Functional Chromatin Extraction: A method to study DNA accessibility in higher-order structures of chromatin

Inside the cell nucleus DNA is compacted through the assembly with histones and other proteins into chromatin. The first layer of DNA packaging is the nucleosome core particle, which consists of DNA wrapped around a histone octamer. Nucleosome core particles are spaced by linker DNA forming a ”beads-on-a-string” structure. According to the textbook model nucleosome arrays are further regularly folded into distinct higher-order structures of chromatin. Since all DNA dependent processes require access to the DNA template, chromatin organization and folding into higher-order structures is thought to regulate genome activity. This thesis investigates how chromatin is structurally organized inside the nucleus to modulate DNA accessibility. A high-throughput approach, called Functional Chromatin Extraction, was developed to analyze DNA accessibility in native chromatin. Therefore, chromatin was digested with different intensities directly inside the nucleus of living cells using the endonuclease MNase. DNA accessibility was assessed on both global and local scale by the differential release rates of nucleosomes from partially (low-MNase) and fully digested chromatin (high-MNase). Thorough analysis of the extracted nucleosomal DNA revealed, that AT rich nucleosomes are prone to over-degradation to sub-nucleosomal fragments in high-MNase. Therefore, nucleosomes of GC rich regions are overrepresented in high-MNase. In contrast, low-MNase results in a homogenous nucleosome distribution not affected by the DNA sequence, thereby obtaining an accurate representation of the global nucleosome landscape. Surprisingly, after correcting for the sequence preferences of MNase, differentially accessible chromatin domains could not be identified. Euchromatin and heterochromatin exhibit similar accessibilities, suggesting that DNA in heterochromatin is in general available for small molecules, like transcription factors. Nevertheless, active regulatory sites, such as promoter and enhancer elements, reveal increased accessibility compared to other regions of the genome and are occupied by fragile nucleosomes showing, that DNA accessibility is modulated locally to regulate gene expression. In summary, the results of this study indicate, that chromatin forms an accessible and dynamic polymer and domains of higher-order structures of chromatin do not exist in human cells. In a second chapter, this thesis focuses on the chromatin architecture of Adenoviruses and dynamic changes during early infection. Similarly to eukaryotic genomes, adenoviral DNA in incoming virions is mainly associated with the structural protein VII (pVII) forming a nucleoprotein complex. However, little is known about the adenoviral chromatin organization and how it relates to viral gene activation during infection. Functional Chromatin Extraction combined with transcriptome sequencing was applied during early infection of human cells. The viral DNA organization into pVII complexes was assessed, showing a defined and functional DNA packaging into nucleosome-like arrays. The chromatin structure of invading viruses correlates with the spatiotemporal activation of viral genes showing an open chromatin conformation with lower pVII densities at early gene loci. Investigation of dynamic chromatin changes within the first four hours of infection, revealed viral chromatin de-condensation and nucleosome assembly preferentially at early gene loci. Remarkably, nucleosomes replace pVII molecules directly at the +1 site of early genes thereby resembling the structure of active host promoter. The time resolved analysis demonstrated, that remodeling of the viral chromatin precedes transcriptional activation and is a prerequisite to generate a transcription competent template

A radial analogue of Poisson's summation formula with applications to powder diffraction and pinwheel patterns

Diffraction images with continuous rotation symmetry arise from amorphous systems, but also from regular crystals when investigated by powder diffraction. On the theoretical side, pinwheel patterns and their higher dimensional generalisations display such symmetries as well, in spite of being perfectly ordered. We present first steps and results towards a general frame to investigate such systems, with emphasis on statistical properties that are helpful to understand and compare the diffraction images. An alternative substitution rule for the pinwheel tiling, with two different prototiles, permits the derivation of several combinatorial and spectral properties of this still somewhat enigmatic example. These results are compared with properties of the square lattice and its powder diffraction.Comment: 16 pages, 8 figure

The origin and maintenance of mammalian peroxisomes involves a de novo PEX16-dependent pathway from the ER

Peroxisomes are ubiquitous organelles that proliferate under different physiological conditions and can form de novo in cells that lack them. The endoplasmic reticulum (ER) has been shown to be the source of peroxisomes in yeast and plant cells. It remains unclear, however, whether the ER has a similar role in mammalian cells and whether peroxisome division or outgrowth from the ER maintains peroxisomes in growing cells. We use a new in cellula pulse-chase imaging protocol with photoactivatable GFP to investigate the mechanism underlying the biogenesis of mammalian peroxisomes. We provide direct evidence that peroxisomes can arise de novo from the ER in both normal and peroxisome-less mutant cells. We further show that PEX16 regulates this process by being cotranslationally inserted into the ER and serving to recruit other peroxisomal membrane proteins to membranes. Finally, we demonstrate that the increase in peroxisome number in growing wild-type cells results primarily from new peroxisomes derived from the ER rather than by division of preexisting peroxisomes

CONTRAST-ENHANCED ULTRASOUND MONITORING OF PERFUSION CHANGES IN HEPATIC NEUROENDOCRINE METASTASES AFTER SYSTEMIC VERSUS SELECTIVE ARTERIAL 177LU/90Y-DOTATOC AND 213BI-DOTATOC RADIOPEPTIDE THERAPY

Radiopeptide therapy with beta emitter labeled 177Lu/90Y- DOTA(0)-Phe(1)-Tyr(3)-octreotide (DOTATOC) and more recently also alpha emitting 213Bi-DOTATOC are promising new treatments for neuroendocrine tumors. No early predictors for treatment response have been recognized and tumor-shrinkage after radiation therapy appears slowly. In some solid tumors a decline in tumor perfusion was found predictive of final treatment response but the gold standard multiphase computed tomography (CT) has a high radiation burden. Therefore we evaluated the ability of contrast-enhanced ultrasound (CEUS) to evaluate tumor perfusion as a response criteria. Materials and Methods: 14 patients with hepatic neuroendocrine tumor (NET) metastases were enrolled in the retrospective study. Eleven patients were treated with beta-emitting 177Lu/90Y-DOTATOC, either intravenous (i.v.) (n = 5) or intra-arterial (i.a.) (n = 6) and three patients received alpha-emitting 213Bi-DOTATOC (i.a.). CEUS and contrast-enhanced CT (CE-CT) were performed before and 3 months after treatment. Results: CE-CT and CEUS presented comparable results in the baseline study and in the assessment of perfusion changes due to the different treatment regimes. A therapy related decrease in tumor perfusion is an early predictor of longterm morphologic response. Conclusion: CEUS is a cheap, ubiquitary available and radiation free technique which showed comparable results for perfusion and diameter of liver metastases compared to CE-CT. Intensity reduction in an arterial phase CEUS can be seen as a positive sign indicating long term tumor response to treatment. Therefore CEUS may be considered as an imaging modality for monitoring early treatment after focal alpha and beta targeted therapy.JRC.E.5-Nuclear chemistr

Different elongation factors distinctly modulate RNA polymerase II transcription in Arabidopsis

Various transcript elongation factors (TEFs) including modulators of RNA polymerase II (RNAPII) activity and histone chaperones tune the efficiency of transcription in the chromatin context. TEFs are involved in establishing gene expression patterns during growth and development in Arabidopsis, while little is known about the genomic distribution of the TEFs and the way they facilitate transcription. We have mapped the genome-wide occupancy of the elongation factors SPT4–SPT5, PAF1C and FACT, relative to that of elongating RNAPII phosphorylated at residues S2/S5 within the carboxyterminal domain. The distribution of SPT4–SPT5 along transcribed regions closely resembles that of RNAPII-S2P, while the occupancy of FACT and PAF1C is rather related to that of RNAPII-S5P. Under transcriptionally challenging heat stress conditions, mutant plants lacking the corresponding TEFs are differentially impaired in transcript synthesis. Strikingly, in plants deficient in PAF1C, defects in transcription across intron/exon borders are observed that are cumulative along transcribed regions. Upstream of transcriptional start sites, the presence of FACT correlates with nucleosomal occupancy. Under stress conditions FACT is particularly required for transcriptional upregulation and to promote RNAPII transcription through +1 nucleosomes. Thus, Arabidopsis TEFs are differently distributed along transcribed regions, and are distinctly required during transcript elongation especially upon transcriptional reprogramming

Distinct role of subunits of the Arabidopsis RNA polymerase II elongation factor PAF1C in transcriptional reprogramming

Transcript elongation by RNA polymerase II (RNAPII) is dynamic and highly regulated, thereby contributing to the implementation of gene expression programs during plant development or in response to environmental cues. The heterohexameric polymerase-associated factor 1 complex (PAF1C) stabilizes the RNAPII elongation complex promoting efficient transcript synthesis. In addition, PAF1C links transcriptional elongation with various post-translational histone modifications at transcribed loci. We have exposed Arabidopsis mutants deficient in the PAF1C subunits ELF7 or CDC73 to elevated NaCl concentrations to provoke a transcriptional response. The growth of elf7 plants was reduced relative to that of wildtype under these challenging conditions, whereas cdc73 plants exhibited rather enhanced tolerance. Profiling of the transcriptional changes upon NaCl exposure revealed that cdc73 responded similar to wildtype. Relative to wildtype and cdc73, the transcriptional response of elf7 plants was severely reduced in accord with their greater susceptibility to NaCl. The data also imply that CDC73 is more relevant for the transcription of longer genes. Despite the fact that both ELF7 and CDC73 are part of PAF1C the strikingly different transcriptional response of the mutants upon NaCl exposure suggests that the subunits have (partially) specific functions

The ‘dark side’ of personal values: Relations to clinical constructs and their implications

Personal values are considered as guiding principles in one's life. Much of previous research on values has consequently focused on its relations with variables that are considered positive, including subjective well-being, personality traits, or behavior (e.g. health-related). However, in this study (N = 366) the negative ‘dark’ side of values is examined. Specifically, the study investigated the relations between Schwartz' (1992) ten value types and four different clinical variables — anxiety, depression, stress, and schizotypy with its subdimensions, unusual experience, cognitive disorganization, introverted anhedonia, and impulsive nonconformity. Positive relations between achievement and depression and stress, and negative relations between anxiety and hedonism and stimulation were predicted and found. Multiple regressions revealed that the ten value types explained the most variance in impulsive nonconformity and the least variance in unusual experience. Overall, values were better in predicting more cognitive clinical variables (e.g., cognitive disorganization) whereas clinical constructs were better in predicted more affective values (e.g., hedonism). Implications of the findings for value research are discussed

Functional involvement of septal miR-132 in extinction and oxytocin-mediated reversal of social fear

Social interactions are critical for mammalian survival and evolution. Dysregulation of social behavior often leads to psychopathologies such as social anxiety disorder, denoted by intense fear and avoidance of social situations. Using the social fear conditioning (SFC) paradigm, we analyzed expression levels of miR-132-3p and miR-124-3p within the septum, a brain region essential for social preference and avoidance behavior, after acquisition and extinction of social fear. Here, we found that SFC dynamically altered both microRNAs. Functional in vivo approaches using pharmacological strategies, inhibition of miR-132-3p, viral overexpression of miR-132-3p, and shRNA-mediated knockdown of miR-132-3p specifically within oxytocin receptor-positive neurons confirmed septal miR-132-3p to be critically involved not only in social fear extinction, but also in oxytocin-induced reversal of social fear. Moreover, Argonaute-RNA-co-immunoprecipitation-microarray analysis and further in vitro and in vivo quantification of target mRNA and protein, revealed growth differentiation factor-5 (Gdf-5) as a target of miR-132-3p. Septal application of GDF-5 impaired social fear extinction suggesting its functional involvement in the reversal of social fear. In summary, we show that septal miR-132-3p and its downstream target Gdf-5 regulate social fear expression and potentially mediate oxytocin-induced reversal of social fear