Rhodamine 19 Alkyl Esters as Effective Antibacterial Agents.

Mitochondria-targeted antioxidants (MTAs) have been studied quite intensively in recent years as potential therapeutic agents and vectors for the delivery of other active substances to mitochondria and bacteria. Their most studied representatives are MitoQ and SkQ1, with its fluorescent rhodamine analog SkQR1, a decyl ester of rhodamine 19 carrying plastoquinone. In the present work, we observed a pronounced antibacterial action of SkQR1 against Gram-positive bacteria, but virtually no effect on Gram-negative bacteria. The MDR pump AcrAB-TolC, known to expel SkQ1, did not recognize and did not pump out SkQR1 and dodecyl ester of rhodamine 19 (C12R1). Rhodamine 19 butyl (C4R1) and ethyl (C2R1) esters more effectively suppressed the growth of ΔtolC Escherichia coli, but lost their potency with the wild-type E. coli pumping them out. The mechanism of the antibacterial action of SkQR1 may differ from that of SkQ1. The rhodamine derivatives also proved to be effective antibacterial agents against various Gram-positive species, including Staphylococcus aureus and Mycobacterium smegmatis. By using fluorescence correlation spectroscopy and fluorescence microscopy, SkQR1 was shown to accumulate in the bacterial membrane. Thus, the presentation of SkQR1 as a fluorescent analogue of SkQ1 and its use for visualization should be performed with caution.

Relationship of Cytotoxic and Antimicrobial Effects of Triphenylphosphonium Conjugates with Various Quinone Derivatives.

Quinone derivatives of triphenylphosphonium have proven themselves to be effective geroprotectors and antioxidants that prevent oxidation of cell components with participation of active free radicals - peroxide (RO2·), alkoxy (RO·), and alkyl (R·) radicals, as well as reactive oxygen species (superoxide anion, singlet oxygen). Their most studied representatives are derivatives of plastoquinone (SkQ1) and ubiquinone (MitoQ), which in addition to antioxidant properties also have a strong antibacterial effect. In this study, we investigated antibacterial properties of other quinone derivatives based on decyltriphenylphosphonium (SkQ3, SkQT, and SkQThy). We have shown that they, just like SkQ1, inhibit growth of various Gram-positive bacteria at micromolar concentrations, while being less effective against Gram-negative bacteria, which is associated with recognition of the triphenylphosphonium derivatives by the main multidrug resistance (MDR) pump of Gram-negative bacteria, AcrAB-TolC. Antibacterial action of SkQ1 itself was found to be dependent on the number of bacterial cells. It is important to note that the cytotoxic effect of SkQ1 on mammalian cells was observed at higher concentrations than the antibacterial action, which can be explained by (i) the presence of a large number of membrane organelles, (ii) lower membrane potential, (iii) spatial separation of the processes of energy generation and transport, and (iv) differences in the composition of MDR pumps. Differences in the cytotoxic effects on different types of eukaryotic cells may be associated with the degree of membrane organelle development, energy status of the cell, and level of the MDR pump expression.

Synthesis of Triphenylphosphonium-Linked Derivative of 3,5-Ditert-butyl-4-hydroxybenzylidene-malononitrile (SF6847) via Knoevenagel Reaction Yields an Effective Mitochondria-Targeted Protonophoric Uncoupler.

Mitochondrial uncouplers are actively sought as potential therapeutics. Here, we report the first successful synthesis of mitochondria-targeted derivatives of the highly potent uncoupler 3,5-ditert-butyl-4-hydroxybenzylidene-malononitrile (SF6847), bearing a cationic alkyl(triphenyl)phosphonium (TPP) group. As a key step of the synthesis, we used condensation of a ketophenol with malononitrile via the Knoevenagel reaction. SF-C5-TPP with a pentamethylene linker between SF6847 and TPP, stimulating respiration and collapsing membrane potential of rat liver mitochondria at submicromolar concentrations, proved to be the most effective uncoupler of the series. SF-C5-TPP showed pronounced protonophoric activity on a model planar bilayer lipid membrane. Importantly, SF-C5-TPP exhibited rather low toxicity in fibroblast cell culture, causing mitochondrial depolarization in cells at concentrations that only slightly affected cell viability. SF-C5-TPP was more effective in decreasing the mitochondrial membrane potential in the cell culture than SF6847, in contrast to the case of isolated mitochondria. Like other zwitterionic uncouplers, SF-C5-TPP inhibited the growth of Bacillus subtilis in the micromolar concentration range.

Methylation of Phenyl Rings in Ester-Stabilized Phosphorus Ylides Vastly Enhances Their Protonophoric Activity.

We have recently discovered that ester-stabilized phosphorus ylides, resulting from deprotonation of a phosphonium salt such as [Ph3PCH2COOR], can transfer protons across artificial and biological membranes. To create more effective cationic protonophores, we synthesized similar phosphonium salts with one ((heptyloxycarbonylmethyl)(p-tolyl)bromide) or two ((butyloxycarbonylmethyl)(3,5-xylyl)osphonium bromide) methyl substituents in the phenyl groups. The methylation enormously augmented both protonophoric activity of the ylides on planar bilayer lipid membrane (BLM) and uncoupling of mammalian mitochondria, which correlated with strongly accelerated flip-flop of their cationic precursors across the BLM.

Nanoparticles of Push-Pull Triphenylamine-Based Molecules for Light-Controlled Stimulation of Neuronal Activity.

Organic semiconductor materials with a unique set of properties are very attractive for interfacing biological objects and can be used for noninvasive therapy or detection of biological signals. Here, we describe the synthesis and investigation of a novel series of organic push-pull conjugated molecules with the star-shaped architecture, consisting of triphenylamine as a branching electron donor core linked through the thiophene π-spacer to electron-withdrawing alkyl-dicyanovinyl groups. The molecules could form stable aqueous dispersions of nanoparticles (NPs) without the addition of any surfactants or amphiphilic polymer matrixes with the average size distribution varying from 40 to 120 nm and absorption spectra very similar to those of human eye retina pigments such as rods and green cones. Variation of the terminal alkyl chain length of the molecules forming NPs from 1 to 12 carbon atoms was found to be an efficient tool to modulate their lipophilic and biological properties. Possibilities of using the NPs as light nanoactuators in biological systems or as artificial pigments for therapy of degenerative retinal diseases were studied both on the model planar bilayer lipid membranes and on the rat cortical neurons. In the planar bilayer system, the photodynamic activity of these NPs led to photoinactivation of ion channels formed by pentadecapeptide gramicidin A. Treatment of rat cortical neurons with the NPs caused depolarization of cell membranes upon light irradiation, which could also be due to the photodynamic activity of the NPs. The results of the work gave more insight into the mechanisms of light-controlled stimulation of neuronal activity and for the first time showed that fine-tuning of the lipophilic affinity of NPs based on organic conjugated molecules is of high importance for creating a bioelectronic interface for biomedical applications.

Zinc-dependent Nucleosome Reorganization by PARP2.

Poly(ADP-ribose)polymerase 2 (PARP2) is a nuclear protein that acts as a DNA damage sensor; it recruits the repair enzymes to a DNA damage site and facilitates formation of the repair complex. Using single particle Förster resonance energy transfer microscopy and electrophoretic mobility shift assay (EMSA) we demonstrated that PARP2 forms complexes with a nucleosome containing different number of PARP2 molecules without altering conformation of nucleosomal DNA both in the presence and in the absence of Mg 2+ or Ca 2+ ions. In contrast, Zn 2+ ions directly interact with PARP2 inducing a local alteration of the secondary structure of the protein and PARP2-mediated, reversible structural reorganization of nucleosomal DNA. AutoPARylation activity of PARP2 is enhanced by Mg 2+ ions and modulated by Zn 2+ ions: suppressed or enhanced depending on the occupancy of two functionally different Zn 2+ binding sites. The data suggest that Zn 2+ /PARP2-induced nucleosome reorganization and transient changes in the concentration of the cations could modulate PARP2 activity and the DNA damage response. Significance Statement: PARP2 recognizes and binds DNA damage sites, recruits the repair enzymes to these sites and facilitates formation of the repair complex. Zn 2+ -induced structural reorganization of nucleosomal DNA in the complex with PARP2, which is reported in the paper, could modulate the DNA damage response. The obtained data indicate the existence of specific binding sites of Mg 2+ and Zn 2+ ions in and/or near the catalytic domain of PARP2, which modulate strongly, differently and ion-specifically PARylation activity of PARP2, which is important for maintaining genome stability, adaptation of cells to stress, regulation of gene expression and antioxidant defense.

The transient character of mitochondrial uncoupling by the popular fungicide fluazinam is specific for liver.

The popular fungicide fluazinam is known to exhibit an unusual cyclic pattern of the protonophoric uncoupling activity in isolated rat liver mitochondria (RLM), with membrane deenergization followed by spontaneous recoupling in the minute scale, which is associated with glutathione conjugation of fluazinam catalyzed by glutathione-S-transferase (GST). Here, we compare the fluazinam effect on RLM with that on rat kidney (RKM) and heart (RHM) mitochondria by monitoring three bioenergetic parameters: oxygen consumption rate, mitochondrial membrane potential and reduction of nucleotides. Only in RLM, the uncoupling activity of fluazinam was transient, i.e. disappeared in a few minutes, whereas in RKM and RHM it was stable in this time scale. We attribute this difference to the increased activity of mitochondrial GST in liver. We report data on the detection of glutathione-fluazinam conjugates by mass-spectrometry, thin layer chromatography and capillary electrophoresis after incubation of fluazinam with RLM but not with RKM, which supports the assumption of the tissue specificity of the conjugation.

Selectivity of cation transport across lipid membranes by the antibiotic salinomycin.

The ionophoric antibiotic salinomycin is in the phase of preclinical tests against several types of malignant tumors including breast cancer. Notwithstanding, the data on its ion selectivity, although being critical for its therapeutic activity, are rather scarce. In the present work, we studied the ability of salinomycin to exert cation/H+-exchange across artificial bilayer lipid membranes (BLM) by measuring electrical potential on planar BLM in the presence of a protonophore and fluorescence responses of the pH-sensitive dye pyranine entrapped in liposomes. The following order of ion selectivity was obtained by these two methods: K+ > Na+ > Rb+ > Cs+ > Li+. Measurements of the monovalent cation-induced quenching of fluorescence of thallium ions in methanol showed that salinomycin effectively binds potassium and calcium but poorly binds sodium and lithium ions. At high concentrations, salinomycin transports Ca2+ through membranes of liposomes and mitochondria, as measured by using the calcium-sensitive dye Fluo-5 N. The data obtained can be used in the mechanistic studies of the anti-tumor activity of salinomycin and its selective cytotoxicity towards cancer stem cells.

Cell-specific expression of the FAP gene is regulated by enhancer elements.

Fibroblast activation protein (FAP) is an integral membrane serine protease that acts as both dipeptidyl peptidase and collagenase. In recent years, FAP has attracted considerable attention due to its specific upregulation in multiple types of tumor cell populations, including cancer cells in various cancer types, making FAP a potential target for therapy. However, relatively few papers pay attention to the mechanisms driving the cell-specific expression of the FAP gene. We found no correlation between the activities of the two FAP promoter variants (short and long) and the endogenous FAP mRNA expression level in several cell lines with different FAP expression levels. This suggested that other mechanisms may be responsible for specific transcriptional regulation of the FAP gene. We analyzed the distribution of known epigenetic and structural chromatin marks in FAP-positive and FAP-negative cell lines and identified two potential enhancer-like elements (E1 and E2) in the FAP gene locus. We confirmed the specific enrichment of H3K27ac in the putative enhancer regions in FAP-expressing cells. Both the elements exhibited enhancer activity independently of each other in the functional test by increasing the activity of the FAP promoter variants to a greater extent in FAP-expressing cell lines than in FAP-negative cell lines. The transcription factors AP-1, CEBPB, and STAT3 may be involved in FAP activation in the tumors. We hypothesized the existence of a positive feedback loop between FAP and STAT3, which may have implications for developing new approaches in cancer therapy.

Synchronization of opening and closing of two gramicidin A channels pulled together by a linker: possible relevance to channel clustering.

The linear 15-mer peptide gramicidin A (gA) produced by Bacillus brevis is known to form the simplest natural ion channel in lipid membranes representing a head-to-head transmembrane dimer. Its incorporation into a planar lipid bilayer manifests itself in regular electrical current transitions. If two gA subunits are tightly connected by a water-soluble, flexible linker of a certain length, the current transitions become heterogeneous: in a part of them, the amplitude is almost twofold higher than that of a single channel, thereby demonstrating the synchronous opening of two single channels. The lifetime, i.e. the open-state duration, of this dual channel is by several orders of magnitude longer than that of the single channel. Here, we used the ideas of the theory of excitons to hypothesize about the mechanism of synchronous opening and closing of two adjacent channels. Two independent (uncoupled) single channels can be described by two independent conformational coordinates q1 and q2, while two closely located channels can exhibit collective behavior, if the coupling between them produces mixing of the individual states (q1,0) and (0,q2). We suppose that a similar phenomenon can occur not only with synthetic derivatives of gA, but also with such natural channel-forming peptide antibiotics and toxins as alamethicin and syringomycin. In particular, channel clustering observed with these peptides may be also associated with formation of collective conductance states, resulting from mixing of their monomeric states, which allows us to explain the fact that clusters of these channels transmit ions and nonelectrolytes of the same size as the original single channels.

Ester-stabilized phosphorus ylides as protonophores on bilayer lipid membranes, mitochondria and chloroplasts.

Triphenylphosphonium ylides are commonly used as key intermediates in the Wittig reaction. Based on the known acidities of stabilized ylide precursors, we proposed that a methylene group adjacent to phosphorus in these compounds can ensure proton shuttling across lipid membranes. Here, we synthesized (decyloxycarbonylmethyl)triphenylphosphonium bromide (CMTPP-C10) by reaction of triphenylphosphine with decyl bromoacetate. This phosphonium salt precursor of the ester-stabilized phosphorus ylide along with its octyl (CMTPP-C8) and dodecyl (CMTPP-C12) analogues was found to be a carrier of protons in mitochondrial, chloroplast and artificial lipid membranes, suggesting that it can reversibly release hydrogen ions and diffuse through the membranes in both zwitterionic (ylide) and cationic forms. The CMTPP-C10-mediated electrical current across planar bilayer lipid membranes exhibited pronounced proton selectivity. Similar to conventional protonophores, known to uncouple electron transport and ATP synthesis, CMTPP-Cn (n = 8, 10, 12) stimulated mitochondrial respiration, while decreasing membrane potential, at micromolar concentrations, thereby showing the classical uncoupling activity in mitochondria. CMTPP-C12 also caused dissipation of transmembrane pH gradient on chloroplast membranes. Importantly, CMTPP-C10 exhibited substantially lower toxicity in cell culture, than C12TPP. Thus, we report the finding of a new class of ylide-type protonophores, which is of substantial interest due to promising therapeutic properties of uncouplers.

Photodynamic activity rather than drilling causes membrane damage by a light-powered molecular nanomotor.

The chase toward endowing chemical compounds with machine-like functions mimicking those of biological molecular machineries has yielded a variety of artificial molecular motors (AMMs). Pharmaceutical applications of photoexcited monomolecular unidirectionally-rotating AMMs have been envisioned in view of their ability to permeabilize biological membranes. Nonetheless, the mechanical properties of lipid membranes render the proposed drilling activity of AMMs doubtful. Here, we show that singlet oxygen released by a photoexcited "molecular drill" oxidized unsaturated lipids composing giant unilamellar vesicles. In contrast, giant liposomes built of saturated lipids were inert to AMM photoactuation. The AMM did not mechanically destroy gramicidin A ion channels in planar bilayer lipid membranes but instead photoinactivated them. Sodium azide, a singlet oxygen quencher, reduced both AMM-mediated light-induced dye release from unsaturated large unilamellar vesicles and protected gramicidin A from photoinactivation. Upon additional consideration of the underlying bilayer mechanics, we conclude that AMMs' envisioned therapeutic and pharmaceutical applications rely on their photodynamic activity rather than their nanomechanical drilling abilities.

Retinoblastoma seeds: impact on American Joint Committee on Cancer clinical staging.

AIM: To investigate whether the American Joint Committee on Cancer (AJCC) clinical category cT2b needs to be subclassified by the type and distribution of retinoblastoma (RB) seeding. METHODS: Multicentre, international registry-based data were collected from RB centres enrolled between January 2001 and December 2013. 1054 RB eyes with vitreous or subretinal seeds from 18 ophthalmic oncology centres, in 13 countries within six continents were analysed. Local treatment failure was defined as the use of secondary enucleation or external beam radiation therapy (EBRT) and was estimated with the Kaplan-Meier method. RESULTS: Clinical category cT2b included 1054 eyes. Median age at presentation was 16.0 months. Of these, 428 (40.6%) eyes were salvaged, and 430 (40.8%) were treated with primary and 196 (18.6%) with secondary enucleation. Of the 592 eyes that had complete data for globe salvage analysis, the distribution of seeds was focal in 143 (24.2%) and diffuse in 449 (75.8%). The 5-year Kaplan-Meier cumulative globe-salvage (without EBRT) was 78% and 49% for eyes with focal and diffuse RB seeding, respectively. Cox proportional hazards regression analysis confirmed a higher local treatment failure risk with diffuse seeds as compared with focal seeds (hazard rate: 2.8; p<0.001). There was insufficient evidence to prove or disprove an association between vitreous seed type and local treatment failure risk(p=0.06). CONCLUSION: This international, multicentre, registry-based analysis of RB eyes affirmed that eyes with diffuse intraocular distribution of RB seeds at diagnosis had a higher risk of local treatment failure when compared with focal seeds. Subclassification of AJCC RB category cT2b into focal vs diffuse seeds will improve prognostication for eye salvage.

Mechanism of curaxin-dependent nucleosome unfolding by FACT.

Human FACT (FACT) is a multifunctional histone chaperone involved in transcription, replication and DNA repair. Curaxins are anticancer compounds that induce FACT-dependent nucleosome unfolding and trapping of FACT in the chromatin of cancer cells (c-trapping) through an unknown molecular mechanism. Here, we analyzed the effects of curaxin CBL0137 on nucleosome unfolding by FACT using spFRET and electron microscopy. By itself, FACT adopted multiple conformations, including a novel, compact, four-domain state in which the previously unresolved NTD of the SPT16 subunit of FACT was localized, apparently stabilizing a compact configuration. Multiple, primarily open conformations of FACT-nucleosome complexes were observed during curaxin-supported nucleosome unfolding. The obtained models of intermediates suggest "decision points" in the unfolding/folding pathway where FACT can either promote disassembly or assembly of nucleosomes, with the outcome possibly being influenced by additional factors. The data suggest novel mechanisms of nucleosome unfolding by FACT and c-trapping by curaxins.

Efficient Selection of Enhancers and Promoters from MIA PaCa-2 Pancreatic Cancer Cells by ChIP-lentiMPRA.

A library of active genome regulatory elements (putative promoters and enhancers) from MIA PaCa-2 pancreatic adenocarcinoma cells was constructed using a specially designed lentiviral vector and a massive parallel reporter assay (ChIP-lentiMPRA). Chromatin immunoprecipitation of the cell genomic DNA by H3K27ac antibodies was used for primary enrichment of the library for regulatory elements. Totally, 11,264 unique genome regions, many of which are capable of enhancing the expression of the CopGFP reporter gene from the minimal CMV promoter, were identified. The regions tend to be located near promoters. Based on the proximity assay, we found an enrichment of highly expressed genes among those associated with three or more mapped distal regions (2 kb distant from the 5'-ends of genes). It was shown significant enrichment of genes related to carcinogenesis or Mia PaCa-2 cell identity genes in this group. In contrast, genes associated with 1-2 distal regions or only with proximal regions (within 2 kbp of the 5'-ends of genes) are more often related to housekeeping functions. Thus, ChIP-lentiMPRA is a useful strategy for creating libraries of regulatory elements for the study of tumor-specific gene transcription.

Bearing Aluminum-Based Alloys: Microstructure, Mechanical Characterizations, and Experiment-Based Modeling Approach.

Due to the engine's start/stop system and a sudden increase in speed or load, the development of alloys suitable for engine bearings requires excellent tribological properties and high mechanical properties. Including additional elements in the Al-rich matrix of these anti-friction alloys should strengthen their tribological properties. The novelty of this work is in constructing a suitable artificial neural network (ANN) architecture for highly accurate modeling and prediction of the mechanical properties of the bearing aluminum-based alloys and thus optimizing the chemical composition for high mechanical properties. In addition, the study points out the impact of soft and more solid phases on the mechanical properties of these alloys. For this purpose, a huge number of alloys (198 alloys) with different chemical compositions combined from Sn, Pb, Cu, Mg, Zn, Si, Ni, Bi, Ti, Mn, Fe, and Al) were cast, annealed, and tested for determining their mechanical properties. The annealed sample microstructure analysis revealed the formation of soft structural inclusions (Sn-rich, Sn-Pb, and Pb-Sn phases) and solid phase inclusions (strengthened phase, Al2Cu). The mechanical properties of ultimate tensile strength (σu), Brinell hardness (HB), and elongation to failure (δ) were used as control responses for constructing the ANN network. The constructed network was optimized by attempting different network architecture designs to reach minimal errors. Besides the excellent tribological characteristics of the designed set of alloys, soft inclusions based on Sn and Pb and solid-phase Cu inclusions fulfilled the necessary level of mechanical properties for anti-friction alloys; the maximum mechanical properties reached were: σu = 197 ± 7 MPa, HB = 77 ± 4, and δ = 20.3 ± 1.0%. The optimal ANN architecture with the lowest errors (correlation coefficient (R) = 0.94, root mean square error (RMSE) = 3.5, and average actual relative error (AARE) = 1.0%) had two hidden layers with 20 neurons. The model was validated by additional experiments, and the characteristics of the new alloys were accurately predicted with a low level of errors: R ≥ 0.97, RMSE = 1-2.65, and AARE ˂ 10%.

Usnic Acid-Mediated Exchange of Protons for Divalent Metal Cations across Lipid Membranes: Relevance to Mitochondrial Uncoupling.

Usnic acid (UA), a unique lichen metabolite, is a protonophoric uncoupler of oxidative phosphorylation, widely known as a weight-loss dietary supplement. In contrast to conventional proton-shuttling mitochondrial uncouplers, UA was found to carry protons across lipid membranes via the induction of an electrogenic proton exchange for calcium or magnesium cations. Here, we evaluated the ability of various divalent metal cations to stimulate a proton transport through both planar and vesicular bilayer lipid membranes by measuring the transmembrane electrical current and fluorescence-detected pH gradient dissipation in pyranine-loaded liposomes, respectively. Thus, we obtained the following selectivity series of calcium, magnesium, zinc, manganese and copper cations: Zn2+ > Mn2+ > Mg2+ > Ca2+ >> Cu2+. Remarkably, Cu2+ appeared to suppress the UA-mediated proton transport in both lipid membrane systems. The data on the divalent metal cation/proton exchange were supported by circular dichroism spectroscopy of UA in the presence of the corresponding cations.

Antibiotic Pyrrolomycin as an Efficient Mitochondrial Uncoupler.

Pyrrolomycins C (Pyr_C) and D (Pyr_D) are antibiotics produced by Actinosporangium and Streptomyces. The mechanism of their antimicrobial activity consists in depolarization of bacterial membrane, leading to the suppression of bacterial bioenergetics through the uncoupling of oxidative phosphorylation, which is based on the protonophore action of these antibiotics [Valderrama et al., Antimicrob. Agents Chemother. (2019) 63, e01450]. Here, we studied the effect of pyrrolomycins on the isolated rat liver mitochondria. Pyr_C was found to be more active than Pyr_D and uncoupled mitochondria in the submicromolar concentration range, which was observed as the mitochondrial membrane depolarization and stimulation of mitochondrial respiration. In the case of mitoplasts (isolated mitochondria with impaired outer membrane integrity), the difference in the action of Pyr_C and Pyr_D was significantly less pronounced. By contrast, in inverted submitochondrial particles (SMPs), Pyr_D was more active as an uncoupler, which caused collapse of the membrane potential even at the nanomolar concentrations. The same ratio of the protonophoric activity of Pyr_D and Pyr_C was obtained by us on liposomes loaded with the pH indicator pyranine. The protonophore activity of Pyr_D in the planar bilayer lipid membranes (BLMs) was maximal at ~pH 9, i.e., at pH values close to pKa of this compound. Pyr_D functions as a typical anionic protonophore; its activity in the BLM could be reduced by the addition of the dipole modifier phloretin. The difference between the protonophore activity of Pyr_C and Pyr_D in the mitochondria and BLMs can be attributed to a higher ability of Pyr_C to penetrate the outer mitochondrial membrane.

N-Terminal Tails of Histones H2A and H2B Differentially Affect Transcription by RNA Polymerase II In Vitro.

Histone N-terminal tails and their post-translational modifications affect various biological processes, often in a context-specific manner; the underlying mechanisms are poorly studied. Here, the role of individual N-terminal tails of histones H2A/H2B during transcription through chromatin was analyzed in vitro. spFRET data suggest that the tail of histone H2B (but not of histone H2A) affects nucleosome stability. Accordingly, deletion of the H2B tail (amino acids 1-31, but not 1-26) causes a partial relief of the nucleosomal barrier to transcribing RNA polymerase II (Pol II), likely facilitating uncoiling of DNA from the histone octamer during transcription. Taken together, the data suggest that residues 27-31 of histone H2B stabilize DNA-histone interactions at the DNA region localized ~25 bp in the nucleosome and thus interfere with Pol II progression through the region localized 11-15 bp in the nucleosome. This function of histone H2B requires the presence of the histone H2A N-tail that mediates formation of nucleosome-nucleosome dimers; however, nucleosome dimerization per se plays only a minimal role during transcription. Histone chaperone FACT facilitates transcription through all analyzed nucleosome variants, suggesting that H2A/H2B tails minimally interact with FACT during transcription; therefore, an alternative FACT-interacting domain(s) is likely involved in this process.

Human PARP1 Facilitates Transcription through a Nucleosome and Histone Displacement by Pol II In Vitro.

Human poly(ADP)-ribose polymerase-1 (PARP1) is a global regulator of various cellular processes, from DNA repair to gene expression. The underlying mechanism of PARP1 action during transcription remains unclear. Herein, we have studied the role of human PARP1 during transcription through nucleosomes by RNA polymerase II (Pol II) in vitro. PARP1 strongly facilitates transcription through mononucleosomes by Pol II and displacement of core histones in the presence of NAD+ during transcription, and its NAD+-dependent catalytic activity is essential for this process. Kinetic analysis suggests that PARP1 facilitates formation of "open" complexes containing nucleosomal DNA partially uncoiled from the octamer and allowing Pol II progression along nucleosomal DNA. Anti-cancer drug and PARP1 catalytic inhibitor olaparib strongly represses PARP1-dependent transcription. The data suggest that the negative charge on protein(s) poly(ADP)-ribosylated by PARP1 interact with positively charged DNA-binding surfaces of histones transiently exposed during transcription, facilitating transcription through chromatin and transcription-dependent histone displacement/exchange.