-seq read densities of H3K4me1 (light blue), H3K4me2 (dark green), H3K4me3 (violet) and H3T3ph (dark blue) on the chromosome 1. Regions shaded in pink (red *) highlight the overlap between H3K4me1 and H3T3ph. D – A magnified view of the red rectangle region from panel C, the TSS of POU2F1 gene. E – Profile plots showing the normalized ChIP-seq signal densities of H3K4me1, H3K4me2, H3K4me3, and H3T3ph around the TSS (TSS ± 20 kb) of protein-coding genes.

Article Snippet: Cells were permeabilized with 0.2% Triton-X100 for 5 min. After washing with PBS, the cells were incubated for 1 hour with 5% BSA in PBS and then with primary antibodies, H3T3ph (16B2; mouse; diluted 1:500 in blocking buffer) and ACA (human; Antibodies Incorporated; 15-234; diluted 1:500 in blocking buffer), in 1% BSA and 0.1% Tween-20 in PBS overnight at 4°C.

Techniques: ChIP-sequencing, Standard Deviation

Journal: bioRxiv

Article Title: A time-resolved atlas of histone modifications during mitotic entry

doi: 10.1101/2025.09.29.679131

Figure Lengend Snippet: A – A Pearson correlation heatmap of ChIP-seq of H3T3ph throughout mitotic entry with H3K9me3 and H3K27me3. B – A violin plot of spike-in calibrated H3T3ph levels at the repetitive centromere of Chr8, as well as its p- and q-arms. C – The same but for Chr5 with the non-repetitive centromere. Statistics for B and C was computed by fitting a linear mixed effect model of log(enrichment). “Timepoint” and “position along the chromosome“ were fit as fixed effects, and “sample” as a random effect. Gaussian distribution of models’ residuals was confirmed. Since T-distribution is symmetric, one-sided p value was calculated for the null hypothesis of no centromeric enrichment of H3T3ph. The hypothesis was rejected in the case of a repetitive centromere on Chr8 (p < 0.0001, ****). The p-values were adjusted with Benjamini-Hochberg procedure. D – A UCSC genome browser view of the magnified centromeric region of Chr8 and Chr5. CENP-H ChIP-seq shows CENP-H (red)-binding peaks (spike-in calibrated) at the centromere of Chr8 (Left) and Chr5 (Right). Other tracks show landscape profile of H3T3ph (dark blue), H3K9me3 (dark pink) and H3K27me3 (dark orange) signal (spike-in calibrated or log 2 (IP/Input)) around the centromeric region.

Article Snippet: Cells were permeabilized with 0.2% Triton-X100 for 5 min. After washing with PBS, the cells were incubated for 1 hour with 5% BSA in PBS and then with primary antibodies, H3T3ph (16B2; mouse; diluted 1:500 in blocking buffer) and ACA (human; Antibodies Incorporated; 15-234; diluted 1:500 in blocking buffer), in 1% BSA and 0.1% Tween-20 in PBS overnight at 4°C.

Techniques: ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Reduced Levels of Lagging Strand Polymerases Shape Stem Cell Chromatin

doi: 10.1101/2024.04.26.591383

Figure Lengend Snippet: ( a-b ) Representative images of: ( a ) an M-phase GSC showing more compact old H3-enriched regions than new H3-enriched regions (positive with a mitotic marker anti-H3S10ph, H3S10P or S10P ; ( b ) an M-phase 8-cell SG showing equally compact old H3-enriched and new H3-enriched regions (positive with S10P) in the control wild-type testes. ( c-d ) Representative images of: ( c ) an M-phase GSC and ( d ) an M-phase 8-cell SG in the pola50 +/- testes, both showing more compact old H3-enriched regions than new H3-enriched regions (positive with a mitotic marker anti-H3T3ph, H3T3P or T3P. ( e ) Compaction index in log 2 scale: Control GSC= 1.27± 0.15 (n=15), Control 8-cell SG= 0.41± 0.05 (n=12), polα50 +/- GSC= 1.21± 0.15 (n=13), and polα50 +/- 8-cell SG= 1.48± 0.14 (n=11). The control compaction index data are from with permission. See for details. All ratios: Mean± SEM. Mann-Whitney test, ****: P < 10 -4 , ns: not significant.

Article Snippet: Primary antibodies used were Armadillo (Arm, 1:100; DSHB N2 7A1), Traffic Jam (Tj, 1:100, from Mark Van Doren, Johns Hopkins University, USA), anti-PCNA (1:100; Santa Cruz sc-56), anti-GFP (1:1,000; Abcam ab 13970), anti-HA (1:200; Sigma-Aldrich H3663), anti-mCherry (1:1,000; Invitrogen M11217), anti-H3K27me3 (1:400; Millipore 07-449), anti-H4K20me2/3 (1:400; Abcam ab78517), anti-H3S10ph (1:2000; Cell Signaling Technology 9701), rabbit anti-H3T3ph (1:200, Millipore 05-746R), and anti-BrdU (1:200; Abcam ab6326).

Techniques: Marker, MANN-WHITNEY