Chromatin structure and DNase I hypersensitivity in the transcriptionally active and inactive porcine tumor necrosis factor gene locus

We have analyzed the chromatin structure of the porcine tumor necrosis factor gene locus (TNF-alpha and TNF-beta). Nuclei from porcine peripheral blood mononuclear cells were digested with different nucleases. As assessed with micrococcal nuclease, the two TNF genes displayed slightly faster digestion kinetics than bulk DNA. Studies with DNaseI revealed distinct DNaseI hypersensitive sites (DH-sites) within the porcine TNF locus. Four DH-sites could be observed in the promoter and mRNA leader regions of the TNF-beta gene. Two DH-sites could be observed for the TNF-alpha gene, one located in the promoter region close to the TATA-box and the other site in intron 3. This pattern of DH-sites was present independently of the activation state of the cells. Interestingly in a porcine macrophage-like cell line, we found that the TNF-alpha promoter DH-site disappeared and another DH-site appeared in the region of intron 1. Additionally, the DH-site of intron 3 could be enhanced by PMA-stimulation in these cells. TNF-beta sites were not detected in this cell line. However, DH-sites were totally absent in fibroblasts (freshly isolated from testicles) and in porcine kidney cells (PK15 cell line) both of which do not transcribe the TNF genes. Therefore, the pattern of DH-sites corresponds to the transcriptional activity of analyzed cells.

Warum Pentose- und nicht Hexose-Nucleinsäuren??. Teil V. (Purin-Purin)-Basenpaarung in der homo-DNS-Reihe: Guanin, Isoguanin, 2,6-Diaminopurin und Xanthin

Why Pentose- and Not Hexose-Nucleic Acids? Purine-Purine Pairing in homo-DNA: Guanine,Isoguanine, 2,6-Diaminopurine, and Xanthine This paper concludes the series of reports in this journal [1–4] on the chemistry of homo-DNA, the constitutionally simplifie dmodel system of hexopyranosyl-(6′ → 4′)-oligonucleotide systems stidued in our laboratory as potentially natural-nucleic-acid alternatives in the context of a chemical aetiology of nucleic-acid structure. The report describes the synthesis and pairing properties of homo-DNA oligonucleotides which contain as nucleobases exclusively purines, and gives, together with part III of the series [3], a survey of what we know today about purine-purine pairingin homo-DNA. In addition, the paper discusses those aspects of the chemistry of homo-DNA which, we think, influence the way how some of the structural features of DNA (and RNA) are to be interpreted on a qualitative level. Purine-purine pairing occurs in the homo-DNA domain in great variety. Most prominent is a novel tridentate Watson-Crick pair between guanine and isoguanine, as well as one between 2,6-diaminopurine and xanthinone, both giving rise to very stable duplexes containing the all-purine strands in antiparallel orientation. For the guanine-isoguanine pair, constitutional assignment is based on temperature-dependent UV and CD spectroscopy of various guanine- and isoguanine-containg duplexes in comparison with duplexes known to be paired in the reverse guanine is replaced by 7-carbauguanine. Isoguanine and 2,6-diaminopurine also have the capability of self-pariring in the reverse-Hoogsteen mode, as previously observed for adenine and guanine [3]. In this type of pairing, the interchangeably. Fig. 36 provides an overall survey of the relative strength of pairing in all possible purine-purine combinations. Watson-Crick pairing of isoguanine with guanine demands the former to participate in its 3H-tautomeric form; hitherto this specific tautomer had not been considered in the pairing chemistry of isoguanine. Whereas (cumulative) purine-purine pairing in DNA (reverse-Hoogsten or Hoogsteen) seems to occur in triplexes and tetrapalexes only, its occurrence in duplexes in a characteristic feature of homo-DNA chemistry. The occurrence of purine-purine Watson-Crick base pairs is probably a consequence of homo-DNA's quasi-linear ladder structure [1][4]. In a double helix, the distance between the two sugar C-atoms, on which a base pair is anchored, is expected to be constrained by the dimensions of the helix; in a linear duplex, however, there would be no restrictions with regard to base-pair length. Homo-DNA's ladder-like model also allows one to recognize one of the reasons why nucleic-acid duplexes prefer to pair in antiparallel, rather than parallel strand orientation: in homo-DNA duplexes, (averaged) backbone and base pair axes are strongly inclined toward one another [4]; the stronger this inclination, the higher the preference for antiparallel strand orientation is expected to be (Fig. 16). In retrospect, homo-DNA turns out to be one of the first artificial oligonucleotide systems (cf. Footnote 65) to demonstrate in a comprehensive way that informational base pairing involving purines and pyrimidines is not a capability unique to ribofuranosyl systems. Stability and helical shape of pairing complexes are not necessary conditions of one another; it is the potential for extensive conformational cooperativity of hte backbone structure with respect to the constellational demands of base pairing and base stacking that determines whether or nor a given type of base-carrying backbone structure is an informational pairing system. From the viewpoint of the chemical aetiology of nucleic-acid structure, which inspired our investigations on hexopyranosyl-(6′ → 4′)-oligonucleotide systems in the first place, the work on homo-DNA is only an extensive model study, because homo-DNA is not to be considered a potential natural-nucleic-acid altenratie. In retrospect, it seems fortunate that the model study was carried out, because without it we could hardly have comprehended the pairing behavior of the proper nucleic-acid alternatives which we have studied later and which will be discussed in Part VI of this series. The English footnotes to Fig. 1–49 provide an extension of this summary.

Modern techniques for the analysis of chromatin and nuclear organization in C. elegans.

In recent years, Caenorhabditis elegans has emerged as a new model to investigate the relationships between nuclear architecture, cellular differentiation, and organismal development. On one hand, C. elegans with its fixed lineage and transparent body is a great model organism to observe gene functions in vivo in specific cell types using microscopy. On the other hand, two different techniques have been applied in nematodes to identify binding sites for chromatin-associated proteins genome-wide: chromatin immunoprecipitation (ChIP), and Dam-mediated identification (DamID). We summarize here all three techniques together as they are complementary. We also highlight strengths and differences of the individual approaches.

Trypanosoma brucei RRM1 is a nuclear RNA-binding protein and modulator of chromatin structure

TbRRM1 of Trypanosoma brucei is a nucleoprotein that was previously identified in a search for splicing factors in T. brucei. We show that TbRRM1 associates with mRNAs and with the auxiliary splicing factor polypyrimidine tract-binding protein 2, but not with components of the core spliceosome. TbRRM1 also interacts with several retrotransposon hot spot (RHS) proteins and histones. RNA immunoprecipitation of a tagged form of TbRRM1 from procyclic (insect) form trypanosomes identified ca. 1,500 transcripts that were enriched and 3,000 transcripts that were underrepresented compared to cellular mRNA. Enriched transcripts encoded RNA-binding proteins, including TbRRM1 itself, several RHS transcripts, mRNAs with long coding regions, and a high proportion of stage-regulated mRNAs that are more highly expressed in bloodstream forms. Transcripts encoding ribosomal proteins, other factors involved in translation, and procyclic-specific transcripts were underrepresented. Knockdown of TbRRM1 by RNA interference caused widespread changes in mRNA abundance, but these changes did not correlate with the binding of the protein to transcripts, and most splice sites were unchanged, negating a general role for TbRRM1 in splice site selection. When changes in mRNA abundance were mapped across the genome, regions with many downregulated mRNAs were identified. Two regions were analyzed by chromatin immunoprecipitation, both of which exhibited increases in nucleosome occupancy upon TbRRM1 depletion. In addition, subjecting cells to heat shock resulted in translocation of TbRRM1 to the cytoplasm and compaction of chromatin, consistent with a second role for TbRRM1 in modulating chromatin structure. IMPORTANCE: Trypanosoma brucei, the parasite that causes human sleeping sickness, is transmitted by tsetse flies. The parasite progresses through different life cycle stages in its two hosts, altering its pattern of gene expression in the process. In trypanosomes, protein-coding genes are organized as polycistronic units that are processed into monocistronic mRNAs. Since genes in the same unit can be regulated independently of each other, it is believed that gene regulation is essentially posttranscriptional. In this study, we investigated the role of a nuclear RNA-binding protein, TbRRM1, in the insect stage of the parasite. We found that TbRRM1 binds nuclear mRNAs and also affects chromatin status. Reduction of nuclear TbRRM1 by RNA interference or heat shock resulted in chromatin compaction. We propose that TbRRM1 regulates RNA polymerase II-driven gene expression both cotranscriptionally, by facilitating transcription and efficient splicing, and posttranscriptionally, via its interaction with nuclear mRNAs.

Paramecium tetraurelia chromatin assembly factor-1-like protein PtCAF-1 is involved in RNA-mediated control of DNA elimination

Genome-wide DNA remodelling in the ciliate Paramecium is ensured by RNA-mediated trans-nuclear crosstalk between the germline and the somatic genomes during sexual development. The rearrangements include elimination of transposable elements, minisatellites and tens of thousands non-coding elements called internally eliminated sequences (IESs). The trans-nuclear genome comparison process employs a distinct class of germline small RNAs (scnRNAs) that are compared against the parental somatic genome to select the germline-specific subset of scnRNAs that subsequently target DNA elimination in the progeny genome. Only a handful of proteins involved in this process have been identified so far and the mechanism of DNA targeting is unknown. Here we describe chromatin assembly factor-1-like protein (PtCAF-1), which we show is required for the survival of sexual progeny and localizes first in the parental and later in the newly developing macronucleus. Gene silencing shows that PtCAF-1 is required for the elimination of transposable elements and a subset of IESs. PTCAF-1 depletion also impairs the selection of germline-specific scnRNAs during development. We identify specific histone modifications appearing during Paramecium development which are strongly reduced in PTCAF-1 depleted cells. Our results demonstrate the importance of PtCAF-1 for the epigenetic trans-nuclear cross-talk mechanism.

Growth Cone Localization of the mRNA Encoding the Chromatin Regulator HMGN5 Modulates Neurite Outgrowth

Neurons exploit local mRNA translation and retrograde transport of transcription factors to regulate gene expression in response to signaling events at distal neuronal ends. Whether epigenetic factors could also be involved in such regulation is not known. We report that the mRNA encoding the high-mobility group N5 (HMGN5) chromatin binding protein localizes to growth cones of both neuron-like cells and of hippocampal neurons, where it has the potential to be translated, and that HMGN5 can be retrogradely transported into the nucleus along neurites. Loss of HMGN5 function induces transcriptional changes and impairs neurite outgrowth, while HMGN5 overexpression induces neurite outgrowth and chromatin decompaction; these effects are dependent on growth cone localization of Hmgn5 mRNA. We suggest that the localization and local translation of transcripts coding for epigenetic factors couple the dynamic neuronal outgrowth process with chromatin regulation in the nucleus.

Na 50 Nachlass Arthur Schopenhauer - 'Schopenhauer-Archiv', 961 - Mitteilung über die Beschädigung und Reparatur des Bilderrahmens

u.a. Schopenhauer-Bild von Julius Hamel; Büste; Portrait; Schopenhauer-Denkmal; 100. Geburtstag; Artikel in der Deutschen Zeitung in Wien; Spenden für ein Denkmal;

Hypoxia-induced transcriptional regulation through chromatin modifications and NC2 function

In response to tumor hypoxia, specific genes that promote angiogenesis, proliferation, and survival are induced. Globally, I find that hypoxia induces a mixed pattern of histone modifications that are typically associated with either transcriptional activation or repression. Furthermore, I find that selective activation of hypoxia-inducible genes occurs simultaneously with widespread repression of transcription. I analyzed histone modifications at the core promoters of hypoxia-repressed and -activated genes and find that distinct patterns of histone modifications correlate with transcriptional activity. Additionally, I discovered that trimethylated H3-K4, a modification generally associated with transcriptional activation, is induced at both hypoxia-activated and repressed genes, suggesting a novel pattern of histone modifications induced during hypoxia. ^ In order to determine the mechanism of hypoxia-induced widespread repression of transcription, I focused my studies on negative cofactor 2 (NC2). Previously, we found that hypoxia-induced repression of the alpha-fetoprotein (AFP) gene occurs during preinitiation complex (PIC) assembly, and I find that NC2, an inhibitor of PIC assembly, is induced during hypoxia. Moreover, I find that the beta subunit of NC2 is essential for hypoxia-mediated repression of AFP, as well as the widespread repression of transcription observed during hypoxia. Previous data in Drosophila and S. cerevisiae indicate that NC2 functions as either an activator or a repressor of transcription. The mechanism of NC2-mediated activation remains unclear; although, Drosophila NC2 function correlates with specific core promoter elements. I tested if NC2 activates transcription in mammalian cells using this core promoter-specific model as a guide. Utilizing site-specific mutagenesis, I find that NC2 function in mammalian cells is not dependent upon specific core promoter elements; however, I do find that mammalian NC2 does function in a gene-specific manner as either an activator or repressor of transcription during hypoxia. Furthermore, I find that binding of the alpha subunit of NC2 specifically correlates with NC2-mediated transcriptional activation. NC2α and NC2β are both required for NC2-mediated transcriptional activation; whereas, NC2β alone is required for hypoxia-induced transcriptional repression. Together, these data indicate that hypoxia mediates changes in gene expression through both chromatin modifications and NC2 function. ^

The INO80 chromatin remodeling complex is involved in the nucleotide excision repair pathway

The genomic DNA of eukaryotic cells is well organized into chromatin structures. However, these repressed structures present barriers that block the access of regulatory factors to the genome during various nuclear events. To overcome the obstacle, two major cellular processes, post-modification of histone tails and ATP-dependent chromatin remodeling, are involved in reconfiguring chromatin structure and creating accessible DNA. Despite the current research progress, much remains to be explored concerning the relationship between chromatin remodeling and DNA repair. Recently, one member of the ATP-dependent chromatin remodeling complexes, INO80, has been found to play a crucial role in DNA damage repair. However, the functions of this complex in higher eukaryotes have yet to be determined. The goal of my study is to generate a human somatic INO80 conditional knockout model and investigate the functions of Ino80 in damage repair.^ By homologous targeting of the INO80 locus in human HCT116 colon epithelial cells, I established a human somatic INO80 conditional knockout model. I have demonstrated that the conditional INO80 cells exhibited a sufficiently viable period when the INO80 protein is removed. Moreover, I found that loss of INO80 resulted in deficient UV lesion repair in response to UV while the protein levels of the NER factors such as XPC, XPA, XPD were not affected. And in vitro repair synthesis assay showed that the NER incision and repair synthesis activities were intact in the absence of INO80. Examination on the damage recognition factor XPC showed its recruitment to damage sites was impaired in the INO80 mutant cells. Loss of INO80 also led to reduced enrichment of XPA at the site of UV lesions. Despite the reduced recruitment of XPC and XPA observed in INO80 mutants, no direct interaction was detected. Meanwhile, direct interaction between INO80 and DDB1, the initial UV lesion detector, was detected by coimmunoprecipitation. UV-induced chromosome relaxation was reduced in cells devoid of INO80. These results demonstrate the INO80 complex may participates in the NER by interacting with DDB1 and having a critical role of in creating DNA accessibility for the nucleotide excision pathway. ^

Regulation of chromatin structure, Smad binding and AFP expression by the Forkhead box transcription factor: Foxa1

Transcription factors must be able to access their DNA binding sites to either activate or repress transcription. However, DNA wrapping and compaction into chromatin occludes most binding sites from ready access by proteins. Pioneer transcription factors are capable of binding their DNA elements within a condensed chromatin context and then reducing the level of nucleosome occupancy so that the chromatin structure is more accessible. This altered accessibility increases the probability of other transcription factors binding to their own DNA binding elements. My hypothesis is that Foxa1, a ‘pioneer’ transcription factor, activates alpha-fetoprotein (AFP) expression by binding DNA in a chromatinized environment, reducing the nucleosome occupancy and facilitating binding of additional transcription factors.^ Using retinoic-acid differentiated mouse embryonic stem cells, we illustrate a mechanism for activation of the tumor marker AFP by the pioneer transcription factor Foxa1 and TGF-β downstream effector transcription factors Smad2 and Smad4. In differentiating embryonic stem cells, binding of the Foxa1 forkhead box transcription factor to chromatin reduces nucleosome occupancy and levels of linker histone H1 at the AFP distal promoter. The more accessible DNA is subsequently bound by the Smad2 and Smad4 transcription factors, concurrent with activation of transcription. Chromatin immunoprecipitation analyses combined with siRNA-mediated knockdown indicate that Smad protein binding and the reduction of nucleosome occupancy at the AFP distal promoter is dependent on Foxa1. In addition to facilitating transcription factor binding, Foxa1 is also associated with histone modifications related to active gene expression. Acetylation of lysine 9 on histone H3, a mark that is associated active transcription, is dependent on Foxa1, while methylation of H3K4, also associated with active transcription, is independent of Foxa1. I propose that Foxa1 potentiates a region of chromatin to respond to Smad proteins, leading to active expression of AFP.^ These studies demonstrate one mechanism whereby a transcription factor can alter the accessibility of additional transcription factors to chromatin, by altering nucleosome positions. Specifically, Foxa1 exposes DNA so that Smad4 can bind to its regulatory element and activate transcription of the tumor-marker gene AFP.^

Detección de intrusiones basada en análisis de eventos del DNS

En este proyecto se hace un análisis en profundidad de las técnicas de ataque a las redes de ordenadores conocidas como APTs (Advanced Persistent Threats), viendo cuál es el impacto que pueden llegar a tener en los equipos de una empresa y el posible robo de información y pérdida monetaria que puede llevar asociada. Para hacer esta introspección veremos qué técnicas utilizan los atacantes para introducir el malware en la red y también cómo dicho malware escala privilegios, obtiene información privilegiada y se mantiene oculto. Además, y cómo parte experimental de este proyecto se ha desarrollado una plataforma para la detección de malware de una red en base a las webs, URLs e IPs que visitan los nodos que la componen. Obtendremos esta visión gracias a la extracción de los logs y registros de DNS de consulta de la compañía, sobre los que realizaremos un análisis exhaustivo. Para poder inferir correctamente qué equipos están infectados o no se ha utilizado un algoritmo de desarrollo propio inspirado en la técnica Belief Propagation (“Propagación basada en creencia”) que ya ha sido usada antes por desarrolladores cómo los de los Álamos en Nuevo México (Estados Unidos) para fines similares a los que aquí se muestran. Además, para mejorar la velocidad de inferencia y el rendimiento del sistema se propone un algoritmo adaptado a la plataforma Hadoop de Apache, por lo que se modifica el paradigma de programación habitual y se busca un nuevo paradigma conocido como MapReduce que consiste en la división de la información en conceptos clave-valor. Por una parte, los algoritmos que existen basados en Belief Propagation para el descubrimiento de malware son propietarios y no han sido publicados completamente hasta la fecha, por otra parte, estos algoritmos aún no han sido adaptados a Hadoop ni a ningún modelo de programación distribuida aspecto que se abordará en este proyecto. No es propósito de este proyecto desarrollar una plataforma comercial o funcionalmente completa, sino estudiar el problema de las APTs y una implementación que demuestre que la plataforma mencionada es factible de implementar. Este proyecto abre, a su vez, un horizonte nuevo de investigación en el campo de la adaptación al modelo MapReduce de algoritmos del tipo Belief Propagation basados en la detección del malware mediante registros DNS. ABSTRACT. This project makes an in-depth investigation about problems related to APT in computer networks nowadays, seeing how much damage could they inflict on the hosts of a Company and how much monetary and information loss may they cause. In our investigation we will find what techniques are generally applied by attackers to inject malware into networks and how this malware escalates its privileges, extracts privileged information and stays hidden. As the main part of this Project, this paper shows how to develop and configure a platform that could detect malware from URLs and IPs visited by the hosts of the network. This information can be extracted from the logs and DNS query records of the Company, on which we will make an analysis in depth. A self-developed algorithm inspired on Belief Propagation technique has been used to infer which hosts are infected and which are not. This technique has been used before by developers of Los Alamos Lab (New Mexico, USA) for similar purposes. Moreover, this project proposes an algorithm adapted to Apache Hadoop Platform in order to improve the inference speed and system performance. This platform replaces the traditional coding paradigm by a new paradigm called MapReduce which splits and shares information among hosts and uses key-value tokens. On the one hand, existing algorithms based on Belief Propagation are part of owner software and they have not been published yet because they have been patented due to the huge economic benefits they could give. On the other hand these algorithms have neither been adapted to Hadoop nor to other distributed coding paradigms. This situation turn the challenge into a complicated problem and could lead to a dramatic increase of its installation difficulty on a client corporation. The purpose of this Project is to develop a complete and 100% functional brand platform. Herein, show a short summary of the APT problem will be presented and make an effort will be made to demonstrate the viability of an APT discovering platform. At the same time, this project opens up new horizons of investigation about adapting Belief Propagation algorithms to the MapReduce model and about malware detection with DNS records.