Journal: bioRxiv

Article Title: Linker Histone H1.5 Contributes to Centromere Integrity in Human Cells

doi: 10.1101/2025.06.03.657682

Figure Lengend Snippet: H1.5 might bind CENP-A mononucleosomes in a non-canonical manner. (A) Schematic representation of in vitro reconstitution of mononucleosomes followed by AFM imaging using tapping mode. (B) Representative AFM images of H3 and CENP-A mononucleosomes with and without H1.5. (C) Boxplot of nucleosome height measurements of H3 and CENP-A mononucleosomes with and without H1.5. (D) Boxplot of the Feret’s diameter measurements of H3 and CENP-A mononucleosomes with and without H1.5. (E) Boxplot of length of DNA fragment, as well as the long and short arm of the entry and exit DNA strands from the nucleosomes. (F) Boxplot of the angle between the long and short DNA arms of H3 and CENP-A mononucleosomes with and without H1.5.

Article Snippet: H3 and CENP-A mononucleosomes (H3/H4 cat#16-0008, CENP-A/H4 cat#16-010, H2A/H2B cat#15-0311, Epicypher) were in vitro reconstituted on a 324-bp DNA fragment (PCR’ed from pGEM-3z601 plasmid from Addgene cat#26656) by stepwise salt-dialysis as described previously ( ).

Techniques: In Vitro, Imaging

Journal: bioRxiv

Article Title: Linker Histone H1.5 Contributes to Centromere Integrity in Human Cells

doi: 10.1101/2025.06.03.657682

Figure Lengend Snippet: H1.5 is selectively reduced upon siRNA knockdown. (A) HPLC chromatograms of acid-extracted histones from siScramble (blue) and siH1.5-treated (red) SVGp12 cells show a selective reduction in H1.5 peak intensity following knockdown. Quantification of H1/nucleosome ratios (normalized to H2B) confirms a significant reduction of total H1 in siH1.5-treated samples (0.29) compared to siScramble control (0.44). (B) RNA-seq quantification of histone H1 mRNA subtypes reveals a significant reduction in H1.5 (p < 0.01) and a mild but significant increase in H1.0 ( p < 0.05 ) upon siH1.5 knockdown. No significant change (ns) was observed in H1.1, H1.2, H1.3, or H1.4 transcripts (n = 2 biological replicates, error bars indicate SEM).

Article Snippet: H3 and CENP-A mononucleosomes (H3/H4 cat#16-0008, CENP-A/H4 cat#16-010, H2A/H2B cat#15-0311, Epicypher) were in vitro reconstituted on a 324-bp DNA fragment (PCR’ed from pGEM-3z601 plasmid from Addgene cat#26656) by stepwise salt-dialysis as described previously ( ).

Techniques: Knockdown, Control, RNA Sequencing

Journal: bioRxiv

Article Title: Linker Histone H1.5 Contributes to Centromere Integrity in Human Cells

doi: 10.1101/2025.06.03.657682

Figure Lengend Snippet: H1.5 interacts with CENP-A mononucleosomes, in vivo . (A) Schematic depicting MNase digestion of HeLa cells followed by CENP-A ChIP. (B) MNase ladder depicting digestion of chromatin to mononucleosomes. ChIP followed by Westerns against H1.5, CENP-A, H3, and H2A histones. 2.5% Input/Unbound and 100% CENP-A ChIP samples loaded. Unbound = supernatant not bound to CENP-A antibody/beads complex. HC=heavy chain of anti-CENP-A ChIP antibody. (C) Venn diagram comparing total shared peaks between native CENP-A and H1.5 histones. (D) Genome browser snapshots of overlapping native CENP-A and H1.5 peaks.

Article Snippet: H3 and CENP-A mononucleosomes (H3/H4 cat#16-0008, CENP-A/H4 cat#16-010, H2A/H2B cat#15-0311, Epicypher) were in vitro reconstituted on a 324-bp DNA fragment (PCR’ed from pGEM-3z601 plasmid from Addgene cat#26656) by stepwise salt-dialysis as described previously ( ).

Techniques: In Vivo

Journal: bioRxiv

Article Title: Linker Histone H1.5 Contributes to Centromere Integrity in Human Cells

doi: 10.1101/2025.06.03.657682

Figure Lengend Snippet: H1.5 associates with CENP-A nucleosomes in vivo. (A) In vivo ChIP verification of chromatin prepared using medium (4 min MNase digestion) or mononucleosome (8 min MNase digestion) conditions, using two bead formats: Sepharose and Dynabeads Protein A. Immunoprecipitation was performed with antibodies against H1.5, CENP-A, or control IgG. Western blotting reveals co-precipitation of H1.5 and CENP-A from chromatin, along with core histones H3 and H2A. Multiplex fluorescent detection of histone bands is shown below the Western panels. Notably, Dynabeads Protein A showed minimal non-specific binding in mock immunoprecipitated samples, and was thus used for all subsequent H1.5 ChIP experiments. (B) Co-IP using anti-CENP-A and anti-H1.5 antibodies from HeLa cells expressing wild-type or truncated H1.5 (Δ157–227-HA). CENP-A robustly co-precipitates with both endogenous and truncated H1.5, suggesting that the C-terminal tail is dispensable for the interaction. (C) Comparative Western blot analysis and fluorescent imaging of histone content in HA-ChIP and CENP-A-ChIP samples confirms the shared presence of CENP-A, H3, and H2A in nucleosome fractions pulled down with H1.5.

Article Snippet: H3 and CENP-A mononucleosomes (H3/H4 cat#16-0008, CENP-A/H4 cat#16-010, H2A/H2B cat#15-0311, Epicypher) were in vitro reconstituted on a 324-bp DNA fragment (PCR’ed from pGEM-3z601 plasmid from Addgene cat#26656) by stepwise salt-dialysis as described previously ( ).

Techniques: In Vivo, Immunoprecipitation, Control, Western Blot, Multiplex Assay, Binding Assay, Co-Immunoprecipitation Assay, Expressing, Imaging