Ubiquitinated histone H2B as gatekeeper of the nucleosome acidic patch

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.


Figure S5 .
Figure S5.Fit of model to map for all structures.Ubiquitin cartoon model showing the fit to cryoEM maps corresponding to: H2BK120ub nucleosome, with ubiquitin in positions 1, 2, 3, and 4 (A-D), H2BK120ub+H3KC79me2 nucleosome with ubiquitin in positions 5 and 6 (E-F), and H2AK119ub nucleosome with ubiquitin in positions 1 and 2 (G-H).

Figure S6 .
Figure S6.Acidic patch -ubiquitin buried surface area calculations for all ubiquitin positions.Full table of values (A) and line chart (up to 10 Å probe radius) (B) showing buried surface area between ubiquitin (all Ub residues) and the nucleosome acidic patch (H2A residues E56, E61, E64, D90, E91, E 92 and H2B residues E105, E113) at all ubiquitin positions.Calculated using "interfaces" command in ChimeraX at a range of probe radii.Solid lines correspond to H2Bub positions while dashed lines and square data points correspond to H2Aub positions.* indicates the H2Bub position was derived from the H2BK120ub+H3KC79me2 nucleosome cryoEM dataset.

Figure S9 .
Figure S9.H2AK119ub nucleosome with ubiquitin in position 1 can accommodate nucleosome stacking without steric clash.CryoEM model of the ubiquitin of H2Aub nucleosome in position 1 (ubiquitin + lower nucleosome), depicted in surface representation, superimposed over an X-ray structure of a tetranucleosome (PDB:1ZBB).Figure is shown in a cut-away surface representation, with the slice halfway through the ubiquitin.

Figure S10 .
Figure S10.Binding of RCC1 to nucleosomes in the presence and absence of LANA peptide.(A) EMSA showing RCC1 (625 nM) binding to unmodified nucleosome (100 nM) in the presence of increasing amount of LANA peptide (0-787 mM).(B) RCC1 binding to nucleosomes containing a native linked H2BK120ub.The nucleosomes in all panels contained 147 bp Widom 601 DNA.All gels stained with SYBR Gold.

Figure S11 .
Figure S11.Arginine 74 of ubiquitin in H2BK120ub nucleosome interacts with the acidic patch formed by histone H2A/H2B.(A) R74 of ubiquitin in position 1 interacts with the nucleosome surface.(B) R74 of ubiquitin in position 2 interacts with the nucleosome surface.

Table S1 . CryoEM data collection, processing, refinement, and validation statistics EC50 relative calculated
as above (4P Logistic; n=2; Graphpad Prism) Max MFI is average MFI at maximum query concentration (n=2) NB: no binding observed