Cryo-EM and biochemical analyses of the nucleosome containing the human histone H3 variant H3.8

Abstract Histone H3.8 is a non-allelic human histone H3 variant derived from H3.3. H3.8 reportedly forms an unstable nucleosome, but its structure and biochemical characteristics have not been revealed yet. In the present study, we reconstituted the nucleosome containing H3.8. Consistent with previous results, the H3.8 nucleosome is thermally unstable as compared to the H3.3 nucleosome. The entry/exit DNA regions of the H3.8 nucleosome are more accessible to micrococcal nuclease than those of the H3.3 nucleosome. Nucleosome transcription assays revealed that the RNA polymerase II (RNAPII) pausing around the superhelical location (SHL) −1 position, which is about 60 base pairs from the nucleosomal DNA entry site, is drastically alleviated. On the other hand, the RNAPII pausing around the SHL(−5) position, which is about 20 base pairs from the nucleosomal DNA entry site, is substantially increased. The cryo-electron microscopy structure of the H3.8 nucleosome explains the mechanisms of the enhanced accessibility of the entry/exit DNA regions, reduced thermal stability and altered RNAPII transcription profile.

In eukaryotic cells, the fundamental structural unit of chromatin is the nucleosome, which is composed of histones H2A, H2B, H3 and H4, with 145 to 147 base pairs (bp) of DNA (1).The core histones are categorized into canonical and variant types (2).Canonical histones are encoded by multiple genes and produced during DNA replication in the S-phase of the cell cycle (3).In contrast, histone variants are produced from a smaller set of genes, and their chromatin incorporation is not restricted to a particular phase of the cell cycle (2,4).Histone variants are considered to have specialized functions in the regulation and maintenance of genomic DNA (2).
The human H3.8 gene is expressed in the ovary, adrenal glands, colon, kidney and thyroid (18).In the present study, we biochemically characterized the H3.8 nucleosome and determined its structure by cryo-electron microscopy (cryo-EM) single particle analysis.

Thermal stability assay of nucleosomes
The thermal stability of the nucleosome containing H3.3 or H3.8 was measured by a thermal stability assay, as described previously (24).The nucleosome (24 pmol) was mixed with 5 × SYPRO Orange dye (Sigma-Aldrich) in 20 μl of reaction mixture (17 mM Tris-HCl buffer (pH 7.5), 0.9 mM dithiothreitol, 4.3% glycerol and 100 mM NaCl).A 19-μl portion of the mixture was subjected to a temperature gradient from 26 to 95 • C, in steps of 1 • C/min, and the fluorescence of SYPRO Orange was detected using a StepOnePlus™ Real-Time PCR system (Applied Biosystems).The fluorescent signals were normalized with the following formula: where F(T) indicates the fluorescence signal intensity at a particular temperature.

Cryo-EM data acquisition of the H3.8 nucleosome
Cryo-EM images of the H3.8 nucleosome were collected by a Krios G4 transmission electron microscope (Thermo Fisher Scientific), operating at 300 kV and equipped with a K3 direct electron detector with a Quantum GIF imaging filter (Gatan), using a slit width of 20 eV.The data collection was performed using the EPU software (Thermo Fisher Scientific), with a pixel size of 1.06 Å and defocus values ranging from −1.0 to −2.5 μm.Each micrograph of the H3.8 nucleosome was recorded with a 4.5-s exposure time, and then fractionated into 40 frames with a total dose of 61.2 electrons per Å 2 .

Image processing
In total, 10,562 micrographs were stacked and motioncorrected using MOTIONCOR2 (30) with dose weighting.The estimation of contrast transfer function (CTF) from the dose-weighted micrographs was performed using CTFFIND4 (31).RELION4 (32) was used for all subsequent image processing.From 9969 micrographs, 9,365,020 particles were automatically picked, based on the 2D class averages generated from 400 micrographs of the collected data set and extracted with a binning factor of 2 (pixel size of 2.12 Å/pixel).In total, 8,822,886 particles were further selected by 2D classification.The crystal structure of a Xenopus laevis nucleosome (PDB ID: 3UT9 (33)) was used as the initial model for 3D classification.After two rounds of 3D classification, 3,900,336 particles were selected and re-extracted without binning for 3D  (35,36).

Results and Discussion
Reconstitution of the H3.8 nucleosome H3.8 contains 11 amino acid substitutions and a 6-amino acid extension at the C-terminus, as compared with H3.3 (Fig. 1A).Detailed structural and biochemical analyses of the H3.8 nucleosome have not been performed, because the H3.8 nucleosome reconstituted with the native α-satellite DNA sequence was extremely unstable (18).To overcome this problem, we reconstituted the H3.8 nucleosome with a palindromic DNA fragment containing the Widom 601L sequence, which forms stably positioned nucleosomes in vitro (33,43).As shown in Fig. 1B and C, the H3.8 and H3.3 nucleosomes were efficiently reconstituted with the 145-bp palindromic Widom 601L DNA.We then performed the thermal stability assay.Consistent with the previous results obtained with the α-satellite DNA, the H3.8 nucleosome with the Widom 601L DNA was less stable than the H3.3 nucleosome (Fig. 1D).In the H3.8 nucleosome with the Widom 601L DNA, the mid-point of the H2A-H2B dissociation was approximately 4 • C higher than that of the H3.8 nucleosome with an α-satellite DNA, as previously reported (18).

Enhanced accessibility of the entry/exit DNA regions of the H3.8 nucleosome
To study the DNA accessibility of the H3.8 nucleosome, we performed a micrococcal nuclease (MNase) treatment assay with the H3.8 and H3.3 nucleosomes and compared the susceptibility of the DNA ends to MNase (Fig. 2A).
MNase is an endo/exonuclease that preferentially digests the DNA detached from the histone surface in the nucleosome (44).The DNA ends of the H3.8 nucleosome were more susceptible to MNase than those of the H3.3 nucleosome (Fig. 2B and Supplementary Fig. S1).This suggested that the replacement of H3.3 with H3.8 renders the entry/exit DNA regions more accessible and/or flexible.

Transcription elongation in the H3.8 nucleosome by RNA polymerase II
The central DNA region located at the dyad axis of the nucleosome is termed superhelical location (SHL) 0. We previously reported that RNA polymerase II (RNAPII) transcribes the DNA wrapped in the nucleosome by gradually peeling the nucleosomal DNA up to the SHL(0) position, which is 73 bp ahead from the entry DNA site (45).
We also found that RNAPII pauses at the SHL(−5) and SHL(−1) positions, which are about 20 and 60 bp from the entry DNA site of the nucleosome, respectively (23,28).
The nucleosome is disassembled when RNAPII transcribes beyond the SHL(0) position, and then reassembled behind the transcribing RNAPII (45).
To determine how RNAPII transcribes the H3.8 nucleosome, which has different DNA wrapping at its entry/exit regions, we reconstituted nucleosomes containing H3.8 or H3.3 with the modified Widom 601 DNA sequence (23).A linker DNA containing a mismatched bubble region (9 bases) was ligated to one end of the reconstituted nucleosome to serve as the RNAPII loading site for transcription (Fig. 3A).The H3.8 and H3.3 nucleosomes reconstituted for the RNAPII transcription were highly purified and contained only a trace amount of naked DNA (Fig. 3B).The nucleosome containing H3.8 or H3.3 was then transcribed by RNAPII in the presence of TFIIS.In the H3.8 nucleosome, the amount of the run-off transcript was slightly increased as compared to the H3.3 nucleosome (Fig. 3C, D and Supplementary Fig. S2).We found that the RNAPII pausing at the SHL(−5) and SHL(−1) positions is conserved in the H3.8 nucleosome (Fig. 3C and Supplementary Fig. S2).In the H3.8 nucleosome, the amount of RNA product corresponding to RNAPII pausing around the SHL(−1) position was drastically decreased as compared to the H3.3 nucleosome (Fig. 3C and Supplementary Fig. S2).On the other hand, the amount of RNA product corresponding to the RNAPII pausing around the SHL(−5) position was substantially increased (Fig. 3C and Supplementary Fig. S2).Therefore, in the H3.8 nucleosome, RNAPII transcribes the DNA around the SHL(−1) position more efficiently than in the H3.3 nucleosome, and the RNAPII pausing around the SHL(−5) position is enhanced.

Cryo-EM structure of the H3.8 nucleosome
We next performed the cryo-electron microscope (cryo-EM) analysis of the H3.8 nucleosome with the 145-bp palindromic Widom 601L DNA.The reconstituted H3.8 nucleosome was prepared by sucrose gradient ultracentrifugation in the presence of glutaraldehyde (GraFix).The cryo-EM structure of the H3.8 nucleosome was determined at 2.3 Å resolution (Fig. 4A, B and Supplementary Fig. S3A-E).
Under tension force, the nucleosomal DNA ends are asymmetrically unwrapped (46).The asymmetric DNA wrapping is also observed in the Giardia lamblia nucleosome with a palindromic Widom 601L DNA (47).We found that the entry/exit DNA regions are asymmetrically wrapped in the H3.8 nucleosome structure (Fig. 4A and Supplementary Fig. S3B).The H3.8-specific His53 residue, corresponding to the H3.3 Arg53 residue, is located near the entry/exit DNA regions (Fig. 5A).Arg has a basic side chain and is the main amino acid residue that directly binds to the DNA backbone in the nucleosome (1,5).Therefore, the Arg-His substitution in H3.8 may reduce the histone-DNA interactions around the entry/exit DNA region of the nucleosome and, thus, render the DNA more accessible.Substitutions at position 53 are also observed in the CENP-A and mouse H3mm18 H3 variants.Interestingly, like the H3.8 nucleosome, the nucleosomes containing CENP-A and H3mm18 also exhibited enhanced DNA end accessibility (11,48,49).The 53rd residue of H3 variants may be important for providing the diversity of the DNA dynamics in the nucleosome.
The H3.3 Ala91 residue is located in a hydrophobic pocket with the H4 Val86, Val87, Leu97 and Phe100 residues (Fig. 5B).Interestingly, in H3.8, the 91st residue is a hydrophilic Thr residue, which drastically changes the orientation of the H4 Phe100 side chain in the H3.8 nucleosome (Fig. 5B).This Ala-Thr substitution in H3.8 may weaken the hydrophobic interaction between H3 and H4 and reduce the stability of the H3.8 nucleosome.We found that the H3.8-specific Thr115 residue, corresponding to the H3.3 Lys115 residue, is located close to the DNA backbone around the SHL(0) position of the nucleosome (Fig. 5C).Thr is a neutral residue and does not form electrostatic interactions with the DNA backbone.Therefore, the Lys-Thr substitution in H3.8 may reduce the histone-DNA interactions around the SHL(0) position and facilitate the alleviation of RNAPII pausing around SHL(−1).
In the H3.8 nucleosome structure, the H3.8-specific Arg86 residue, corresponding to the H3.3 Ser86 residue, is located near the DNA backbone around the SHL(−5) region (Fig. 5D).The Arg residue may bind to the DNA backbone and may stabilize the local histone-DNA contact.This H3.8-specific Arg86 residue may explain the mechanism by which the RNAPII pausing around the SHL(−5) position is enhanced by the addition local histone-DNA interaction in the H3.8 nucleosome.

Conclusion
In the present study, we successfully reconstituted the nucleosome containing the human histone H3.8 variant.In comparison with the H3.3 nucleosome, we found that (i) the H3.8 nucleosome is less stable, (ii) the entry/exit DNA regions of the H3.8 nucleosome are more accessible, (iii) the RNAPII pausing around the SHL(−1) position of the nucleosome is alleviated and (iv) the RNAPII pausing around the SHL(−5) position of the nucleosome is enhanced.These biochemical characteristics may be explained by the H3.8-specific amino acid residues, such as His53, Thr91, Thr115 and Arg86, which are located in the contact surfaces with the entry/exit DNA regions, a hydrophobic core with H4, the SHL(0) DNA region and the SHL(−5) region, respectively, as revealed by the cryo-EM structure of the H3.8 nucleosome.These biochemical and structural features of the H3.8 nucleosome form a basis to elucidate its biological relevance and provide new insights into understanding the roles of histone variants in cells.

Fig. 2 .
Fig. 2. DNA end flexibility of the H3.8 nucleosome.(A) Schematic diagram of the micrococcal nuclease (MNase) treatment assay.The DNA ends of the nucleosome are preferentially digested by MNase.After deproteinization, the resulting DNA fragments were analyzed by non-denaturing PAGE.(B) A representative gel image of the MNase treatment assay.The nucleosomes containing H3.3 (lanes 2-7) or H3.8 (lanes 8-13) were incubated in the presence of MNase for 0, 3, 6, 9, 12 and 15 min.The resulting DNA fragments were analyzed by non-denaturing PAGE with EtBr staining.The results were confirmed to be reproducible by two additional independent experiments (shown in Supplementary Fig. S1).

Fig. 3 .
Fig. 3. RNAPII transcription on the H3.8 nucleosome.(A) Schematic diagram of the RNAPII transcription assay.(B) Non-denaturing PAGE analysis of purified nucleosomes containing a 198-bp DNA for transcription.The gel was stained by EtBr.(C) Representative gel image of the RNAPII transcription assay.The nucleosomes containing H3.3 (lanes 3-8) or H3.8 (lanes 9-14) were incubated in the presence of RNAPII, TFIIS, dNTPs and DY647 fluorescently labeled RNA primer, and the transcription reaction was conducted for 0 min (lanes 3 and 9), 3 min (lanes 4 and 10), 6 min (lanes 5 and 11), 9 min (lanes 6 and 12), 12 min (lanes 7 and 13) and 15 min (lanes 8 and 14).After termination of the transcription reaction, the resulting elongated RNA fragments were fractionated by denaturing PAGE and detected by DY647 fluorescence.The results were confirmed to be reproducible by two additional independent experiments (shown in Supplementary Fig. S2).(D) Graphical representation of the RNAPII transcription assay.The band intensities corresponding to the run-off transcripts in the H3.3 and H3.8 nucleosomes were quantitated, and the run-off transcripts (%) relative to that of the naked DNA template were plotted against the reaction time.The error bars indicate standard deviations (n = 3).

Fig. 5 .
Fig. 5. Structural comparison of the nucleosomes containing H3.3 and H3.8.(A) Close-up views around the entry/exit DNA regions of the H3.8 nucleosome (left panel) and the H3.3 nucleosome (right panel).The position 53 residues of H3.3 and H3.8 are shown in cyan with side chains.The cryo-EM map of the H3.8 αN helix region is overlaid on the cartoon model.(B) Structural comparison around position 91 in the H3.8 nucleosome (left panel) and the H3.3 nucleosome (right panel).The position 91 residues of H3.3 and H3.8 are shown in cyan with side chains.The van der Waals surfaces of the side chain atoms around the position 91 residues of H3 are represented as spheres.(C) Close-up views around SHL(0) of the H3.8 nucleosome (left panel) and the H3.3 nucleosome (right panel).The position 115 residues of H3.3 and H3.8 are shown in cyan with side chains.The cryo-EM map of the H3.8L2 loop region is overlaid on the cartoon model.(D) Close-up views around SHL(−5) of the H3.8 nucleosome (left panel) and the H3.3 nucleosome (right panel).The position 86 residues of H3.3 and H3.8 are shown in cyan with side chains.The cryo-EM map of the H3.8 Arg86 is overlaid on the cartoon model.The PDB ID of the H3.3 nucleosome structure shown in this figure is 5X7X.