Local and global analysis of the structure-function relation in chromatin

Every cell in the human body contains the identical hereditary information, encoded in the DNA. The sequence of the human genome was deciphered one decade ago as part of the Human Genome Project (International Human Genome Sequencing Consortium, 2001, 2004; Venter et al., 2001). Remarkably, knowledge of the sequence could not explain the spatial and temporal differences in gene expression, which result in the various tissues composing a human body. It becomes more and more evident that several epigenetic pathways are required to regulate transcription in a time- and context-dependent manner. This thesis investigates one particular mechanism in detail, the functional effect of chromatin architecture on gene regulation. In eukaryotes, DNA is compacted into chromatin, with nucleosomes being the basic packaging unit. In general, chromatin can be classified into two major states, the transcriptionally active euchromatin and the repressed heterochromatin (Woodcock and Ghosh, 2010). Gene expression is closely linked to the structural and spatial arrangement of the chromatin domains in the nucleus, with silent genomic loci tethered to the nuclear periphery and active regions located in the center (Takizawa et al., 2008). To allow access of DNA-binding proteins involved in transcription, replication or repair, the nucleoprotein complex is capable of dynamically changing its accessibility upon physiological cues. Chromatin dynamics are modulated by a number of different factors, e.g. ATP-dependent chromatin remodeling machines, histone chaperones and histone-modifying enzymes (Margueron and Reinberg, 2010). This study aimed to elucidate the structure-function relation of chromatin in mammalian cells on both a global and a local scale. First, chromatin dynamics were analyzed on a genome-wide level. Several high-throughput assays were developed, including formalde-hyde-assisted isolation of regulatory elements (FAIRE)-Seq to monitor the tissue-specific usage of regulatory elements in human blood cells. In addition, micrococcal nuclease (MNase)-Seq was applied to study global re-positioning of nucleosomes and to identify functionally different chromatin domains according to their accessibility. High-throughput sequencing of mono- and dinucleosomal DNA from HeLa cells reveals a condensed chromatin structure at promoters of repressed genes and a defined, open structure around the transcription start sites (TSSs) of highly expressed genes. Further characterization of the determined chromatin domains uncovers correlation of very accessible, open structures with increased occupancy of RNA polymerase (RNAP) II, CTCF and activating histone marks. In contrast, less accessible chromatin is associated with low gene density and expression. Remarkably, the two states differ in nucleosome spacing, directly reflecting accessibility and the respective higher-order organization. Furthermore, MNase-Seq of primary human cells treated with the proinflammatory cytokine TNFα reveals genome-wide re-positioning of nucleosomes. Strikingly, chromatin structures around responsive TSSs but also throughout the gene body open up gradually, as monitored at 10 and 30 min after stimulation. In addition, defined nucleosome positioning and a distinct histone PTM signature are observed at exon/intron junctions, in particular around novel recursive splicing sites. The global opening precedes transcription and is most pronounced at ‘hotspots’ carrying binding sites for NF-κB and associated proteins. Exposure of the binding sites is probably achieved by TNFα-specific chromatin remodeling and occurs predominantly at promoter regions, exons and Alu repeats. Finally, this thesis links altered chromatin structures in Down syndrome patients to upregulated expression of histone clusters and chromatin-associated proteins. In a second chapter, this thesis focuses on the local chromatin architecture of ribosomal RNA (rRNA) genes in mouse. Previous studies showed that actively transcribed or poised rRNA genes form promoter-terminator chromatin loops (Németh et al., 2008). Here, a hamster cell line harboring one repeat of mouse rDNA is introduced as a new model system to elucidate functional dynamics in chromatin looping on a single gene scale. Both promoter and terminator comprise binding sites for the Transcription Termination Factor for RNA polymerase I (TTF-I), therefore, the impact of this protein on chromatin looping is investigated in detail. Notably, TTF-I binds cooperatively to the target site cluster at the rRNA gene terminator in a chromatin-dependent manner. Furthermore, the cluster enhances gene expression directly by increasing the loading of RNAPI and transcription factors to the gene. In addition, the results indicate that TTF-I binding is indispensible for establishing the chromatin loop by mediating promoter-terminator interactions. According to the ribomotor model, the loop structure promotes efficient recycling of polymerases and associated proteins, explaining the observed effects in factor occupancy and gene expression. In summary, the chromatinized TTF-I binding site cluster at the rRNA gene terminator acts as an enhancer element by physically interacting with the promoter, up-regulating transcription levels by transcription factor recruitment and exhibiting a defined chromatin signature linked to active regulatory elements