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.

Graphical Abstract

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.

Figure 2.

Two discrete ubiquitin positions in cryoEM maps of nucleosomes containing H2BK120ub and H3K_C79me2. (A) CryoEM map of nucleosome containing H2BK120ub and H3K_C79me2 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 H3K_C79me2 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 H3K_C79me2 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.

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).

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.

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 K_D 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 K_D and standard deviation shown (n = 2).

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).