BACKGROUND: Many plant secondary metabolites (PSMs) were shown to intercalate into DNA helix or interact with DNA grooves. This may influence histone-DNA interactions changeing chromatin structure and genome functioning.
METHODS: Nucleosome stability and linker histone H1.2, H1.4 and H1.5 localizations were studied in HeLa cells after the treatment with 15 PSMs, which are DNA-binders and possess anticancer activity according to published data. Chromatin remodeler CBL0137 was used as a control. Effects of PSMs were studied using fluorescent microscopy, flowcytometry, quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), western-blotting.
RESULTS: We showed that 1-hour treatment with CBL0137 strongly inhibited DNA synthesis and caused intensive linker histone depletion consistent with nucleosome destabilization. None of PSMs caused nucleosome destabilization, while most of them demonstrated significant influence on linker histone localizations. In particular, cell treatment with 11 PSMs at non-toxic concentrations induced significant translocation of the histone H1.5 to nucleoli and most of PSMs caused depletion of the histones H1.2 and H1.4 from chromatin fraction. Curcumin, resveratrol, berberine, naringenin, and quercetin caused significant redistribution of all three variants of the studied linker histones showing some overlap of PSM effects on linker histone DNA-binding. We demonstrated that PSMs, which induced the most significant redistribution of the histone H1.5 (berberine, curcumin and naringenin), influence the proportion of cells synthesizing DNA, expressing or non-expressing cyclin B and influence cell cycle distribution. Berberine induction of H1.5 translocations to nucleoli was shown to occur independently on the phases of cell cycle (metaphase was not analyzed).
CONCLUSIONS: For the first time we revealed PSM influence on linker histone location in cell nuclei that opens a new direction of PSM research as anticancer agents.

The incorporation of histone variants has structural ramifications on nucleosome dynamics and stability. Due to their unique sequences, histone variants can alter histone-histone or histone-DNA interactions, impacting the folding of DNA around the histone octamer and the overall higher-order structure of chromatin fibers. These structural modifications alter chromatin compaction and accessibility of DNA by transcription factors and other regulatory proteins to influence gene regulatory processes such as DNA damage and repair, as well as transcriptional activation or repression. Histone variants can also generate a unique interactome composed of histone chaperones and chromatin remodeling complexes. Any of these perturbations can contribute to cellular plasticity and the progression of human diseases. Here, we focus on a frequently overlooked group of histone variants lying within the four human histone gene clusters and their contribution to breast cancer.

Chronic hepatitis B virus (HBV) infection is an incurable global health threat responsible for causing liver disease and hepatocellular carcinoma. During the genesis of infection, HBV establishes an independent minichromosome consisting of the viral covalently closed circular DNA (cccDNA) genome and host histones. The viral X gene must be expressed immediately upon infection to induce degradation of the host silencing factor, Smc5/6. However, the relationship between cccDNA chromatinization and X gene transcription remains poorly understood. Establishing a reconstituted viral minichromosome platform, we found that nucleosome occupancy in cccDNA drives X transcription. We corroborated these findings in cells and further showed that the chromatin destabilizing molecule CBL137 inhibits X transcription and HBV infection in hepatocytes. Our results shed light on a long-standing paradox and represent a potential new therapeutic avenue for the treatment of chronic HBV infection.

Nucleosome positioning and stability affect gene regulation in eukaryotic chromatin. Histone H2A.Z is an evolutionally conserved histone variant that forms mobile and unstable nucleosomes in vivo and in vitro. In the present study, we reconstituted nucleosomes containing human H2A.Z.1 mutants, in which the N-terminal or C-terminal half of H2A.Z.1 was replaced by the corresponding canonical H2A region. We found that the N-terminal portion of H2A.Z.1 is involved in flexible nucleosome positioning, whereas the C-terminal portion leads to weak H2A.Z.1-H2B association in the nucleosome. These results indicate that the N-terminal and C-terminal portions are independently responsible for the H2A.Z.1 nucleosome characteristics.

The human FACT (facilitates chromatin transcription) complex, composed of two subunits SPT16 (Suppressor of Ty 16) and SSRP1 (Structure-specific recognition protein-1), plays essential roles in nucleosome remodeling. However, the molecular mechanism of FACT reorganizing the nucleosome still remains elusive. In this study, we demonstrate that FACT displays dual functions in destabilizing the nucleosome and maintaining the original histones and nucleosome integrity at the single-nucleosome level. We found that the subunit SSRP1 is responsible for maintenance of nucleosome integrity by holding the H3/H4 tetramer on DNA and promoting the deposition of the H2A/H2B dimer onto the nucleosome. In contrast, the large subunit SPT16 destabilizes the nucleosome structure by displacing the H2A/H2B dimers. Our findings provide mechanistic insights by which the two subunits of FACT coordinate with each other to fulfill its functions and suggest that FACT may play essential roles in preserving the original histones with epigenetic identity during transcription or DNA replication.

BACKGROUND: The histone variant H3.3 plays a critical role in maintaining the pluripotency of embryonic stem cells (ESCs) by regulating gene expression programs important for lineage specification. H3.3 is deposited by various chaperones at regulatory sites, gene bodies, and certain heterochromatic sites such as telomeres and centromeres. Using Tet-inhibited expression of epitope-tagged H3.3 combined with ChIP-Seq we undertook genome-wide measurements of H3.3 dissociation rates across the ESC genome and examined the relationship between H3.3-nucleosome turnover and ESC-specific transcription factors, chromatin modifiers, and epigenetic marks.
RESULTS: Our comprehensive analysis of H3.3 dissociation rates revealed distinct H3.3 dissociation dynamics at various functional chromatin domains. At transcription start sites, H3.3 dissociates rapidly with the highest rate at nucleosome-depleted regions (NDRs) just upstream of Pol II binding, followed by low H3.3 dissociation rates across gene bodies. H3.3 turnover at transcription start sites, gene bodies, and transcription end sites was positively correlated with transcriptional activity. H3.3 is found decorated with various histone modifications that regulate transcription and maintain chromatin integrity. We find greatly varying H3.3 dissociation rates across various histone modification domains: high dissociation rates at active histone marks and low dissociation rates at heterochromatic marks. Well- defined zones of high H3.3-nucleosome turnover were detected at binding sites of ESC-specific pluripotency factors and chromatin remodelers, suggesting an important role for H3.3 in facilitating protein binding. Among transcription factor binding sites we detected higher H3.3 turnover at distal cis-acting sites compared to proximal genic transcription factor binding sites. Our results imply that fast H3.3 dissociation is a hallmark of interactions between DNA and transcriptional regulators.
CONCLUSIONS: Our study demonstrates that H3.3 turnover and nucleosome stability vary greatly across the chromatin landscape of embryonic stem cells. The presence of high H3.3 turnover at RNA Pol II binding sites at extragenic regions as well as at transcription start and end sites of genes, suggests a specific role for H3.3 in transcriptional initiation and termination. On the other hand, the presence of well-defined zones of high H3.3 dissociation at transcription factor and chromatin remodeler binding sites point to a broader role in facilitating accessibility.

Don Crothers, Mikael Kubista, Jon Widom, and their teams have been first to look for strong nucleosomes, in a bid to reveal the nucleosome positioning pattern(s) carried by the nucleosome DNA sequences. They were first to demonstrate that the nucleosome stability correlates with 10-11 base sequence periodicity, and that the strong nucleosomes localize preferentially in centromeres. This review describes these findings and their connection to recent discovery of the strong nucleosomes (SNs) with visibly periodic nucleosome DNA sequences.