DNA loop domain rearrangements in blast transformed human lymphocytes and lymphoid leukaemic Jurkat T cells

Chromatin loops are important elements of both chromatin higher-order structure and transcription regulation system. Our previous works have shown that several features of the loop domain organization could be investigated by single cell gel electrophoresis (the comet assay) using the kinetic approach. In this study we applied this technique to study DNA loop domain organization in lymphoid cells: human lymphocytes, lymphoblasts cultivated during 24 h and 44 h, and T cells of Jurkat cell line. Two features of the loop domain organization were found to depend on the cell functional state. First, DNA fraction in the loops of large sizes (more than ~200 kb) was essentially increased in proliferating (de-differentiated) cells in comparison with terminally differentiated lymphocytes. Second, the linear density of the loops not larger than ~200 kb was decreased in transcriptionally active cells and was increased upon their inactivation

Structural plasticity of single chromatin fibers revealed by torsional manipulation

Magnetic tweezers are used to study the mechanical response under torsion of single nucleosome arrays reconstituted on tandem repeats of 5S positioning sequences. Regular arrays are extremely resilient and can reversibly accommodate a large amount of supercoiling without much change in length. This behavior is quantitatively described by a molecular model of the chromatin 3-D architecture. In this model, we assume the existence of a dynamic equilibrium between three conformations of the nucleosome, which are determined by the crossing status of the entry/exit DNAs (positive, null or negative). Torsional strain, in displacing that equilibrium, extensively reorganizes the fiber architecture. The model explains a number of long-standing topological questions regarding DNA in chromatin, and may provide the ground to better understand the dynamic binding of most chromatin-associated proteins.Comment: 18 pages, 7 figures, Supplementary information available at http://www.nature.com/nsmb/journal/v13/n5/suppinfo/nsmb1087_S1.htm

Rad51 Polymerization Reveals a New Chromatin Remodeling Mechanism

Rad51 protein is a well known protagonist of homologous recombination in eukaryotic cells. Rad51 polymerization on single-stranded DNA and its role in presynaptic filament formation have been extensively documented. Rad51 polymerizes also on double-stranded DNA but the significance of this filament formation remains unclear. We explored the behavior of Saccharomyces cerevisiae Rad51 on dsDNA and the influence of nucleosomes on Rad51 polymerization mechanism to investigate its putative role in chromatin accessibility to recombination machinery. We combined biochemical approaches, transmission electron microscopy (TEM) and atomic force microscopy (AFM) for analysis of the effects of the Rad51 filament on chromatinized templates. Quantitative analyses clearly demonstrated the occurrence of chromatin remodeling during nucleoprotein filament formation. During Rad51 polymerization, recombinase proteins moved all the nucleosomal arrays in front of the progressing filament. This polymerization process had a powerful remodeling effect, as Rad51 destabilized the nucleosomes along considerable stretches of DNA. Similar behavior was observed with RecA. Thus, recombinase polymerization is a powerful mechanism of chromatin remodeling. These remarkable features open up new possibilities for understanding DNA recombination and reveal new types of ATP-dependent chromatin dynamics

Promoter prediction using physico-chemical properties of DNA

The ability to locate promoters within a section of DNA is known to be a very difficult and very important task in DNA analysis. We document an approach that incorporates the concept of DNA as a complex molecule using several models of its physico-chemical properties. A support vector machine is trained to recognise promoters by their distinctive physical and chemical properties. We demonstrate that by combining models, we can improve upon the classification accuracy obtained with a single model. We also show that by examining how the predictive accuracy of these properties varies over the promoter, we can reduce the number of attributes needed. Finally, we apply this method to a real-world problem. The results demonstrate that such an approach has significant merit in its own right. Furthermore, they suggest better results from a planned combined approach to promoter prediction using both physicochemical and sequence based techniques

An All-Atom Model of the Chromatin Fiber Containing Linker Histones Reveals a Versatile Structure Tuned by the Nucleosomal Repeat Length

In the nucleus of eukaryotic cells, histone proteins organize the linear genome into a functional and hierarchical architecture. In this paper, we use the crystal structures of the nucleosome core particle, B-DNA and the globular domain of H5 linker histone to build the first all-atom model of compact chromatin fibers. In this 3D jigsaw puzzle, DNA bending is achieved by solving an inverse kinematics problem. Our model is based on recent electron microscopy measurements of reconstituted fiber dimensions. Strikingly, we find that the chromatin fiber containing linker histones is a polymorphic structure. We show that different fiber conformations are obtained by tuning the linker histone orientation at the nucleosomes entry/exit according to the nucleosomal repeat length. We propose that the observed in vivo quantization of nucleosomal repeat length could reflect nature's ability to use the DNA molecule's helical geometry in order to give chromatin versatile topological and mechanical properties

G+C content dominates intrinsic nucleosome occupancy

<p>Abstract</p> <p>Background</p> <p>The relative preference of nucleosomes to form on individual DNA sequences plays a major role in genome packaging. A wide variety of DNA sequence features are believed to influence nucleosome formation, including periodic dinucleotide signals, poly-A stretches and other short motifs, and sequence properties that influence DNA structure, including base content. It was recently shown by Kaplan et al. that a probabilistic model using composition of all 5-mers within a nucleosome-sized tiling window accurately predicts intrinsic nucleosome occupancy across an entire genome <it>in vitro</it>. However, the model is complicated, and it is not clear which specific DNA sequence properties are most important for intrinsic nucleosome-forming preferences.</p> <p>Results</p> <p>We find that a simple linear combination of only 14 simple DNA sequence attributes (G+C content, two transformations of dinucleotide composition, and the frequency of eleven 4-bp sequences) explains nucleosome occupancy <it>in vitro </it>and <it>in vivo </it>in a manner comparable to the Kaplan model. G+C content and frequency of AAAA are the most important features. G+C content is dominant, alone explaining ~50% of the variation in nucleosome occupancy <it>in vitro</it>.</p> <p>Conclusions</p> <p>Our findings provide a dramatically simplified means to predict and understand intrinsic nucleosome occupancy. G+C content may dominate because it both reduces frequency of poly-A-like stretches and correlates with many other DNA structural characteristics. Since G+C content is enriched or depleted at many types of features in diverse eukaryotic genomes, our results suggest that variation in nucleotide composition may have a widespread and direct influence on chromatin structure.</p

Несостоятельность колоректального анастомоза после передней резекции прямой кишки: частота, факторы риска

Purpose of the study. To study the frequency and risk factors for the development of colorectal anastomosis leakage after anterior rectal resection for cancer.Materials and Methods. In a retrospective cohort study between April 2011 and February 2018. The data of the case histories of patients who underwent combined treatment for rectal cancer were analyzed. The study included patients who underwent neoadjuvant chemoradiotherapy followed by surgery. The influence of risk factors on the incidence of AN was assessed based on multivariate analysis using the logistic regression method.Results. The study was conducted in 474 patients who underwent anterior resection for rectal cancer. Depending on the outcome of the operation, the patients were divided into two groups: with the formation of a preventive intestinal stoma (main group, n = 344) and without formation (control group, n = 140) The overall incidence of postoperative complications was 20.6 % in patients with preventive stoma (PS) and 26.1 % without PS (p = 0.198). Colorectal anastomosis (RCA) leaks were detected in 40 (11.6 %) patients in the PS group and in 9 (6.9 %) patients in the non-PS group (р = 0,134). This complication was detected on the 7th day (2–12 days) after the operation in 5 (9.8 %) patients, while in patients with PS on the 10th day (11.6 %) and without PS on 3 (6.9 %) %), (p = 0.134). According to the classification of NCA severity, grade A was detected in 12 patients, B – at 24, C – in 11 patients. The frequency of reoperations in patients with PS was 10.8 %, while in patients with PS it was 15 % (p &lt;0.001). The most common reason for reoperations in patients with LCA in the group without PS was the development of peritonitis (5.4 %, p &lt;0.001). In a statistical multivariate analysis, the most significant risk factors for the development of NCA were: the presence of preoperative chemoradiotherapy (p = 0.001), diabetes mellitus (p = 0.031) and stage III–IV of the disease (p = 0.051).Conclusions. The formation of a preventive intestinal stoma after anterior resections does not reduce the incidence of colorectal anastomosis leaks (p = 0.134), however, it reduces the degree of manifestation of AN in the severity of the course of the postoperative postoperative period (p = 0.029). The frequency of reoperations in patients without a PS is significantly higher than in patients with a stoma (p &lt;0.0001). Diabetes mellitus, chemoradiotherapy, and stage III–IV of the disease are factors influencing the development of leakage.Цель исследования – изучить частоту и факторы риска развития несостоятельности колоректального анастомоза (НКА) после передней резекции прямой кишки по поводу рака.Материалы и методы. В ретроспективном когортном исследовании за период с апреля 2011 по февраль 2018 г. проанализированы данные историй болезни пациентов, которым проведено комбинированное лечение по поводу рака прямой кишки. В исследование были включены пациенты, которым проведена неоадъювантная химиолучевая терапия с последующим хирургическим вмешательством. Оценивали влияние факторов риска на частоту развития несостоятельности анастомоза на основании многофакторного анализа методом логистической регрессии.Результаты. Исследование было проведено у 474 пациентов, перенесших переднюю резекцию по поводу рака прямой кишки. В зависимости от исхода операции больные были разделены на 2 группы: с формированием кишечной превентивной стомы (ПС) (основная группа, n = 344) и без формирования ПС (контрольная группа, n = 140). Общая частота послеоперационных осложнения составила 20,6 % у больных с ПС и 26,1 % – без ПС (р = 0,198). НКА была выявлена у 40 (11,6 %) пациентов в группе с ПС и у 9 (6,9 %) – в группе без ПС (р = 0,134). Данное осложнение выявлялось на 7-е (медиана) сутки (2–12 сут) после операции у 5 (9,8 %) больных, при этом у больных с ПС (11,6 %) – на 10-е сутки и без ПС (6,9 %) на 3-и, (р = 0,134). По классификации тяжести НКА степень A выявлена у 12 больных, B – у 24, C – у 11 пациентов. Частота повторных операций у больных с ПС составила 10,8 %, в то время как у больных без ПС – 15 % (р &lt;0,001). Наиболее частой причиной повторных операций у больных с НКА в группе без ПС являлось развитие перитонита (5,4 %, р &lt;0,001). При многофакторном анализе наиболее значимыми факторами риска развития НКА являлись наличие предоперационной химиолучевой терапии (р = 0,001), сахарный диабет (р = 0,031), а также III–IV стадия заболевания (р = 0,051).Выводы. Формирование превентивной кишечной стомы после передних резекций не снижает частоту НКА (р = 0,134), однако снижает степень проявления НКА в послеоперационном периоде. Частота повторных операций у больных без ПС достоверно выше, чем у больных с наличием ПС. Сахарный диабет, проведенная химиолучевая терапия, а также III–IV стадия заболевания являются факторами, влияющими на развитие НКА

Molecular Mechanics of the α-Actinin Rod Domain: Bending, Torsional, and Extensional Behavior

α-Actinin is an actin crosslinking molecule that can serve as a scaffold and maintain dynamic actin filament networks. As a crosslinker in the stressed cytoskeleton, α-actinin can retain conformation, function, and strength. α-Actinin has an actin binding domain and a calmodulin homology domain separated by a long rod domain. Using molecular dynamics and normal mode analysis, we suggest that the α-actinin rod domain has flexible terminal regions which can twist and extend under mechanical stress, yet has a highly rigid interior region stabilized by aromatic packing within each spectrin repeat, by electrostatic interactions between the spectrin repeats, and by strong salt bridges between its two anti-parallel monomers. By exploring the natural vibrations of the α-actinin rod domain and by conducting bending molecular dynamics simulations we also predict that bending of the rod domain is possible with minimal force. We introduce computational methods for analyzing the torsional strain of molecules using rotating constraints. Molecular dynamics extension of the α-actinin rod is also performed, demonstrating transduction of the unfolding forces across salt bridges to the associated monomer of the α-actinin rod domain

Nucleosomes in gene regulation: theoretical approaches

This work reviews current theoretical approaches of biophysics and bioinformatics for the description of nucleosome arrangements in chromatin and transcription factor binding to nucleosomal organized DNA. The role of nucleosomes in gene regulation is discussed from molecular-mechanistic and biological point of view. In addition to classical problems of this field, actual questions of epigenetic regulation are discussed. The authors selected for discussion what seem to be the most interesting concepts and hypotheses. Mathematical approaches are described in a simplified language to attract attention to the most important directions of this field

Nucleosome conformational flexibility in experiments with single chromatin fibers

Studies on the chromatin nucleosome organization play an ever increasing role in our comprehension of mechanisms of the gene activity regulation. This minireview describes the results on the nucleosome conformational flexibility, which were obtained using magnetic tweezers to apply torsion to oligonucleosome fibers reconstituted on single DNA molecules. Such an approach revealed a new structural form of the nucleosome, the reversome, in which DNA is wrapped in a right-handed superhelix around a distorted histone octamer. Molecular mechanisms of the nucleosome structural flexibility and its biological relevance are discussed