Structural insight into BRCA1-BARD1 complex recruitment to damaged chromatin.

The BRCA1-BARD1 complex directs the DNA double-strand break (DSB) repair pathway choice to error-free homologous recombination (HR) during the S-G2 stages. Targeting BRCA1-BARD1 to DSB-proximal sites requires BARD1-mediated nucleosome interaction and histone mark recognition. Here, we report the cryo-EM structure of BARD1 bound to a ubiquitinated nucleosome core particle (NCPUb) at 3.1 Å resolution and illustrate how BARD1 simultaneously recognizes the DNA damage-induced mark H2AK15ub and DNA replication-associated mark H4K20me0 on the nucleosome. In vitro and in vivo analyses reveal that the BARD1-NCPUb complex is stabilized by BARD1-nucleosome interaction, BARD1-ubiquitin interaction, and BARD1 ARD domain-BARD1 BRCT domain interaction, and abrogating these interactions is detrimental to HR activity. We further identify multiple disease-causing BARD1 mutations that disrupt BARD1-NCPUb interactions and hence impair HR. Together, this study elucidates the mechanism of BRCA1-BARD1 complex recruitment and retention by DSB-flanking nucleosomes and sheds important light on cancer therapeutic avenues.

Structural basis of nucleosome dynamics modulation by histone variants H2A.B and H2A.Z.2.2.

Nucleosomes are dynamic entities with wide-ranging compositional variations. Human histone variants H2A.B and H2A.Z.2.2 play critical roles in multiple biological processes by forming unstable nucleosomes and open chromatin structures, but how H2A.B and H2A.Z.2.2 confer these dynamic features to nucleosomes remains unclear. Here, we report cryo-EM structures of nucleosome core particles containing human H2A.B (H2A.B-NCP) at atomic resolution, identifying large-scale structural rearrangements in the histone octamer in H2A.B-NCP. H2A.B-NCP compacts approximately 103 bp of DNA wrapping around the core histones in approximately 1.2 left-handed superhelical turns, in sharp contrast to canonical nucleosome encompassing approximately 1.7 turns of DNA. Micrococcal nuclease digestion assay reveals that nineteen H2A.B-specific residues, including a ROF ("regulating-octamer-folding") sequence of six consecutive residues, are responsible for loosening of H2A.B-NCPs. Unlike H2A.B-NCP, the H2A.Z.2.2-containing nucleosome (Z.2.2-NCP) adopts a less-extended structure and compacts around 125 bp of DNA. Further investigation uncovers a crucial role for the H2A.Z.2.2-specific ROF in both H2A.Z.2.2-NCP opening and SWR1-dependent histone replacement. Taken together, these first high-resolution structure of unstable nucleosomes induced by histone H2A variants elucidate specific functions of H2A.B and H2A.Z.2.2 in enhancing chromatin dynamics.

Role of a DEF/Y motif in histone H2A-H2B recognition and nucleosome editing.

The SWR complex edits the histone composition of nucleosomes at promoters to facilitate transcription by replacing the two nucleosomal H2A-H2B (A-B) dimers with H2A.Z-H2B (Z-B) dimers. Swc5, a subunit of SWR, binds to A-B dimers, but its role in the histone replacement reaction was unclear. In this study, we showed that Swc5 uses a tandem DEF/Y motif within an intrinsically disordered region to engage the A-B dimer. A 2.37-Å X-ray crystal structure of the histone binding domain of Swc5 in complex with an A-B dimer showed that consecutive acidic residues and flanking hydrophobic residues of Swc5 form a cap over the histones, excluding histone-DNA interaction. Mutations in Swc5 DEF/Y inhibited the nucleosome editing function of SWR in vitro. Swc5 DEF/Y interacts with histones in vivo, and the extent of this interaction is dependent on the remodeling ATPase of SWR, supporting a model in which Swc5 acts as a wedge to promote A-B dimer eviction. Given that DEF/Y motifs are found in other evolutionary unrelated chromatin regulators, this work provides the molecular basis for a general strategy used repeatedly during eukaryotic evolution to mobilize histones in various genomic functions.

NMR investigations on H2A-H2B heterodimer dynamics conferred by histone variant H2A.Z.

H2A.Z, a highly conserved histone H2A variant in eukaryotes, plays critical roles in multiple nuclear events. H2A.Z forms a heterodimer with H2B when incorporated into nucleosomes. The heterodimer dynamics has been implicated in H2A.Z functions. To gain insights into H2A.Z dynamics, we analyzed yeast H2A.Z-H2B dimer (ZB) and yeast H2A-H2B dimer (AB) using solution NMR spectroscopy. First, we measured the 1H-15N heteronuclear NOE ratio of ZB and showed that the H2A.Z αC-helix region (residues 100-118) undergoes less structure fluctuation than its H2A counterpart. Strikingly, substituting H2A residues G99N100V101 with H2A.Z counterparts R106A107 reduced the fluctuation of H2A αC-helix, suggesting that H2A.Z dynamics play an important role in αC-helix extension and H2A.Z-chaperones recognition. We next measured the hydrogen-deuterium exchange (HX) rate of ZB and verified that the H2A.Z α2 helix and H2B α2, α3 helices are mostly protected. Notably, we observed nearly identical HX profiles for dimerized ZB and AB, suggesting that they have similar solution structures and dynamic characters. Together, our study gains first insight into H2A.Z-H2B dimer dynamics and sheds light on how its dynamics affect the structure and function of H2A.Z variant.

Crystal structure of the histone heterodimer containing histone variant H2A.Bbd.

H2A.Bbd, the most divergent histone variant among all known H2A type histones, is involved in gene transcription, spermiogenesis, DNA replication and RNA splicing. Incorporation of H2A.Bbd-H2B dimer, a fundamental unit of H2A.Bbd nucleosome, modulate structures of nucleosome or chromatin, but the underlying mechanism remains elusive. Here we determined a crystal structure of H2A.Bbd-H2B dimer at 2.6 Šresolution. Although the H2A.Bbd-H2B dimer structure largely resembles that of H2A-H2B, substitution of H2A αC helix residues by H2A.Bbd counterparts lead to the transition of a long αC-helix to the short 310-helix, likely owing to the rearrangement of the hydrogen-bond network. Moreover, structural comparison revealed a strikingly altered electrostatic potential surface for H2A.Bbd-H2B dimer displaying a diminished DNA binding capability. Our study provides the first high-resolution structure of histone variant H2A.Bbd and shed a light on biological function of H2A.Bbd.

Recognition of the inherently unstable H2A nucleosome by Swc2 is a major determinant for unidirectional H2A.Z exchange.

The multisubunit chromatin remodeler SWR1/SRCAP/p400 replaces the nucleosomal H2A-H2B dimer with the free-form H2A.Z-H2B dimer, but the mechanism governing the unidirectional H2A-to-H2A.Z exchange remains elusive. Here, we perform single-molecule force spectroscopy to dissect the disassembly/reassembly processes of the H2A nucleosome and H2A.Z nucleosome. We find that the N-terminal 1-135 residues of yeast SWR1 complex protein 2 (previously termed Swc2-Z) facilitate the disassembly of nucleosomes containing H2A but not H2A.Z. The Swc2-mediated nucleosome disassembly/reassembly requires the inherently unstable H2A nucleosome, whose instability is conferred by three H2A α2-helical residues, Gly47, Pro49, and Ile63, as they selectively weaken the structural rigidity of the H2A-H2B dimer. It also requires Swc2-ZN (residues 1-37) that directly anchors to the H2A nucleosome and functions in the SWR1-catalyzed H2A.Z replacement in vitro and yeast H2A.Z deposition in vivo. Our findings provide mechanistic insights into how the SWR1 complex discriminates between the H2A nucleosome and H2A.Z nucleosome, establishing a simple paradigm for the governance of unidirectional H2A.Z exchange.

Anp32e, a higher eukaryotic histone chaperone directs preferential recognition for H2A.Z.

H2A.Z is a highly conserved histone variant in all species. The chromatin deposition of H2A.Z is specifically catalyzed by the yeast chromatin remodeling complex SWR1 and its mammalian counterpart SRCAP. However, the mechanism by which H2A.Z is preferentially recognized by non-histone proteins remains elusive. Here we identified Anp32e, a novel higher eukaryote-specific histone chaperone for H2A.Z. Anp32e preferentially associates with H2A.Z-H2B dimers rather than H2A-H2B dimers in vitro and in vivo and dissociates non-nucleosomal aggregates formed by DNA and H2A-H2B. We determined the crystal structure of the Anp32e chaperone domain (186-232) in complex with the H2A.Z-H2B dimer. In this structure, the region containing Anp32e residues 214-224, which is absent in other Anp32 family proteins, specifically interacts with the extended H2A.Z αC helix, which exhibits an unexpected conformational change. Genome-wide profiling of Anp32e revealed a remarkable co-occupancy between Anp32e and H2A.Z. Cells overexpressing Anp32e displayed a strong global H2A.Z loss at the +1 nucleosomes, whereas cells depleted of Anp32e displayed a moderate global H2A.Z increase at the +1 nucleosomes. This suggests that Anp32e may help to resolve the non-nucleosomal H2A.Z aggregates and also facilitate the removal of H2A.Z at the +1 nucleosomes, and the latter may help RNA polymerase II to pass the first nucleosomal barrier.