Peeling and Sliding in Nucleosome Repositioning

We investigate the mechanisms of histone sliding and detachment with a stochastic model that couples thermally-induced, passive histone sliding with active motor-driven histone unwrapping. Analysis of a passive loop or twist defect-mediated histone sliding mechanism shows that diffusional sliding is enhanced as larger portions of the DNA is peeled off the histone. The mean times to histone detachment and the mean distance traveled by the motor complex prior to histone detachment are computed as functions of the intrinsic speed of the motor. Fast motors preferentially induce detachment over sliding. However, for a fixed motor speed, increasing the histone-DNA affinity (and thereby decreasing the passive sliding rate) increases the mean distance traveled by the motor.Comment: 5 pp, 4 fig

Das Cystein-reiche LIM-Protein 4 (CRP4) beeinflusst patho-/physiologische Funktionen vaskulärer Glattmuskelzellen der Aorta

CRP-Proteine zählen zu den LIM-only Proteinen und sind in der Lage über LIM-Domänen Protein-Protein-Interaktionen zu vermitteln, sowie als Adapterproteine zu fungieren. Dies befähigt CRP-Proteine und mutmaßlich auch CRP4 verschiedene zelluläre Funktionen im vaskulären System, wie z.B. die Regulation der Zellmotilität, Genexpression und Differenzierung von vaskulären Glattmuskelzellen (VSMC), auszuüben. Gegenstand dieser Dissertation war sowohl die Aufklärung der putativen physiologischen, als auch pathophysiologischen Funktionen von vaskulärem CRP4. Dabei lag ein Fokus auf der Untersuchung der möglichen Bedeutung von CRP4, als vaskuläres Substrat der zyklischen Guanosin-3‘,5‘-monophosphat (cGMP)-abhängigen Proteinkinase Typ I (cGKI), für den Stickstoffmonoxid (NO)/cGMP/cGKI-Signalweg und cGMP-modulierte Zell-/Gefäßfunktionen. Im Rahmen dieser Dissertation konnte gezeigt werden, dass CRP4 sowohl die Proteinexpression bedeutsamer Faktoren des NO/cGMP/cGKI-Signalwegs in VSMCs reguliert, als auch die Phosphorylierungslevel cGKI-spezifischer Substratproteine beeinflusst. Darüber hinaus führte eine CRP4-Ablation zu einer reduzierten Ca2+-Sensitivität der Myofilamente in VSMCs und einer reduzierten maximalen Kontraktilität in CRP4 KO Aortenringen. Trotz einer verminderten cGKI-vermittelten Hemmung der [Ca2+]i-Transienten in VSMCs resultierte eine CRP4-Defizienz in einem erhöhten Relaxationsvermögen der Aortenringe und einer stärkeren Blutdruckabnahme in vivo als Antwort auf cGMP-erhöhende Substanzen. Zusammenfassend implizieren die Befunde, dass die Ablation von CRP4 zur Disinhibition des endogenen, vaskulären NO/cGMP/cGKI-Signalwegs führt. Des Weiteren wurde der Einfluss von CRP4 auf die Entwicklung einer Atherosklerose, bei der VSMCs eine große Rolle spielen, analysiert. Die in vivo Befunde dieser Dissertation zeigten einen pro-atherogenen Effekt für CRP4 im atherogenen ApoE KO Mausmodell mit einem höheren Migrationsvermögen kultivierter CRP4 WT VSMCs. In fortgeschrittenen Plaques beeinflusste CRP4 zudem die Stabilität der Plaques und führte zu einer höheren Oxidation von LDL, sowie einer erhöhten Anfälligkeit für Kalzifizierungen der VSMCs in CRP4 WT/ApoE KO Läsionen. Diese Effekte werden möglicherweise über eine Hemmung der Proteinexpression des anti-oxidativen Enzyms Peroxiredoxin 4 (PRDX4) in synthetischen VSMCs durch CRP4 vermittelt. Des Weiteren zeigte CRP4 eine anti-proliferative Wirkung auf synthetische VSMCs in vitro und innerhalb fortgeschrittener atherosklerotischer Plaques. Zusammenfassend resultieren die pro-atherogenen Effekte von CRP4 in einem milderen atherosklerotischen Phänotyp in ApoE KO CRP4-defizienten Mäusen

Effekte transkranieller Gleichstromstimulation des Präfrontalkortex auf Aufmerksamkeit, Reaktionsinhibition und kognitive Flexibilität

Exekutive Funktionen (EF) dienen dem Menschen dazu, auf Reize schnell und adäquat zu reagieren und sich so in seiner Umwelt zurecht zu finden. Der linke dorsolaterale Präfrontalkortex (dlPFC) bietet dafür eine der wesentlichen funktionell-neuroanatomische Grundlage. Mit der transkraniellen Gleichstromstimulation (tDCS) besteht die Möglichkeit, nichtinvasiv Einfluss auf die kortikale Erregbarkeit zu nehmen. Diese Technik konnte schon bei gestörten EF genutzt werden, um diese zu verbessern. Ziel dieser Studie war es nun, bei gesunden Probanden die EF Reaktionsinhibition, kognitive Flexibilität und Daueraufmerksamkeit durch tDCS zu optimieren. In einem doppelverblindeten randomisierten cross-over Design erhielten 50 gesunde Probanden eine sham und eine verum Stimulation im Abstand von zwei Tagen. Stimuliert wurde anodal mit 1mA für 20 min am linken dlPFC. Die Referenzelektrode wurde kontralateral supraorbital fixiert. Zur Testung der EF wurde der Parametric Go/No-Go (PGNG) Test verwendet, zudem wurde mithilfe eines Fragebogens (Positive and Negative Affect Schedule; PANAS) die momentane Stimmung vor und nach der Stimulation erhoben. In keinem der Zielparameter zeigten sich signifikante Unterschiede zwischen den Leistungen während verum- und sham-tDCS. Ferner wurde die Stimmung nicht von der Stimulation beeinflusst. Es sind keine gravierenden Nebenwirkungen aufgetreten und keiner der Probanden hat den Versuch abgebrochen. Die Studie kann die in anderen Studien gezeigte Wirkung der tDCS auf EF bei gesunden Probanden nicht bestätigen. Sie zeigt auf, wie wichtig es ist, weitere Parameter zu finden, die im Voraus Aufschluss auf die Effekte der Stimulation liefern können

DNA sequence encoded repression of rRNA gene transcription in chromatin

Eukaryotic genomes are packaged into nucleosomes that occlude DNA from interacting with most DNA-binding proteins. Nucleosome positioning and chromatin organization is critical for gene regulation. We have investigated the mechanism by which nucleosomes are positioned at the promoters of active and silent rRNA genes (rDNA). The reconstitution of nucleosomes on rDNA results in sequence-dependent nucleosome positioning at the rDNA promoter that mimics the chromatin structure of silent rRNA genes in vivo, suggesting that active mechanisms are required to reorganize chromatin structure upon gene activation. Nucleosomes are excluded from positions observed at active rRNA genes, resulting in transcriptional repression on chromatin. We suggest that the repressed state is the default chromatin organization of the rDNA and gene activation requires ATP-dependent chromatin remodelling activities that move the promoter-bound nucleosome about 22-bp upstream. We suggest that nucleosome remodelling precedes promoter-dependent transcriptional activation as specific inhibition of ATP-dependent chromatin remodelling suppresses the initiation of RNA Polymerase I transcription in vitro. Once initiated, RNA Polymerase I is capable of elongating through reconstituted chromatin without apparent displacement of the nucleosomes. The results reveal the functional cooperation of DNA sequence and chromatin remodelling complexes in nucleosome positioning and in establishing the epigenetic active or silent state of rRNA genes

Effect of Mercury on Membrane Proteins, Anionic Transport and Cell Morphology in Human Erythrocytes.

Mercury (Hg) is a heavy metal widespread in all environmental compartments as one of the most hazardous pollutants. Human exposure to this natural element is detrimental for several cellular types including erythrocytes (RBC) that accumulate Hg mainly bound to the SH groups of different cellular components, including protein cysteine residues. The cellular membrane represents a major target of Hg-induced damage in RBC with loss of physiological phospholipid asymmetry, due to phosphatidylserine (PS) exposure to the external membrane leaflet. To investigate Hg-induced cytotoxicity at the molecular level, the possible interaction of this heavy metal with RBC membrane proteins was investigated. Furthermore, Hg-induced alterations in band 3 protein (B3p) transport function, PS-exposing macrovesicle (MVs) formation and morphological changes were assessed. For this aim, human RBC were treated in vitro with different HgCl <sub>2</sub> concentrations (range 10-40 µM) and the electrophoretic profile of membrane proteins as well as the expression levels of Ankyrin and Flottilin-2 evaluated by SDS-PAGE and Western blot, respectively. The effect of alterations in these proteins on RBC morphology was evaluated by digital holographic microscopy and anionic transport efficiency of B3p was evaluated as sulphate uptake. Finally, PS- bearing MVs were quantified by annexin-V binding using FACS analysis. Findings presented in this paper indicate that RBC exposure to HgCl <sub>2</sub> induces modifications in the electrophoretic profile of membrane protein fraction. Furthermore, our study reveals the Hg induced alterations of specific membrane proteins, such as Ankyrin, a protein essential for membrane-cytoskeleton linkage and Flotillin-2, a major integral protein of RBC lipid rafts, likely responsible for decreased membrane stability and increased fragmentations. Accordingly, under the same experimental conditions, RBC morphological changes and PS-bearing MVs release are observed. Finally, RBC treatment significantly affects the B3p-mediated anionic transport, that we report reduced upon HgCl <sub>2</sub> treatment in a dose dependent manner. Altogether, the findings reported in this paper confirm that RBC are particularly vulnerable to Hg toxic effect and provide new insight in the Hg-induced protein modification in human RBC affecting the complex biological system of cellular membrane. In particular, Hg could induce dismantle of vertical cohesion between the plasma membrane and cytoskeleton as well as destabilization of lateral linkages of functional domains. Consequently, decreased membrane deformability could impair RBC capacity to deal with the shear forces in the circulation increasing membrane fragmentations. Furthermore, findings described in this paper have also significant implication in RBC physiology, particularly related to gas exchanges

Footprint traversal by ATP-dependent chromatin remodeler motor

ATP-dependent chromatin remodeling enzymes (CRE) are bio-molecular motors in eukaryotic cells. These are driven by a chemical fuel, namely, adenosine triphosphate (ATP). CREs actively participate in many cellular processes that require accessibility of specific segments of DNA which are packaged as chromatin. The basic unit of chromatin is a nucleosome where 146 bp $\sim$ 50 nm of a double stranded DNA (dsDNA) is wrapped around a spool formed by histone proteins. The helical path of histone-DNA contact on a nucleosome is also called "footprint". We investigate the mechanism of footprint traversal by a CRE that translocates along the dsDNA. Our two-state model of a CRE captures effectively two distinct chemical (or conformational) states in the mechano-chemical cycle of each ATP-dependent CRE. We calculate the mean time of traversal. Our predictions on the ATP-dependence of the mean traversal time can be tested by carrying out {\it in-vitro} experiments on mono-nucleosomes.Comment: 11 pages, 12 figures; minor revision of tex

Purine– and pyrimidine–triple-helix-forming oligonucleotides recognize qualitatively different target sites at the ribosomal DNA locus

Triplexes are noncanonical DNA structures, which are functionally associated with regulation of gene expression through ncRNA targeting to chromatin. Based on the rules of Hoogsteen base-pairing, polypurine sequences of a duplex can potentially form triplex structures with single-stranded oligonucleotides. Prediction of triplex-forming sequences by bioinformatics analyses have revealed enrichment of potential triplex targeting sites (TTS) at regulatory elements, mainly in promoters and enhancers, suggesting a potential function of RNA – DNA triplexes in transcriptional regulation. Here, we have quantitatively evaluated the potential of different sequences of human and mouse ribosomal RNA genes (rDNA) to form triplexes at different salt and pH conditions. We show by biochemical and biophysical approaches that some of these predicted sequences form triplexes with high affinity, following the canonical rules for triplex formation. We further show that RNA triplex-forming oligos (TFOs) are more stable than their DNA counterpart, and point mutations strongly affect triplex formation. We further show differential sequence requirements of pyrimidine and purine TFO sequences for efficient binding, depending on the G–C content of the TTS. The unexpected sequence specificity, revealing distinct sequence requirements for purine and pyrimidine TFOs, shows that in addition to the Hoogsteen pairing rules, a sequence code and mutations have to be taken into account to predict genomic TTS

DNMT1 but not its interaction with the replication machinery is required for maintenance of DNA methylation in human cells

DNA methylation plays a central role in the epigenetic regulation of gene expression in vertebrates. Genetic and biochemical data indicated that DNA methyltransferase 1 (Dnmt1) is indispensable for the maintenance of DNA methylation patterns in mice, but targeting of the DNMT1 locus in human HCT116 tumor cells had only minor effects on genomic methylation and cell viability. In this study, we identified an alternative splicing in these cells that bypasses the disrupting selective marker and results in a catalytically active DNMT1 protein lacking the proliferating cell nuclear antigen–binding domain required for association with the replication machinery. Using a mechanism-based trapping assay, we show that this truncated DNMT1 protein displays only twofold reduced postreplicative DNA methylation maintenance activity in vivo. RNA interference–mediated knockdown of this truncated DNMT1 results in global genomic hypomethylation and cell death. These results indicate that DNMT1 is essential in mouse and human cells, but direct coupling of the replication of genetic and epigenetic information is not strictly required

Nucleosomes protect DNA from DNA methylation in vivo and in vitro

Positioned nucleosomes limit the access of proteins to DNA. However, the impact of nucleosomes on DNA methylation in vitro and in vivo is poorly understood. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the de novo methyltransferases. We show that compared to linker DNA, nucleosomal DNA is largely devoid of CpG methylation. ATP-dependent chromatin remodelling frees nucleosomal CpG dinucleotides and renders the remodelled nucleosome a 2-fold better substrate for Dnmt3a methyltransferase compared to free DNA. These results reflect the situation in vivo, as quantification of nucleosomal DNA methylation levels in HeLa cells shows a 2-fold decrease of nucleosomal DNA methylation levels compared to linker DNA. Our findings suggest that nucleosomal positions are stably maintained in vivo and nucleosomal occupancy is a major determinant of global DNA methylation patterns in vivo

Nucleosome repositioning via loop formation

Active (catalysed) and passive (intrinsic) nucleosome repositioning is known to be a crucial event during the transcriptional activation of certain eucaryotic genes. Here we consider theoretically the intrinsic mechanism and study in detail the energetics and dynamics of DNA-loop-mediated nucleosome repositioning, as previously proposed by Schiessel et al. (H. Schiessel, J. Widom, R. F. Bruinsma, and W. M. Gelbart. 2001. {\it Phys. Rev. Lett.} 86:4414-4417). The surprising outcome of the present study is the inherent nonlocality of nucleosome motion within this model -- being a direct physical consequence of the loop mechanism. On long enough DNA templates the longer jumps dominate over the previously predicted local motion, a fact that contrasts simple diffusive mechanisms considered before. The possible experimental outcome resulting from the considered mechanism is predicted, discussed and compared to existing experimental findings