Histone Tetrasome Dynamics Affects Chromatin Transcription.

During various DNA-centered processes in the cell nucleus, the minimal structural units of chromatin organization, nucleosomes, are often transiently converted to hexasomes and tetrasomes missing one or both H2A/H2B histone dimers, respectively. However, the structural and functional properties of the subnucleosomes and their impact on biological processes in the nuclei are poorly understood. Here, using biochemical approaches, molecular dynamics simulations, single-particle Förster resonance energy transfer (spFRET) microscopy and NMR spectroscopy, we have shown that, surprisingly, removal of both dimers from a nucleosome results in much higher mobility of both histones and DNA in the tetrasome. Accordingly, DNase I footprinting shows that DNA-histone interactions in tetrasomes are greatly compromised, resulting in formation of a much lower barrier to transcribing RNA polymerase II than nucleosomes. The data suggest that tetrasomes are remarkably dynamic structures and their formation can strongly affect various biological processes.

[The Role of the WGR Domain in the Functions of PARP1 and PARP2].

The PARP1 and PARP2 proteins are members of the poly(ADP-ribose) polymerase family involved in the regulation of DNA repair and replication, RNA processing, ribosome biogenesis, transcription, cell division, and cell death. PARP1 and PARP2 are promising targets for the development of anticancer drugs and can be used in the treatment of cardiovascular, neurodegenerative, and other disorders. The WGR domain has been shown to play a central role in the functioning of PARP1 and PARP2 proteins. This review considers the mechanisms of functioning of WGR domains in the PARP1 and PARP2 proteins, which have several similar and specialized properties. Understanding these processes is of great interest to fundamental science and can contribute to the development of more effective and selective inhibitors of PARP1 and PARP2.

[Guanine Quadruplexes in Cell Nucleus Metabolism].

Cell metabolism depends, to a large extent, on correct regulation of gene expression. One of the mechanisms of regulation is the formation of nucleic acid secondary structures, among which guanine quadruplexes (G-quadruplexes, or G4) are of particular importance. G-quadruplexes are dynamic structures whose stability is determined by their size, ionic composition, and the nature of the nucleic acids forming them. They are regulated by various protein factors. Guanine quadruplexes play an important role in the regulation of many processes occurring in DNA and RNA, from maintaining telomere homeostasis to determining the ribosome landing site on mRNA. Therefore, these structures are considered a promising target for antitumor therapy, and their detailed study is important to modern biology. This review is focused on the structure and thermodynamic properties of G-quadruplexes together with their interaction with some nuclear proteins.

Poly(ADP-Ribosyl) Code Functions.

Poly(ADP-ribosyl)ation plays a key role in cellular metabolism. Covalent poly(ADP-ribosyl)ation affects the activity of the proteins engaged in DNA repair, chromatin structure regulation, gene expression, RNA processing, ribosome biogenesis, and protein translation. Non-covalent PAR-dependent interactions are involved in the various types of cellular response to stress and viral infection, such as inflammation, hormonal signaling, and the immune response. The review discusses how structurally different poly(ADP-ribose) (PAR) molecules composed of identical monomers can differentially participate in various cellular processes acting as the so-called "PAR code." The article describes the ability of PAR polymers to form functional biomolecular clusters through a phase-separation in response to various signals. This phase-separation contributes to rapid spatial segregation of biochemical processes and effective recruitment of the necessary components. The cellular PAR level is tightly controlled by a network of regulatory proteins: PAR code writers, readers, and erasers. Impaired PAR metabolism is associated with the development of pathological processes causing oncological, cardiovascular, and neurodegenerative diseases. Pharmacological correction of the PAR level may represent a new approach to the treatment of various diseases.

[Opposite Effects of Histone H1 and HMGN5 Protein on Distant Interactions in Chromatin].

Transcriptional enhancers in the cell nuclei typically interact with the target promoters in cis over long stretches of chromatin, but the mechanism of this communication remains unknown. Previously we have developed a defined in vitro system for quantitative analysis of the rate of distant enhancer-promoter communication (EPC) and have shown that the chromatin fibers maintain efficient distant EPC in cis. Here we investigate the roles of linker histone H1 and HMGN5 protein in EPC. A considerable negative effect of histone H1 on EPC depending on its C- and N-tails was shown. Protein HMGN5 that affects chromatin compaction and is associated with active chromatin counteracts EPC inhibition by H1. The data suggest that the efficiency of the interaction between the enhancer and the promoter depends on the structure and dynamics of the chromatin fiber localized between them and can be regulated by proteins associated with chromatin.

Histone Tails Promote PARP1-Dependent Structural Rearrangements in Nucleosomes.

PARP 1 alters the wrapping of nucleosomal DNA on the histone octamer, thereby modulating the accessibility of different genome sites to nuclear protein factors. Here, we show that non-structured histone tails are involved in the PARP1-induced structural rearrangements in nucleosomes, facilitate and stabilize them, but do not affect the enzymatic activity of PARP1.

[HMGB Proteins as DNA Chaperones That Modulate Chromatin Activity].

HMGB proteins are involved in structural rearrangements caused by regulatory chromatin remodeling factors. Particular interest is attracted to a DNA chaperone mechanism, suggesting that the HMGB proteins introduce bends into the double helix, thus rendering DNA accessible to effector proteins and facilitating their activity. The review discusses the role that the HMBG proteins play in key intranuclear processes, including assembly of the preinitiation complex during transcription of ribosomal genes; transcription by RNA polymerases I, II, and III; recruitment of the SWI/SNF complex during transcription of nonribosomal genes; DNA repair; etc. The functions of the HMGB proteins are considered in detail with the examples of yeast HMO1 and NHP6. The two proteins possess unique features in adition to properties characteristic of the HMGB proteins. Thus, NHP6 stimulates a large-scale ATP-independent unwrapping of nucleosomal DNA by the FACT complex, while in its absence FACT stabilizes the nucleosome. HMO1 acts as an alternative linker histone. Both HMO1 and NHP6 are of applied interest primarly because they are homologs of human HMGB1, an important therapeutic target of anticancer and anti-inflammatory treatments.

[Inhibiting the pro-tumor and transcription factor FACT: Mechanisms].

Conventional antitumor therapy is often complicated by the emergence of the so-called cancer stem cells (CSCs), which are characterized by low metabolic rates and high resistance to almost all existing therapies. Many problems of clinical oncology and a poor efficacy of current treatments in particular are ascribed to CSCs. Therefore, it is important to develop new compounds capable of eliminating both rapidly proliferating tumor cells and standard treatment-resistant CSCs. Curaxins have been demonstrated to manifest various types of antitumor activity. Curaxins simultaneously affect at least three key molecular cascades involved in tumor development, including the p53, NF-κB, and HSF1 metabolic pathways. In addition, studies of some curaxins indicate that they can inhibit the transcriptional induction of the genes for matrix metalloproteinases 1 and 8 (MMP1 and MMP8); the PI3K/AKT/mTOR signaling cascades; cIAP-1 (apoptosis protein 1) inhibitor activity; topoisomerase II; and a number of oncogenes, such as c-MYC and others. In vivo experiments have shown that the CSC population increases on gemcitabine monotherapy and is reduced on treatment with curaxin CBL0137. The data support the prospective use of FACT inhibitors as new anticancer drugs with multiple effects on cell metabolism.

Structure and Functions of Linker Histones.

Linker histones such as variants H1, H5, and other similar proteins play an important role in regulation of chromatin structure and dynamics. However, interactions of linker histones with DNA and proteins, as well as specific functions of their different variants, are poorly studied. This is because they acquire tertiary structure only when interacting with a nucleosome, and because of limitations of currently available methods. However, deeper investigation of linker histones and their interactions with other proteins will address a number of important questions - from structure of compacted chromatin to regulation of early embryogenesis. In this review, structures of histone H1 variants and its interaction with chromatin DNA are considered. A possible functional significance of different H1 variants, a role of these proteins in maintaining interphase chromatin structure, and interactions of linker histones with other cellular proteins are also discussed.

Transcription-induced DNA supercoiling: New roles of intranucleosomal DNA loops in DNA repair and transcription.

RNA polymerase II (Pol II) transcription through chromatin is accompanied by formation of small intranucleosomal DNA loops. Pol II captured within a small loop drives accumulation of DNA supercoiling, facilitating further transcription. DNA breaks relieve supercoiling and induce Pol II arrest, allowing detection of DNA damage hidden in chromatin structure.

[Structural studies of chromatin remodeling factors].

Changes of chromatin structure require participation of chromatin remodeling factors (CRFs), which are ATP-dependent multisubunit complexes that change the structure of the nucleosome without covalently modifying its components. CRFs act together with other protein factors to regulate the extent of chromatin condensation. Four CRF families are currently distinguished based on their structural and biochemical characteristics: SWI/SNF, ISWI, Mi-2/CHD, and SWR/INO80. X-ray diffraction analysis and electron microscopy are the main methods to obtain structural information about macromolecules. CRFs are difficult to obtain in crystal because of their large sizes and structural heterogeneity, and transmission electron microscopy (TEM) is mostly employed in their structural studies. The review considers all structures obtained for CRFs by TEM and discusses several models of CRF-nucleosome interactions.

[Structure and function of histone chaperone FACT].

FACT is heterodimer protein complex and histone chaperone that plays an important role in maintaining and modifying chromatin structure during various DNA-dependent processes. FACT is involved in nucleosome assembly de novo and in the preservation and recovery of the nucleosome structure during and after transcription, replication and repair of DNA. During transcript elongation FACT reduces the height of the nucleosome barrier and supports survival of the nucleosomes during and after passage of RNA polymerase II. In this process FACT interacts with histone H2A-H2B dimer in nucleosomes, thus facilitating uncoiling of nucleosomal DNA from the octamer of histones; it also facilitates subsequent recovery of the canonical structure of the nucleosome after transcription. FACT also plays an important role in transformation of human cells and in maintaining the viability of the tumor cells.

Nucleosome positioning and composition modulate in silico chromatin flexibility.

The dynamic organization of chromatin plays an essential role in the regulation of gene expression and in other fundamental cellular processes. The underlying physical basis of these activities lies in the sequential positioning, chemical composition, and intermolecular interactions of the nucleosomes-the familiar assemblies of ∼150 DNA base pairs and eight histone proteins-found on chromatin fibers. Here we introduce a mesoscale model of short nucleosomal arrays and a computational framework that make it possible to incorporate detailed structural features of DNA and histones in simulations of short chromatin constructs. We explore the effects of nucleosome positioning and the presence or absence of cationic N-terminal histone tails on the 'local' inter-nucleosomal interactions and the global deformations of the simulated chains. The correspondence between the predicted and observed effects of nucleosome composition and numbers on the long-range communication between the ends of designed nucleosome arrays lends credence to the model and to the molecular insights gleaned from the simulated structures. We also extract effective nucleosome-nucleosome potentials from the simulations and implement the potentials in a larger-scale computational treatment of regularly repeating chromatin fibers. Our results reveal a remarkable effect of nucleosome spacing on chromatin flexibility, with small changes in DNA linker length significantly altering the interactions of nucleosomes and the dimensions of the fiber as a whole. In addition, we find that these changes in nucleosome positioning influence the statistical properties of long chromatin constructs. That is, simulated chromatin fibers with the same number of nucleosomes exhibit polymeric behaviors ranging from Gaussian to worm-like, depending upon nucleosome spacing. These findings suggest that the physical and mechanical properties of chromatin can span a wide range of behaviors, depending on nucleosome positioning, and that care must be taken in the choice of models used to interpret the experimental properties of long chromatin fibers.

Structural Analysis of the Key Intermediate Formed during Transcription through a Nucleosome.

Transcription through chromatin by different RNA polymerases produces different biological outcomes and is accompanied by either nucleosome survival at the original location (Pol II-type mechanism) or backward nucleosome translocation along DNA (Pol III-type mechanism). It has been proposed that differences in the structure of the key intermediates formed during transcription dictate the fate of the nucleosomes. To evaluate this possibility, structure of the key intermediate formed during transcription by Pol III-type mechanism was studied by DNase I footprinting and molecular modeling. The Pol III-type mechanism is characterized by less efficient formation of the key intermediate required for nucleosome survival (Ø-loop, Pol II-type mechanism), most likely due to steric interference between the RNA polymerase and DNA in the Ø-loop. The data suggest that the lower efficiency of Ø-loop formation induces formation of a lower nucleosomal barrier and nucleosome translocation during transcription by Pol III-type mechanism.

Role of C-terminal domain phosphorylation in RNA polymerase II transcription through the nucleosome.

End-initiated transcription of a 256 base-pair (bp) template containing a single uniquely positioned nucleosome by yeast and calf thymus nuclear RNA polymerases II (pol II) was analyzed in vitro. The nucleosome-specific pausing pattern is similar to the pattern observed in the case of transcription of the same nucleosome by yeast RNA polymerase III. However, the pausing pattern is clearly different from the patterns observed previously during transcription by promoter-initiated and assembled pol II. This suggests that end-initiated and promoter-initiated RNA polymerases differ in the way they progress through the nucleosome. The rates of transcription through the nucleosome by pol II are significantly lower than the rates observed in the case of SP6 polymerase and RNA polymerase III. Using calf thymus pol II, we have investigated the possibility that phosphorylation of the C-terminal domain (CTD) facilitates transcription through the nucleosome. The rates of transcription through the nucleosome by phosphorylated (IIO) and nonphosphorylated (IIA) forms of calf thymus pol II are very similar. This suggests that CTD phosphorylation is not sufficient to facilitate transcription through the nucleosome by end-initiated pol II.

DNA supercoiling allows enhancer action over a large distance.

Enhancers are regulatory DNA elements that can activate their genomic targets over a large distance. The mechanism of enhancer action over large distance is unknown. Activation of the glnAp2 promoter by NtrC-dependent enhancer in Escherichia coli was analyzed by using a purified system supporting multiple-round transcription in vitro. The data suggest that DNA supercoiling is an essential requirement for enhancer action over a large distance (2,500 bp) but not over a short distance (110 bp). DNA supercoiling facilitates functional enhancer-promoter communication over a large distance, probably by bringing the enhancer and promoter into close proximity.

Facilitated transcription through the nucleosome at high ionic strength occurs via a histone octamer transfer mechanism.

The rate of transcription through the nucleosome, the fine structure of the nucleosomal barrier, and the fate of the nucleosome during transcription at different salt concentrations were analyzed using linear 227-base pair mononucleosomal templates containing a uniquely positioned nucleosome core. At lower ionic strength (30 mm NaCl), the nucleosome constitutes a strong barrier for SP6 RNA polymerase. At higher ionic strength (330 mm NaCl), the rates of transcription on nucleosomal and histone-free DNA templates are very similar. At both higher and lower ionic strengths, the complete histone octamer is transferred over the same distance by fundamentally similar mechanisms. The data indicate that even at the rate of transcription characteristic of histone-free DNA, the transfer intermediates can be formed quite efficiently. This suggests possible mechanisms that could facilitate transcription through the nucleosome at physiological ionic strength.

The nature of the nucleosomal barrier to transcription: direct observation of paused intermediates by electron cryomicroscopy.

Transcribing SP6 RNA polymerase was arrested at unique positions in the nucleosome core, and the complexes were analyzed using biochemical methods and electron cryomicroscopy. As the polymerase enters the nucleosome, it disrupts DNA-histone interactions behind and up to approximately 20 bp ahead of the elongation complex. After the polymerase proceeds 30-40 bp into the nucleosome, two intermediates are observed. In one, only the DNA ahead of the polymerase reassociates with the octamer. In the other, DNA both ahead of and behind the enzyme reassociates. These intermediates present a barrier to elongation. When the polymerase approaches the nucleosome dyad, it displaces the octamer, which is transferred to promoter-proximal DNA.