Chromatin structure-dependent histone incorporation revealed by a genome-wide deposition assay.

In eukaryotes, histone variant distribution within the genome is the key epigenetic feature. To understand how each histone variant is targeted to the genome, we developed a new method, the RhIP (Reconstituted histone complex Incorporation into chromatin of Permeabilized cell) assay, in which epitope-tagged histone complexes are introduced into permeabilized cells and incorporated into their chromatin. Using this method, we found that H3.1 and H3.3 were incorporated into chromatin in replication-dependent and -independent manners, respectively. We further found that the incorporation of histones H2A and H2A.Z mainly occurred at less condensed chromatin (open), suggesting that condensed chromatin (closed) is a barrier for histone incorporation. To overcome this barrier, H2A, but not H2A.Z, uses a replication-coupled deposition mechanism. Our study revealed that the combination of chromatin structure and DNA replication dictates the differential histone deposition to maintain the epigenetic chromatin states.

Nuclear Long Non-Coding RNAs as Epigenetic Regulators in Cancer.

BACKGROUND: Transcriptome analyses have revealed the presence of numerous long non-coding RNAs (lncRNAs) in mammalian cells. Many lncRNAs are expressed in development-, differentiation-, and disease-specific manners, suggesting their importance as cell regulators. Some nuclear lncRNAs are bound to specific genomic loci, either near or distant from their own transcription sites, and regulate gene expression in cis or trans. These lncRNAs recruit epigenetic factors, including the DNA methyl transferase and histone modification complex, and mediate both the 3D genome structure and nuclear domains. LncRNAs are now considered as an emerging member of epigenetic regulators. LncRNAs are dysregulated in various types of cancer and act as either oncogenic or tumor- suppressing factors. They are involved in virtually all of the cancer hallmarks and are potential diagnostic markers and therapeutic targets. OBJECTIVE: In this review, we describe several representative lncRNAs and provide a current overview of the mechanisms by which lncRNAs participate in epigenetic regulation and contribute to cancer development.

Nuclear microenvironment in cancer: Control through liquid-liquid phase separation.

The eukaryotic nucleus is not a homogenous single-spaced but a highly compartmentalized organelle, partitioned by various types of membraneless structures, including nucleoli, PML bodies, paraspeckles, DNA damage foci and RNA clouds. Over the past few decades, these nuclear structures have been implicated in biological reactions such as gene regulation and DNA damage response and repair, and are thought to provide "microenvironments," facilitating these reactions in the nucleus. Notably, an altered morphology of these nuclear structures is found in many cancers, which may relate to so-called "nuclear atypia" in histological examinations. While the diagnostic significance of nuclear atypia has been established, its nature has remained largely enigmatic and awaits characterization. Here, we review the emerging biophysical principles that govern biomolecular condensate assembly in the nucleus, namely, liquid-liquid phase separation (LLPS), to investigate the nature of the nuclear microenvironment. In the nucleus, LLPS is typically driven by multivalent interactions between proteins with intrinsically disordered regions, and is also facilitated by protein interaction with nucleic acids, including nuclear non-coding RNAs. Importantly, an altered LLPS leads to dysregulation of nuclear events and epigenetics, and often to tumorigenesis and tumor progression. We further note the possibility that LLPS could represent a new therapeutic target for cancer intervention.

Gene regulation by non-coding RNAs in the 3D genome architecture.

Appropriate gene expression is essential for producing the correct amount of proteins at the right time, which is critical for living organisms. In the three-dimensional (3D) space of the nucleus, genomes are folded into higher order chromatin structures that are intimately associated with epigenetic factors, including histone modifications and nuclear long non-coding RNAs (lncRNAs). LncRNAs regulate transcription for both activation and repression, either in cis or in trans. Many ncRNAs are expressed in development-specific, differentiation-specific, and disease-specific manners, suggesting that they are critical regulators for organ generation and maintenance. In this review, we mainly describe the following ncRNAs: Xist, involved in X chromosome inactivation, Firre, which serves as a platform for trans-chromosomal associations, and UMLILO and ELEANORS, which co-regulate genes involved in the immune response and breast cancer, respectively. These ncRNAs are gene regulators in the context of the 3D genome structure.

Nucleosome destabilization by nuclear non-coding RNAs.

In the nucleus, genomic DNA is wrapped around histone octamers to form nucleosomes. In principle, nucleosomes are substantial barriers to transcriptional activities. Nuclear non-coding RNAs (ncRNAs) are proposed to function in chromatin conformation modulation and transcriptional regulation. However, it remains unclear how ncRNAs affect the nucleosome structure. Eleanors are clusters of ncRNAs that accumulate around the estrogen receptor-α (ESR1) gene locus in long-term estrogen deprivation (LTED) breast cancer cells, and markedly enhance the transcription of the ESR1 gene. Here we detected nucleosome depletion around the transcription site of Eleanor2, the most highly expressed Eleanor in the LTED cells. We found that the purified Eleanor2 RNA fragment drastically destabilized the nucleosome in vitro. This activity was also exerted by other ncRNAs, but not by poly(U) RNA or DNA. The RNA-mediated nucleosome destabilization may be a common feature among natural nuclear RNAs, and may function in transcription regulation in chromatin.

Incorporation and influence of Leishmania histone H3 in chromatin.

Immunopathologies caused by Leishmania cause severe human morbidity and mortality. This protozoan parasite invades and persists inside host cells, resulting in disease development. Leishmania modifies the epigenomic status of the host cells, thus probably averting the host cell defense mechanism. To accomplish this, Leishmania may change the host cell chromatin structure. However, the mechanism by which the parasite changes the host cell chromatin has not been characterized. In the present study, we found that ectopically produced Leishmania histone H3, LmaH3, which mimics the secreted LmaH3 in infected cells, is incorporated into chromatin in human cells. A crystallographic analysis revealed that LmaH3 forms nucleosomes with human histones H2A, H2B and H4. We found that LmaH3 was less stably incorporated into the nucleosome, as compared to human H3.1. Consistently, we observed that LmaH3-H4 association was remarkably weakened. Mutational analyses revealed that the specific LmaH3 Trp35, Gln57 and Met98 residues, which correspond to the H3.1 Tyr41, Arg63 and Phe104 residues, might be responsible for the instability of the LmaH3 nucleosome. Nucleosomes containing LmaH3 resisted the Mg2+-mediated compaction of the chromatin fiber. These distinct physical characteristics of LmaH3 support the possibility that histones secreted by parasites during infection may modulate the host chromatin structure.

Endocrine therapy-resistant breast cancer model cells are inhibited by soybean glyceollin I through Eleanor non-coding RNA.

Long-term estrogen deprivation (LTED) of an estrogen receptor (ER) α-positive breast cancer cell line recapitulates cancer cells that have acquired estrogen-independent cell proliferation and endocrine therapy resistance. Previously, we have shown that a cluster of non-coding RNAs, Eleanors (ESR1 locus enhancing and activating non-coding RNAs) formed RNA cloud and upregulated the ESR1 gene in the nuclei of LTED cells. Eleanors were inhibited by resveratrol through ER. Here we prepared another polyphenol, glyceollin I from stressed soybeans, and identified it as a major inhibitor of the Eleanor RNA cloud and ESR1 mRNA transcription. The inhibition was independent of ER, unlike one by resveratrol. This was consistent with a distinct tertiary structure of glyceollin I for ER binding. Glyceollin I preferentially inhibited the growth of LTED cells and induced apoptosis. Our results suggest that glyceollin I has a novel role in LTED cell inhibition through Eleanors. In other words, LTED cells or endocrine therapy-resistant breast cancer cells may be ready for apoptosis, which can be triggered with polyphenols both in ER-dependent and ER-independent manners.

Stable complex formation of CENP-B with the CENP-A nucleosome.

CENP-A and CENP-B are major components of centromeric chromatin. CENP-A is the histone H3 variant, which forms the centromere-specific nucleosome. CENP-B specifically binds to the CENP-B box DNA sequence on the centromere-specific repetitive DNA. In the present study, we found that the CENP-A nucleosome more stably retains human CENP-B than the H3.1 nucleosome in vitro. Specifically, CENP-B forms a stable complex with the CENP-A nucleosome, when the CENP-B box sequence is located at the proximal edge of the nucleosome. Surprisingly, the CENP-B binding was weaker when the CENP-B box sequence was located in the distal linker region of the nucleosome. This difference in CENP-B binding, depending on the CENP-B box location, was not observed with the H3.1 nucleosome. Consistently, we found that the DNA-binding domain of CENP-B specifically interacted with the CENP-A-H4 complex, but not with the H3.1-H4 complex, in vitro. These results suggested that CENP-B forms a more stable complex with the CENP-A nucleosome through specific interactions with CENP-A, if the CENP-B box is located proximal to the CENP-A nucleosome. Our in vivo assay also revealed that CENP-B binding in the vicinity of the CENP-A nucleosome substantially stabilizes the CENP-A nucleosome on alphoid DNA in human cells.

DNA binding properties of the actin-related protein Arp8 and its role in DNA repair.

Actin and actin-related proteins (Arps), which are members of the actin family, are essential components of many of these remodeling complexes. Actin, Arp4, Arp5, and Arp8 are found to be evolutionarily conserved components of the INO80 chromatin remodeling complex, which is involved in transcriptional regulation, DNA replication, and DNA repair. A recent report showed that Arp8 forms a module in the INO80 complex and this module can directly capture a nucleosome. In the present study, we showed that recombinant human Arp8 binds to DNAs, and preferentially binds to single-stranded DNA. Analysis of the binding of adenine nucleotides to Arp8 mutants suggested that the ATP-binding pocket, located in the evolutionarily conserved actin fold, plays a regulatory role in the binding of Arp8 to DNA. To determine the cellular function of Arp8, we derived tetracycline-inducible Arp8 knockout cells from a cultured human cell line. Analysis of results obtained after treating these cells with aphidicolin and camptothecin revealed that Arp8 is involved in DNA repair. Together with the previous observation that Arp8, but not γ-H2AX, is indispensable for recruiting INO80 complex to DSB in human, results of our study suggest an individual role for Arp8 in DNA repair.

Nap1 stimulates homologous recombination by RAD51 and RAD54 in higher-ordered chromatin containing histone H1.

Homologous recombination plays essential roles in mitotic DNA double strand break (DSB) repair and meiotic genetic recombination. In eukaryotes, RAD51 promotes the central homologous-pairing step during homologous recombination, but is not sufficient to overcome the reaction barrier imposed by nucleosomes. RAD54, a member of the ATP-dependent nucleosome remodeling factor family, is required to promote the RAD51-mediated homologous pairing in nucleosomal DNA. In higher eukaryotes, most nucleosomes form higher-ordered chromatin containing the linker histone H1. However, the mechanism by which RAD51/RAD54-mediated homologous pairing occurs in higher-ordered chromatin has not been elucidated. In this study, we found that a histone chaperone, Nap1, accumulates on DSB sites in human cells, and DSB repair is substantially decreased in Nap1-knockdown cells. We determined that Nap1 binds to RAD54, enhances the RAD54-mediated nucleosome remodeling by evicting histone H1, and eventually stimulates the RAD51-mediated homologous pairing in higher-ordered chromatin containing histone H1.

Mislocalization of the centromeric histone variant CenH3/CENP-A in human cells depends on the chaperone DAXX.

Centromeres are essential for ensuring proper chromosome segregation in eukaryotes. Their definition relies on the presence of a centromere-specific H3 histone variant CenH3, known as CENP-A in mammals. Its overexpression in aggressive cancers raises questions concerning its effect on chromatin dynamics and contribution to tumorigenesis. We find that CenH3 overexpression in human cells leads to ectopic enrichment at sites of active histone turnover involving a heterotypic tetramer containing CenH3-H4 with H3.3-H4. Ectopic localization of this particle depends on the H3.3 chaperone DAXX rather than the dedicated CenH3 chaperone HJURP. This aberrant nucleosome occludes CTCF binding and has a minor effect on gene expression. Cells overexpressing CenH3 are more tolerant of DNA damage. Both the survival advantage and CTCF occlusion in these cells are dependent on DAXX. Our findings illustrate how changes in histone variant levels can disrupt chromatin dynamics and suggests a possible mechanism for cell resistance to anticancer treatments.

Gas-phase structure of the histone multimers characterized by ion mobility mass spectrometry and molecular dynamics simulation.

The minimum structural unit of chromatin is the nucleosome core particle (NCP), consisting of 146 bp of DNA wrapped around a histone octamer, which itself contains two H2A/H2B dimers and one (H3/H4)2 tetramer. These multimers possess functionally important tail regions that are intrinsically disordered. In order to elucidate the mechanisms behind NCP assembly and disassembly processes, which are highly related to gene expression, structural characterization of the H2A/H2B dimer and (H3/H4)2 tetramer will be of importance. In the present study, human histone multimers with disordered tail regions were characterized by electrospray ionization (ESI) ion mobility-mass spectrometry (IM-MS) and molecular dynamics (MD) simulation. Experimentally obtained arrival times of these histone multimer ions showed rather wide distributions, implying that multiple conformers exist for each histone multimer in the gas phase. To examine their structures, MD simulations of the histone multimers were performed first in solution and then in vacuo at four temperatures, resulting in a variety of histone multimer structures. Theoretical collision cross-section (CCS) values calculated for the simulated structures revealed that structural models with smaller CCS values had more compact tail regions than those with larger CCS values. This implied that variation of the CCS values of the histone multimers were primarily due to the random behaviors of the tail regions in the gas phase. The combination of IM-MS and MD simulation enabled clear and comprehensive characterization of the gas-phase structures of histone multimers containing disordered tails.

Nap1 regulates proper CENP-B binding to nucleosomes.

CENP-B is a widely conserved centromeric satellite DNA-binding protein, which specifically binds to a 17-bp DNA sequence known as the CENP-B box. CENP-B functions positively in the de novo assembly of centromeric nucleosomes, containing the centromere-specific histone H3 variant, CENP-A. At the same time, CENP-B also prevents undesired assembly of the CENP-A nucleosome through heterochromatin formation on satellite DNA integrated into ectopic sites. Therefore, improper CENP-B binding to chromosomes could be harmful. However, no CENP-B eviction mechanism has yet been reported. In the present study, we found that human Nap1, an acidic histone chaperone, inhibited the non-specific binding of CENP-B to nucleosomes and apparently stimulated CENP-B binding to its cognate CENP-B box DNA in nucleosomes. In human cells, the CENP-B eviction activity of Nap1 was confirmed in model experiments, in which the CENP-B binding to a human artificial chromosome or an ectopic chromosome locus bearing CENP-B boxes was significantly decreased when Nap1 was tethered near the CENP-B box sequence. In contrast, another acidic histone chaperone, sNASP, did not promote CENP-B eviction in vitro and in vivo and did not stimulate specific CENP-B binding to CENP-A nucleosomes in vitro. We therefore propose a novel mechanism of CENP-B regulation by Nap1.

Histone chaperone activity of Fanconi anemia proteins, FANCD2 and FANCI, is required for DNA crosslink repair.

Fanconi anaemia (FA) is a rare hereditary disorder characterized by genomic instability and cancer susceptibility. A key FA protein, FANCD2, is targeted to chromatin with its partner, FANCI, and plays a critical role in DNA crosslink repair. However, the molecular function of chromatin-bound FANCD2-FANCI is still poorly understood. In the present study, we found that FANCD2 possesses nucleosome-assembly activity in vitro. The mobility of histone H3 was reduced in FANCD2-knockdown cells following treatment with an interstrand DNA crosslinker, mitomycin C. Furthermore, cells harbouring FANCD2 mutations that were defective in nucleosome assembly displayed impaired survival upon cisplatin treatment. Although FANCI by itself lacked nucleosome-assembly activity, it significantly stimulated FANCD2-mediated nucleosome assembly. These observations suggest that FANCD2-FANCI may regulate chromatin dynamics during DNA repair.

HIV-1 Vpr induces DNA double-strand breaks.

Recent observations imply that HIV-1 infection induces chromosomal DNA damage responses. However, the precise molecular mechanism and biological relevance are not fully understood. Here, we report that HIV-1 infection causes double-strand breaks in chromosomal DNA. We further found that Vpr, an accessory gene product of HIV-1, is a major factor responsible for HIV-1-induced double-strand breaks. The purified Vpr protein promotes double-strand breaks when incubated with isolated nuclei, although it does not exhibit endonuclease activity in vitro. A carboxyl-terminally truncated Vpr mutant that is defective in DNA-binding activity is less capable of Vpr-dependent double-strand break formation in isolated nuclei. The data suggest that double-strand breaks induced by Vpr depend on its DNA-binding activity and that Vpr may recruit unknown nuclear factor(s) with positive endonuclease activity to chromosomal DNA. This is the first direct evidence that Vpr induces double-strand breaks in HIV-1-infected cells. We discuss the possible roles of Vpr-induced DNA damage in HIV-1 infection and the involvement of Vpr in further acquired immunodeficiency syndrome-related tumor development.

Location of tyrosine 315, a target for phosphorylation by cAbl tyrosine kinase, at the edge of the subunit-subunit interface of the human Rad51 filament.

Rad51 is a key element of recombinational DNA repair and its activity is regulated by phosphorylation of the tyrosine residue at position 315 by cAbl kinase. This phosphorylation could be involved in the resistance of cancer cells to chemotherapy. We have investigated the role of this residue by comparing the three-dimensional structures of human Rad51 and its prokaryotic homologue, Escherichia coli RecA. The residue appeared to be on the edge of the subunit-subunit interacting site. The fluorescence intensity of the tryptophan residue inserted at position 315 of human Rad51 in the place of tyrosine was decreased by adding 3 M urea, although the protein was not unfolded as there was no large change in the fluorescence peak position or circular dichroism signal. This change in fluorescence occurred at a lower urea concentration when the protein was diluted, which favours dissociation. These results indicate that the change is related to the dissociation of Rad51 polymer and that residue 315 is close to the subunit-subunit interacting site. ATP and ADP, which affect the filament structure, caused a blue shift in the fluorescence peak. These nucleotides probably altered the subunit-subunit contacts and may thus affect the filament structure. Phosphorylation of this residue could therefore affect the formation and structure of the Rad51 filament. Correct prediction of subunit-subunit interface of Rad51 by simple comparison of structures of Rad51 and RecA supports the idea that Rad51 forms the filament in a similar way as does RecA.