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The ATRX-DAXX histone chaperone complex incorporates the histone variant H3.3 at heterochromatic regions in a replication-independent manner. Here, we present a high-resolution x-ray crystal structure of an interaction surface between ATRX and DAXX. We use single amino acid substitutions in DAXX that abrogate formation of the complex to explore ATRX-dependent and ATRX-independent functions of DAXX. We find that the repression of specific murine endogenous retroviruses is dependent on DAXX, but not on ATRX. In support, we reveal the existence of two biochemically distinct DAXX-containing complexes: the ATRX-DAXX complex involved in gene repression and telomere chromatin structure, and a DAXX-SETDB1-KAP1-HDAC1 complex that represses endogenous retroviruses independently of ATRX and H3.3 incorporation into chromatin. We find that histone H3.3 stabilizes DAXX protein levels and can affect DAXX-regulated gene expression without incorporation into nucleosomes. Our study demonstrates a nucleosome-independent function for the H3.3 histone variant.

The histone H3.3 chaperone DAXX is implicated in formation of heterochromatin and transcription silencing, especially for newly infecting DNA virus genomes entering the nucleus. Epstein-Barr virus (EBV) can efficiently establish stable latent infection as a chromatinized episome in the nucleus of infected cells. The EBV tegument BNRF1 is a DAXX-interacting protein required for the establishment of selective viral gene expression during latency. Here we report the structure of BNRF1 DAXX-interaction domain (DID) in complex with DAXX histone-binding domain (HBD) and histones H3.3-H4. BNRF1 DID contacts DAXX HBD and histones through non-conserved loops. The BNRF1-DAXX interface is responsible for BNRF1 localization to PML-nuclear bodies typically associated with host-antiviral resistance and transcriptional repression. Paradoxically, the interface is also required for selective transcription activation of viral latent cycle genes required for driving B-cell proliferation. These findings reveal molecular details of virus reprogramming of an antiviral histone chaperone to promote viral latency and cellular immortalization.

After DNA replication, chromosomal processes including DNA repair and transcription take place in the context of sister chromatids. While cell cycle regulation can guide these processes globally, mechanisms to distinguish pre- and post-replicative states locally remain unknown. Here we reveal that new histones incorporated during DNA replication provide a signature of post-replicative chromatin, read by the human TONSL–MMS22L homologous recombination complex. We identify the TONSL ankyrin repeat domain (ARD) as a reader of histone H4 tails unmethylated at K20 (H4K20me0), which are specific to new histones incorporated during DNA replication and mark post-replicative chromatin until the G2/M phase of the cell cycle. Accordingly, TONSL–MMS22L binds new histones H3–H4 both before and after incorporation into nucleosomes, remaining on replicated chromatin until late G2/M. H4K20me0 recognition is required for TONSL–MMS22L binding to chromatin and accumulation at challenged replication forks and DNA lesions. Consequently, TONSL ARD mutants are toxic, compromising genome stability, cell viability and resistance to replication stress. Together, these data reveal a histone-reader-based mechanism for recognizing the post-replicative state, offering a new angle to understand DNA repair with the potential for targeted cancer therapy.

Rtt107 (regulator of Ty1 transposition 107; Esc4) is a DNA repair protein from Saccharomyces cerevisiae that can restore stalled replication forks following DNA damage. There are six BRCT (BRCA1 C-terminal) domains in Rtt107 that act as binding sites for other recruited proteins during DNA repair. Several Rtt107 binding partners have been identified, including Slx4, Rtt101, Rad55, and the Smc5/6 (structural maintenance of chromosome) protein complex. Rtt107 can reportedly be recruited to chromatin in the presence of Rtt101 and Rtt109 upon DNA damage, but the chromatin-binding site of Rtt107 has not been identified. Here, we report our investigation of the interaction between phosphorylated histone H2A (γH2A) and the C-terminal tandem BRCT repeats (BRCT(5)-BRCT(6)) of Rtt107. The crystal structures of BRCT(5)-BRCT(6) alone and in a complex with γH2A reveal the molecular basis of the Rtt107-γH2A interaction. We used in vitro mutagenesis and a fluorescence polarization assay to confirm the location of the Rtt107 motif that is crucial for this interaction. In addition, these assays indicated that this interaction requires the phosphorylation of H2A. An in vivo phenotypic analysis in yeast demonstrated the critical role of BRCT(5)-BRCT(6) and its interaction with γH2A during the DNA damage response. Our results shed new light on the molecular mechanism by which Rtt107 is recruited to chromatin in response to stalled DNA replication forks.

Spt6 is a highly conserved transcription elongation factor and histone chaperone. It binds directly to the RNA polymerase II C-terminal domain (RNAPII CTD) through its C-terminal region that recognizes RNAPII CTD phosphorylation. In this study, we determined the solution structure of the C-terminal region of Saccharomyces cerevisiae Spt6, and we discovered that Spt6 has two SH2 domains in tandem. Structural and phylogenetic analysis revealed that the second SH2 domain was evolutionarily distant from canonical SH2 domains and represented a novel SH2 subfamily with a novel binding site for phosphoserine. In addition, NMR chemical shift perturbation experiments demonstrated that the tandem SH2 domains recognized Tyr(1), Ser(2), Ser(5), and Ser(7) phosphorylation of RNAPII CTD with millimolar binding affinities. The structural basis for the binding of the tandem SH2 domains to different forms of phosphorylated RNAPII CTD and its physiological relevance are discussed. Our results also suggest that Spt6 may use the tandem SH2 domain module to sense the phosphorylation level of RNAPII CTD.

Prp20p is the homolog of mammalian RCC1 (regulator of chromosome condensation 1) in Saccharomyces cerevisiae, which acts as the guanine nucleotide exchange factor (GEF) for Gsp1p (yeast Ran). Prp20p plays multiple roles in mRNA metabolism, nucleocytoplasmic transport and mitosis regulation. Prp20p also functions as a linker between chromatin and nuclear pore complex (NPC) which regulates the NPC-mediated boundary activity (BA). Prp20p contains an N-terminal nuclear localization signal (NLS) and a typical RCC1-like domain (RLD). Here we present the 1.9Å crystal structure of the RCC1-like domain of Prp20p, which exhibits a classical seven-bladed β-propeller. We also proved that the additional β-wedge in Prp20p is essential for the interaction between Prp20p and Gsp1p. Based on this structure, we built a complex model of Prp20p and Gsp1p which was optimized by molecular dynamics (MD) simulations. Our model reveals that Prp20p and RCC1 share similar Ran GTPase binding mode. In addition, we also studied the histone-binding property of Prp20p in vitro., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Brd4 belongs to the BET family. It is a multifunctional protein involved in transcription, replication, the signal transduction pathway, and cell cycle progression. All of these functions are linked to its association with acetylated chromatin. With its tandem bromodomains, Brd4 avidly binds to diacetylated H4-AcK5/K12 and H3-AcK9/K14 peptides. Solution structure of the second bromodomain (BD) is reported in this research. In addition to the piD-helix, which is special for BET members, an incompact alphaZ' distinct from Brd2 BD2 is found, although they have identical sequences in this region. Both BD1 and BD2 bind to monoacetylated H4-AcK5 and H4-AcK12 peptides, but with subtle differences. An NMR perturbation study and mutational analysis identified the binding interface and revealed several residues important for binding specificity. By molecular dynamics simulations, a complex model composed of H4-AcK5/K12 and two molecules of BD2 is presented. Relaxation data and internal motions of BD2 are also discussed. Unlike Brd2 BD1, the two bromodomains of Brd4 are mainly monomeric in solution. They do not form heterodimers like TAFII250. It suggests that Brd4 should have its own mechanism to reinforce its chromatin association both in mitotic retention and related cellular regulation.

BRD7 is an important protein tightly associated with Nasopharyngeal carcinoma (NPC). Overexpression of BRD7 inhibits NPC cell growth and cell cycle by transcriptionally regulating the cell cycle related genes. BRD7 contains a bromodomain that is found in many chromatin-associated proteins and in nearly all known nuclear histone acetyltransferases (HATs) and plays an important role in chromatin remodeling and transcriptional activation. Here, we report the solution structure of BRD7 bromodomain determined by NMR spectroscopy, and its binding specificity revealed by NMR titration with several acetylated histone peptides. We find that BRD7 bromodomain contains the typical left-handed four-helix bundle topology, and can bind with weak affinity to lysine-acetylated peptides derived from histone H3 with K9 or K14 acetylated and from histone H4 with K8, K12 or K16 acetylated. Our results show that BRD7 bromodomain lacks inherent binding specificity when binding to histones in vitro.