Replacement of canonical histones with specialized histone variants promotes altering of chromatin structure and function. The essential histone variant H2A.Z affects various DNA-based processes via poorly understood mechanisms. Here, we determine the comprehensive interactome of H2A.Z and identify PWWP2A as a novel H2A.Z-nucleosome binder. PWWP2A is a functionally uncharacterized, vertebrate-specific protein that binds very tightly to chromatin through a concerted multivalent binding mode. Two internal protein regions mediate H2A.Z-specificity and nucleosome interaction, whereas the PWWP domain exhibits direct DNA binding. Genome-wide mapping reveals that PWWP2A binds selectively to H2A.Z-containing nucleosomes with strong preference for promoters of highly transcribed genes. In human cells, its depletion affects gene expression and impairs proliferation via a mitotic delay. While PWWP2A does not influence H2A.Z occupancy, the C-terminal tail of H2A.Z is one important mediator to recruit PWWP2A to chromatin. Knockdown of PWWP2A in results in severe cranial facial defects, arising from neural crest cell differentiation and migration problems. Thus, PWWP2A is a novel H2A.Z-specific multivalent chromatin binder providing a surprising link between H2A.Z, chromosome segregation, and organ development.

Mutations of the Glu76 residue of canonical histone H2B are frequently found in cancer cells. However, it is quite mysterious how a single amino acid substitution in one of the multiple H2B genes affects cell fate. Here we found that the H2B E76K mutation, in which Glu76 is replaced by Lys (E76K), distorted the interface between H2B and H4 in the nucleosome, as revealed by the crystal structure and induced nucleosome instability in vivo and in vitro. Exogenous production of the H2B E76K mutant robustly enhanced the colony formation ability of the expressing cells, indicating that the H2B E76K mutant has the potential to promote oncogenic transformation in the presence of wild-type H2B. We found that other cancer-associated mutations of histones, H3.1 E97K and H2A.Z.1 R80C, also induced nucleosome instability. Interestingly, like the H2B E76K mutant, the H3.1 E97K mutant was minimally incorporated into chromatin in cells, but it enhanced the colony formation ability. In contrast, the H2A.Z.1 R80C mutant was incorporated into chromatin in cells, and had minor effects on the colony formation ability of the cells. These characteristics of histones with cancer-associated mutations may provide important information toward understanding how the mutations promote cancer progression.

Nucleotide excision repair (NER) is a versatile DNA repair pathway, which can remove an extremely broad range of base lesions from the genome. In mammalian global genomic NER, the XPC protein complex initiates the repair reaction by recognizing sites of DNA damage, and this depends on detection of disrupted/destabilized base pairs within the DNA duplex. A model has been proposed that XPC first interacts with unpaired bases and then the XPD ATPase/helicase in concert with XPA verifies the presence of a relevant lesion by scanning a DNA strand in 5′-3′ direction. Such multi-step strategy for damage recognition would contribute to achieve both versatility and accuracy of the NER system at substantially high levels. In addition, recognition of ultraviolet light (UV)-induced DNA photolesions is facilitated by the UV-damaged DNA-binding protein complex (UV-DDB), which not only promotes recruitment of XPC to the damage sites, but also may contribute to remodeling of chromatin structures such that the DNA lesions gain access to XPC and the following repair proteins. Even in the absence of UV-DDB, however, certain types of histone modifications and/or chromatin remodeling could occur, which eventually enable XPC to find sites with DNA lesions. Exploration of novel factors involved in regulation of the DNA damage recognition process is now ongoing.

We found that vertebrate H2A.Z-2 is involved in the regulation of the DNA damage response at a very early stage, via the damaged chromatin reorganization required for RAD51 focus formation.

H2A.Z is one of the most evolutionally conserved histone variants. In vertebrates, this histone variant has two isoforms, H2A.Z.1 and H2A.Z.2, each of which is coded by an individual gene.H2A.Z is involved in multiple epigenetic regulations, and in humans, it also has relevance to carcinogenesis. In this study, we utilized the H2A.Z DKO cells, in which both H2A.Z isoform genes could be inducibly knocked out, for the functional analysis of H2A.Z by a genetic complementation assay, as the first example of its kind in vertebrates. Ectopically expressed wild-type H2A.Z and two N-terminal mutants, a non-acetylable H2A.Z mutant and a chimera in which the N-terminal tail of H2A.Z.1 was replaced with that of the canonical H2A, complemented the mitotic defects of H2A.Z DKO cells similarly, suggesting that both acetylation and distinctive sequence of the N-terminal tail of H2A.Z are not required for mitotic progression. On the other hand, each one of these three forms of H2A.Z complemented the transcriptional defects of H2A.Z DKO cells differently. These results suggest that the N-terminal tail of vertebrate H2A.Z make distinctively different contributions to these epigenetic events. Our results also imply that this genetic complementation system is a novel and useful tool for the functional analysis of H2A.Z. INTRODUTIONIn eukaryotes, the genomic DNA is packaged into chromatin. The basal unit of chromatin, known as nucleosome, is composed of 146 base pairs of DNA wrapped around a core of histone Author ManuscriptThis article is protected by copyright. All rights reserved proteins consisting of H2A, H2B, H3 and H4 (Luger et al., 1997;Kornberg et al., 1999).Changes in chromatin structure are fundamental to epigenetic regulation. Multiple factors, including chemical modifications of histones and deposition of histone variants, cause changes in the chromatin structure (Anna et al., 2010). Except histone H4, all canonical hitones have multiple H2A.Z is reportedly involved in multiple epigenetic events, including transcription, chromosome segregation and heterochromatin organization (Bönisch and Hake, 2012;Millar, 2013;Chambers et al., 2012). Consistent with these functions of H2A.Z, it was observed that H2A.Z is localized to the promoter regions of genes and pericentric heterochromatin (Guillemette et al., 2005. Rangasamy et al., 2003. H2A.Z is also reported to play important roles in various biological phenomena. For example, H2A.Z is required for ES cell pluripotency and differentiation (Creyghton et al., 2008;Wang et al., 2015), and over-expression of H2A.Z has been observed in several types of cancer cells, including breast cancer, bladder cancer, and malignant melanoma cells (Hua et al., 2008;Kim et al., 2013;Vardabasso et al., 2015).Although certain features of H2A.Z, which are distinct from those of the canonical H2A, are thought to contribute to various functions orchestrated by H2A.Z, their identities remained unknown so far.non-allelic histone variants (Talbert et al., 2010; Talbert et al., 2012). Although t...

Histone exchange and histone post-translational modifications play important roles in the regulation of DNA metabolism, by re-organizing the chromatin configuration. We previously demonstrated that the histone variant H2A.Z-2 is rapidly exchanged at damaged sites after DNA double strand break induction in human cells. In yeast, the small ubiquitin-like modifier (SUMO) modification of H2A.Z is involved in the DNA damage response. However, whether the SUMO modification regulates the exchange of human H2A.Z-2 at DNA damage sites remains unclear. Here, we show that H2A.Z-2 is SUMOylated in a damage-dependent manner, and the SUMOylation of H2A.Z-2 is suppressed by the depletion of the SUMO E3 ligase, PIAS4. Moreover, PIAS4 depletion represses the incorporation and eviction of H2A.Z-2 at damaged sites. These findings demonstrate that the PIAS4-mediated SUMOylation regulates the exchange of H2A.Z-2 at DNA damage sites.

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The ubiquitin–proteasome system (UPS) plays crucial roles in regulation of various biological processes, including DNA repair. In mammalian global genome nucleotide excision repair (GG-NER), activation of the DDB2-associated ubiquitin ligase upon UV-induced DNA damage is necessary for efficient recognition of lesions. To date, however, the precise roles of UPS in GG-NER remain incompletely understood. Here, we show that the proteasome subunit PSMD14 and the UPS shuttle factor RAD23B can be recruited to sites with UV-induced photolesions even in the absence of XPC, suggesting that proteolysis occurs at DNA damage sites. Unexpectedly, sustained inhibition of proteasome activity results in aggregation of PSMD14 (presumably with other proteasome components) at the periphery of nucleoli, by which DDB2 is immobilized and sequestered from its lesion recognition functions. Although depletion of PSMD14 alleviates such DDB2 immobilization induced by proteasome inhibitors, recruitment of DDB2 to DNA damage sites is then severely compromised in the absence of PSMD14. Because all of these proteasome dysfunctions selectively impair removal of cyclobutane pyrimidine dimers, but not (6–4) photoproducts, our results indicate that the functional integrity of the proteasome is essential for the DDB2-mediated lesion recognition sub-pathway, but not for GG-NER initiated through direct lesion recognition by XPC.