Abstract (English)
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 ...
Abstract (English)
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.
Translation of the abstract (German)
In der Zelle liegt DNA verpackt mit Proteine vor. Der Komplex aus DNA und Proteinen wird als Chromatin bezeichnet. Der erste Verpackungsgrad der DNA ist das Nukleosom, das aus einem Histonoktamer besteht, um den sich die DNA wickelt. Die Nuklesomenfaser ist wie eine "Perlenkette" geformt. Gemäß den Lehrbuchmodellen wird die Nukleosomenfaser weiter zu regulären Chromatinstrukturen höherer Ordnung ...
Translation of the abstract (German)
In der Zelle liegt DNA verpackt mit Proteine vor. Der Komplex aus DNA und Proteinen wird als Chromatin bezeichnet. Der erste Verpackungsgrad der DNA ist das Nukleosom, das aus einem Histonoktamer besteht, um den sich die DNA wickelt. Die Nuklesomenfaser ist wie eine "Perlenkette" geformt. Gemäß den Lehrbuchmodellen wird die Nukleosomenfaser weiter zu regulären Chromatinstrukturen höherer Ordnung gefaltet. Da alle DNA abhängigen Prozesse Zugang zur DNA benötigen, wird vermutet, dass die Chromatinorganisation und -verpackung genomische Prozesse beeinflusst und reguliert. In dieser Arbeit wird untersucht welchen Einfluss die strukturelle Verpackung von Chromatin auf die DNA Zugänglichkeit hat. Dazu wurde eine Hochdurchsatzmethode "Funktionelle Chromatin Extraktion" entwickelt um die DNA Zugänglichkeit unter nativen Bedingungen zu untersuchen. Hierbei wird ein partieller (low-MNase) mit einem starken (high-MNase) Chromatinverdau verglichen. Überraschenderweise, konnten keine Unterschiede in der DNA Zugänglichkeit in Euchromatin und Heterochromatin festgestellt werden. Vielmehr wird die DNA-Zugägnlichkeit lokal auf gezielten regulatorischen Bereichen, wie Enhancer-Elemente oder Promotoren, moduliert.
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