Behaviors of nucleosomes with mutant histone H4s in euchromatic domains of living human cells

doi:10.21203/rs.3.rs-3953132/v1

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Behaviors of nucleosomes with mutant histone H4s in euchromatic domains of living human cells

https://doi.org/10.21203/rs.3.rs-3953132/v1

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published 14 May, 2024

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Since Robert Feulgen first stained DNA in the cell, visualizing genome chromatin has been a central issue in cell biology to uncover how chromatin is organized and behaves in the cell. To approach this issue, we have developed single-molecule imaging of nucleosomes, a basic unit of chromatin, to unveil local nucleosome behavior in living cells. In this study, we investigated behaviors of nucleosomes with various histone H4 mutants in living HeLa cells to address the role of H4 tail acetylation, including H4K16Ac and others, which are generally associated with more transcriptionally active chromatin regions. We ectopically expressed wild-type (wt) or mutated H4s (H4K16 point, H4K5,8,12,16 quadruple, and H4 tail deletion) fused with HaloTag in HeLa cells. Expressed wtH4-Halo, H4K16-Halo mutants, and multiple H4-Halo mutants had the euchromatin-concentrated distribution. Consistently, the genomic regions of the wtH4-Halo nucleosomes corresponded to Hi-C contact domains with active chromatin marks (A-compartment). Utilizing single-nucleosome imaging, we found that none of the H4 deacetylation or acetylation mimicked H4 mutants altered the overall local nucleosome motion. This finding suggests that H4 mutant nucleosomes embedded in the condensed euchromatic domains with excess endogenous H4 nucleosomes cannot cause an observable change in the local motion. Interestingly, H4 with four lysine-to-arginine mutations displayed a substantial freely diffusing fraction in the nucleoplasm, whereas H4 with a truncated N-terminal tail was incorporated in heterochromatic regions as well as euchromatin. Our study indicates the power of single-nucleosome imaging to understand individual histone/nucleosome behavior reflecting chromatin environments in living cells.

single-nucleosome imaging

histone H4

nucleosome

acetylation

H4K16Ac

chromatin

Exactly a century ago, Robert Feulgen described the first method to visualize DNA chains, where the genetic information is stored inside the cells (Benedum and Meusch 1999), long before the double-helical structure of DNA was revealed (Watson and Crick 1953). DNA is a negatively charged polymer (Maeshima et al. 2021) and is wrapped around the octamer of basic core histone proteins (two copies each of H2A, H2B, H3, and H4) via electrostatic interactions to form a nucleosome (Olins and Olins 2003; Luger et al. 1997; McGinty and Tan 2015; Koyama and Kurumizaka 2018). A string of nucleosomes is rather irregularly folded in the condensed chromatin domains in both euchromatin and heterochromatin in the nucleus (Misteli 2020) (Cremer et al. 2020; Miron et al. 2020; Zakirov et al. 2021; Maeshima et al. 2024). Recent studies have revealed that nucleosomes fluctuate (Iida et al. 2022) and behave like a liquid in the condensed domains of living cells (Nozaki et al. 2023).

In the nucleosome, all core histones have the intrinsically disordered N-terminal tail and globular histone fold domains (Luger et al. 1997; McGinty and Tan 2015; Koyama and Kurumizaka 2018). The N-terminal tail domain protrudes away from the nucleosome particle (Fig. 1a) and is crucial to binding other nucleosomes (Dorigo et al. 2003; Pepenella et al. 2014; Hansen et al. 2021). The N-terminal tail is often subjected to various post-translational modifications, which play a role in establishing or quenching higher-order chromatin structures (Mersfelder and Parthun 2006; Millan-Zambrano et al. 2022). Histone tail acetylation at numerous lysine residues neutralizes their positive charge, thereby weakening their attraction to DNA or acidic patches of neighboring nucleosomes, which in turn causes chromatin decompaction (Mann and Grunstein 1992) (Grunstein 1997) (Dion et al. 2005; Millan-Zambrano et al. 2022).

Although there are several acetylation sites within the N-terminal tail, acetylation of histone H4 lysine-16 (H4K16Ac) is enough to disrupt the condensate formation of 12-mer nucleosome arrays (Shogren-Knaak et al. 2006; Robinson et al. 2008; Allahverdi et al. 2011). The effect of H4K16Ac was similar to that of a complete removal of the N-terminal tail of H4 (H4Δ1–19) in vitro (Shogren-Knaak et al. 2006). Since histone acetylation has been linked to more ‘open’ and dynamic chromatin states, H4K16Ac is thought to be involved in gene transcription and DNA damage response in vivo (Grunstein 1997). H4K16Ac is also targeted to the X chromosome of male flies and is important for dosage compensation (Bone et al. 1994). Li et al. showed that depletion of MOF/KAT8, which is the major enzyme to catalyze H4K16Ac, led to massive chromatin compaction with a significant increase in densely stained heterochromatin in the mouse ES cell (mESC) (Li et al. 2012). However, Taylor et al. reported that loss of H4K16Ac did not alter higher-order chromatin compaction during mouse ESCs differentiation (Taylor et al. 2013). Thus, the functional roles of H4K16Ac in regulating chromatin organization in the cell remain unclear. It is intriguing to investigate whether H4K16Ac can make the nucleosome locally more dynamic and mobile in human cells.

To approach this issue, we investigated behaviors of nucleosomes with various histone H4 mutants in living human cells using single-nucleosome imaging/tracking (Hihara et al. 2012; Nozaki et al. 2017; Lerner et al. 2020; Ide et al. 2022; Lakadamyali 2022). We stably expressed wild-type (wt) or various H4 mutants tagged with HaloTag in HeLa cells (i.e., wtH4 or H4K16 point, H4K5,8,12,16 quadruple, and H4 tail deletion mutants). Localizations of the ectopically expressed wt and mutant H4s were concentrated into euchromatic regions. Consistently, the genome-wide analysis showed that the genomic regions of the wtH4-Halo nucleosomes corresponded to Hi-C contact domains with active chromatin marks (A-compartment). Using single-nucleosome imaging, we found that all H4 mutants had similar local chromatin behavior to the one displayed by wtH4, suggesting that nucleosomes with mutant H4 are embedded in the condensed domains with an excess of endogenous H4 nucleosomes and that mutations within the N-terminal tail of H4 are insufficient to cause an observable change in the local motion. Interestingly, the nuclear localization of mutant H4-Halo was more uniform in cells expressing H4 with four lysine-to-arginine mutations (H4R-quad) or H4 mutants with a truncated N-terminal tail (H4Δ1–19). H4R-quad displayed a substantial freely diffusing fraction in the nucleoplasm, whereas H4Δ1–19 was also incorporated in heterochromatic regions. This study underscores the utility of single-nucleosome imaging in unraveling the behavior of individual histones/nucleosomes within the complex chromatin environment of living cells.

Construction of histone H4-HaloTag expression plasmids

The human histone H4 (H4C1) coding region was amplified from the total cDNA library of RPE-1 cells and displaced H2B in PB-EF1a-H2B-HaloTag-IRES-Puro previously made in our laboratory. The plasmid for tailless histone H4-HaloTag, H4(∆1–19)-HaloTag, was obtained from PB-EF1a-H4-HaloTag-IRES-Neo by incomplete amplification of the H4 coding region, excluding amino acid residues 1–19. Later, PB-EF1a-H4(∆1–19)-HaloTag-IRES-Neo was used to make the constructs to express histone H4 quadruple mutants, H4K(5,8,12,16)mut-HaloTag, by reconstruction of the H4 N-terminal tail with PCR primers including four identical pre-synthesized mutations: AAG (Lys) -> AGG (Arg), CAG (Gln), or GCG (Ala). To achieve stable and comparable ectopic expression levels of the fusion protein in HeLa cells, the coding regions for H4-HaloTag, H4(∆1–19)-HaloTag, and H4K(5,8,12,16)mut-HaloTag were all transferred to the Flp-In system vector (V602020; Invitrogen). The resulting plasmids were named pEF5-H4-HaloTag-FRT, pEF5-H4(∆1–19)-HaloTag-FRT, and pEF5-H4K(5,8,12,16)mut-HaloTag-FRT, respectively.

H4K16 point substitution mutants, H4K16R- and H4K16A-HaloTag, were obtained through PCR amplification of pEF5-H4-HaloTag-FRT with appropriate pairs of mutagenic PCR primers. H4K16Q-HaloTag was first obtained from PCR amplification of PB-EF1a-H4-HaloTag-IRES-Puro with mutagenic PCR primers. Then the H4K16Q-HaloTag fragment was transferred to the Flp-In system vector to obtain pEF5-H4K16Q-HaloTag-FRT.

Establishment of stable cell lines

HeLa cells containing the flippase recognition target (FRT) site in their genome, HeLa FRT-bla, were used as the model cell line. HeLa cells (Maeshima et al. 2006) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, D5796-500ML; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, ABB213138; HyClone) at 37°C in 5% CO₂.

To establish HeLa cells with stable ectopic expression of H4-HaloTag or mutant H4-HaloTag, the corresponding plasmids were co-transfected with pOG44 (V600520; Thermo Fischer Scientific) using the Effectene Transfection Reagent kit (301425; QIAGEN) in a 12-well plate. Two days following the transfection, the cell populations were expanded on 100-mm dishes with the medium containing 200 µg/mL hygromycin B (10687010; Thermo Fischer Scientific) for positive selection. The medium was refreshed every 3–4 days. After about 2 weeks, the clones were isolated. For stable ectopic expression of H4(Δ1–19)-HaloTag with PiggyBac system, PB-EF1a-H4(∆1–19)-HaloTag-IRES-Neo was co-transfected with pCMV-hyPBase (Yusa et al. 2011) using the Effectene Transfection Reagent kit in a 12-well plate. The selection was performed with 800 µg/mL G418 (08973-14; Nacalai Tesque).

Expression and localization of H4-HaloTag in cells

To assess the localization of H4-HaloTag as well as mutant H4-HaloTag in HeLa cells, the cells were seeded on 1 mg/mL poly-L-lysine-coated (P1524-500MG; Sigma-Aldrich) coverslips (C018001; Matsunami). The HaloTag was labeled with an excess amount of ligand, 5 nM tetramethylrhodamine (TMR, 8251; Promega), for 30 min at 37°C in 5% CO₂. The rest of the procedures were conducted at room temperature.

After briefly rinsing with 1×PBS, the cells on coverslips were fixed with 1.85% formaldehyde (064–00406; Wako) in 1×PBS for 15 min. Excess molecular crosslinking and fixation were quenched with 50 mM glycine (077–00735; Wako) in 1×PBS for 5 min. The cells were then permeabilized with 0.5% Triton X-100 (T-9284; Sigma-Aldrich) in 1×PBS for 5 min. After briefly rinsing with 1×PBS, the DNA in cells was stained with 0.5 µg/mL 4’,6-diamidino-2-phenylindole (DAPI, 10236276001; Roche) in 1×PBS for 5 min. Finally, after rinsing with 1×PBS twice, the coverslips were mounted onto micro slide glasses (S011120; Matsunami) in p-phenylenediamine (PPDI; 20 mM Hepes [pH 7.4], 1 mM MgCl₂, 100 mM KCl, 78% glycerol, and 1 mg/mL paraphenylene diamine [695106-1G; Sigma-Aldrich]) and sealed with nail polish (T and B; Shiseido).

Z-stack images (every 0.4 µm along the Z axis, 20 sections in total) of the cells were taken using a DeltaVision Elite (or DeltaVision Ultra) epifluorescence microscope (Applied Precision) equipped with an Olympus PlanApoN 60× NA 1.42 objective, sCMOS camera, InsightSSI light (~ 50 mW), and the four-color standard filter set. DeltaVision image acquisition software, SoftWorx 7.X, was used to capture and perform deconvolution of acquired images in all cases. Image fusion, projection over the entire nucleus, and quantification were done using Fiji (ImageJ).

H4-Halo labeled nucleosome pull-down, purification of nucleosomal DNA, and library preparation.

Cell nuclei were isolated as previously described (Lewis and Laemmli 1982; Shimamoto et al. 2017) with minor modifications. Briefly, HeLa cells were maintained at 37°C and 5% CO₂ in DMEM supplemented with 10% FBS on a cell culture plate. Collected cells were washed with nuclei isolation buffer composed of 3.75 mM Tris-HCl (pH 7.5), 20 mM KCl, 0.5 mM EDTA, 0.05 mM spermine, 0.125 mM spermidine, 0.1% Trasylol (14716; Cayman Chemical), and 0.1 mM phenylmethylsulfonyl fluoride (PMSF; P7626-1G; Millipore Sigma) by two cycles of centrifugal spins at 193×g for 7 min at 23°C. The pellets were then resuspended in nuclei isolation buffer containing 0.05% Empigen BB detergent (45165-50ML; Sigma-Aldrich) (nuclei isolation buffer+) and immediately homogenized with 10 downward strokes using a tight Dounce pestle. After a 5-min centrifugation of the cell lysates at 440×g, the pellets were washed with nuclei isolation buffer+. After centrifugation at 440×g at 4°C for 5 min, the supernatant was removed and resuspended in 10 ml of nuclei isolation buffer+. This step was repeated four times before the samples were ready for nucleosome purification.

Chromatin purification was carried out as described by (Ura and Kaneda 2001; Iida et al. 2023), with some modifications. The nuclei (equivalent to ~ 2 mg of DNA) in nuclei isolation buffer (10 mM Tris–HCl, pH 7.5, 1.5 mM MgCl₂, 1.0 mM CaCl₂, 0.25 M sucrose, and 0.1 mM PMSF) were digested with 50 U of micrococcal nuclease (LS004797; Worthington) at 30°C for 1 h. In this digestion step, 1 µM HaloTag PEG-Biotin Ligand (G859A; Promega) or an equivalent amount of DMSO was added. After centrifugation at 440×g at 4°C for 5 min, the nuclei were lysed with lysis buffer (10 mM Tris–HCl, pH 8.0, 5 mM EDTA, and 0.1 mM PMSF) on ice for 5 min. The lysate was dialyzed against dialysis buffer (10 mM HEPES-NaOH, pH 7.5, 0.1 mM EDTA, and 0.1 mM PMSF) at 4°C overnight using Slide-A-Lyzer (66380; Thermo Fisher Scientific). The dialyzed lysate was centrifuged at 20,400×g at 4°C for 10 min. The supernatant was recovered and used as the purified nucleosome fraction (mainly mono nucleosomes). To verify complete MNase digestion, DNA was purified, electrophoresed on a 1.5% agarose gel, and visualized by staining with ethidium bromide (Fig. 2b).

The biotinylated or untreated nucleosome fraction (28 µg) was diluted with an equal volume of 2× binding buffer (10mM HEPES-NaOH pH 7.5, 200 mM NaCl, 2 mM DTT, and cOmplete™ EDTA-free protease inhibitor cocktail (11873580001; Roche). As shown previously (Ide et al. 2021), the chromatin solution was added to streptavidin-FG beads (TAS8848 N1170, Tamagawa-Seiki) pre-equilibrated with coating buffer (PBS, 2.5% BSA, 0.05% Tween 20) for 1 h. The mixtures were incubated at 5 ºC for 2 h on a shaker. The beads were collected with a magnetic rack and subjected to the washing procedure as described by Gatto et al. (Gatto et al. 2022). Briefly, the beads were washed once with 1 mL of cold 1× binding buffer (10 mM HEPES-NaOH pH 7.5, 100 mM NaCl, 1 mM DTT,), twice with 1 mL of cold wash buffer 1 (HEPES-NaOH pH 7.5, 140 mM NaCl, 1% Triton X-100, 0.5% NP40, 0.1% SDS), twice with 1 mL of cold wash buffer 2 (HEPES-NaOH pH 7.5, 360 mM NaCl, 1% Triton X-100, 0.5% NP40, 0.1% SDS), twice with 1 mL of cold wash buffer 3 (HEPES-NaOH pH 7.5, 250 mM LiCl, 0.5% Triton X-100, 0.5% NP40) and once with TE buffer (10 mM Tris–HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 50 µL TE buffer.

For DNA extraction, 40 µL of beads underwent RNase digestion (4 µL RNase A, 1 mg/mL, 30 min at 37°C) and subsequent proteinase K digestion in the presence of SDS (4 µL of 20 mg/mL proteinase K, and 10 µL of 10% SDS, at 1,200 rpm and 50 ºC on shaker for 1 h). Beads were removed by magnetic separation and DNA extracted using AMPure XP beads (A63880, Beckman Coulter) according to the manufacturer’s instructions, and eluted in Milli-Q water. Total DNA content of each sample was measured using Qubit (Q32851, Thermo), and the quality of DNA samples was assessed by an Agilent 2100 Bioanalyzer using an Agilent High Sensitivity DNA kit (5067 − 4626, Agilent). cDNA libraries were synthesized by the ThuPLEX DNA-seq Kit (R400675, Takara). The size distributions of the libraries were checked by an Agilent 2100 Bioanalyzer using an Agilent High Sensitivity DNA kit. Pooled amplicon library was sequenced with paired-end 2 × 100 bp reads on the Illumina NovaSeq 6000 platform.

For protein analysis of the HaloTag pull-down fraction (Fig. 2C), the remaining 10 µL of beads were resuspended with an equal volume of 2× Laemmli sample buffer (Laemmli 1970) supplemented with 10% 2-mercaptoethanol (133–1457; Wako) and incubated at 99 ºC for 10 min. Beads were removed by centrifugation at 10,000×g for 3 min. The Input sample and the pull-down samples from the biotinylated and untreated nucleosome (negative control) were separated using SDS–polyacrylamide gel electrophoresis and were subjected to western blotting by anti-HaloTag antibody (G9211, Promega).

For data analysis of purified H4-Halo labeled nucleosomal DNA, the nf-core (Ewels et al. 2020, 2022) ChIP-seq pipeline (nfcore/chipseq: version 2.0.0) was utilized, employing a Docker configuration profile and executed using the default parameters. The human genome hg19, retrieved from Illumina’s iGenomes, was used for the reference genome. Peaks were detected using the broad mode of MACS2 (Zhang et al. 2008). To evaluate the overlap between the H4-HaloTag labeled regions and contact domains (i.e., Hi-C A-compartment and histone modification patterns), we compared the H4-HaloTag regions with previously published data as described in (Nozaki et al. 2023). The contact domain data from HeLa defined by (Rao et al. 2014) were downloaded from Gene Expression Omnibus, accession number GSE63525 (GSE63525_HeLa_Arrowhead_domainlist.txt). Compartment annotations on the HeLa cell genome are SNIPER predictions obtained from (Xiong and Ma 2019). Peak lists on hg19 genome defined by the ChIP-seq analysis for the histone modifications were obtained from the ENCODE portal (Consortium 2012) (Davis et al. 2018) with the following identifiers: H3K4me2 (ENCFF108DAJ), H3K4me3 (ENCFF447CLK), H3K9ac (ENCFF723WDR), H3K27ac (ENCFF144EOJ), H3K79me2 (ENCFF916VLX), H3K36me3 (ENCFF001SVY), H3K4me1 (ENCFF162RSB), and H3K27me3 (ENCFF252BLX).

Pre-extraction of freely diffusing histones

After staining the HaloTag with an excess amount of TMR, the cells were briefly washed with cytoskeletal buffer (CSK; 0.01 M PIPES-KOH, pH 7.0, 0.1M NaCl, 0.3 M sucrose, 3 mM MgCl₂, protease inhibitors) before and after incubation in 0.5% TritonX-100 in CSK buffer (CSK containing 0.5% tritonX-100 and 1mM PMSF) for 5 min (Maeshima et al. 2006). After briefly rinsing with 1×PBS, the cells on coverslips were fixed with 3.7% formaldehyde in 1×PBS for 5 min. The cells were then permeabilized with 1% Triton X-100 in 1×PBS for 20 min. DAPI staining, mounting onto slide glasses, and fluorescence imaging were performed as described above.

Single-nucleosome imaging microscopy

To assess the local nucleosome behavior of mutant H4-HaloTag as well as wtH4-HaloTag in living HeLa cells, the cells were seeded on 1 mg/mL poly-L-lysine-coated glass-base dishes (3970-035; Iwaki). The HaloTag was sparsely labeled with 50 pM (for MSD analysis) or 25 pM (for Spot-On analysis) TMR in phenol red-free DMEM (21063-029; Thermo Fisher Scientific) with 10% FBS for 20 min at 37°C in 5% CO₂. Then, the cells were washed with Hank’s balanced salt solution (1×HBSS, H1387; Sigma Aldrich) three times and kept in phenol red-free DMEM with 10% FBS at 37°C in 5% CO₂ using an INU-TIZ-F1 (Tokai Hit) live cell chamber and GM-8000 digital gas mixer (Tokai Hit) during imaging. Single H4-HaloTag-TMR nucleosomes were visualized using Nikon Eclipse Ti inverted microscope with a 100-mW Sapphire 561-nm laser (Coherent) and sCMOS ORCA-BT camera (Hamamatsu Photonics). TMR was excited by the 561-nm laser through Nikon 100× PlanApo TIRF NA 1.49 objective and detected at 575–710 nm. Oblique illumination with a thin optical sheet achieved high signal-to-noise ratio for single-nucleosome imaging (Tokunaga et al. 2008). Sequential image frames (150 for MSD analysis or 300 for Spot-On analysis) were captured using MetaMorph software (Molecular Devices) at 50 ms (for MSD analysis) or 10 ms (for Spot-On analysis) of continuous laser exposure.

Single nucleosome tracking and analysis

Image processing and preparation for single nucleosome tracking were performed using ImageJ Fiji. Sequential 16-bit images of single H4-HaloTag molecules were kept (for Spot-On analysis) or reduced to an 8-bit grayscale (for MSD analysis). The background signal was subtracted using the rolling ball method (radius = 50 pixels). The region corresponding to the cell nucleus was manually extracted using the “Polygon selections” method. The centroids of detected fluorescent dots in each image were determined, and their trajectories were calculated using u-track (MATLAB package (Jaqaman et al. 2008); for MSD analysis) or TrackMate (Fiji package (Tinevez et al. 2017); for Spot-On analysis). The u-track trajectories were then used to calculate the displacement distributions and the mean squared displacements (MSD) of fluorescent dots in Python. The originally calculated 2D MSD values were multiplied by 1.5 (4 to 6 Dt) to obtain 3D MSD values. MSD plots with appropriate statistical analyses between various phenotypes were made on Python.

For angle distribution analysis (Iida et al. 2022; Izeddin et al. 2014), first, the consecutive tracked positions {(x₀, y₀), (x₁, y₁), ⋯, (x_n, y_n), ⋯} of single nucleosomes on the XY plane were converted into a set of displacement vectors ∆r_n = (x_{n + 1} − x_n, y_{n + 1} − y_n)^t. Then, the angle between two vectors ∆r_n and ∆r_{n + 1} was calculated for every trajectory in our experiments, and the normalized polar histogram was plotted in Python. The angle distribution was normalized by 2, and the magnitudes correspond to the probability density. The color spectrum indicates each angle in a circular range from 0॰ to 360॰.

The TrackMate trajectories were submitted for the 2-state Spot-On analysis as described in (Hansen et al. 2018). The frame rate was set at 10 ms, the number of time points was set at 10, and the maximum diffusion coefficient of the bound fraction, D_bound (max), was set at 0.03 µm²/s.

Cellular protein preparation and immunoblotting

HeLa cells were lysed in hot Laemmli buffer (Laemmli 1970) containing 0.1M dithiothreitol (DTT). The resulting lysate was incubated at 95°C for 10 min to denature proteins and then sonicated. The proteins from the lysate were resolved on a 12% polyacrylamide SDS-gel at 150 V for 1 h. Then they were transferred to the 0.45 µm polyvinylidene difluoride (PVDF) Immobilon-P membrane (IPVH00010, Merck) at a constant current of 110 mA for 45 min using the semi-dry method (ATTO; 25 mM tris, 5% methanol). After blocking in 5% bovine serum albumin (BAC65-1150, Equitech-Bio) in phosphate-buffered saline (PBS) with 0.1% Tween 20 for 1 h at room temperature, the membrane-bound proteins were probed by anti-H4 (1:4000 dilution; ab10158, Abcam) and anti-HaloTag (1:1000 dilution; G9211, Promega) antibodies, followed by the appropriate secondary antibody: anti-rabbit (1:5000 dilution; 170–6515, Bio-Rad) or anti-mouse (1:5000 dilution; 170–6516, Bio-Rad) horseradish peroxidase (HRP)–conjugated goat antibody. Chemiluminescence reactions were used (WBKLS0100, Millipore) and detected by EZ-Capture MG (AE-9300H-CSP, ATTO).

Ectopic expression of wild-type and various mutant H4s with HaloTag in HeLa cells

The C-terminus of human wild-type (wt) and various H4 mutants was fused to the HaloTag protein, which is driven by the EF-1α promoter (Mizushima and Nagata 1990), to study how they affect the local behavior of nucleosomes in HeLa cells. C-terminal tagging was chosen to minimize potential influences the HaloTag may have had on the molecular behavior of the H4 N-terminal tail that contains crucial amino acid residues, including K16 (Fig. 1a). To ensure comparable expression levels of the wt and mutant H4s (Fig. S1), the construct was inserted into the FRT site in HeLa cells, which had been previously introduced into their genome (Maeshima et al. 2006) via Flp recombinase-mediated DNA recombination. With proper recombination, the cells became hygromycin-resistant and blasticidin-sensitive (Fig. 1b).

Expressed H4-HaloTag (H4-Halo) protein can be labeled with a fluorescent HaloTag ligand, such as tetramethylrhodamine (TMR). The localization of wtH4-Halo in HeLa cells was assessed by labeling HaloTag with a saturating amount of TMR (i.e., 5 nM for 30 min; Fig. 1c). H4-HaloTag-TMR was in the nucleus with notable signal depletion at the nuclear and nucleolar peripheries, presumably heterochromatin rich regions (Fig. 1d). This H4-HaloTag-TMR localization is distinctive from that of DAPI DNA staining or H2B-HaloTag-TMR, which are incorporated genome-wide (Fig. 1d) (Nagashima et al. 2019; Iida et al. 2022). The localization of ectopically expressed H4-HaloTag-TMR seemed to be biased toward euchromatic regions, consistent with a previous report (Kimura and Cook 2001).

Euchromatic localization of wtH4-Halo labeled nucleosomes

We next investigated the genomic localizations of nucleosomes, into which ectopically expressed wtH4-Halo was incorporated, to assess their euchromatic-biased localizations in more detail. H4-Halo was conjugated with a biotin PEG ligand during the nucleosome purification from HeLa cells expressing H4-Halo (Fig. 2a). The nucleosomes with H4-Halo-biotin (Fig. 2b) were separated using streptavidin (Fig. 2c) and subjected to sequencing (Fig. 3a). We identified 99,111 H4-Halo nucleosome peaks in the annotated HeLa S3 genome (hg19) (Fig. 3a and 3d and Fig. S2a), while control H2B-Halo nucleosome data did not identify a considerable number of peaks (788, Fig. 3a and 3d and Fig. S2a), indicating a more genome-wide incorporation of H2B-Halo compared to that of H4-Halo. We next compared H4-Halo nucleosome regions to available data of Hi-C contact domains (Rao et al. 2014), active chromatin marks (Zhang et al. 2020), and inactive chromatin marks (Zhang et al. 2020). A total of 78.6% of H4-Halo-labeled regions overlapped with Hi-C contact/loop domains (or TADs) (Fig. 3b and 3d, Fig. S2a, and Fig. S3a). A total of 97.4% of the H4-Halo-labeled regions belonged to the A-compartment (Fig. 3c and 3d, Fig. S2a, and Fig. S3b). H4-Halo-labeled regions were significantly enriched with active chromatin marks (H3K4me1–3, H3K9ac, H3K27ac, H3K36me3, and H3K79me2) and excluded from an inactive chromatin mark (H3K27me3) (Fig. 3d, Fig. S2a, and Fig. S3c-j). These results indicated that H4-Halo labeled nucleosome regions correspond to Hi-C contact domains with active chromatin marks (A-compartment). This finding is consistent with our previous imaging data (Fig. 1c-d) and (Kimura and Cook 2001). Nucleosome labeling by ectopically expressed H4-Halo allows us to focus on nucleosome behavior in euchromatic contact domains.

Single-nucleosome imaging of euchromatic H4-Halo

We performed single-nucleosome imaging by labeling H4-Halo with a low concentration of TMR (Fig. 4a). Sparse labeling of nucleosomes allowed individual H4-Halo dots to be clearly resolved (Fig. 4b; Movie S1) using oblique illumination microscopy (Fig. 4c) (Tokunaga et al. 2008; Nozaki et al. 2017; Ide et al. 2022), which allowed illumination of a thin optical region within a nucleus. We acquired continuous image sequences of the dots at 50 ms per frame (150 frames, 7.5 s total). Each fluorescent dot displayed a single-step photobleaching pattern, a characteristic of single-molecules. (Fig. 4d). The detected dots in each image were fitted to an assumed Gaussian function to precisely define the center of each dot beyond the diffraction limit (Betzig et al. 2006; Rust et al. 2006; Selvin et al. 2007). The trajectories of each dot were then tracked using u-track software (Fig. 4e) (Jaqaman et al. 2008) for a period of 100 frames (5 s total). The position determination accuracy of H4-Halo was 12.5 nm (Fig. S4a).

Because free H4-Halo-TMR molecules moved too quickly to be tracked with a 50 ms/frame image acquisition, we could only characterize and quantify the behavior of the nucleosomes into which H4-Halo was stably incorporated. Observed H4-Halo motion exhibited about 80 nm of displacement for 100 ms of observation time (Fig. 4f). Their calculated mean squared displacement (MSD) described a subdiffusive motion of H4-Halo, which was higher than that of H2B-Halo (Fig. 4g-h, S4b-c) (Iida et al. 2022). The higher MSD values for H4-Halo are explained by its euchromatic localization, predominantly marking more mobile genomic regions (Nozaki et al. 2023). Note that the MSD value of H2B-Halo reflects the averaged movement of nucleosomes in whole chromatin.

Chemical crosslinking of chromatin with 2% formaldehyde (FA) severely suppressed fluctuations of H4-Halo (Fig. 4g-f). The radii of constraint (Rc; p = 6/5 × Rc²) (Dion and Gasser 2013) of H4-Halo and H2B-Halo were calculated by taking the square root of the MSD plateaus, and were estimated to be 140.87 ± 15.75 nm (mean ± SD) and 132.49 ± 15.03 nm, respectively. Moreover, their MSD exponents were α = 0.52 (H4-Halo) and α = 0.45 (H2B-Halo) in the time range between 0 and 0.5 s (Fig. 4h, S4c) and the asymmetry coefficient (AC = -1.029; Fig. 4j) of the motion angle distribution (Fig. 4i-k) of H4-Halo was greater than those of H2B-Halo (α = 0.45; AC = − 1.336) (Iida et al. 2022). Collectively, these results indicate that the local chromatin behavior of euchromatin-concentrated H4-Halo is more mobile and less constrained than that of genome-wide H2B-Halo. These findings are in good agreement with our recent studies on Cy3-labeled nucleotides belonging to early DNA replication foci representing the euchromatin (Nozaki et al. 2023).

Similar behaviors between wtH4 and H4K16 mutant nucleosomes

To determine how acetylation status at lysine-16 of H4 (H4K16Ac) affects local nucleosome behavior, we introduced substitution mutations at the codon corresponding to K16 in wtH4-Halo (Fig. 5a). The K16R mutation replaces lysine with another positively charged amino acid residue, arginine, which is the phenocopy of the deacetylated state of K16 (Fig. 5a, right). In contrast, the K16Q mutation approximates constitutive acetylation (Fig. 5a, right). The K16A mutation introduces alanine that is neither charged nor acetylated (Fig. 5a, right). The expression levels of all three H4K16 mutants expressed in HeLa cells were comparable to wtH4 (Fig. S1a). The localizations of all three H4K16 mutants were biased to euchromatin regions of the nucleus, like wtH4-Halo (Fig. 5b).

We investigated the local nucleosome behaviors of the three H4K16 mutants in HeLa cells (Fig. 5c; Movie S2-4). The substitutions at K16 did not impair incorporation of the mutants into nucleosomes, as we did not observe an increase in freely moving H4K16 mutants (Fig. 5c; Movie S2-4). In principle, constitutively charged and uncharged states of K16 might change local nucleosome behavior. However, the observed MSD values for the mimicked charged (arginine) and uncharged (glutamine) states of H4K16 were not different from wtH4 (Fig. 5d). In addition to MSD, AC values were like those of wtH4 (Fig. 5e), confirming that H4K16 mutations do not alter the local nucleosome motion in euchromatin.

Behaviors of H4 with multiple mutations

As the H4K16 point mutation did not alter local nucleosome behavior, we wondered if a single amino acid substitution was not enough to witness a change in nucleosome motion. To exaggerate the mutant phenotypes, we developed a series of H4 quadruple mutants, by replacing all the lysines at positions 5 (K5), 8 (K8), 12 (K12) and 16 (K16) with arginine, glutamine, or alanine (Fig. 6a). The H4Q quadruple mutant (H4Q-quad) and H4A quadruple mutant (H4A-quad) also showed euchromatic localizations like that of wtH4 (Fig. 6b). However, the H4R-quadruple mutant (H4R-quad) had a more uniform localization within the nucleus in the FA-fixed cells (Fig. 6b).

We assessed the local motion of H4 quadruple mutants (Fig. 6c and 6d, Movies S5-S7). Notably, as compared with other mutants, H4R-quad showed an increase in its freely moving fraction in living cells (Movie S5), suggesting a reduced capacity for nucleosome incorporation. However, the calculated MSD of all H4 quadruple mutant nucleosomes showed subdiffusive curves like wtH4 (Fig. 6d), as we could track only nucleosome-incorporated H4 quadruple mutants at this frame rate (50 ms/frame). AC values for nucleosomes with H4Q-quad or H4A-quad were comparable to those of wtH4 (Fig. 6e), suggesting that multiple Q or A mutations in the N-terminal tail of H4 do not alter their nucleosome behaviors in euchromatin. Interestingly, H4R-quad had a somewhat lower AC value of -1.224 (Fig. 6e), implying that nucleosomes with H4R-quad are more constrained.

Nucleosomes with H4 quadruple lysine mutants exhibit similar behavior to that of wtH4 nucleosomes.

We also tracked the wtH4 and mutant H4s over a shorter exposure time, 10 ms/frame, to capture freely diffusing H4 mutants (Movies S8-S11). Spot-On analysis (Hansen et al. 2018) suggested that the proportion of bound H4R-quad is only 37.4%, whereas it is 87.9% for wtH4 and over 80% for all other H4 multiple mutants (Figs. 6f and S5). Long jumps of H4 with more than 0.2 µm in 10 ms, representing the fraction not incorporated into the nucleosomes, were much more frequent in the H4R-quad (Fig. S5). Consistently, when we pre-extracted the cells to remove free H4R-quad before fixation (Fig. S6), the total intensity of the TMR signal decreased more than 2 times (Fig. S6d), suggesting the existence of a larger fraction of free H4R-quad that were not incorporated into nucleosomes.

Behavior of the H4 tailless mutant

We deleted the first 19 amino acids of H4 to yield the so-called ‘tailless’ H4 mutant (H4Δ1–19, Fig. 7a), expecting a truncated tail would completely abolish its contribution to internucleosomal interactions. The expression level of H4Δ1–19 was more than 3 times higher than those of wtH4 and other H4 mutants (Fig. S1a), probably due to increased protein stability. To avoid the artifactual effect of over-expression, we used a PiggyBac transposition system (Yusa et al. 2011) and obtained a comparable expression level of H4Δ1–19 to wtH4 (Fig. S1b). Interestingly, H4Δ1–19 had a more “DAPI-like” localization within the nucleus than wtH4 (Fig. 7b). The calculated MSD of H4Δ1–19 was significantly lower than that of wtH4 (Fig. 7d; Movie S12). AC values of H4Δ1–19 were also lower than those of wtH4 (Fig. 7e). Spot-On analysis suggested the presence of an 84% bound fraction in tailless H4 (Fig. 7f; Movie S13), indicating that the truncated H4 mutant does not have problems incorporating into chromatin, unlike H4R-quad. These results suggest that deleting the H4 tail facilitates the incorporation of the ectopically expressed H4 into both euchromatin and heterochromatin.

Using single-nucleosome imaging, we investigated the behaviors of nucleosomes with mutant H4s tagged with Halo in living HeLa cells. The H4 mutant constructs were inserted into a unique FRT site of the HeLa genome and expressed at similar levels, which allowed us to compare their behavior by single-nucleosome imaging. Ectopically expressed wtH4-Halo localized to euchromatin in HeLa cells and had a more dynamic behavior than H2B-Halo, which was incorporated genome-wide. Consistently, genomic regions of the wtH4-Halo nucleosomes corresponded to Hi-C contact domains with active chromatin marks (A-compartment). Our studies of H4 mutant nucleosomes were focused in euchromatic domains, where histone acetylation marks are more relevant and enriched.

H4-Halo was distributed mainly in euchromatin (HiC A-compartment), while H2B-Halo was located genome-wide, like “DNA staining” (Figs. 1 and 3; Fig. S2). Why? As Kimura and Cook discussed (Kimura and Cook 2001), a possible mechanism is as follows. Although endogenous H4 and H2B are expressed strictly in a DNA replication-dependent manner, the H4-Halo and H2B-Halo are under the constitutively active (unregulated) control of the EF1α promoter (Mizushima and Nagata 1990). The resulting mRNAs are polyadenylated, which do not occur in the endogenous ones. H4-Halo and H2B-Halo will accumulate during the G1 phase as a soluble pool and will inevitably be incorporated into euchromatin as soon as replication begins. Consistent with this observation, H4-Halo labeled genomic regions were 82.9% overlapped with the early replicated regions labeled by Cy3-dCTP incorporation (Fig. S7) (Nozaki et al. 2023). As the S phase progresses, endogenous levels of H4 and H2B may dominate while H4-Halo and H2B-Halo incorporation becomes negligible. Once H4-Halo is incorporated into nucleosomes, it is stably maintained throughout the cell cycle (Kimura and Cook 2001). A continuous exchange happens in the case of H2B-Halo and it incorporates genome-wide.

Nucleosomes with any of the three H4K16 point mutations (H4K16Q, H4K16R, and H4K16A) in euchromatin had a similar local behavior to that of wtH4. Although H4K16Ac, a famous histone acetylation mark, is associated with the decompaction of chromatin in vitro (Shogren-Knaak et al. 2006; Robinson et al. 2008; Allahverdi et al. 2011), we could not observe motion changes in H4K16 mutant nucleosomes in living human cells. Like H4K16 mutants, nucleosomes with H4Q-quad or H4A-quad mutations had both similar euchromatin-biased localizations and local chromatin behaviors to that of wtH4.

Our results are consistent with recent reports, including ours, suggesting that euchromatin consists of condensed chromatin domains where only very limited regions are open (Miron et al. 2020; Cremer et al. 2020; Zakirov et al. 2021; Nozaki et al. 2023; Maeshima et al. 2024). Nucleosomes containing mutated H4s were also surrounded by nucleosomes containing endogenous H4. Therefore, the effect of an H4 mutation could be embedded in the chromatin domains that behaved as a unit (Fig. 8). In this context, expression levels of H4 mutants may matter to detect their effects. While the strong EF1α promoter (Mizushima and Nagata 1990) used for expressing H4 mutants achieved stable ectopic expressions, the FRT system provided only a single unique site on the genome of HeLa cells. Expression levels of the mutant H4 were still far lower than that of endogenous histone H4 with its multicopy loci. Although more robust expression levels of these H4 mutants could enhance their effects, stronger expression of the tagged histone may be highly cytotoxic, as Kimura suggested (Kimura and Cook 2001).

Interestingly, the H4R-quad mutant had a larger freely diffusing fraction in the nucleoplasm than wtH4 and other mutants. We suggest that the H4R-quad mutant had a severe problem inserting into the nucleosomes. One explanation is that newly synthesized histone H4 proteins require acetylation marks at K5 and K12, which are crucial for DNA replication-coupled incorporation of nucleosomes (Parthun 2007). In agreement with this, the H4Q-quad mutant (i.e., the constitutive acetylation mimetic) did not have the same incorporation problem as the H4R-quad mutant (i.e., the deacetylation mimetic). It is intriguing that the H4A-quad mutant, which is not an acetylation mimetic, did not have an incorporation problem. Because H4A is like H4Ac only by net charge, the charge neutralization at K5 and K12 may be more critical for the incorporation process than the chemical structure of the KAc side chain.

Tailless H4 had a more “DAPI-like” localization. However, unlike the H4R-quad mutant, single-nucleosome imaging revealed that this pattern was not caused by freely diffusing mutant histones. We saw a subset of tailless H4 nucleosome dots at the nuclear periphery region, where heterochromatin is enriched. Lower MSD values of the tailless H4 supported the presence of tailless H4 in the less mobile heterochromatic regions. The underlying mechanism of how tailless H4 incorporated into heterochromatin remains unclear. Because the H4 tail truncation severely impairs nucleosome-nucleosome interactions and compromises any compensatory post-translational modifications, we infer that the mutant nucleosomes have a greater tendency to be incorporated in the more silent heterochromatic regions. In such inert and inactive genomic regions, the mutant nucleosomes exert less deleterious effects.

In conclusion, using single-nucleosome imaging, we investigated the behaviors of nucleosomes with H4K16 mutations incorporated into the euchromatic domains of living HeLa cells. While a point mutation at H4K16 and some multiple lysine mutations did not change the motion of nucleosomes within condensed euchromatin domains, we found the H4R-quad mutant diffused freely in the nucleoplasm, suggesting it had a nucleosome incorporation problem. Furthermore, the tailless H4 mutant was incorporated into heterochromatin, as well as euchromatin. Our results indicate that single-nucleosome imaging is a very powerful tool that can be used to analyze mutant histones. Some mutations in core histones act as oncogenic drivers, leading to the term “oncohistones” (Bennett et al. 2019; Nacev et al. 2019). It would be intriguing to examine the behavior of nucleosomes containing oncohistones in further studies.

Competing interests

The authors declare that they have no competing interests, financial or otherwise.

Data and materials availability

The data have been deposited with links to BioProject accession numbers PRJDB17378 (H2B-HaloTag pull-down) and PRJDB17379 (H4-HaloTag pull-down) in the DDBJ BioProject database.

Author Contribution

A. S. and K. Maeshima designed the project. A. S. constructed mutants of histone H4 and created the cell lines with help of S. Ide and S. Iida. A. S. and S. Iida performed single-nucleosome imaging, tracking, and analysis. A. S. performed the cell biology experiments with help of S. Iida. S. T. and K. Minami purified H4-Halo labeled nucleosomal DNA and prepared sequencing library with help of S. Ide. A. T. performed sequencing. K. H. and K. K. analyzed the sequencing data. A. S., S. Iida., and K. Maeshima wrote the manuscript with input from all the authors.

Acknowledgments

We are grateful to Dr. K. M. Marshall for critical reading and editing of this manuscript. We thank Dr. H. Kimura for providing his antibodies, Dr. K. Hibino, Mr. M. A. Shimazoe, and Maeshima laboratory members for their helpful discussions and support. This work was supported by JSPS grants JP21H02453 (K. Maeshima), JP20H05936 (K. Maeshima), JP22H05606 (S. Ide), JP23K17398 (K. Maeshima, S. Ide), JP21H02535 (S. Ide), 22H04925 (PAGS) (K. Maeshima), Takeda Science Foundation (K. Maeshima). A. S. is a MEXT Scholar. S. Iida and K. Minami were SOKENDAI Special Researchers (JST SPRING JPMJSP2104) and are currently JSPS Fellows (S. Iida, JP23KJ0996; K. Minami, JP23KJ0998).

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Behaviors of nucleosomes with mutant histone H4s in euchromatic domains of living human cells

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and methods

Construction of histone H4-HaloTag expression plasmids

Establishment of stable cell lines

Expression and localization of H4-HaloTag in cells

Pre-extraction of freely diffusing histones

Single-nucleosome imaging microscopy

Single nucleosome tracking and analysis

Cellular protein preparation and immunoblotting

Results

Ectopic expression of wild-type and various mutant H4s with HaloTag in HeLa cells

Euchromatic localization of wtH4-Halo labeled nucleosomes

Single-nucleosome imaging of euchromatic H4-Halo

Similar behaviors between wtH4 and H4K16 mutant nucleosomes

Behaviors of H4 with multiple mutations

Behavior of the H4 tailless mutant

Discussion

Declarations

Competing interests

Data and materials availability

Author Contribution

Acknowledgments

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1