
==== Front
Nucleic Acids Res
Nucleic Acids Res
nar
Nucleic Acids Research
0305-1048
1362-4962
Oxford University Press

39149911
10.1093/nar/gkae698
gkae698
AcademicSubjects/SCI00010
Structural Biology
Ubiquitinated histone H2B as gatekeeper of the nucleosome acidic patch
Hicks Chad W Department of Biophysics & Biophysical Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA

Rahman Sanim Department of Biophysics & Biophysical Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA

Gloor Susan L EpiCypher Inc., 6 Davis Drive, Suite 755, Durham, NC 27709, USA

Fields James K Department of Biophysics & Biophysical Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA

Husby Natalia Ledo EpiCypher Inc., 6 Davis Drive, Suite 755, Durham, NC 27709, USA

Vaidya Anup EpiCypher Inc., 6 Davis Drive, Suite 755, Durham, NC 27709, USA

Maier Keith E EpiCypher Inc., 6 Davis Drive, Suite 755, Durham, NC 27709, USA

Morgan Michael Department of Biophysics & Biophysical Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA

Keogh Michael-Christopher EpiCypher Inc., 6 Davis Drive, Suite 755, Durham, NC 27709, USA

https://orcid.org/0000-0001-8578-2969
Wolberger Cynthia Department of Biophysics & Biophysical Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA

To whom correspondence should be addressed. Tel: +1 410 955 0728; Email: cwolberg@jhmi.edu
09 9 2024
16 8 2024
16 8 2024
52 16 99789995
01 8 2024
15 7 2024
16 2 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.
2024
https://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Monoubiquitination of histones H2B-K120 (H2BK120ub) and H2A-K119 (H2AK119ub) play opposing roles in regulating transcription and chromatin compaction. H2BK120ub is a hallmark of actively transcribed euchromatin, while H2AK119ub is highly enriched in transcriptionally repressed heterochromatin. Whereas H2BK120ub is known to stimulate the binding or activity of various chromatin-modifying enzymes, this post-translational modification (PTM) also interferes with the binding of several proteins to the nucleosome H2A/H2B acidic patch via an unknown mechanism. Here, we report cryoEM structures of an H2BK120ub nucleosome showing that ubiquitin adopts discrete positions that occlude the acidic patch. Molecular dynamics simulations show that ubiquitin remains stably positioned over this nucleosome region. By contrast, our cryoEM structures of H2AK119ub nucleosomes show ubiquitin adopting discrete positions that minimally occlude the acidic patch. Consistent with these observations, H2BK120ub, but not H2AK119ub, abrogates nucleosome interactions with acidic patch-binding proteins RCC1 and LANA, and single-domain antibodies specific to this region. Our results suggest a mechanism by which H2BK120ub serves as a gatekeeper to the acidic patch and point to distinct roles for histone H2AK119 and H2BK120 ubiquitination in regulating protein binding to nucleosomes.

Graphical Abstract

Graphical Abstract

National Institute of General Medical Sciences 10.13039/100000057 R35GM130393 National Cancer Institute 10.13039/100000054 F31CA261154 F31CA271743 National Institutes of Health 10.13039/100000002 R43GM134834 R44GM119893 Frederick National Laboratory for Cancer Research 10.13039/100012728 HSSN261200800001E
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pmcIntroduction

The nucleosome is the fundamental organizational unit of chromatin and comprises two copies each of histones H2A, H2B, H3, and H4 wrapped by ∼147 base pairs (bp) of DNA. Nuclear proteins interact with the nucleosome at various sites including the histone core, histone tails, nucleosomal DNA, and extra-nucleosomal linker DNA (1,2). The core histones are subject to a wide array of post-translational modifications (PTMs) that play a central role in regulating transcription (3), DNA replication (4), chromatin compaction (5), and the DNA damage response (6). PTMs ranging from small chemical groups, such as methyl, acetyl, and phosphoryl, to the attachment of 76-amino acid ubiquitin (7), modulate protein binding to chromatin and impact higher-order chromatin structure (8,9), thereby regulating downstream biological processes (10,11).

The nucleosome contains a conserved cluster of negatively charged residues in histones H2A and H2B, known as the nucleosome acidic patch (12), that is a hotspot for interactions with proteins that regulate transcription, the cell cycle, and the DNA damage response (reviewed in (2)). Structural studies have shown that proteins primarily engage the nucleosome acidic patch with arginine residues (2), as first seen in the crystal structures of the viral peptide, LANA, bound to nucleosome (13) and the Ran guanine nucleotide exchange factor (Ran GEF), RCC1, bound to a nucleosome (14). A recent proteomics study identified many additional proteins that bind the acidic patch and are involved in epigenetic regulation, transcription, and cell-cycle regulation (15).

Monoubiquitination of histones H2B-K120 (H2BK120ub) and H2A-K119 (H2AK119ub) play opposing roles in transcription regulation and chromatin compaction (16,17). H2BK120ub is enriched in actively transcribed genes (18,19), where it stimulates methylation of histone H3-K4 (18,20,21) and H3-K79 (19,22,23). H2BK120ub is dynamically regulated throughout the cell cycle (24), serving as a signal for the DNA damage response (25) and chromatin decompaction (8). H2AK119ub is enriched at heterochromatic regions (26), where it stimulates trimethylation of H3K27 (27–29), a repressive mark (30), and promotes chromatin compaction (31,32).

There have been several reports that H2BK120ub can interfere with the binding of proteins to nucleosomes, including human RCC1, yeast Sir3, and Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen (LANA) peptide (33–35). All three proteins contact the nucleosome acidic patch, which raises the possibility that the ubiquitin conjugated to H2BK120 interferes with their binding to this surface (13,14,36). However, a role for H2BK120ub in blocking protein binding seems at odds with the presumed mobility of ubiquitin, given the flexibility of its carboxy-terminal tail and of the aliphatic lysine side-chain to which it is covalently linked (37). Such flexibility could, in principle, allow ubiquitin to adopt positions on the nucleosome surface that would not clash with protein binding.

To gain insight to the structural basis by which H2BK120ub could interfere with protein binding to the nucleosome, we determined cryoEM structures of nucleosomes containing ubiquitin conjugated to H2B-K120. We found that the ubiquitin adopts several distinct positions that partially occlude the nucleosome acidic patch. Molecular dynamics (MD) simulations indicate that the ubiquitin conjugated to H2B-K120 remains stably associated over the nucleosome acidic patch, in positions that would be expected to interfere with protein binding. We also determined cryoEM structures of H2AK119ub nucleosomes and found that, while the ubiquitin adopts two distinct positions, neither significantly occludes the acidic patch. In vitro studies showed that H2BK120ub, but not H2AK119ub, dramatically reduces nucleosome binding by RCC1, LANA peptide, and single domain antibodies that specifically contact the acidic patch. Our results establish a mechanism by which H2BK120ub can regulate protein binding to the nucleosome acidic patch, thereby impacting a major hub of chromatin interactions.

Materials and methods

Expression and purification of ubiquitin

Ubiquitin (G76C) was expressed and purified as previously described (38).

Expression and purification of histones

Expression plasmids for Xenopus laevis histones H2A, H2B, H3, and H4 were a gift from Gregory Bowman (Johns Hopkins University). Histones were expressed and purified as previously described (39), with the following modifications. Thawed cell pellet was lysed with multiple passes through a Microfluidizer (Microfluidics). After lysis, centrifugation, and multiple washes using a Triton X-100 containing buffer, washed cell pellets were resuspended in a buffer containing 20 mM HEPES pH 7.5, 7 M Guanidine HCl, 10 mM dithiothreitol (DTT). Resuspended washed cell pellets were size exclusion purified using a buffer containing 10 mM Tris pH 8.0, 7 M urea, 1 mM EDTA, 5 mM β -mercaptoethanol (BME). Fractions were pooled and injected onto a 2-column series with a HiTrap Q-XL column connected upstream of a HiTrap SP-XL column using a buffer containing 20 mM Tris pH 7.8, 7 M urea, 1 mM EDTA, 5 mM BME). The Q-XL column was removed from the system, and then the histones were eluted from the SP-XL column with a gradient of 0–1 M NaCl on an ÄKTA Pure (Cytiva) chromatography system.

Preparation of ubiquitinated histone H2BK120 and H2AK119

Dichloroacetone (DCA)-linked H2BK120ub and H2AK119ub were prepared as previously described (38).

Preparation and purification of dimethylated histone H3-K79 (H3K79me2)

A methyl-lysine analog of H3K79me2 (H3KC79me2) was prepared by alkylating the cysteine in histone H3K79C as previously described (40) and lyophilized. To purify the reaction product, the lyophilized sample was dissolved in filtered 7 M guanidine HCl and loaded onto a PROTO 300 C4 reverse-phase column (Higgins Analytical) with a buffer containing 0.1% TFA and eluted with a gradient of 0–90% acetonitrile on an HPLC (Waters). The peaks corresponding to unreacted histone H3-K79C and H3KC79me2 were collected and identified. Identity and purity were determined by digesting a sample with trypsin and analyzing its mass/charge ratio using Matrix Assisted Laser Desorption/Ionization–Mass Spectrometry (MALDI-MS) (Supplementary Figure S1). The H3KC79me2 sample was dialyzed into 5 mM BME, lyophilized, and stored at –20°C.

Purification of Widom 601 DNA

The Widom 601 DNA sequence (147 bp) (41) was expressed in Escherichia coli XL-1 Blue using the pST55-16 × 601 plasmid (14). The 601 sequence was expressed, purified, and isolated as described (42). The 601 sequence used is:

5'−ATCGGATGTATATATCTGACACGTGCCTGGAGACTAGGGAGTAATCCCCTTGGCGGTTAAAACGCGGGGGACAGCGCGTACGTGCGTTTAAGCGGTGCTAGAGCTGTCTACGACCAATTGAGCGGCCTCGGCACCGGGATTCTCGAT−3′.

601 DNA flanked by 19 bp of linker DNA (185 bp; 19-N-19) was amplified using polymerase chain reaction (PCR) using primers (IDT DNA) containing overhangs. The primers used were:

Forward: 5′−biosg−GTCGCTGTTCGCGACCGGCAATCGATGTATATATCTGACACGTGCC−3′

Reverse: 5′−GACCCTATACGCGGCCGCCCATCAGAATCCCGGTGCCGAG−3′

Phusion polymerase was used to amplify 100 μl reaction volumes with standard PCR parameters. The PCR product was precipitated by combining 100% EtOH, PCR mix, and 3 M sodium acetate pH 5.2 (10:1:20) at –80°C for 1 h. Precipitated DNA was collected by centrifugation and the supernatant was removed. The pellet was washed with 70% EtOH and air dried before resuspending in TE Buffer (10 mM Tris pH 8.0, 1 mM EDTA). The PCR product was purified with PCI (phenol:chloroform:isoamyl alcohol 25:24:1). PCI was added to an equal volume of resuspended PCR product pellet, vortexed, and centrifuged to separate the phases. The organic phase was removed and the aqueous phase was extracted twice more the same way. All of the removed organic phases were pooled, back-extracted with TE buffer to collect any PCR product left behind in the organic phase, and all the aqueous phases were combined and saved as the purified PCR product. Purified PCR product, 3 M sodium acetate pH 5.2, and 100% EtOH was combined in a 10:1:20 ratio and placed at –20°C overnight. The precipitated purified PCR product was then centrifuged, the pellet washed with 70% EtOH, centrifuged again, and allowed to dry. This pellet of purified 185bp 601 DNA was then resuspended in MilliQ (Sigma) water and stored at –20°C.

Preparation of nucleosomes

Nucleosomes for structural studies (unmod-Nuc 147/185 bp, H2AK119ub Nuc 147/185 bp, H2BK120ub Nuc 147/185 bp, H2BK120ub + H3KC79me2 Nuc 185 bp) were reconstituted as previously described (39), with the modifications described below.

For each sample, histone octamer and Widom 601 DNA was combined in an octamer:DNA molar ratio of 1.2:1 in a buffer containing 10 mM Tris pH 7.5, 2 M KCl, 1 mM EDTA, 1 mM DTT, such that final DNA concentration was 6 μM. Nucleosomes were then assembled by salt gradient dialysis, reducing the salt concentration to 0.25 M KCl over 24 h. Precipitate was removed by centrifugation and nucleosome purity assessed by native gel electrophoresis. Nucleosome samples that showed excess free DNA or higher-order species were further purified by loading onto a SK DEAE-5PW column (TOSOH biosciences) equilibrated in buffer containing 10 mM Tris pH 7.5, 0.25 M KCl, 0.5 mM EDTA, 1 mM DTT and eluted with a gradient of 0.25–0.6 M KCl on an Agilent HPLC instrument. Purified nucleosome was dialyzed into a buffer containing 20 mM HEPES pH 7.5, 25 mM KCl, 1 mM EDTA, 1 mM DTT, 20% glycerol, flash frozen in liquid nitrogen, and stored at –80°C.

Nucleosomes for dCypher™ Luminex nucleosome binding assays (unmodified (EpiCypher 16-0006); acidic patch mutant H2A(E61A) (EpiCypher 16-0029); H2AK119ub1 (EpiCypher 16–0395); or H2BK120ub1 (EpiCypher 16-0396)) were generated on 5′ biotinylated DNA (147 bp of 601 nucleosome positioning sequence) as previously described (43–45) and confirmed by SDS-PAGE, immunoblotting and mass spectrometry. The H2B-K120 and H2A-K119 ubiquitinated histones used for these binding studies possess a native gamma-lysine isopeptide linkage (44,46).

Purification of RCC1

A plasmid encoding RCC1 fused to an N-terminal hexahistidine (6xHis) tag was transformed into E. coli BL21(DE3)Rosetta2-pLysS cells. The colonies were used to inoculate 5 ml volumes of media, which were expanded to 1 L volumes for full-scale growth. Cultures were grown at 37°C and 200 RPM in 2× yeast extract tryptone (2× YT) media supplemented with ampicillin and chloramphenicol. Cultures were induced by addition of 1 mM isopropyl-ß-d-thiogalactopyranoside (IPTG) when they reached an OD600 of 0.4–0.6, and were grown for an additional 18 h at 18°C. Cells were harvested by centrifugation, resuspended in buffer containing 20 mM HEPES pH 7.5, 300 mM NaCl, 50 mM imidazole, 10% glycerol, 1 mM DTT, 1 tablet/50 ml Complete Protease Inhibitor Cocktail (Millipore Sigma #11836153001), flash-frozen in liquid nitrogen, and stored at –80°C.

The frozen cell pellet suspension was thawed in a water bath and an equal volume of buffer containing 20 mM HEPES pH 7.5, 300 mM NaCl, 50 mM imidazole, 1 mM DTT, and 0.2 mM PMSF added. Diluted cell suspension was lysed by sonication for three rounds each of 1 min total processing time (5 s on/10 s off) at 40% power. The resulting whole cell extract was centrifuged at 17000 RPM and the supernatant filtered using a 1.1 μm filter. The resulting clarified extract was loaded onto a 5 mL HisTrap HP (Cytiva) column equilibrated in 20 mM HEPES pH 7.5, 300 mM NaCl, 50 mM imidazole, 1 mM DTT and eluted with a gradient of 0.05–1 M imidazole on an ÄKTA Pure instrument (Cytiva). Eluted protein was diluted with 20 mM HEPES pH 7.5, 10% glycerol, 1 mM DTT, 0.2 mM PMSF to a final salt concentration of 50 mM NaCl and filtered using a 1.1 μm filter. The resulting protein was loaded onto 5 mL HiTrap SP-HP (Cytiva) cation exchange column equilibrated in 20 mM HEPES pH 7.5, 50 mM NaCl, 10% glycerol, 1 mM DTT, 0.2 mM PMSF and eluted with a gradient of 0.05–2 M NaCl. Eluted protein was dialyzed overnight in 20 mM HEPES, 150 mM NaCl, 10% glycerol, 1 mM DTT, and 0.2 mM PMSF, concentrated, flash-frozen in liquid nitrogen, and stored at –80°C.

CryoEM sample preparation

Nucleosome containing H2BK120ub and 147 bp Widom 601 DNA was thawed on ice and buffer exchanged into nucleosome storage buffer (20 mM HEPES pH 7.8, 50 mM NaCl, 1 mM DTT) using a Zeba Spin Desalting Column (Thermo #89882). Quantifoil R 2/2 copper 200 mesh grids (Electron Microscopy Sciences #Q2100CR2) were glow-discharged for 45 s at 15 mA using a PELCO easiGLOW Glow Discharge System to apply a negative charge to their surface. Then 3 μl of sample at 0.52 mg/ml was applied to the grid, immediately blotted for 3.5 s with a blot force of 5, and plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher) set at 100% humidity and 4°C.

Nucleosome containing histone H2BK120ub, dimethylated histone H3K79 analog (H3KC79me2) and the 147 bp Widom 601 DNA with 19 bp linkers (185 bp) was thawed on ice and buffer exchanged into nucleosome storage buffer. Quantifoil R 2/2 copper 200 mesh grids were glow-discharged for 30 s at 15 mA, after which 3 μl of sample at 1.50 mg/ml was applied to the grid, left to adsorb for 90 s, then blotted for 3 s with a blot force of 5, and plunge-frozen in liquid ethane using a Vitrobot Mark IV apparatus set to 100% humidity and 4°C.

Nucleosome containing histone H2AK119ub and the 147 bp Widom 601 DNA was buffer exchanged into nucleosome storage buffer. Quantifoil R 2/2 copper 200 mesh grids were glow-discharged for 45 s at 15 mA, after which 3 μl of sample at 0.82 mg/ml was applied to the grid, immediately blotted for 3.5 s with a blot force of 5, and plunge-frozen in liquid ethane using a Vitrobot Mark IV apparatus set to 100% humidity and 4°C.

CryoEM data collection

Data on H2BK120ub nucleosomes were collected at the Beckman Center for Cryo-EM at the Johns Hopkins University School of Medicine using a Thermo Fisher Titan Krios 300 kV electron microscope equipped with a Falcon 4 direct electron detector and a Selectris energy filter. A dataset of 8392 exposures was collected in counting mode and recorded in Electron Event Representation (EER) format using a magnification of 130kx, pixel size of 0.97 Å, nominal dose of 40 e−/Å2, dose rate of 6.42 e−/px/s, a defocus range of –0.5 to –2.5 μm, and an energy filter slit width of 10 eV. A multi-shot imaging strategy was used to collect 8 shots per hole, utilizing beam image shift to move between each target.

Data on H2BK120ub + H3KC79me2 nucleosomes were collected at the National Cryo-EM Facility (NCEF) at the Frederick National Laboratory for Cancer Research, using a Thermo Fisher Titan Krios 300 kV electron microscope equipped with a Gatan K3 camera and an energy filter. A dataset of 7782 exposures was collected in super-resolution mode using a magnification of 105kx, pixel size of 0.436 Å, nominal dose of 50 e−/Å2, dose rate of 12.33 e−/px/s, 40 frames per exposure, a defocus range of –0.75 to –1.75 μm, and an energy filter slit width of 20 eV. A multi-shot imaging strategy was used to collect three shots per hole, utilizing beam image shift to move between each target.

Data on H2AK119ub nucleosomes were collected at the Beckman Center for Cryo-EM at Johns Hopkins University School of Medicine using a Titan Krios 300 kV electron microscope equipped with a Falcon 4 direct electron detector and Selectris energy Filter. Two datasets were collected and combined. The first dataset of 5479 exposures was collected in counting mode and recorded in Electron Event Representation (EER) format using a magnification of 130kx, pixel size of 0.97 Å, nominal dose of 40 e−/Å2, dose rate of 6.42 e−/px/s, a defocus range of –0.2 to –5.0 μm, and an energy filter slit width of 10 eV. A multi-shot imaging strategy was used to collect 9 shots per hole, utilizing beam image shift to move between each target. A second dataset of 6732 exposures was collected in counting mode and recorded in EER format using a magnification of 130kx, pixel size of 0.97 Å, nominal dose of 40 e−/Å2, dose rate of 6.46 e−/px/s, a defocus range of –0.5 to –3.0 μm, and an energy filter slit width of 10 eV. A multi-shot imaging strategy was used to collect 9 shots per hole, utilizing beam image shift to move between each target.

CryoEM data processing

Data on H2BK120ub nucleosomes were processed in cryoSPARC v4.1 (47). Exposures were motion-corrected and cropped to one-half of their original resolution using Patch Motion Correction. The contrast transfer function (CTF) correction was performed using Patch Motion Correction. Poor quality micrographs were removed using Manually Curate Exposures, yielding 8149 high-quality micrographs. An initial round of particle picking was performed using Blob Picker and Inspect Picks, then extracted using Extract from Micrographs. A set of 2D templates was created by first performing 2D classification on the extracted particles, then selecting representative views using Select 2D classes. These templates were used to perform a second round of particle picking using Template Picker and Inspect Picks, then extracted using Extract from Micrographs to yield an uncleaned particle stack of 754004 particles. One round of 2D Classification and Select 2D Classes was performed to remove junk particles by discarding distinctly poor quality 2D classes to yield a partially cleaned particle stack of 550718 particles. Additional particle cleaning was performed using four parallel 4-structure Ab-Initio Reconstruction jobs, keeping particles from the good classes, which yielded a cleaned particle stack of 329531 particles. These particles were aligned along their C2 symmetry axis using the Non-Uniform Refinement job (48) and then symmetry-expanded to double the effective number of particles to 659062 particles. One round of focused 3D Classification was performed with a spherical focus mask centered on the area of blurred ubiquitin density to yield four distinct ubiquitin positions. Individual particle CTF was refined with Local CTF Refinement, image group CTF was refined with Global CTF Refinement, and structures were refined with local refinement to produce H2BK120 ubiquitin position 1 (76770 particles, 3.32 Å resolution), position 2 (71307 particles, 3.33 Å resolution), position 3 (73431 particles, 3.34 Å resolution), and position 4 (65167 particles, 3.36 Å resolution). The final maps were sharpened with Local Filtering.

Data on H2BK120ub + H3KC79me2 nucleosomes were processed in cryoSPARC v3.3 (47). Exposures were motion-corrected and cropped to one-half of their original resolution using Patch Motion Correction. The CTF correction was performed using Patch CTF Estimation. Poor quality micrographs were removed using Manually Curate Exposures, yielding 7324 high-quality micrographs. An initial round of particle picking was performed using Blob Picker and Inspect Picks, then extracted using Extract from Micrographs. A set of 2D templates was created by first performing 2D Classification on the extracted particles then selecting representative views using Select 2D classes. These templates were used to perform a second round of particle picking using Template Picker and Inspect Picks, then extracted using Extract from Micrographs to yield an uncleaned particle stack of 2288109 particles. Two rounds of 2D Classification and Select 2D Classes was performed to remove junk particles by discarding distinctly poor quality 2D classes, yielding a partially cleaned particle stack of 1034537 particles. Additional particle cleaning was performed using multiple iterations of Ab-Initio Reconstruction jobs, yielding a cleaned particle stack of 628403 particles. The particles were aligned along their C2 symmetry axis using the Non-Uniform Refinement job (48) and then symmetry-expanded to double the effective number of particles to 1256806 particles. Three rounds of focused 3D Classification were performed with a large spherical focus mask centered on H2BK120 on one face of the nucleosome to yield two distinct ubiquitin positions. Individual particle CTF was refined with Local CTF refinement, and structures were refined with Local Refinement to yield maps showing H2BK120 ubiquitin positions 5 (818874 particles, 2.93 Å resolution) and 6 (282239 particles, 3.06 Å resolution). The final maps were sharpened with Local Filtering.

Data on H2AK119ub nucleosomes were processed in cryoSPARC v3.3 (47). Exposures from both datasets were imported with an EER upsampling factor of 2 and motion corrected using Patch Motion Correction. The CTF correction was performed using Patch Motion Correction. Poor quality micrographs were removed using Manually Curate Exposures, yielding 10018 high-quality micrographs. An initial round of particle picking was performed using Blob Picker and Inspect Picks, then extracted using Extract from Micrographs. A set of 2D templates was created by first performing 2D Classification on the extracted particles then selecting representative views using Select 2D Classes. These templates were used to perform a second round of particle picking using Template Picker and Inspect Picks, then extracted using Extract from Micrographs to yield an uncleaned particle stack of 1824939 particles. One round of 2D Classification and Select 2D Classes was performed to remove junk particles by discarding distinctly poor quality 2D classes to yield a cleaned particle stack of 895614 particles. We note that iterative Ab-Initio Reconstruction jobs for particle cleaning did not improve resolution, so were therefore not used for additional particle cleaning. The particles were aligned along their C2 symmetry axis using the Non-Uniform Refinement job (48) and then symmetry-expanded, doubling the effective number of particles to 1791228 particles. These particles were used in a local refinement 3D reconstruction to produce a structure with poor density for ubiquitin, showing a blur of lower resolution density across the surface of the nucleosome. A round of focused 3D classification was performed with a cylindrical focus mask centered on the area of blurred ubiquitin density to yield two distinct ubiquitin positions. An additional round of focused 3D classification was performed for each ubiquitin position using a spherical focus mask centered on each ubiquitin position. Individual particle CTF was refined with Local CTF Refinement, image group CTF was refined with Global CTF Refinement, and structures were refined with local refinement to produce H2AK119 ubiquitin position 1 (102259 particles, 3.41 Å resolution) and position 2 (68407 particles, 3.47 Å resolution). The final maps were sharpened with Local Filtering.

Model building and refinement

Initial models of nucleosomes containing H2BK120ub, H2BK120ub + H3KC79me2, and H2AK119ub were constructed by rigid-body fitting models for an unmodified nucleosome (PDB: 4ZUX) and ubiquitin (PDB: 1UBQ) into density maps using ChimeraX (49), and refined using all-atom flexible refinement with strong restraints in Coot 0.9.6 (50). Since the particles were subjected to C2 symmetry expansion and 3D classified according to the position of only one of the ubiquitin molecules, the ubiquitin was modeled onto only one side of the nucleosome. Histone tails were extended where density was visible. C-terminal tail residues of ubiquitin were omitted where density was not visible. The modeled ubiquitin includes residues 1–72 in H2Bub position 4, 1–74 in H2Bub position 5, 1–73 in H2Bub position 6, and 1–73 in H2Aub positions 1 and 2. The DNA of H2BK120ub + H3KC79me2 nucleosomes was extended from 145 to 157 bp where density was visible to account for the for the extra-nucleosomal linker DNA present in this sample. All models were further refined in PHENIX (51) using phenix.real_space_refine (52) and validated using the CryoEM Comprehensive Validation module in PHENIX running MolProbity (53). Figures were generated with ChimeraX (49).

Molecular dynamics simulations

The H2B-ubiquitinated nucleosome system was built using the full-length crystal structure of the X. laevis nucleosome (1KX5) (54) and the crystal structure of ubiquitin (1UBQ) (55). Following the approach of Carroll et al. (56), the backbone of the ubiquitin-linked K48 in chain B of PDB entry 3NS8 (57) was aligned with the lysine backbone of histone H2BK120. Next, the sidechain of H2BK120 was rebuilt to have the same geometry as K48 in 3NS8. The ubiquitin C-terminal glycine in chain A of 3NS8 (post-alignment) was then used as the C-terminal glycine of 1UBQ. The unstructured C-terminus of 1UBQ (residues 71–75) was refined using the Modloop server (58) to connect and refine the C-terminal glycine to the rest of 1UBQ. The covalent bond between H2BK120 and the C-terminal glycine of ubiquitin was built using the LEaP program in AMBER20 (59), resulting in a bond between the neutralized lysine sidechain nitrogen and the glycine carbonyl carbon. The structure was solvated in a truncated octahedron box with periodic boundary conditions set to 1.25 nm away from the ubiquitinated nucleosome. Sodium and chloride ions were then added to neutralize the system charge and to reach a final concentration of 150 mM.

All simulations were performed using the AMBER ff14SB (60) force field, including parmbsc1 DNA and CUFIX ion parameter corrections (61,62). These force field parameters have been shown to effectively capture nucleosome dynamics in all-atom MD simulations (63). All water molecules were described using a TIP3P water model. For the peptide bond between H2BK120 and the C-terminal glycine of ubiquitin, we employed the force field parameters parameterized by Carroll et al (56).

Simulations were carried out using the CPU version of Particle-Mesh Ewald Molecular Dynamics (PMEMD) in the AMBER20 software (59). All systems were energy-minimized using a steepest descent gradient method for 10000 steps with a 500 kJ mol−1 nm−2 positional restraint applied to all heavy atoms. Systems were then equilibrated as described by Armeev et al. (63): (i) 100 ps with positional restraints of 500 kJ mol−1 nm−2 with 0.5 fs time step; (ii) 200 ps with positional restraints of 50 kJ mol−1 nm−2 with 2 fs time step (and further the same); (iii) 200 ps with positional restraints of 5 kJ mol−1 nm−2 and (iv) 200 ps with positional restraints of 0.5 kJ mol−1 nm−2; (v) 200 ps of unrestrained simulations. Systems were equilibrated in the canonical (NVT) ensemble, at 300 K and 1 bar using the Langevin thermostat and Parrinello-Rahman barostat. Systems were then simulated with no positional restraints for 1 ns in an isobaric-isothermal (NPT) ensemble at 300 K and 1 bar, using a Langevin thermostat and Monte Carlo barostat (same for production run simulations). All simulations used a 4 fs time step using hydrogen mass repartitioning. Hydrogen bond lengths were constrained using the SHAKE algorithm. The cutoff for non-bonded interactions were set to 12.0 Å, and long-range electrostatic interactions were computed using the particle mesh EWALD (PME) method. Trajectory frames were written every 50 ps. The production run was simulated in duplicate for 400 ns, producing a total of 800 ns of simulation time.

The AmberTools CPPTRAJ package (64) was used to center trajectories. Simulations were visualized using Visual Molecular Dynamics (VMD) (65) and PyMOL. Distance calculations were performed using the MDAnalysis package (66). To calculate the distance between ubiquitin and the nucleosome acidic patch, the distance between the center of mass of each group was used. The center of mass of ubiquitin was based on the backbone atoms of regions with secondary structure. The acidic patch center of mass was based on the backbone heavy atoms of the following residues of histone H2A: E61, E64, D90, E91 and E92. The ubiquitin molecules on both faces of the nucleosome were used from each trajectory. The distances sampled between ubiquitin and the acidic patch are presented as a probability distribution function using the seaborn package in python. To generate ubiquitin clusters, we used the K-means clustering algorithm available in the AmberTools CPPTRAJ package (64). Using both trajectory replicates, we generated 10 clusters based on the RMSD of the backbone heavy atoms of ubiquitin containing secondary structure. Since there are two ubiquitin molecules on the nucleosome, the ubiquitin molecule that had the highest RMSD was used to generate the RMSD clusters. For the calculation of ubiquitin RMSD, all trajectory frames were aligned to the backbone heavy atoms of the histone octamer core in the cryoEM structure of the ubiquitinated nucleosome.

Electrophoretic mobility shift assays

Binding reactions were prepared in 12 μl volumes by combining RCC1 and nucleosome in binding buffer (20 mM HEPES pH 7.6, 50 mM NaCl, 5% sucrose, 1 mM DTT, 2.5 mM MgCl2, 0.1 mg/ml BSA). In competitive binding experiments LANA peptide (1–23) was also added as a reaction component. Nucleosome was always added last. Prepared reactions were equilibrated on ice for 1 h. Prior to sample loading, 6% TBE gels were run at 150 V for 60 min at 4°C in 0.25× Tris–borate–EDTA (TBE) buffer. Then, 10 μl of each equilibrated binding reaction was loaded on a 6% TBE gel and run at 150 V for 100–120 min at 4°C in 0.25× TBE buffer. Gels were stained in the dark on a rotating shaker for 20 min with SYBR Gold (Invitrogen) DNA-intercalating stain diluted to 1:5000, then imaged.

Surface plasmon resonance

The affinity of RCC1 for nucleosome was measured on a Biacore 8K biosensor (GE Healthcare) by surface plasmon resonance (SPR). Streptavidin from Streptomyces avidinii (Millipore Sigma) corresponding to 2000 response units (RU) was amine-coupled on the utilized flow channels of a CM5 sensor chip. Approximately 200 RU of 5′ biotinylated 185 bp nucleosome (unmodified, H2AK119ub, or H2BK120ub) was captured directly on flow cell 2 in separate channels. Binding experiments were carried out in HBS-EP + buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20), where RCC1 was used as the analyte and titrated overflow cells 1 and 2 in 2-fold dilutions. Following a 600 s dissociation, an additional 300 s injection of HBS-EP + buffer served to regenerate the sensor surface. Sensorgrams were double-referenced against the control flow cell and buffer injections. Data from two replicates were fit to a 1:1 steady-state affinity binding model using Biacore Insight Evaluation software and plotted in GraphPad Prism. Mean and standard deviation values are shown.

Development of single domain antibodies (VHH) to the nucleosome acidic patch

Immunizations were performed at Eurogentec, phage display selection and recombinant expression at QVQ, and final characterization at EpiCypher. In brief, two llamas were immunized with nucleosomes and Freund's Complete adjuvant, and boosted on days 28, 43 and 52 with additional immunogen and Freund's incomplete adjuvant. RNA was harvested from peripheral blood lymphocytes on day 52, reverse transcribed, single domain antibody (also termed VHH; Variable Heavy Domain of a Heavy chain antibody) cDNAs isolated by PCR, and cloned to VCSM13 phage-display libraries of 2 × 109 and 3 × 109 transformants. After three rounds of library selection to nucleosomes, single-colony phage were isolated and ELISA screened to confirm target binding. Candidate VHH were Sanger sequenced and categorized to families. Chosen VHH (C-terminal 6His-tagged) were sequence verified, expressed as recombinants in BL21 E. coli, purified by Ni-NTA chromatography, and purity confirmed by SDS-PAGE. VHH epitopes were identified / characterized using fully defined nucleosomes (WT, PTM+, mutants) in dCypher Alpha (67) and dCypher Luminex (unpublished data).

dCypher™ luminex nucleosome binding assays

Semi-synthetic nucleosomes (Unmodified, Kub1-modified and acidic patch mutated) were separately coupled to saturation to distinct magnetic avidin-coated xMAP bead regions (Luminex). All handling and incubations of MagPlex beads were performed under subdued lighting. Nucleosome: bead region complexes were adjusted to 1 million beads/ml, multiplexed, and exchanged into long term storage buffer (10 mM Cacodylate pH 7.5, 0.01% BSA, 0.01% Tween-20, 1 mM EDTA, 10 mM b-mercaptoethanol, 50% glycerol) for storage at –20°C. Panel balance and Nucleosome: bead region identity were confirmed using anti-dsDNA (EMD Millipore #MAB030; 1/5, 1/50 and 1/500), anti-H3.1/2 (Active Motif #61629; 1/250, 1/1000 and 1/4000) and anti-PTM (EMD Millipore #04–263, CST #8240S; both 1/250, 1/1000 and 1/4000).

For the dCypher Luminex assay, the binding of Queries (GST-2XLANA-His6 (hereafter GST-LANA) or VHH), to multiplexed nucleosomes (1000 beads/well: the Targets) was tested by serially diluting the Queries (two-fold: GST-LANA, 1.3 μM–40 pM final; VHH, 2 μM–2 nM final). In brief, 50 μl of multiplexed nucleosome panel was combined with 50 μl query in Luminex buffer (final: 20 mM Tris pH 7.5, 100 mM NaCl, 0.01% NP40, 0.01% BSA, 1 mM DTT) in a black, flat-bottom 96-well plate (GreinerBio #655900) and incubated under subdued lighting for one hr with shaking (800 RPM). Beads were captured / washed twice (100 μl of Luminex buffer) on a plate-based magnet. 100 μl of either anti-GST (Fortis Life Sciences #A190-122A; 1/1000) or Luminex buffer was respectively added to GST-LANA or VHH and incubated for 30 min with shaking (800 RPM). Beads were captured/washed twice (100 μl of Luminex buffer) on a plate-based magnet. 100 μl of PE donkey anti-rabbit IgG (BioLegend #406421; 2 ug/ml) for GST-LANA, or PE goat anti-alpaca IgG VHH domain (Jackson ImmunoResearch Laboratories #128–115-232; 1/400) for VHH, was added and incubated for 30 min with shaking (800 rpm). After two additional washes, beads were resuspended in 100 μl Luminex buffer and Median Fluorescence Intensity (MFI) read on a FlexMap3D instrument (PerkinElmer), counting a minimum of 50-events per Target. Data were analyzed and visualized in GraphPad Prism 10. Data are plotted as the mean and standard deviation of duplicates. A sigmoidal, four-parameter logistic fit of log transformed VHH (M) fit was used. Representative experiments are shown in figures.

The GST-LANA construct used was GST-TEVrs-2XLANA-2XGGGS-SrtAss-6HIS:

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSIEENLYFQSMAPPGMRLRSGRSTGAPLTRGSGGGGSMAPPGMRLRSGRSTGAPLTRGSGGGGSGGGGSLPATGGEGKSSGSGSESKSTGGHHHHHH

Results

Ubiquitin conjugated to histone H2B-K120 occludes the nucleosome acidic patch

To investigate whether H2BK120ub adopts positions that could interfere with the binding of proteins that interact with the nucleosome acidic patch, we determined cryoEM structures of nucleosome core particles containing Widom 601 DNA (147 bp) and ubiquitinated histone H2B-K120 (Supplementary Table S1). The density maps revealed four distinct positions of ubiquitin on the nucleosome surface, denoted H2Bub positions 1, 2, 3, and 4 (Figure 1A, Supplementary Figure S2). In H2Bub position 1, ubiquitin sits nearest the center of the histone octamer core, and progressively further away in positions 2–4. The global resolution estimates for the EM maps for H2Bub positions 1, 2, 3, and 4 were calculated to be 3.32, 3.33, 3.34, and 3.36 Å, respectively (Supplementary Figures S3A–D). The local resolution of the nucleosome core, at ∼3 Å, was higher than that of the conjugated ubiquitin, at ∼6 Å, indicating greater conformational heterogeneity of the ubiquitin compared to the nucleosome core (Supplementary Figures S4A–D). The secondary structure elements of ubiquitin in H2Bub positions 1–3 were sufficiently resolved to unambiguously position and orient the ubiquitin within the electron density maps (Supplementary Figures S5A–C). In all three positions, the ubiquitin beta sheet is oriented towards the nucleosome surface, while the alpha helix faces away (Figure 1B). Given its proximity to the other positions, we modeled the ubiquitin in H2Bub position 4 in a similar orientation relative to the nucleosome surface (Supplementary Figure S5D).

Figure 1. Four discrete ubiquitin positions in cryoEM maps of nucleosomes containing H2BK120ub. (A) CryoEM Maps of H2BK120ub nucleosome showing ubiquitin in four discrete positions. (B) Superposition of the four ubiquitin positions depicted in cartoon representation (colors as in panel A). (C) Electrostatic surface representation of the histone octamer core showing the nucleosome acidic patch. (D) Superposition of the four ubiquitin positions (colors as in A) over an electrostatic surface representation of the histone octamer core.

The ubiquitin in H2Bub positions 1 and 2 occludes large portions of the nucleosome acidic patch, as is evident when viewed in projection (Figure 1C, D). To quantitate the extent to which ubiquitin in each position occludes the acidic patch, we calculated the buried surface area between the acidic patch and ubiquitin using a range of probe radius values (see Methods). At a probe radius of 5 Å, the acidic patch surface area occluded by ubiquitin in H2Bub positions 1, 2, and 3 is 1235, 891, and 405 Å2, respectively (Supplementary Figure S6A). This trend is generally consistent across buried surface calculations performed using larger and smaller probe radii (Supplementary Figures S6A, B).

Since methylation of histone H3K79 by DOT1L depends upon prior ubiquitination of histone H2BK120 (68), both modifications can co-occur on the same nucleosome (69). To see if this methyl modification affects H2BK120ub positioning on the nucleosome surface, we determined cryoEM structures of a nucleosome containing H2BK120ub, di-methylated histone H3-K79 (H3KC79me2), and Widom 601 DNA with 19 bp linkers (185 bp) (Supplementary Table S1). The cryoEM dataset revealed two additional distinct ubiquitin positions on the nucleosome surface, denoted H2Bub positions 5 and 6 (Figures 2A, B, Supplementary Figure S7). The global resolution estimates for these EM maps were 2.93 and 3.06 Å, respectively (Supplementary Figures S3E, F), with a local resolution of ∼2.5 Å for the nucleosome core and ∼5 Å for ubiquitin (Supplementary Figures S4E, F). The ubiquitin in position 5 lies between previously determined H2Bub positions 2 and 3 (Figure 2C). While the ubiquitin in H2Bub position 6 lies closer to histone H3 than any of the other H2Bub positions, it does not interact directly with H3KC79me2. The density corresponding to ubiquitin in positions 5 and 6 was sufficiently well-resolved to unambiguously orient the ubiquitin with its beta sheet oriented towards the nucleosome surface (Figures 2A, B, Supplementary Figures S5E, F). As observed for H2Bub positions 1 and 2, the ubiquitin in positions 5 and 6 occludes large portions of the nucleosome acidic patch (Figure 2D). At a probe radius of 5 Å, the occluded surface area between the acidic patch and ubiquitin in H2Bub positions 5 and 6 is 890 Å2 and 1119 Å2, respectively (Supplementary Figures S6A, B).

Figure 2. Two discrete ubiquitin positions in cryoEM maps of nucleosomes containing H2BK120ub and H3KC79me2. (A) CryoEM map of nucleosome containing H2BK120ub and H3KC79me2 with ubiquitin in position 5. Inset at right shows a cartoon model of H2Bub in position 5. (B) CryoEM map of nucleosome containing H2BK120ub and H3KC79me2 with ubiquitin in position 6. Inset at right shows a cartoon model of H2Bub in position 6. (C) The six ubiquitin positions observed in H2BK120ub nucleosomes with and without H3KC79me2 overlaid on a nucleosome with the histone octamer colored using an electrostatic surface representation. (D) Surface models of H2Bub in positions 5 and 6 overlaid on a nucleosome with the histone octamer colored using an electrostatic surface representation.

Molecular dynamics simulations reveal stability of ubiquitin conjugated to H2B-K120

While our density maps indicate that ubiquitin conjugated to H2BK120 can adopt several discrete positions, these structural snapshots do not provide information on the dynamic motion of ubiquitin on the nucleosome surface. We therefore used molecular dynamics (MD) simulations to investigate the mobility of ubiquitin in H2BK120ub nucleosomes. We built the H2BK120ub nucleosome system with the initial ubiquitin position at H2Bub position 5, which is approximately halfway between H2Bub positions 2 and 3. To gauge whether ubiquitin remained stably positioned over the acidic patch, we monitored the distance between their centers of mass over a 400 ns trajectory (Figure 3A). Over the course of the simulation, the distance between ubiquitin and the acidic patch varied between 24 and 36 Å (Figure 3B). Importantly, the ubiquitin primarily sampled conformations near the acidic patch and the distance between the ubiquitin and acidic patch largely remained within 1 Å of the starting ubiquitin position (Figure 3B). We note that ubiquitin samples some conformations farther from the acidic patch over the course of the simulation, as apparent from the local minima at ∼34 Å. This is consistent with our cryoEM data, where H2Bub can sample orientations that occlude the acidic patch to a lesser extent.

Figure 3. Molecular dynamics simulations of H2BK120ub nucleosome. (A) Diagram depicting calculation between the center of mass (COM) of ubiquitin and of the nucleosome acidic patch. The representative structure shows H2BK120ub nucleosome in the initial ubiquitin position for the MD simulations. (B) Probability density of finding ubiquitin at a specific distance from the acidic patch calculated over the entire simulation (black solid line). Initial starting position of ubiquitin indicated by a red dashed line. (C) K-means cluster analysis of ubiquitin showing the 10 most representative structures from one face of the nucleosome over the entire simulation (blue) and the ubiquitin cluster position relative to the nucleosome acidic patch (red), and ubiquitin in H2Bub positions 3 (green), 4 (yellow) and 5 (orange).

To visualize representative structures in the MD simulation, we used K-means clustering of ubiquitin throughout the trajectory to obtain representative snapshots consistent with H2Bub positions 3, 4, and 5 (Figure 3C). We did not see representative ubiquitin positions corresponding to H2Bub cryoEM positions 1, 2, and 6, likely because of limited sampling time. Overall, the MD simulations are consistent with our structural studies showing that ubiquitin conjugated to H2Bub occludes access to the acidic patch.

Ubiquitin conjugated to histone H2A-K119 minimally occludes the nucleosome acidic patch

Ubiquitinated histone H2A-K119 (H2AK119ub) is one of the most abundant PTMs in higher eukaryotes (70), but its effect on protein binding to the nucleosome has not been explored. To see whether ubiquitin conjugated to H2A-K119 adopts discrete positions, we determined cryoEM structures of the nucleosome core particle containing ubiquitinated histone H2A-K119 and Widom 601 DNA (147 bp) (Supplementary Table S1). The cryoEM dataset revealed two distinct ubiquitin positions, denoted H2Aub positions 1 and 2 (Figures 4A, B, Supplementary Figure S8), with global resolution estimates for the corresponding EM maps of 3.41 and 3.47 Å, respectively (Supplementary Figures S3G, H). Similar to the maps for H2BK120ub nucleosome, the local resolution of the H2AK119ub nucleosome core, at ∼3 Å, was higher than for the conjugated ubiquitin, at ∼6 Å (Supplementary Figures S4G, H). However, the map resolution was sufficient to unambiguously orient ubiquitin within the EM maps (Supplementary Figures S5G, H).

Figure 4. Two discrete ubiquitin positions in cryoEM maps of nucleosomes containing H2AK119ub. (A) CryoEM map of H2AK119ub nucleosome with ubiquitin in position 1. Inset shows a cartoon model of H2Aub in position 1. (B) CryoEM map of H2AK119ub nucleosome with ubiquitin in position 2. Inset shows a cartoon model of H2Aub in position 2. (C) CryoEM surface models of H2AK119ub nucleosome with both ubiquitin positions overlaid on a nucleosome with the histone octamer colored using an electrostatic surface representation.

The ubiquitin in H2Aub position 1 binds to the nucleosome surface over histones H3 and H4, immediately adjacent to the acidic patch (Figure 4C). In this position, ubiquitin partially overlaps the acidic patch, with an occluded surface area of 480 Å2 as measured with a 5 Å probe radius (Supplementary Figures S6A, B). The ubiquitin in H2Aub position 2 sits at the nucleosome dyad bound to the nucleosomal DNA terminus, end-on, with the ubiquitin beta-sheet oriented towards the DNA (Figure 4B). While the resolution is not sufficient to discern side chains, the ubiquitin position is consistent with van der Waals interactions between the hydrophobic patch on the ubiquitin beta sheet and the terminal base pair of the wrapping DNA. This ubiquitin position would be incompatible with a nucleosome embedded within a longer DNA duplex. We note that our attempts to determine the structure of an H2AK119ub nucleosome containing an additional 19 bp of flanking linker DNA (185 bp), which would be expected to prevent association of ubiquitin with the DNA end, were unsuccessful due to aggregation of the particles on cryoEM grids.

Since heterochromatic regions are characterized by chromatin that is condensed and enriched in H2AK119ub (26), we explored whether the ubiquitin in H2Aub position 1 could be accommodated within higher-order nucleosome packing. While the precise nature of inter-nucleosomal interactions in condensed chromatin remains an area of active investigation (71), solution-based experiments, X-ray crystal structures, and cryoEM maps have revealed stacked nucleosomes (72–75). Modeling using the structure of a compacted tetranucleosome (72) shows that the ubiquitin in H2Aub position 1 can be readily accommodated between two stacked nucleosomes without steric clash (Supplementary Figure S9). Ubiquitin in this position can also accommodate binding of the linker histone H1, which binds to the nucleosome dyad, promotes chromatin condensation and is abundant in heterochromatic regions (76,77). Two ubiquitin molecules from opposing H2AK119ub nucleosomes do not fit between two stacked nucleosomes (PDB: 1ZBB) without steric clash. However, a repositioning of the ubiquitin molecules or stacked nucleosomes may allow two ubiquitin molecules to fit between two stacked nucleosomes.

Ubiquitin conjugated to histone H2B but not H2A competes with RCC1 binding to nucleosomes

RCC1, a Ran guanine nucleotide exchange factor (Ran GEF) (78), interacts with the nucleosome acidic patch and the adjacent DNA in a multivalent fashion (14). H2BK120ub has been shown to interfere with RCC1 binding to the nucleosome as assayed in pulldown experiments coupled with mass spectrometry analysis (33,34). It is therefore possible that H2BK120ub occlusion of the acidic patch observed in our structural studies might account for reduced nucleosome binding by RCC1. While the effect of H2AK119ub on RCC1 binding to nucleosomes has not been explored, we speculated this modification would have a minimal effect based on minimal occlusion of the acidic patch by H2AK119ub in our cryoEM structure (Figure 4).

We first quantitated the effect of H2BK120ub on RCC1 binding to nucleosomes using electrophoretic mobility shift assays (EMSA). On unmodified nucleosomes, increasing concentrations of RCC1 resulted in the formation of two discrete higher molecular weight bands (Figure 5A), which presumably correspond to 1:1 and 2:1 RCC1:nucleosome complexes. We confirmed the identify of these bands by showing that they disappear in the presence of increasing concentrations of LANA peptide, which binds to the acidic patch (13) and has been shown to abrogate RCC1 binding (79) (Supplementary Figure S10A). At the highest concentrations of RCC1 (in the absence of LANA), we observed a high molecular weight smear resulting from non-specific aggregates containing RCC1 and nucleosome (Figure 5A). We next assayed RCC1 binding to H2BK120ub nucleosomes and found no discrete high molecular weight bands corresponding to RCC1:nucleosome complexes, although a high molecular weight smear corresponding to potential aggregates was visible at the highest RCC1 concentrations (Figure 5C). To rule out the possibility of artifacts due to the use of a non-native DCA linkage to attach ubiquitin to H2B-K120 (38), we confirmed that ubiquitin covalently linked with a native isopeptide bond also disrupts formation of the 1:1 and 2:1 RCC1:nucleosome bands (Supplementary Figure S10B).

Figure 5. Binding of RCC1 to nucleosomes with ubiquitin conjugated to H2BK120 and H2AK119. (A) Electrophoretic mobility shift assay (EMSA) showing binding of the indicated concentrations of RCC1 to unmodified nucleosome (100 nM). Complexes visualized with DNA stain, SYBR Gold. (B) Representative sensorgram of surface plasmon resonance (SPR) assay of RCC1 binding to unmodified nucleosome with average KD and standard deviation shown (n = 2). (C) EMSA showing binding of RCC1 to H2BK120ub nucleosome (100 nM). (D) Representative sensorgram of SPR assay of RCC1 binding to H2BK120ub nucleosome. (E) EMSA showing binding of RCC1 to H2AK119ub nucleosome (100 nM). (F) SPR assay of RCC1 binding to H2AK119ub nucleosome with average KD and standard deviation shown (n = 2).

To obtain a more precise measure of the impact of H2BK120ub on RCC1 binding to nucleosomes, we measured equilibrium dissociation constants by surface plasmon resonance (SPR). We found that RCC1 binds unmodified nucleosome with a KD of 730 ± 100 nM (Figure 5B), while the affinity for H2BK120ub nucleosomes is far lower, with an estimated KD greater than 10000 nM (Figure 5D). This far lower affinity is consistent with the reported role of H2BK120ub in interfering with RCC1 binding to nucleosomes (33,34).

To test whether it is simply the presence of ubiquitin that interferes with binding to nucleosomes, we assayed RCC1 binding to H2AK119ub nucleosomes by EMSA. Increasing concentrations of RCC1 results in formation of discrete bands corresponding to 1:1 and 2:1 RCC1:H2AK119ub nucleosome complexes (Figure 5E), similar to those observed in assays of binding to unmodified nucleosome (Figure 5A). The KD of RCC1 for nucleosomes containing H2AK119ub was measured by SPR to be 540 ± 90 nM (Figure 5F), similar to the KD of RCC1 for unmodified nucleosomes (Figure 5B). These results indicate that ubiquitin conjugated to H2AK119 does not interfere with RCC1 binding to the nucleosome, consistent with our cryoEM data showing that ubiquitin in H2AK119ub nucleosomes only partly occludes the nucleosome acidic patch (Figure 4C).

To further explore the ability of H2BK120ub or H2AK119ub to interfere with nucleosome binding, we compared the effect of these PTMs on additional acidic patch-binding entities. Nucleosome binding by a glutathione-S-transferase-LANA fusion protein (GST-LANA) decreases in the presence of H2BK120ub (35), while the impact of H2AK119ub has not been explored. We used dCypher™ Luminex (see Materials and methods) to compare the binding of GST-LANA to an unmodified, H2BK120ub, H2AK119ub, and acidic patch mutant H2A(E61A) nucleosome panel in multiplex. As expected, the binding affinity of GST-LANA was significantly weaker on H2BK120ub or H2A(E61A) nucleosomes than on unmodified nucleosomes (Figure 6A, Supplementary Table S2). As observed for RCC1 (Figure 5E), GST-LANA binding to H2AK119ub nucleosomes was indistinguishable from unmodified nucleosomes (Figure 6A).

Figure 6. H2BK120ub but not H2AK119ub inhibits interactions with the nucleosome acidic patch. Acidic patch mutation H2A(E61A) and H2BK120ub, but not H2AK119ub, interferes with the binding of GST-LANA (A) (35) and chromatibody VHH (B) to nucleosome (80) in dCypher-Luminex assays (see Methods); measured using median fluorescence intensity (MFI) units. (C–E) H2A(E61A) and H2BK120ub, but not H2AK119ub, also interfere with the binding of three newly generated nucleosome acidic patch specific VHH (clones 1E9, 1G1 and 1B2: see Materials and methods).

We next assayed the effect of H2BK120ub or H2AK119ub on the binding of four single domain antibodies (aka. Variable Heavy Domain of a Heavy chain antibodies; VHH) that specifically bind the nucleosome acidic patch (see Materials and methods). As shown in Figures 6B–E, the H2A(E61A) acidic patch substitution abrogated nucleosome binding by all four VHH (chromatibody, 1E9, 1G1, 1B2), confirming the importance of acidic patch contacts. As observed for RCC1 and GST-LANA, the presence of H2BK120ub substantially reduced VHH-nucleosome binding, while binding to H2AK119ub nucleosomes was indistinguishable from unmodified nucleosomes (Figures 6B–E). Note that, while the apparent EC50 of the chromatibody is similar for unmodified and ubiquitinated nucleosomes (Supplementary Table S2), the median fluorescence intensity is far lower for H2BK120ub nucleosomes as compared to unmodified or H2AK119ub nucleosomes. Taken together, these results indicate that H2BK120ub, but not H2AK119ub, generally interferes with nucleosome engagement by acidic patch binding entities.

Discussion

Our results provide a mechanism by which ubiquitin conjugated to histone H2B-K120 can exclude proteins from the nucleosome. There had been reports that nucleosome binding by human RCC1, yeast SIR3, and the viral LANA peptide was disrupted by monoubiquitination of histone H2B-K120 (33–35). These proteins bind the nucleosome acidic patch (13,14,36), leading to the speculation that H2BK120ub may act as a gatekeeper to this cluster of acidic residues in histones H2A/H2B (33,35,81,82). Our cryoEM maps unexpectedly showed ubiquitin positioned on the nucleosome in multiple distinct locations that occlude the nucleosome acidic patch, particularly H2Bub positions 1, 2, and 5 (Figures 1A, 2A, Supplementary Figure S6). The resulting steric clash could explain how H2BK120ub interferes with binding of select proteins to the nucleosome acidic patch (2). It is also possible that H2BK120ub may inhibit protein binding to other adjacent nucleosome surfaces, including the H2B C-terminal helix. By contrast, cryoEM maps of H2AK119ub nucleosomes revealed only one stable ubiquitin position on the face of the nucleosome, which only partly overlaps the H2A/H2B acidic patch (Figure 4A, Supplementary Figure S6). The observation of multiple stable positions of H2BK120ub that occlude the acidic patch suggests that ubiquitin conjugated to H2BK120 is more likely to interfere with protein binding to the acidic patch than ubiquitin conjugated to H2AK119. The effect of H2B-K120 ubiquitination in disrupting nucleosome binding of unrelated proteins is consistent with the idea that any gatekeeping function of H2BK120ub is steric, and not related to particular folds or sequence motifs.

Although the precise nature of the contacts between ubiquitin and the nucleosome could not be resolved, the EM maps for nucleosomes containing H2Bub position 1 and 2 show weak density for ubiquitin residue R74 interacting with nucleosome acidic patch residues H2A-E56 and H2B-E113 (Supplementary Figures S11A, B). It is possible these interactions help position the ubiquitin on the nucleosome, although we note that there is no density for R74 interaction in density maps corresponding to H2Bub positions 3 through 6. Ubiquitin residue R72 is also in a position where it could potentially interact with the acidic patch, particularly in positions 1 and 2. The ubiquitin hydrophobic patch, composed of beta sheet residues L8, I44, and V70 (83), is a common site of interaction with ubiquitin binding proteins (83–85). In our cryoEM structures of H2BK120ub nucleosomes, the ubiquitin hydrophobic patch is oriented towards the nucleosome surface (Figures 1B and 2A, B) and could also potentially mediate van der Waals contacts with histone residues. H2Bub position 3, in particular, positions the I44 hydrophobic patch near H2B residues 45–48. Further investigations will be needed to determine which ubiquitin residues are most important for positioning ubiquitin on the nucleosome surface.

Density for ubiquitin conjugated to H2B-K120 had been previously observed in complexes containing additional proteins bound to the nucleosome. In reported structures of proteins that bind to both the histone octamer and the ubiquitin in H2BK120ub, ubiquitin adopts different positions that depend, in each case, on direct interactions with the bound protein (86–90) (Supplementary Figure S12A). A crystal structure of a nucleosome containing H2BK120ub alone did not show density for ubiquitin (91), indicating either that ubiquitin is conformationally heterogeneous in the absence of a bound protein, or that it adopts multiple positions that could not be resolved in the electron density maps. However, a cryoEM structure of Set2 histone methyltransferase bound to H2BK120ub nucleosome showed EM density for ubiquitin over the acidic patch (92), in close proximity to H2Bub position 1 in the present study (Supplementary Figure S12B). A cryoEM structure of the Chd1 chromatin remodeler bound to H2BK120ub nucleosome with partially unwrapped DNA shows EM density for ubiquitin partially occluding the acidic patch (81) (Supplementary Figure S12B). In those cases, it was not clear if the presence of either Set2 or Chd1, or the partially unwrapped DNA in the latter structure, contributed to ubiquitin positioning, although we note that the density maps show no evidence of direct interactions between the bound protein, or DNA, and ubiquitin.

The H2BK120ub used in our structural studies contained a non-native dichloroacetone (DCA) linkage between ubiquitin and H2B (see Methods), which is one bond length longer than a native isopeptide linkage (93). We therefore cannot rule out the possibility that the positioning of ubiquitin observed in our structural studies may be due, in part, to unique characteristics of the DCA linkage. We note, however, that binding of RCC1 to nucleosomes is disrupted to the same extent by isopeptide-linked H2BK120ub and DCA-linked H2BK120ub (Supplementary Figure S10B). Their similar behavior in solution experiments indicates that native-linked ubiquitin behaves in a manner similar to that of DCA-linked ubiquitin in occluding the nucleosome acidic patch.

We note that the relative residence time of ubiquitin at each observed position cannot be determined from cryoEM maps. While it is most likely that ubiquitin coupled to H2B-K120 has many degrees of freedom and can adopt a large number of positions relative to the nucleosome, our ability to capture discrete snapshots at sufficient resolution to orient the ubiquitin suggests it has sufficient residence time at or near the acidic patch to interfere with protein binding. Molecular dynamics simulations of H2BK120ub nucleosomes support our observation that ubiquitin largely remains near the acidic patch (Figures 3A, B). Although our MD simulations were able to recapitulate H2Bub positions 3, 4, and 5 observed in our cryoEM maps, we did not capture positions 1, 2, and 6, suggesting that further sampling is required to observe them. Future work using longer simulations or enhanced techniques will be needed to recapitulate other H2Bub positions, and potentially identify metastable states that cannot be captured by cryoEM. Nevertheless, our MD simulations interrogate the dynamics of H2BK120ub and show that the ubiquitin remains positioned at or near the nucleosome acidic patch, thereby occluding this interaction hub.

It is important to note that the histone methyltransferases, DOT1L, Dot1, and COMPASS, are able to bind to the nucleosome acidic patch in the presence of ubiquitinated H2B-K120, as they also contain specific elements that bind ubiquitin patch (35,86,87,94–96). The ubiquitin is thus repositioned through energetically favorable interactions with each enzyme, clearing the way for other parts of the protein to contact the nucleosome acidic patch. Such multivalent binding to both ubiquitin and the nucleosome could explain why DOT1L, Dot1, and COMPASS bind specifically to nucleosomes containing H2BK120ub, while RCC1, Sir3, and LANA, which do not bind ubiquitin, are excluded from the nucleosome acidic patch by this PTM.

How might the ability of H2BK120ub to control access to the nucleosome acidic patch help orchestrate the complex series of events that take place during transcription initiation and elongation? Histone H2BK120 is dynamically ubiquitinated and deubiquitinated during transcription (97), which enables the conjugated ubiquitin to serve a transient gating function that can be switched off by removal of the ubiquitin (98). While the precise point at which ubiquitin is conjugated and subsequently removed is not known, the transient presence of H2BK120ub can delimit the period during which certain acidic patch binders cannot be recruited to the nucleosome. Most chromatin remodeling enzymes must bind to the nucleosome acidic patch to reposition nucleosomes and activate transcription (99), and can therefore potentially be regulated by ubiquitination of H2B. An example is yeast SWR1C, which exchanges the canonical H2A for histone variant, H2A.Z, and contacts the nucleosome acidic patch, as well as the C-terminus of histone H2B (100). Intriguingly, ubiquitination of H2B and incorporation of the histone variant, H2A.Z, at the +1 nucleosome are anticorrelated (101), consistent with a model in which ubiquitin disrupts SWR1C activity (100). The remodeling enzyme, Chd1, is stimulated by H2BK120ub, although the stimulation mechanism may be related to a role for ubiquitin in favoring the unwrapping of nucleosomal DNA in the presence of Chd1 (81). Further studies will be needed to address the mechanisms underlying the complex roles that H2B ubiquitination plays in transcription, DNA replication, and DNA repair.

Monoubiquitination of histones H2A and H2B play opposing roles in regulating transcription through their interaction with enzymes that specifically recognize these modifications, and by promoting or disrupting chromatin compaction (16,17). Our studies shed light on a potential additional mechanism in which H2BK120ub, but not H2AK119ub, regulates binding to the acidic patch, a hub of nucleosome engagement for diverse chromatin interactors.

Supplementary Material

gkae698_Supplemental_File

Acknowledgements

We thank Duncan Sousa and Dazhong (David) Ding for advice and support with sample preparation and data collection at the Beckman Center for Cryo-EM at Johns Hopkins. We thank Edvin Pozharskiy for advice and support with cryoEM grid screening at the Maryland Center for Advanced Molecular Analysis (M-CAMA). We thank Wolberger lab members for their insights and discussions on the manuscript.

Author contributions: Chad W. Hicks: Conceptualization, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization, Writing—original draft. Sanim Rahman: Formal analysis, Investigation, Methodology, Validation, Visualization, Writing—review & editing. Susan L. Gloor: Formal analysis, Investigation, Methodology, Validation, Visualization. James K. Fields: Investigation, Formal analysis, Methodology, Validation. Natalia Ledo Husby: Resources, Validation. Anup Vaidya: Resources, Validation. Keith E. Maier: Resources, Validation. Michael Morgan: Resources. Michael-Christopher Keogh: Project administration, Funding acquisition, Supervision, Writing – review & editing. Cynthia Wolberger: Project administration, Funding acquisition, Supervision, Writing—review & editing.

Data availability

Models and cryoEM maps were deposited in the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB) under the following accession codes: H2BK120ub nucleosome ubiquitin position 1 (PDB: 8V25, EMDB: 42898), position 2 (PDB: 8V26, EMDB: 42899), position 3 (PDB: 8V27, EMDB: 42900), position 4 (PDB: 8V28, EMDB: 42901); H2BK120ub + H3K79me2 nucleosome ubiquitin position 5 (PDB: 8G6G, EMDB: 29767), position 6 (PDB: 8G6H, EMDB: 29769); H2AK119ub nucleosome ubiquitin position 1 (PDB: 8G6Q, EMDB: 29778), position 2 (PDB: 8G6S, EMDB: 29781).

Raw cryoEM movies were deposited in the EMPIAR database under the following accession codes: H2BK120ub nucleosome (EMPIAR-11972), H2BK120ub + H3KC79me2 nucleosome (EMPIAR-12022), H2AK119ub nucleosome (EMPIAR-11970).

Supplementary data

Supplementary Data are available at NAR Online.

Funding

National Institute of General Medical Sciences [R35GM130393 to C.W.]; National Cancer Institute (NCI) [F31CA261154 to C.W.H., F31CA271743 to S.R.] of the National Institutes of Health (NIH); EpiCypher is supported by the NIH [R43GM134834 and R44GM119893]; NCI National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research [HSSN261200800001E, in part]. Funding for open access charge: National Institutes of Health.

Conflict of interest statement. EpiCypher is a commercial developer and supplier of reagents (e.g. semi-synthetic nucleosomes) and platforms (e.g. dCypher-Luminex) used in this study. SLG, NLH, AV, KEMS.L.G., N.L.H., A.V., K.E.M. and M.-C.K. own shares in EpiCypher and M.-C.K. is a board member of same. The authors declare no other competing interests.

Notes

Present address: Michael Morgan. Stablix Inc., 201 Brookline Avenue, Suite 1, Boston, MA 02215, USA.
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References

1. McGinty R.K. , TanS. Nucleosome structure and function. Chem. Rev. 2015; 115 :2255–2273.25495456
2. McGinty R.K. , TanS. Principles of nucleosome recognition by chromatin factors and enzymes. Curr. Opin. Struct. Biol. 2021; 71 :16–26.34198054
3. Li B. , CareyM., WorkmanJ.L. The role of chromatin during transcription. Cell. 2007; 128 :707–719.17320508
4. Lai W.K. , PughB.F. Understanding nucleosome dynamics and their links to gene expression and DNA replication. Nat. Rev. Mol. Cell Biol. 2017; 18 :548–562.28537572
5. Luger K. , DechassaM.L., TremethickD.J. New insights into nucleosome and chromatin structure: an ordered state or a disordered affair?. Nat. Rev. Mol. Cell Biol. 2012; 13 :436–447.22722606
6. Hauer M.H. , GasserS.M. Chromatin and nucleosome dynamics in DNA damage and repair. Genes Dev. 2017; 31 :2204–2221.29284710
7. Jenuwein T. , AllisC.D. Translating the histone code. Science. 2001; 293 :1074–1080.11498575
8. Fierz B. , ChatterjeeC., McGintyR.K., Bar-DaganM., RaleighD.P., MuirT.W. Histone H2B ubiquitylation disrupts local and higher-order chromatin compaction. Nat. Chem. Biol. 2011; 7 :113–119.21196936
9. Shogren-Knaak M. , IshiiH., SunJ.-M., PazinM.J., DavieJ.R., PetersonC.L. Histone H4-K16 acetylation controls chromatin structure and protein interactions. Science. 2006; 311 :844–847.16469925
10. Martire S. , BanaszynskiL.A. The roles of histone variants in fine-tuning chromatin organization and function. Nat. Rev. Mol. Cell Biol. 2020; 21 :522–541.32665685
11. Kouzarides T. Chromatin modifications and their function. Cell. 2007; 128 :693–705.17320507
12. Luger K. , MäderA.W., RichmondR.K., SargentD.F., RichmondT.J. Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature. 1997; 389 :251–260.9305837
13. Barbera A.J. , ChodaparambilJ.V., Kelley-ClarkeB., JoukovV., WalterJ.C., LugerK., KayeK.M. The nucleosomal surface as a docking station for Kaposi's sarcoma herpesvirus LANA. Science. 2006; 311 :856–861.16469929
14. Makde R.D. , EnglandJ.R., YennawarH.P., TanS. Structure of RCC1 chromatin factor bound to the nucleosome core particle. Nature. 2010; 467 :562–566.20739938
15. Skrajna A. , GoldfarbD., KedzioraK.M., CousinsE.M., GrantG.D., SpanglerC.J., BarbourE.H., YanX., HathawayN.A., BrownN.G.et al . Comprehensive nucleosome interactome screen establishes fundamental principles of nucleosome binding. Nucleic Acids Res. 2020; 48 :9415–9432.32658293
16. Cao J. , YanQ. Histone ubiquitination and deubiquitination in transcription, DNA damage response, and cancer. Front. Oncol. 2012; 2 :26.22649782
17. Meas R. , MaoP. Histone ubiquitylation and its roles in transcription and DNA damage response. DNA Repair. 2015; 36 :36–42.26422137
18. Sun Z.-W. , AllisC.D. Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast. Nature. 2002; 418 :104–108.12077605
19. Briggs S.D. , XiaoT., SunZ.-W., CaldwellJ.A., ShabanowitzJ., HuntD.F., AllisC.D., StrahlB.D. Trans-histone regulatory pathway in chromatin. Nature. 2002; 418 :498.12152067
20. Dover J. , SchneiderJ., Tawiah-BoatengM.A., WoodA., DeanK., JohnstonM., ShilatifardA. Methylation of histone H3 by COMPASS requires ubiquitination of histone H2B by Rad6. J. Biol. Chem. 2002; 277 :28368–28371.12070136
21. Shahbazian M.D. , ZhangK., GrunsteinM. Histone H2B ubiquitylation controls processive methylation but not monomethylation by Dot1 and Set1. Mol. Cell. 2005; 19 :271–277.16039595
22. Ng H.H. , XuR.-M., ZhangY., StruhlK. Ubiquitination of histone H2B by Rad6 is required for efficient Dot1-mediated methylation of histone H3 lysine 79. J. Biol. Chem. 2002; 277 :34655–34657.12167634
23. McGinty R.K. , KimJ., ChatterjeeC., RoederR.G., MuirT.W. Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation. Nature. 2008; 453 :812–816.18449190
24. Mueller R.D. , YasudaH., HatchC.L., BonnerW.M., BradburyE.M. Identification of ubiquitinated histones 2A and 2B in Physarum polycephalum. Disappearance of these proteins at metaphase and reappearance at anaphase. J. Biol. Chem. 1985; 260 :5147–5153.2985575
25. Nakamura K. , KatoA., KobayashiJ., YanagiharaH., SakamotoS., OliveiraD.V., ShimadaM., TauchiH., SuzukiH., TashiroS.et al . Regulation of homologous recombination by RNF20-dependent H2B ubiquitination. Mol. Cell. 2011; 41 :515–528.21362548
26. Barbour H. , DaouS., HendzelM., AffarE.B. Polycomb group-mediated histone H2A monoubiquitination in epigenome regulation and nuclear processes. Nat. Commun. 2020; 11 :5947.33230107
27. Kalb R. , LatwielS., BaymazH.I., JansenP.W., MüllerC.W., VermeulenM., MüllerJ. Histone H2A monoubiquitination promotes histone H3 methylation in Polycomb repression. Nat. Struct. Mol. Biol. 2014; 21 :569–571.24837194
28. Cooper S. , GrijzenhoutA., UnderwoodE., AncelinK., ZhangT., NesterovaT.B., Anil-KirmizitasB., BassettA., KooistraS.M., AggerK.et al . Jarid2 binds mono-ubiquitylated H2A lysine 119 to mediate crosstalk between polycomb complexes PRC1 and PRC2. Nat. Commun. 2016; 7 :13661.27892467
29. Kasinath V. , BeckC., SauerP., PoepselS., KosmatkaJ., FainiM., TosoD., AebersoldR., NogalesE. JARID2 and AEBP2 regulate PRC2 in the presence of H2AK119ub1 and other histone modifications. Science. 2021; 371 :eabc3393.33479123
30. Cao R. , WangL., WangH., XiaL., Erdjument-BromageH., TempstP., JonesR.S., ZhangY. Role of histone H3 lysine 27 methylation in polycomb-group silencing. Science. 2002; 298 :1039–1043.12351676
31. Lavigne M. , FrancisN.J., KingI.F., KingstonR.E. Propagation of silencing: recruitment and repression of naive chromatin in trans by Polycomb repressed chromatin. Mol. Cell. 2004; 13 :415–425.14967148
32. Francis N.J. , KingstonR.E., WoodcockC.L. Chromatin compaction by a polycomb group protein complex. Science. 2004; 306 :1574–1577.15567868
33. Dann G.P. , LiszczakG.P., BagertJ.D., MüllerM.M., NguyenU.T., WojcikF., BrownZ.Z., BosJ., PanchenkoT., PihlR.et al . ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference. Nature. 2017; 548 :607–611.28767641
34. Shema-Yaacoby E. , NikolovM., Haj-YahyaM., SimanP., AllemandE., YamaguchiY., MuchardtC., UrlaubH., BrikA., OrenM.et al . Systematic identification of proteins binding to chromatin-embedded ubiquitylated H2B reveals recruitment of SWI/SNF to regulate transcription. Cell Rep. 2013; 4 :601–608.23933260
35. Anderson C.J. , BairdM.R., HsuA., BarbourE.H., KoyamaY., BorgniaM.J., McGintyR.K. Structural basis for recognition of ubiquitylated nucleosome by Dot1L methyltransferase. Cell Rep. 2019; 26 :1681–1690.30759380
36. Armache K.-J. , GarlickJ.D., CanzioD., NarlikarG.J., KingstonR.E. Structural basis of silencing: sir3 BAH domain in complex with a nucleosome at 3.0 Å resolution. Science. 2011; 334 :977–982.22096199
37. Vijay-Kumar S. , BuggC.E., WilkinsonK.D., CookW.J. Three-dimensional structure of ubiquitin at 2.8 A resolution. Proc. Natl. Acad. Sci. 1985; 82 :3582–3585.2987935
38. Morgan M. , JbaraM., BrikA., WolbergerC. Semisynthesis of ubiquitinated histone H2B with a native or nonhydrolyzable linkage. Methods in Enzymology. 2019; 618 :Elsevier 1–27.30850047
39. Luger K. , RechsteinerT.J., RichmondT.J. Preparation of nucleosome core particle from recombinant histones. Methods in Enzymology. 1999; 304 :Elsevier 3–19.10372352
40. Simon M.D. Installation of site-specific methylation into histones using methyl lysine analogs. Curr. Protoc. Mol. Biol. 2010; Chapter 21 :21.18.1–21.18.10.
41. Lowary P.T. , WidomJ. New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning. J. Mol. Biol. 1998; 276 :19–42.9514715
42. Dyer P.N. , EdayathumangalamR.S., WhiteC.L., BaoY., ChakravarthyS., MuthurajanU.M., LugerK. Reconstitution of nucleosome core particles from recombinant histones and DNA. Methods in Enzymology. 2003; 375 :Elsevier 23–44.
43. Schachner L.F. , JooßK., MorganM.A., PiuntiA., MeinersM.J., KafaderJ.O., LeeA.S., IwanaszkoM., CheekM.A., BurgJ.M.et al . Decoding the protein composition of whole nucleosomes with Nuc-MS. Nat. Methods. 2021; 18 :303–308.33589837
44. Thomas J.F. , Valencia-SánchezM.I., TamburriS., GloorS.L., RustichelliS., Godínez-LópezV., De IoannesP., LeeR., Abini-AgbomsonS., GretarssonK.et al . Structural basis of histone H2A lysine 119 deubiquitination by Polycomb Repressive deubiquitinase BAP1/ASXL1. Sci. Adv. 2023; 9 :eadg9832.37556531
45. Marunde M.R. , FuchsH.A., BurgJ.M., PopovaI.K., VaidyaA., HallN.W., WeinzapfelE.N., MeinersM.J., WatsonR., GillespieZ.B.et al . Nucleosome conformation dictates the histone code. 2024; 13 :e78866.
46. Bi X. , YangR., FengX., RhodesD., LiuC.-F. Semisynthetic UbH2A reveals different activities of deubiquitinases and inhibitory effects of H2A K119 ubiquitination on H3K36 methylation in mononucleosomes. Org. Biomol. Chem. 2016; 14 :835–839.26615908
47. Punjani A. , RubinsteinJ.L., FleetD.J., BrubakerM.A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods. 2017; 14 :290–296.28165473
48. Punjani A. , ZhangH., FleetD.J. Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction. Nat. Methods. 2020; 17 :1214–1221.33257830
49. Pettersen E.F. , GoddardT.D., HuangC.C., MengE.C., CouchG.S., CrollT.I., MorrisJ.H., FerrinT.E. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 2021; 30 :70–82.32881101
50. Emsley P. , LohkampB., ScottW.G., CowtanK. Features and development of Coot. Acta Crystallogr. Sect. D Biol. Crystallogr. 2010; 66 :486–501.20383002
51. Liebschner D. , AfonineP.V., BakerM.L., BunkócziG., ChenV.B., CrollT.I., HintzeB., HungL.-W., JainS., McCoyA.J.et al . Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. Sect. D: Struct. Biol. 2019; 75 :861–877.31588918
52. Afonine P.V. , PoonB.K., ReadR.J., SobolevO.V., TerwilligerT.C., UrzhumtsevA., AdamsP.D. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. Sect. D: Struct. Biol. 2018; 74 :531–544.29872004
53. Williams C.J. , HeaddJ.J., MoriartyN.W., PrisantM.G., VideauL.L., DeisL.N., VermaV., KeedyD.A., HintzeB.J., ChenV.B.et al . MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 2018; 27 :293–315.29067766
54. Davey C.A. , SargentD.F., LugerK., MaederA.W., RichmondT.J. Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 Å resolution. J. Mol. Biol. 2002; 319 :1097–1113.12079350
55. Vijay-Kumar S. , BuggC.E., CookW.J. Structure of ubiquitin refined at 1.8 åresolution. J. Mol. Biol. 1987; 194 :531–544.3041007
56. Carroll E.C. , LatorracaN.R., LindnerJ.M., MaguireB.C., PeltonJ.G., MarquseeS. Mechanistic basis for ubiquitin modulation of a protein energy landscape. Proc. Natl. Acad. Sci. U.S.A. 2021; 118 :e2025126118.33723075
57. Lai M.-Y. , ZhangD., LaRonde-LeBlancN., FushmanD. Structural and biochemical studies of the open state of Lys48-linked diubiquitin. Biochim. Biophys. Acta. 2012; 1823 :2046–2056.22542781
58. Fiser A. , SaliA. ModLoop: automated modeling of loops in protein structures. Bioinformatics. 2003; 19 :2500–2501.14668246
59. Case D.A. , AktulgaH.M., BelfonK., Ben-ShalomI.Y., BrozellS.R., CeruttiD.S., CheathamT.E.III, CisnerosG.A., CruzeiroV.W., DardenT.Aet al . Amber 2021. 2021; San Francisco University of California.
60. Maier J.A. , MartinezC., KasavajhalaK., WickstromL., HauserK.E., SimmerlingC. ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory Comput. 2015; 11 :3696–3713.26574453
61. Ivani I. , DansP.D., NoyA., PérezA., FaustinoI., HospitalA., WaltherJ., AndrioP., GoñiR., BalaceanuA.et al . Parmbsc1: a refined force field for DNA simulations. Nat. Methods. 2016; 13 :55–58.26569599
62. Yoo J. , AksimentievA. New tricks for old dogs: improving the accuracy of biomolecular force fields by pair-specific corrections to non-bonded interactions. Phys. Chem. Chem. Phys. 2018; 20 :8432–8449.29547221
63. Armeev G.A. , KniazevaA.S., KomarovaG.A., KirpichnikovM.P., ShaytanA.K. Histone dynamics mediate DNA unwrapping and sliding in nucleosomes. Nat. Commun. 2021; 12 :2387.33888707
64. Roe D.R. , CheathamT.E.III PTRAJ and CPPTRAJ: software for processing and analysis of molecular dynamics trajectory data. J. Chem. Theory Comput. 2013; 9 :3084–3095.26583988
65. Humphrey W. , DalkeA., SchultenK. VMD: visual molecular dynamics. J. Mol. Graphics. 1996; 14 :33–38.
66. Michaud-Agrawal N. , DenningE.J., WoolfT.B., BecksteinO. MDAnalysis: a toolkit for the analysis of molecular dynamics simulations. J. Comput. Chem. 2011; 32 :2319–2327.21500218
67. Marunde M.R. , PopovaI.K., WeinzapfelE.N., KeoghM.-C. The dCypher approach to interrogate chromatin reader activity against posttranslational modification-defined histone peptides and nucleosomes. Chromatin: Methods and Protocols. 2022; Springer 231–255.
68. McGinty R.K. , KimJ., ChatterjeeC., RoederR.G., MuirT.W. Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation. Nature. 2008; 453 :812–816.18449190
69. Wojcik F. , DannG.P., BehL.Y., DebelouchinaG.T., HofmannR., MuirT.W. Functional crosstalk between histone H2B ubiquitylation and H2A modifications and variants. Nat. Commun. 2018; 9 :1394.29643390
70. West M.H. , BonnerW.M. Histone 2A, a heteromorphous family of eight protein species. Biochemistry. 1980; 19 :3238–3245.7407044
71. Lobbia V.R. , SanchezM.C.T., van IngenH. Beyond the nucleosome: nucleosome-protein interactions and higher order chromatin structure. J. Mol. Biol. 2021; 433 :166827.33460684
72. Schalch T. , DudaS., SargentD.F., RichmondT.J. X-ray structure of a tetranucleosome and its implications for the chromatin fibre. Nature. 2005; 436 :138–141.16001076
73. Song F. , ChenP., SunD., WangM., DongL., LiangD., XuR.-M., ZhuP., LiG. Cryo-EM study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units. Science. 2014; 344 :376–380.24763583
74. Zhou B.-R. , JiangJ., GhirlandoR., NorouziD., YadavK.S., FengH., WangR., ZhangP., ZhurkinV., BaiY. Revisit of reconstituted 30-nm nucleosome arrays reveals an ensemble of dynamic structures. J. Mol. Biol. 2018; 430 :3093–3110.29959925
75. Dombrowski M. , EngeholmM., DienemannC., DodonovaS., CramerP. Histone H1 binding to nucleosome arrays depends on linker DNA length and trajectory. Nat. Struct. Mol. Biol. 2022; 29 :493–501.35581345
76. Willcockson M.A. , HealtonS.E., WeissC.N., BartholdyB.A., BotbolY., MishraL.N., SidhwaniD.S., WilsonT.J., PintoH.B., MaronM.I.et al . H1 histones control the epigenetic landscape by local chromatin compaction. Nature. 2021; 589 :293–298.33299182
77. Bednar J. , Garcia-SaezI., BoopathiR., CutterA.R., PapaiG., ReymerA., SyedS.H., LoneI.N., TonchevO., CrucifixC.et al . Structure and dynamics of a 197 bp nucleosome in complex with linker histone H1. Mol. Cell. 2017; 66 :384–397.28475873
78. Carazo-Salas R.E. , GuarguagliniG., GrussO.J., SegrefA., KarsentiE., MattajI.W. Generation of GTP-bound Ran by RCC1 is required for chromatin-induced mitotic spindle formation. Nature. 1999; 400 :178–181.10408446
79. England J.R. , HuangJ., JenningsM.J., MakdeR.D., TanS. RCC1 uses a conformationally diverse loop region to interact with the nucleosome: a model for the RCC1–nucleosome complex. J. Mol. Biol. 2010; 398 :518–529.20347844
80. Jullien D. , VignardJ., FedorY., BéryN., OlichonA., CrozatierM., ErardM., CassardH., DucommunB., SallesB.et al . Chromatibody, a novel non-invasive molecular tool to explore and manipulate chromatin in living cells. J. Cell Sci. 2016; 129 :2673–2683.27206857
81. Sundaramoorthy R. , HughesA.L., El-MkamiH., NormanD.G., FerreiraH., Owen-HughesT. Structure of the chromatin remodelling enzyme Chd1 bound to a ubiquitinylated nucleosome. eLife. 2018; 7 :e35720.30079888
82. Spangler C.J. , YadavS.P., LiD., GeilC.N., SmithC.B., WangG.G., LeeT.-H., McGintyR.K. DOT1L activity in leukemia cells requires interaction with ubiquitylated H2B that promotes productive nucleosome binding. Cell Rep. 2022; 38 :110369.35172132
83. Beal R. , DeverauxQ., XiaG., RechsteinerM., PickartC. Surface hydrophobic residues of multiubiquitin chains essential for proteolytic targeting. Proc. Natl. Acad. Sci. U.S.A. 1996; 93 :861–866.8570649
84. Hicke L. , SchubertH.L., HillC.P. Ubiquitin-binding domains. Nat. Rev. Mol. Cell Biol. 2005; 6 :610–621.16064137
85. Sloper-Mould K.E. , JemcJ.C., PickartC.M., HickeL. Distinct functional surface regions on ubiquitin. J. Biol. Chem. 2001; 276 :30483–30489.11399765
86. Worden E.J. , HoffmannN.A., HicksC.W., WolbergerC. Mechanism of cross-talk between H2B ubiquitination and H3 methylation by Dot1L. Cell. 2019; 176 :1490–1501.30765112
87. Hsu P.L. , ShiH., LeonenC., KangJ., ChatterjeeC., ZhengN. Structural basis of H2B ubiquitination-dependent H3K4 methylation by COMPASS. Mol. Cell. 2019; 76 :712–723.31733991
88. Morgan M.T. , Haj-YahyaM., RingelA.E., BandiP., BrikA., WolbergerC. Structural basis for histone H2B deubiquitination by the SAGA DUB module. Science. 2016; 351 :725–728.26912860
89. Wang H. , DienemannC., StützerA., UrlaubH., CheungA., CramerP. Structure of the transcription coactivator SAGA. Nature. 2020; 577 :717–720.31969703
90. Xue H. , YaoT., CaoM., ZhuG., LiY., YuanG., ChenY., LeiM., HuangJ. Structural basis of nucleosome recognition and modification by MLL methyltransferases. Nature. 2019; 573 :445–449.31485071
91. Machida S. , SekineS., NishiyamaY., HorikoshiN., KurumizakaH. Structural and biochemical analyses of monoubiquitinated human histones H2B and H4. Open Biology. 2016; 6 :160090.27335322
92. Bilokapic S. , HalicM. Nucleosome and ubiquitin position Set2 to methylate H3K36. Nat. Commun. 2019; 10 :3795.31439846
93. Yin L. , KrantzB., RussellN.S., DeshpandeS., WilkinsonK.D. Nonhydrolyzable diubiquitin analogues are inhibitors of ubiquitin conjugation and deconjugation. Biochemistry. 2000; 39 :10001–10010.10933821
94. Valencia-Sánchez M.I. , De IoannesP.D., WangM., VasilyevN., ChenR., NudlerE., ArmacheJ.-P., ArmacheK.-J. Structural basis of Dot1L stimulation by histone H2B lysine 120 ubiquitination. Mol. Cell. 2019; 74 :1010–1019.30981630
95. Worden E.J. , ZhangX., WolbergerC. Structural basis for COMPASS recognition of an H2B-ubiquitinated nucleosome. eLife. 2020; 9 :e53199.31922488
96. Valencia-Sánchez M.I. , De IoannesP., WangM., TruongD.M., LeeR., ArmacheJ.-P., BoekeJ.D., ArmacheK.-J. Regulation of the Dot1 histone H3K79 methyltransferase by histone H4K16 acetylation. Science. 2021; 371 :eabc6663.33479126
97. Henry K.W. , WyceA., LoW.S., DugganL.J., EmreN.C., KaoC.F., PillusL., ShilatifardA., OsleyM.A., BergerS.L. Transcriptional activation via sequential histone H2B ubiquitylation and deubiquitylation, mediated by SAGA-associated Ubp8. Genes Dev. 2003; 17 :2648–2663.14563679
98. Nune M. , MorganM.T., ConnellZ., McCulloughL., JbaraM., SunH., BrikA., FormosaT., WolbergerC. FACT and Ubp10 collaborate to modulate H2B deubiquitination and nucleosome dynamics. eLife. 2019; 8 :e40988.30681413
99. Reyes A.A. , MarcumR.D., HeY. Structure and function of chromatin remodelers. J. Mol. Biol. 2021; 433 :166929.33711345
100. Baier A.S. , GioacchiniN., EekP., LeithE.M., TanS., PetersonC.L. Dual engagement of the nucleosomal acidic patches is essential for deposition of histone H2A.Z by SWR1C. eLife. 2024; 13 :RP94869.38809771
101. Rhee H.S. , BatailleA.R., ZhangL., PughB.F. Subnucleosomal structures and nucleosome asymmetry across a genome. Cell. 2014; 159 :1377–1388.25480300
