Interactions of Nucleosomes with Acidic Patch-Binding Peptides: A Combined Structural Bioinformatics, Molecular Modeling, Fluorescence Polarization, and Single-Molecule FRET Study.

In eukaryotic organisms, genomic DNA associates with histone proteins to form nucleosomes. Nucleosomes provide a basis for genome compaction, epigenetic markup, and mediate interactions of nuclear proteins with their target DNA loci. A negatively charged (acidic) patch located on the H2A-H2B histone dimer is a characteristic feature of the nucleosomal surface. The acidic patch is a common site in the attachment of various chromatin proteins, including viral ones. Acidic patch-binding peptides present perspective compounds that can be used to modulate chromatin functioning by disrupting interactions of nucleosomes with natural proteins or alternatively targeting artificial moieties to the nucleosomes, which may be beneficial for the development of new therapeutics. In this work, we used several computational and experimental techniques to improve our understanding of how peptides may bind to the acidic patch and what are the consequences of their binding. Through extensive analysis of the PDB database, histone sequence analysis, and molecular dynamic simulations, we elucidated common binding patterns and key interactions that stabilize peptide-nucleosome complexes. Through MD simulations and FRET measurements, we characterized changes in nucleosome dynamics conferred by peptide binding. Using fluorescence polarization and gel electrophoresis, we evaluated the affinity and specificity of the LANA1-22 peptide to DNA and nucleosomes. Taken together, our study provides new insights into the different patterns of intermolecular interactions that can be employed by natural and designed peptides to bind to nucleosomes, and the effects of peptide binding on nucleosome dynamics and stability.

Electron microscopy analysis of ATP-independent nucleosome unfolding by FACT.

FACT is a histone chaperone that participates in nucleosome removal and reassembly during transcription and replication. We used electron microscopy to study FACT, FACT:Nhp6 and FACT:Nhp6:nucleosome complexes, and found that all complexes adopt broad ranges of configurations, indicating high flexibility. We found unexpectedly that the DNA binding protein Nhp6 also binds to the C-terminal tails of FACT subunits, inducing more open geometries of FACT even in the absence of nucleosomes. Nhp6 therefore supports nucleosome unfolding by altering both the structure of FACT and the properties of nucleosomes. Complexes formed with FACT, Nhp6, and nucleosomes also produced a broad range of structures, revealing a large number of potential intermediates along a proposed unfolding pathway. The data suggest that Nhp6 has multiple roles before and during nucleosome unfolding by FACT, and that the process proceeds through a series of energetically similar intermediate structures, ultimately leading to an extensively unfolded form.

Molecular Mechanisms of Oncogenesis through the Lens of Nucleosomes and Histones.

At the cellular level, cancer is the disease of both the genome and the epigenome, and the interplay between genetic mutations and epigenetic states may occur at the level of elementary chromatin units, the nucleosomes. They are formed by a segment of DNA wrapped around an octamer of histone proteins. In this review, we survey various mechanisms of cancer etiology and progression mediated by histones and nucleosomes. In particular, we discuss the effects of mutations in histones, changes in their expression and slicing on epigenetic dysregulation and carcinogenesis. The links between cancer phenotypes and differential expression of histone variants and isoforms are summarized. Finally, we discourse the geometric and steric effects of DNA compaction in nucleosomes on DNA mutation rate, interactions with transcription factors, including pioneer transcription factors, and prospects of cancer cells' genome and epigenome editing.

Histone Octamer Structure Is Altered Early in ISW2 ATP-Dependent Nucleosome Remodeling.

Nucleosomes are the fundamental building blocks of chromatin that regulate DNA access and are composed of histone octamers. ATP-dependent chromatin remodelers like ISW2 regulate chromatin access by translationally moving nucleosomes to different DNA regions. We find that histone octamers are more pliable than previously assumed and distorted by ISW2 early in remodeling before DNA enters nucleosomes and the ATPase motor moves processively on nucleosomal DNA. Uncoupling the ATPase activity of ISW2 from nucleosome movement with deletion of the SANT domain from the C terminus of the Isw2 catalytic subunit traps remodeling intermediates in which the histone octamer structure is changed. We find restricting histone movement by chemical crosslinking also traps remodeling intermediates resembling those seen early in ISW2 remodeling with loss of the SANT domain. Other evidence shows histone octamers are intrinsically prone to changing their conformation and can be distorted merely by H3-H4 tetramer disulfide crosslinking.

Genomic profiling of multiple sequentially acquired tumor metastatic sites from an "exceptional responder" lung adenocarcinoma patient reveals extensive genomic heterogeneity and novel somatic variants driving treatment response.

We used next-generation sequencing to identify somatic alterations in multiple metastatic sites from an "exceptional responder" lung adenocarcinoma patient during his 7-yr course of ERBB2-directed therapies. The degree of heterogeneity was unprecedented, with ∼1% similarity between somatic alterations of the lung and lymph nodes. One novel translocation, PLAG1-ACTA2, present in both sites, up-regulated ACTA2 expression. ERBB2, the predominant driver oncogene, was amplified in both sites, more pronounced in the lung, and harbored an L869R mutation in the lymph node. Functional studies showed increased proliferation, migration, metastasis, and resistance to ERBB2-directed therapy because of L869R mutation and increased migration because of ACTA2 overexpression. Within the lung, a nonfunctional CDK12, due to a novel G879V mutation, correlated with down-regulation of DNA damage response genes, causing genomic instability, and sensitivity to chemotherapy. We propose a model whereby a subclone metastasized early from the primary site and evolved independently in lymph nodes.

Large-scale ATP-independent nucleosome unfolding by a histone chaperone.

DNA accessibility to regulatory proteins is substantially influenced by nucleosome structure and dynamics. The facilitates chromatin transcription (FACT) complex increases the accessibility of nucleosomal DNA, but the mechanism and extent of its nucleosome reorganization activity are unknown. Here we determined the effects of FACT from the yeast Saccharomyces cerevisiae on single nucleosomes by using single-particle Förster resonance energy transfer (spFRET) microscopy. FACT binding results in dramatic ATP-independent, symmetrical and reversible DNA uncoiling that affects at least 70% of the DNA within a nucleosome, occurs without apparent loss of histones and proceeds via an 'all-or-none' mechanism. A mutated version of FACT is defective in uncoiling, and a histone mutation that suppresses phenotypes caused by this FACT mutation in vivo restores the uncoiling activity in vitro. Thus, FACT-dependent nucleosome unfolding modulates the accessibility of nucleosomal DNA, and this activity is an important function of FACT in vivo.

Peptide nanofibrils boost retroviral gene transfer and provide a rapid means for concentrating viruses.

Inefficient gene transfer and low virion concentrations are common limitations of retroviral transduction. We and others have previously shown that peptides derived from human semen form amyloid fibrils that boost retroviral gene delivery by promoting virion attachment to the target cells. However, application of these natural fibril-forming peptides is limited by moderate efficiencies, the high costs of peptide synthesis, and variability in fibril size and formation kinetics. Here, we report the development of nanofibrils that self-assemble in aqueous solution from a 12-residue peptide, termed enhancing factor C (EF-C). These artificial nanofibrils enhance retroviral gene transfer substantially more efficiently than semen-derived fibrils or other transduction enhancers. Moreover, EF-C nanofibrils allow the concentration of retroviral vectors by conventional low-speed centrifugation, and are safe and effective, as assessed in an ex vivo gene transfer study. Our results show that EF-C fibrils comprise a highly versatile, convenient and broadly applicable nanomaterial that holds the potential to significantly facilitate retroviral gene transfer in basic research and clinical applications.