Journal: bioRxiv

Article Title: Roles of histone chaperone Nap1 and histone acetylation in regulating phase-separation of chromatin arrays

doi: 10.1101/2025.05.09.653121

Figure Lengend Snippet: a Experimental scheme to form chromatin condensates: Dodecamer nucleosome arrays at 200 nM incubated in a buffer containing 150 mM NaCl result in condensate droplets. The scale bar is 10 μm. b Droplets fuse with each other in a few seconds timescale, confirming their liquid-like nature. The scale bars are 3μm. c Fluorescence recovery after photobleaching (FRAP) also confirms the liquid-like nature of the droplets. The scale bars are 3μm.

Article Snippet: Two short adapter DNA fragments (Integrated DNA Technologies, Coralville, IA) were ligated to the sticky ends of the purified nucleosome array DNA with T4 DNA ligase (New England Biolabs) at 16 °C for 14 hours followed by inactivation at 65 °C for 20 minutes.

Techniques: Incubation, Fluorescence

Journal: bioRxiv

Article Title: Roles of histone chaperone Nap1 and histone acetylation in regulating phase-separation of chromatin arrays

doi: 10.1101/2025.05.09.653121

Figure Lengend Snippet: a Nucleosome arrays with the H4 tail acetylation mimic (H4KQ) do not form condensates, but those with the H3 tail acetylation mimic (H3KQ) form spherical phase-separated droplets. Nucleosome arrays with H4 or H3 tails truncated (gH4 or gH3) form solid or gel-like aggregates. The scale bars are 10 μm. b In situ H4 tail acetylation by Piccolo NuA4 histone acetyltransferase dissolves the phase-separated droplets already formed with nucleosome arrays with no modifications, confirming the effect shown in a . Incubation with the enzyme and coenzyme A (CoA) instead of acetyl-CoA (Ac-CoA) does not dissolve the condensates, confirming that the effect is acetylation-dependent. The white scale bars are 3 μm. c In situ H3 tail acetylation with Ada2/Ada3/Gcn5 histone acetyltransferase does not affect phase separation of nucleosome arrays. The scale bars are 10 μm.

Article Snippet: Two short adapter DNA fragments (Integrated DNA Technologies, Coralville, IA) were ligated to the sticky ends of the purified nucleosome array DNA with T4 DNA ligase (New England Biolabs) at 16 °C for 14 hours followed by inactivation at 65 °C for 20 minutes.

Techniques: In Situ, Incubation

Journal: bioRxiv

Article Title: Roles of histone chaperone Nap1 and histone acetylation in regulating phase-separation of chromatin arrays

doi: 10.1101/2025.05.09.653121

Figure Lengend Snippet: a Histone chaperone Nap1 dissolves pre-formed chromatin aggregates with nucleosome arrays containing tailless histone H3 (gH3) at a molar ratio of 1:2 (nucleosome:Nap1). b Histone chaperone Nap1 pre-mixed with gH3 nucleosome arrays at a molar ratio of 1:2 (nucleosome:Nap1) before adding 150 mM NaCl significantly inhibits the formation of solid or gel-like chromatin aggregates. c The effect of Nap1 presented in a is not observed with spherical droplets formed with arrays with no modification (WT) up to a molar ratio of 1:8 (nucleosome:Nap1) for 30 min. All scale bars are 10 μm.

Article Snippet: Two short adapter DNA fragments (Integrated DNA Technologies, Coralville, IA) were ligated to the sticky ends of the purified nucleosome array DNA with T4 DNA ligase (New England Biolabs) at 16 °C for 14 hours followed by inactivation at 65 °C for 20 minutes.

Techniques: Modification

Journal: bioRxiv

Article Title: Roles of histone chaperone Nap1 and histone acetylation in regulating phase-separation of chromatin arrays

doi: 10.1101/2025.05.09.653121

Figure Lengend Snippet: a FRAP recovery times and recovery fractions are plotted for the condensates formed with unmodified nucleosome arrays (WT) labeled at the histone H4 E63C (Tet) and at H2B T115C (Di), unmodified arrays in the presence of Nap1 (Nap1) labeled at Tet and Di, and H3KQ arrays (H3KQ) labeled at Tet and Di. The recovery times of unmodified nucleosome arrays labeled at the histone H4 E63C (Nap1 Tet) and at H2B T115C (Nap1 Di) in the presence of Nap1 are faster than those in its absence, indicating that Nap1 enhances nucleosome dynamics in condensates. The FRAP recovery times of unmodified nucleosome arrays labeled at histone H4 E63C in the presence and absence of Nap1 (WT Tet and Nap1 Tet) are slower than those of unmodified nucleosome arrays labeled at histone H2B T115C (WT Di and Nap1 Di), indicating that histone H2A-H2B dimers diffuse faster than nucleosomes. Nucleosome arrays with H3KQ show dramatically faster FRAP recovery compared to the unmodified arrays (WT). FRAP recovery fractions are in the range of ∼50 - ∼60 % in all cases. A higher FRAP recovery fraction of the Di-labeled unmodified arrays (WT Di) than that of the Tet-labeled WT arrays (WT Tet) is noted. The same trend is noted with H3KQ arrays (H3KQ Tet vs H3KQ Di). The significances shown are from two-sided student’s t-test (*: p ≤0.05, **: p≤0.01, ***: p≤0.001, ****: p≤0.0001, ns: not significant). The sample size n = 12, 16, 17, 15, 9, and 17, respectively for the WT Tet, WT Di, Nap1 Tet, Nap1 Di, H3KQ Tet, and H3KQ Di cases. The sample size refers to the number of distinct droplets tested in each case. The samples and the measurements were made on at least two different days. The values marked on the box plots represent mean ± standard deviation. b Concentrations of the nucleosome arrays in the three cases (WT, Nap1, and H3KQ) reveal an elevated concentration of nucleosomes in the presence of Nap1. The concentrations in the WT and H3KQ droplets are the same within error. The scale bars are 3 μm. The sample size n = 15, 18, and 21 respectively for the WT Tet, Nap1 Tet, and H3KQ Tet cases. The sample size refers to the number of distinct droplets tested in each case. The samples and the measurements were made on at least two different days. c The effect of Nap1 in elevating the nucleosome concentration in chromatin condensates does not result in droplet formation with H4KQ arrays up to two hours.

Article Snippet: Two short adapter DNA fragments (Integrated DNA Technologies, Coralville, IA) were ligated to the sticky ends of the purified nucleosome array DNA with T4 DNA ligase (New England Biolabs) at 16 °C for 14 hours followed by inactivation at 65 °C for 20 minutes.

Techniques: Labeling, Standard Deviation, Concentration Assay

Journal: bioRxiv

Article Title: Roles of histone chaperone Nap1 and histone acetylation in regulating phase-separation of chromatin arrays

doi: 10.1101/2025.05.09.653121

Figure Lengend Snippet: Fluorescently labeled nucleosome arrays at H4 E63C were imaged with STORM. The “Total” images for 10 s represent the nucleosome particles identified during a 10-sec period of STORM imaging. The “Clusters” images for 10 s represent five or more histone H4 molecules clustered together within 110 nm from each other during a 10-sec period. The “Total” images for 400 s represent the overlap of the 40 consecutive 10-s “Total” images. The “Clusters” images for 400 s represent the overlap of the 40 consecutive 10-s “Clusters” images. These STORM images of both WT and H3KQ arrays confirm a structural scaffold of nucleosome arrays that are relatively immobile on the timescale of 10 seconds. The z-position of the localized nucleosomes are color coded as shown in the color scale.

Article Snippet: Two short adapter DNA fragments (Integrated DNA Technologies, Coralville, IA) were ligated to the sticky ends of the purified nucleosome array DNA with T4 DNA ligase (New England Biolabs) at 16 °C for 14 hours followed by inactivation at 65 °C for 20 minutes.

Techniques: Labeling, Imaging

Journal: bioRxiv

Article Title: Roles of histone chaperone Nap1 and histone acetylation in regulating phase-separation of chromatin arrays

doi: 10.1101/2025.05.09.653121

Figure Lengend Snippet: a Experiments were set up based on optical tweezers to measure the dynamic modulus of nucleosome array droplets. A 1 μm polystyrene bead is brought inside a condensate whose diameter is at least 4 μm. The bead is sinusoidally oscillated at 7 different frequencies (left). The oscillation amplitude of the bead and the phase lag between the bead and the trapping beam are monitored (center). The lag and the attenuated amplitude of the bead are fit with a Burgers model with two Maxwell components (right). b Examples of dynamic modulus fitting and results for unmodified arrays (WT), WT arrays with Nap1 (Nap1), and arrays with the H3 tail acetylation mimic (H3KQ). c The fitting results are summarized for the two relaxation time components τ 0 and τ 1 , the two viscosity components η 0 and η 1 , and their sum η tot , the two corresponding elastic modulus μ 0 and μ 1 , and their sum μ tot . The longer time relaxation component and its viscosity is significantly shorter and lower in the Nap1 and H3KQ cases than the WT case. As the longer relaxation component is the dominant component, the total viscosity is also significantly lower with the Nap1 and H3KQ cases, revealing less viscous and more fluidic condensates induced by these changes. The sample size n = 19, 20, and 16, respectively for the WT, Nap1, and H3KQ cases. The sample size refers to the number of distinct droplets tested in each case. The samples and the measurements were made on at least two different days. The values marked on the box plots represent mean ± standard deviation. The significances shown are from two-sided student’s t-test (*: p ≤0.05, **: p≤0.01, ***: p≤0.001, ****: p≤0.0001, ns: not significant).

Article Snippet: Two short adapter DNA fragments (Integrated DNA Technologies, Coralville, IA) were ligated to the sticky ends of the purified nucleosome array DNA with T4 DNA ligase (New England Biolabs) at 16 °C for 14 hours followed by inactivation at 65 °C for 20 minutes.

Techniques: Viscosity, Standard Deviation

Journal: eLife

Article Title: Concerted regulation of ISWI by an autoinhibitory domain and the H4 N-terminal tail

doi: 10.7554/eLife.21477

Figure Lengend Snippet: ( A ) N-terminal truncation mutants of Drosophila ISWI. ( B ) DNA- and nucleosome-stimulated ATP turnover. ATPase rates were measured in the presence of saturating concentrations of ATP (1 mM), DNA (0.2 g/l) or nucleosomes (0.1 g/l). Errors for nucleosome-stimulated rates of ISWI deletion mutants are minimal and maximal values of two independent measurements, and s.d. for all other measurements (n ≥ 4). ATPase rates in absence of nucleic acids were <0.022 s −1 for all ISWI variants (data not shown). ( C ) Remodeling activity was determined by measuring the accessibility changes of a unique KpnI restriction site in a 25-mer nucleosomal array (100 nM nucleosomes, 300 nM enzyme). Errors are s.d. (n ≥ 3) except for ISWI ΔppHSA; ΔAT-hook for which minimal and maximal values of two independent measurements are shown. Raw data of the remodeling assay can be found in . Color code as in panel B . DOI: http://dx.doi.org/10.7554/eLife.21477.007

Article Snippet: DNA for 25-mer nucleosomal arrays used in remodeling assays was excised from pFMP233 with EcoRI HF, HincII and AseI (NEB) and purified by phenol/chloroform extraction and ethanol precipitation.

Techniques: Activity Assay

Journal: bioRxiv

Article Title: High-resolution dissection of human cell type-specific enhancers in cis and trans activities

doi: 10.1101/2023.09.23.559140

Figure Lengend Snippet: (A) Experimental design and procedures of ChIP-STARR-seq in cis and trans condition. K C K D – K562 cells with K562 DNA; A C A D – A549 cells with A549 DNA; A C K D – A549 cells with K562 DNA; K C A D – K562 cells with A549 DNA. (B) Volcano plot showing the identified active enhancers in each condition. (C) Example genome browser snapshot showing the reporter RNA FPKM signals, isolated plasmid DNA FPKM signals and the calculated enhancer activities measured by the log2 fold change between RNA signals and DNA signals. (D) Genomic distribution of the active enhancers identified in each condition. (E) Distance distribution of the active enhancers to their nearest transcription start site (TSS) in each condition.

Article Snippet: Dinucleosome ChIP DNA fragments were selected using Pippin Prep with 1.5% agarose gel cassette and dye-free internal marker K (Sage Science CDF1510).

Techniques: Isolation, Plasmid Preparation