The PCNA-Pol δ complex couples lagging strand DNA synthesis to parental histone transfer for epigenetic inheritance.

Inheritance of epigenetic information is critical for maintaining cell identity. The transfer of parental histone H3-H4 tetramers, the primary carrier of epigenetic modifications on histone proteins, represents a crucial yet poorly understood step in the inheritance of epigenetic information. Here, we show the lagging strand DNA polymerase, Pol δ, interacts directly with H3-H4 and that the interaction between Pol δ and the sliding clamp PCNA regulates parental histone transfer to lagging strands, most likely independent of their roles in DNA synthesis. When combined, mutations at Pol δ and Mcm2 that compromise parental histone transfer result in a greater reduction in nucleosome occupancy at nascent chromatin than mutations in either alone. Last, PCNA contributes to nucleosome positioning on nascent chromatin. On the basis of these results, we suggest that the PCNA-Pol δ complex couples lagging strand DNA synthesis to parental H3-H4 transfer, facilitating epigenetic inheritance.

Studying Structure and Functions of Nucleosomes with Atomic Force Microscopy.

Chromatin is an epigenetic platform for implementation of DNA-dependent processes. Nucleosome, as a basic level of chromatin compaction, largely determines its properties and structure. In the study of nucleosomes structure and functions physicochemical tools are actively used, such as magnetic and optical "tweezers", "DNA curtains", nuclear magnetic resonance, X-ray crystallography, and cryogenic electron microscopy, as well as optical methods based on Förster resonance energy transfer. Despite the fact that these approaches make it possible to determine a wide range of structural and functional characteristics of chromatin and nucleosomes with high spatial and time resolution, atomic force microscopy (AFM) complements the capabilities of these methods. The results of structural studies of nucleosome focusing on the AFM method development are presented in this review. The possibilities of AFM are considered in the context of application of other physicochemical approaches.

Genome wide nucleosome landscape shapes 3D chromatin organization.

The hierarchical chromatin organization begins with formation of nucleosomes, which fold into chromatin domains punctuated by boundaries and ultimately chromosomes. In a hierarchal organization, lower levels shape higher levels. However, the dependence of higher-order 3D chromatin organization on the nucleosome-level organization has not been studied in cells. We investigated the relationship between nucleosome-level organization and higher-order chromatin organization by perturbing nucleosomes across the genome by deleting Imitation SWItch (ISWI) and Chromodomain Helicase DNA-binding (CHD1) chromatin remodeling factors in budding yeast. We find that changes in nucleosome-level properties are accompanied by changes in 3D chromatin organization. Short-range chromatin contacts up to a few kilo-base pairs decrease, chromatin domains weaken, and boundary strength decreases. Boundary strength scales with accessibility and moderately with width of nucleosome-depleted region. Change in nucleosome positioning seems to alter the stiffness of chromatin, which can affect formation of chromatin contacts. Our results suggest a biomechanical "bottom-up" mechanism by which nucleosome distribution across genome shapes 3D chromatin organization.

The importance of DNA sequence for nucleosome positioning in transcriptional regulation.

Nucleosome positioning is a key factor for transcriptional regulation. Nucleosomes regulate the dynamic accessibility of chromatin and interact with the transcription machinery at every stage. Influences to steer nucleosome positioning are diverse, and the according importance of the DNA sequence in contrast to active chromatin remodeling has been the subject of long discussion. In this study, we evaluate the functional role of DNA sequence for all major elements along the process of transcription. We developed a random forest classifier based on local DNA structure that assesses the sequence-intrinsic support for nucleosome positioning. On this basis, we created a simple data resource that we applied genome-wide to the human genome. In our comprehensive analysis, we found a special role of DNA in mediating the competition of nucleosomes with cis-regulatory elements, in enabling steady transcription, for positioning of stable nucleosomes in exons, and for repelling nucleosomes during transcription termination. In contrast, we relate these findings to concurrent processes that generate strongly positioned nucleosomes in vivo that are not mediated by sequence, such as energy-dependent remodeling of chromatin.

Native and tagged CENP-A histones are functionally inequivalent.

BACKGROUND: Over the past several decades, the use of biochemical and fluorescent tags has elucidated mechanistic and cytological processes that would otherwise be impossible. The challenging nature of certain nuclear proteins includes low abundancy, poor antibody recognition, and transient dynamics. One approach to get around those issues is the addition of a peptide or larger protein tag to the target protein to improve enrichment, purification, and visualization. However, many of these studies were done under the assumption that tagged proteins can fully recapitulate native protein function. RESULTS: We report that when C-terminally TAP-tagged CENP-A histone variant is introduced, it undergoes altered kinetochore protein binding, differs in post-translational modifications (PTMs), utilizes histone chaperones that differ from that of native CENP-A, and can partially displace native CENP-A in human cells. Additionally, these tagged CENP-A-containing nucleosomes have reduced centromeric incorporation at early G1 phase and poorly associates with linker histone H1.5 compared to native CENP-A nucleosomes. CONCLUSIONS: These data suggest expressing tagged versions of histone variant CENP-A may result in unexpected utilization of non-native pathways, thereby altering the biological function of the histone variant.

MNase-Seq Analysis for Identifying Stress-Altered Nucleosome Occupancy in Plants.

Nucleosome occupancy plays an important role in chromatin compaction, affecting biological processes by hampering the binding of cis-acting elements such as transcription factors, RNA polymerase machinery, and coregulatory. Accessible regions allow for cis-acting elements to bind DNA and regulate transcription. Here, we detail our protocol to profile nucleosome occupancy and chromatin structure dynamics under drought stress at the genome-wide scale using micrococcal nuclease (MNase) digestion. Combining variable MNase concentration treatments and high-throughput sequencing, we investigate the changes in the overall chromatin state using bread wheat samples from an exemplary drought experiment.

Angle between DNA linker and nucleosome core particle regulates array compaction revealed by individual-particle cryo-electron tomography.

The conformational dynamics of nucleosome arrays generate a diverse spectrum of microscopic states, posing challenges to their structural determination. Leveraging cryogenic electron tomography (cryo-ET), we determine the three-dimensional (3D) structures of individual mononucleosomes and arrays comprising di-, tri-, and tetranucleosomes. By slowing the rate of condensation through a reduction in ionic strength, we probe the intra-array structural transitions that precede inter-array interactions and liquid droplet formation. Under these conditions, the arrays exhibite irregular zig-zag conformations with loose packing. Increasing the ionic strength promoted intra-array compaction, yet we do not observe the previously reported regular 30-nanometer fibers. Interestingly, the presence of H1 do not induce array compaction; instead, one-third of the arrays display nucleosomes invaded by foreign DNA, suggesting an alternative role for H1 in chromatin network construction. We also find that the crucial parameter determining the structure adopted by chromatin arrays is the angle between the entry and exit of the DNA and the corresponding tangents to the nucleosomal disc. Our results provide insights into the initial stages of intra-array compaction, a critical precursor to condensation in the regulation of chromatin organization.

Interplay between histone variants and chaperones in plants.

Histone chaperones and histone variants play crucial roles in DNA replication, gene transcription, and DNA repair in eukaryotes. Histone chaperones reversibly promote nucleosome assembly and disassembly by incorporating or evicting histones and histone variants to modulate chromatin accessibility, thereby altering the chromatin states and modulating DNA-related biological processes. Cofactors assist histone chaperones to target specific chromatin regions to regulate the exchange of histones and histone variants. In this review, we summarize recent progress in the interplay between histone variants and chaperones in plants. We discuss the structural basis of chaperone-histone complexes and the mechanisms of their cooperation in regulating gene transcription and plant development.

FACT maintains chromatin architecture and thereby stimulates RNA polymerase II pausing during transcription in vivo.

Facilitates chromatin transcription (FACT) is a histone chaperone that supports transcription through chromatin in vitro, but its functional roles in vivo remain unclear. Here, we analyze the in vivo functions of FACT with the use of multi-omics analysis after rapid FACT depletion from human cells. We show that FACT depletion destabilizes chromatin and leads to transcriptional defects, including defective promoter-proximal pausing and elongation, and increased premature termination of RNA polymerase II. Unexpectedly, our analysis revealed that promoter-proximal pausing depends not only on the negative elongation factor (NELF) but also on the +1 nucleosome, which is maintained by FACT.

To slide or not to slide: key role of the hexasome in chromatin remodeling revealed.

Hexasomes are non-canonical nucleosomes that package DNA with six instead of eight histones. First discovered 40 years ago as a consequence of transcription, two near-atomic-resolution cryo-EM structures of the hexasome in complex with the chromatin remodeler INO80 have now started to unravel its mechanistic impact on the regulatory landscape of chromatin. Loss of one histone H2A-H2B dimer converts inactive nucleosomes into distinct and favorable substrates for ATP-dependent chromatin remodeling.

Systematic assessment of ISWI subunits shows that NURF creates local accessibility for CTCF.

Catalytic activity of the imitation switch (ISWI) family of remodelers is critical for nucleosomal organization and DNA binding of certain transcription factors, including the insulator protein CTCF. Here we define the contribution of individual subcomplexes by deriving a panel of isogenic mouse stem cell lines, each lacking one of six ISWI accessory subunits. Individual deletions of subunits of either CERF, RSF, ACF, WICH or NoRC subcomplexes only moderately affect the chromatin landscape, while removal of the NURF-specific subunit BPTF leads to a strong reduction in chromatin accessibility and SNF2H ATPase localization around CTCF sites. This affects adjacent nucleosome occupancy and CTCF binding. At a group of sites with reduced chromatin accessibility, CTCF binding persists but cohesin occupancy is reduced, resulting in decreased insulation. These results suggest that CTCF binding can be separated from its function as an insulator in nuclear organization and identify a specific role for NURF in mediating SNF2H localization and chromatin opening at bound CTCF sites.

Anticancer Drugs of Lysine Specific Histone Demethylase-1 (LSD1) Display Variable Inhibition on Nucleosome Substrates.

Lysine specific demethylase-1 (LSD1) serves as a regulator of transcription and represents a promising epigenetic target for anticancer treatment. LSD1 inhibitors are in clinical trials for the treatment of Ewing's sarcoma (EWS), acute myeloid leukemia, and small cell lung cancer, and the development of robust inhibitors requires accurate methods for probing demethylation, potency, and selectivity. Here, the inhibition kinetics on the H3K4me2 peptide and nucleosome substrates was examined, comparing the rates of demethylation in the presence of reversible [CC-90011 (PD) and SP-2577 (SD)] and irreversible [ORY-1001 (ID) and tranylcypromine (TCP)] inhibitors. Inhibitors were also subject to viability studies in three human cell lines and Western blot assays to monitor H3K4me2 nucleosome levels in EWS (TC-32) cells, enabling a correlation of drug potency, inhibition in vitro, and cell-based studies. For example, SP-2577, a drug in clinical trials for EWS, inhibits activity on small peptide substrates (Ki = 60 ± 20 nM) using an indirect coupled assay but does not inhibit demethylation on H3K4me2 peptides or nucleosomes using direct Western blot approaches. In addition, the drug has no effect on H3K4me2 levels in TC-32 cells. These data show that SP-2577 is not an LSD1 enzyme inhibitor, although the drug may function independent of demethylation due to its cytotoxic selectivity in TC-32 cells. Taken together, this work highlights the pitfalls of using coupled assays to ascribe a drug's mode of action, emphasizes the use of physiologically relevant substrates in epigenetic drug targeting strategies, and provides insight into the development of substrate-selective inhibitors of LSD1.

Inseparable RNA binding and chromatin modification activities of a nucleosome-interacting surface in EZH2.

Polycomb repressive complex 2 (PRC2) interacts with RNA in cells, but there is no consensus on how RNA regulates PRC2 canonical functions, including chromatin modification and the maintenance of transcription programs in lineage-committed cells. We assayed two separation-of-function mutants of the PRC2 catalytic subunit EZH2, defective in RNA binding but functional in methyltransferase activity. We find that part of the RNA-binding surface of EZH2 is required for chromatin modification, yet this activity is independent of RNA. Mechanistically, the RNA-binding surface within EZH2 is required for chromatin modification in vitro and in cells, through interactions with nucleosomal DNA. Contrarily, an RNA-binding-defective mutant exhibited normal chromatin modification activity in vitro and in lineage-committed cells, accompanied by normal gene repression activity. Collectively, we show that part of the RNA-binding surface of EZH2, rather than the RNA-binding activity per se, is required for the histone methylation in vitro and in cells, through interactions with the substrate nucleosome.

Acute depletion of BRG1 reveals its primary function as an activator of transcription.

The mammalian SWI/SNF-like BAF complexes play critical roles during animal development and pathological conditions. Previous gene deletion studies and characterization of human gene mutations implicate that the complexes both repress and activate a large number of genes. However, the direct function of the complexes in cells remains largely unclear due to the relatively long-term nature of gene deletion or natural mutation. Here we generate a mouse line by knocking in the auxin-inducible degron tag (AID) to the Smarca4 gene, which encodes BRG1, the essential ATPase subunit of the BAF complexes. We show that the tagged BRG1 can be efficiently depleted by osTIR1 expression and auxin treatment for 6 to 10 h in CD4 + T cells, hepatocytes, and fibroblasts isolated from the knock-in mice. The acute depletion of BRG1 leads to decreases in nascent RNAs and RNA polymerase II binding at a large number of genes, which are positively correlated with the loss of BRG1. Further, these changes are correlated with diminished accessibility at DNase I Hypersensitive Sites (DHSs) and p300 binding. The acute BRG1 depletion results in three major patterns of nucleosome shifts leading to narrower nucleosome spacing surrounding transcription factor motifs and at enhancers and transcription start sites (TSSs), which are correlated with loss of BRG1, decreased chromatin accessibility and decreased nascent RNAs. Acute depletion of BRG1 severely compromises the Trichostatin A (TSA) -induced histone acetylation, suggesting a substantial interplay between the chromatin remodeling activity of BRG1 and histone acetylation. Our data suggest BRG1 mainly plays a direct positive role in chromatin accessibility, RNAPII binding, and nascent RNA production by regulating nucleosome positioning and facilitating transcription factor binding to their target sites.

Nucleosomal DNA has topological memory.

One elusive aspect of the chromosome architecture is how it constrains the DNA topology. Nucleosomes stabilise negative DNA supercoils by restraining a DNA linking number difference (∆Lk) of about -1.26. However, whether this capacity is uniform across the genome is unknown. Here, we calculate the ∆Lk restrained by over 4000 nucleosomes in yeast cells. To achieve this, we insert each nucleosome in a circular minichromosome and perform Topo-seq, a high-throughput procedure to inspect the topology of circular DNA libraries in one gel electrophoresis. We show that nucleosomes inherently restrain distinct ∆Lk values depending on their genomic origin. Nucleosome DNA topologies differ at gene bodies (∆Lk = -1.29), intergenic regions (∆Lk = -1.23), rDNA genes (∆Lk = -1.24) and telomeric regions (∆Lk = -1.07). Nucleosomes near the transcription start and termination sites also exhibit singular DNA topologies. Our findings demonstrate that nucleosome DNA topology is imprinted by its native chromatin context and persists when the nucleosome is relocated.

Dual engagement of the nucleosomal acidic patches is essential for deposition of histone H2A.Z by SWR1C.

The yeast SWR1C chromatin remodeling enzyme catalyzes the ATP-dependent exchange of nucleosomal histone H2A for the histone variant H2A.Z, a key variant involved in a multitude of nuclear functions. How the 14-subunit SWR1C engages the nucleosomal substrate remains largely unknown. Studies on the ISWI, CHD1, and SWI/SNF families of chromatin remodeling enzymes have demonstrated key roles for the nucleosomal acidic patch for remodeling activity, however a role for this nucleosomal epitope in nucleosome editing by SWR1C has not been tested. Here, we employ a variety of biochemical assays to demonstrate an essential role for the acidic patch in the H2A.Z exchange reaction. Utilizing asymmetrically assembled nucleosomes, we demonstrate that the acidic patches on each face of the nucleosome are required for SWR1C-mediated dimer exchange, suggesting SWR1C engages the nucleosome in a 'pincer-like' conformation, engaging both patches simultaneously. Loss of a single acidic patch results in loss of high affinity nucleosome binding and nucleosomal stimulation of ATPase activity. We identify a conserved arginine-rich motif within the Swc5 subunit that binds the acidic patch and is key for dimer exchange activity. In addition, our cryoEM structure of a Swc5-nucleosome complex suggests that promoter proximal, histone H2B ubiquitylation may regulate H2A.Z deposition. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition.

Nonequilibrium switching of segmental states can influence compaction of chromatin.

Knowledge about the dynamic nature of chromatin organization is essential to understand the regulation of processes like DNA transcription and repair. The existing models of chromatin assume that protein organization and chemical states along chromatin are static and the 3D organization is purely a result of protein-mediated intra-chromatin interactions. Here we present a new hypothesis that certain nonequilibrium processes, such as switching of chemical and physical states due to nucleosome assembly/disassembly or gene repression/activation, can also simultaneously influence chromatin configurations. To understand the implications of this inherent nonequilibrium switching, we present a block copolymer model of chromatin, with switching of its segmental states between two states, mimicking active/repressed or protein unbound/bound states. We show that competition between switching timescale Tt, polymer relaxation timescale τp, and segmental relaxation timescale τs can lead to non-trivial changes in chromatin organization, leading to changes in local compaction and contact probabilities. As a function of the switching timescale, the radius of gyration of chromatin shows a non-monotonic behavior with a prominent minimum when Tt ≈ τp and a maximum when Tt ≈ τs. We find that polymers with a small segment length exhibit a more compact structure than those with larger segment lengths. We also find that the switching can lead to higher contact probability and better mixing of far-away segments. Our study also shows that the nature of the distribution of chromatin clusters varies widely as we change the switching rate.

Triple lysine and nucleosome-binding motifs of the viral IE19 protein are required for human cytomegalovirus S-phase infections.

Herpesvirus genomes are maintained as extrachromosomal plasmids within the nuclei of infected cells. Some herpesviruses persist within dividing cells, putting the viral genome at risk of being lost to the cytoplasm during mitosis because karyokinesis (nuclear division) requires nuclear envelope breakdown. Oncogenic herpesviruses (and papillomaviruses) avoid genome loss during mitosis by tethering their genomes to cellular chromosomes, thereby ensuring viral genome uptake into newly formed nuclei. These viruses use viral proteins with DNA- and chromatin-binding capabilities to physically link viral and cellular genomes together in a process called tethering. The known viral tethering proteins of human papillomavirus (E2), Epstein-Barr virus (EBNA1), and Kaposi's sarcoma-associated herpesvirus (LANA) each contain two independent domains required for genome tethering, one that binds sequence specifically to the viral genome and another that binds to cellular chromatin. This latter domain is called a chromatin tethering domain (CTD). The human cytomegalovirus UL123 gene encodes a CTD that is required for the virus to productively infect dividing fibroblast cells within the S phase of the cell cycle, presumably by tethering the viral genome to cellular chromosomes during mitosis. The CTD-containing UL123 gene product that supports S-phase infections is the IE19 protein. Here, we define two motifs in IE19 required for S-phase infections: an N-terminal triple lysine motif and a C-terminal nucleosome-binding motif within the CTD.IMPORTANCEThe IE19 protein encoded by human cytomegalovirus (HCMV) is required for S-phase infection of dividing cells, likely because it tethers the viral genome to cellular chromosomes, thereby allowing them to survive mitosis. The mechanism through which IE19 tethers viral genomes to cellular chromosomes is not understood. For human papillomavirus, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus, viral genome tethering is required for persistence (latency) and pathogenesis (oncogenesis). Like these viruses, HCMV also achieves latency, and it modulates the properties of glioblastoma multiforme tumors. Therefore, defining the mechanism through which IE19 tethers viral genomes to cellular chromosomes may help us understand, and ultimately combat or control, HCMV latency and oncomodulation.

DNA methylation-based high-resolution mapping of long-distance chromosomal interactions in nucleosome-depleted regions.

3C-based methods have significantly advanced our understanding of 3D genome organization. However, it remains a formidable task to precisely capture long-range chromosomal interactions between individual loci, such as those between promoters and distal enhancers. Here, we present Methyltransferase Targeting-based chromosome Architecture Capture (MTAC), a method that maps the contacts between a target site (viewpoint) and the rest of the genome in budding yeast with high resolution and sensitivity. MTAC detects hundreds of intra- and inter-chromosomal interactions within nucleosome-depleted regions (NDRs) that cannot be captured by 4C, Hi-C, or Micro-C. By applying MTAC to various viewpoints, we find that (1) most long-distance chromosomal interactions detected by MTAC reflect tethering by the nuclear pore complexes (NPCs), (2) genes co-regulated by methionine assemble into inter-chromosomal clusters near NPCs upon activation, (3) mediated by condensin, the mating locus forms a highly specific interaction with the recombination enhancer (RE) in a mating-type specific manner, and (4) correlation of MTAC signals among NDRs reveal spatial mixing and segregation of the genome. Overall, these results demonstrate MTAC as a powerful tool to resolve fine-scale long-distance chromosomal interactions and provide insights into the 3D genome organization.

Chromatin regulates alternative polyadenylation via the RNA polymerase II elongation rate.

The RNA polymerase II (Pol II) elongation rate influences poly(A) site selection, with slow and fast Pol II derivatives causing upstream and downstream shifts, respectively, in poly(A) site utilization. In yeast, depletion of either of the histone chaperones FACT or Spt6 causes an upstream shift of poly(A) site use that strongly resembles the poly(A) profiles of slow Pol II mutant strains. Like slow Pol II mutant strains, FACT- and Spt6-depleted cells exhibit Pol II processivity defects, indicating that both Spt6 and FACT stimulate the Pol II elongation rate. Poly(A) profiles of some genes show atypical downstream shifts; this subset of genes overlaps well for FACT- or Spt6-depleted strains but is different from the atypical genes in Pol II speed mutant strains. In contrast, depletion of histone H3 or H4 causes a downstream shift of poly(A) sites for most genes, indicating that nucleosomes inhibit the Pol II elongation rate in vivo. Thus, chromatin-based control of the Pol II elongation rate is a potential mechanism, distinct from direct effects on the cleavage/polyadenylation machinery, to regulate alternative polyadenylation in response to genetic or environmental changes.