There is a genome in a cell, as everyone knows, but there is also an epigenome. The epigenome regulates the transcription of the underlying genome. In the last decade, it was discovered that the epigenome state and its regulation are important for differentiation and development. Correlation studies with aging samples had led to the hypothesis that misregulation of the epigenome causes aging and cancer. Furthermore, diseases were identified which are caused by errors in the epigenome state and its regulation. Identification of erroneous epigenome states and misregulation requires the prior knowledge of the common state. Several studies aim at measuring epigenome states in different organisms and cell types and thus, provide a huge amount of data. In this dissertation, a pipeline is developed to analyze and characterize histone modifications with respect to different cell types. Application of this pipeline is shown for a published data set of mouse consisting of data for H3K4me3, H3K27me3, and H3K9me3 measured in embryonic stem cells, embryonic fibroblasts and neuronal progenitors. Furthermore, methods for the detection of the epigenetic patterns are presented in this dissertation. Therefore, a segmentation method is developed to segment the genome guided by the data sets. Based on this segmentation, the epigenome states as well as epigenetic variation can be studied. Different visualization methods are developed to highlight the epigenetic patterns in the segmentation data. Application of the segmentation AND visualization methods to the mouse data set had resulted in not only colorful squares but also in biological conclusions! It demonstrate the power of the developed methods. Although the studied data set in this dissertation contains only ordinary tissue cells, the methods are not restricted to study the reference epigenome state. Comparison of normal and disease cells as well as comparison with aged cells are possible with all of the methods. Finally, the methods are compared based on the obtained results. It shows that all methods highlight different aspects of the data. Thus, applying all methods to the same data sets, deep insights into the epigenome in murine embryonic stem cells, embryonic fibroblasts and neuronal progenitor cells are gained. For example, it had been found that several mechanisms exist setting H3K4me3 marks. Furthermore, not all mechanisms are found in all cell types. Strong evidence had been found that catalysis of H3K4me3 and H3K27me3 is coupled.

There is a genome in a cell, as everyone knows, but there is also an epigenome. The epigenome regulates the transcription of the underlying genome. In the last decade, it was discovered that the epigenome state and its regulation are important for differentiation and development. Correlation studies with aging samples had led to the hypothesis that misregulation of the epigenome causes aging and cancer. Furthermore, diseases were identified which are caused by errors in the epigenome state and its regulation. Identification of erroneous epigenome states and misregulation requires the prior knowledge of the common state. Several studies aim at measuring epigenome states in different organisms and cell types and thus, provide a huge amount of data. In this dissertation, a pipeline is developed to analyze and characterize histone modifications with respect to different cell types. Application of this pipeline is shown for a published data set of mouse consisting of data for H3K4me3, H3K27me3, and H3K9me3 measured in embryonic stem cells, embryonic fibroblasts and neuronal progenitors. Furthermore, methods for the detection of the epigenetic patterns are presented in this dissertation. Therefore, a segmentation method is developed to segment the genome guided by the data sets. Based on this segmentation, the epigenome states as well as epigenetic variation can be studied. Different visualization methods are developed to highlight the epigenetic patterns in the segmentation data. Application of the segmentation AND visualization methods to the mouse data set had resulted in not only colorful squares but also in biological conclusions! It demonstrate the power of the developed methods. Although the studied data set in this dissertation contains only ordinary tissue cells, the methods are not restricted to study the reference epigenome state. Comparison of normal and disease cells as well as comparison with aged cells are possible with all of the methods. Finally, the methods are compared based on the obtained results. It shows that all methods highlight different aspects of the data. Thus, applying all methods to the same data sets, deep insights into the epigenome in murine embryonic stem cells, embryonic fibroblasts and neuronal progenitor cells are gained. For example, it had been found that several mechanisms exist setting H3K4me3 marks. Furthermore, not all mechanisms are found in all cell types. Strong evidence had been found that catalysis of H3K4me3 and H3K27me3 is coupled.

Endotoxin-tolerante Zellen zeigen über mehrere Tage eine verminderte Produktion pro-inflammatorischer Zytokine, sodass epigenetische Veränderungen ein Grund für die Endotoxintoleranz sein könnte. Im 1. Teil wurden epigenetische Veränderungen an gezielten LPS-tolerisierbaren Genen mithilfe eines in-vitro-Modells mit humanen Monozyten untersucht. Die Gene kodierend für TNF und CXCL10 zeigten eine Reduktion der transkriptionsaktivierenden Histonmarker H3K27ac und H4ac, die durch eine stark reduzierte Genexpression in toleranten Monozyten begleitet wurde. Demgegenüber wiesen Gene wie IL6 und IL1B eine Zunahme an H4ac und H3K27ac auf, während ihre Genexpression in widersprüchlicher Weise reduziert war. Repressive epigenetische Marker (H3K9me2, H3K27me3, H4K20me3, DNA-Methylierung) konnten in den untersuchten Genen nicht nachgewiesen werden. Zudem war die IL6-Genexpression verstärkt abhängig von der Signaltransduktion toleranter Monozyten, was auf unterschiedliche Repressionsmechanismen schließen lässt. Im 2. Teil konnte gezeigt werden, dass die genomweite transkriptionelle Reprogrammierung durch eine globale Verschiebung von aktiven H3K27ac und H4ac in naiven Monozyten zu repressiven H3K9me2, H3K27me3 und H4K20me3 in toleranten, restimulierten Zellen einherging. Mehr als 10000 Genombereiche wiesen Veränderungen an Histonmarkern auf, obwohl nur 3638 Gene unterschiedlich exprimiert waren. Circa 27% der differentiell exprimierten Gene zeigten ein Expressionsmuster, welches mit Veränderungen an aktiven und/oder repressiven Markern innerhalb der Promoterregion korrelierte. Zudem zeigten intergenische Regionen einen verstärkten Anstieg an repressiven Histonmarkern, was auf eine mögliche regulatorische Funktion dieser Bereiche in der Endotoxintoleranz schließen lässt. Die Studie zeigt, dass die Epigenetik der Monozyten stark von der Endotoxintoleranzinduktion betroffen ist, wenn auch nicht alle Veränderungen dem beobachteten Genexpressionsmuster zugeordnet werden konnten. / Endotoxin-tolerant cells show a reduced ability to produce pro-inflammatory cytokines for several days, which assumes an impact of epigenetic changes in endotoxin tolerance induction. Using an in vitro model with human monocytes, the first part focused on the analysis of epigenetic changes in specific LPS-tolerizable genes. The genes encoding for TNF and CXCL10 showed a reduction in the transcription-activating histone marks H3K27ac and H4ac in tolerant monocytes, which was accompanied by a strongly reduced gene expression. In contrast, the IL6 and IL1B genes showed an increase in activating histone modifications, while their gene expressions were moderately reduced. Repressive epigenetic marks (H3K9me2, H3K27me3, H4K20me3, DNA methylation) were not specifically enhanced in the genes studied. Particularly the IL6 gene expression was more susceptible to the signaling strength in tolerant monocytes implying distinct mechanisms in the repression of the genes analyzed. Within the second part, genome-wide reprogramming of tolerant monocytes was accompanied by a global shift from activating H3K27ac and H4ac in naive monocytes to repressive H3K9me2, H3K27me3 and H4K20me3 in tolerant cells treated with LPS. More than 10000 genomic regions were distinctly regulated by histone marks, though only 3638 genes were differentially expressed. Correlation analyses identified 27 % of the differentially expressed genes that showed a transcriptional level consistent with changes in activating and/or repressive histone marks within their promoter regions. Intergenic regions were highly enriched for repressive histone marks in LPS-tolerant monocytes implying a regulatory function in endotoxin tolerance. The data indicate that the epigenetic environment of monocytes is highly affected by endotoxin tolerance induction, though not all changes are directly linked to the gene expression pattern observed.

In dieser Dissertation wurde eine isolierte Brachydaktylie vom Typ E (BDE) untersucht. Grundlage war eine Familie mit autosomal-dominanten Erbgang BDE. Der genetische Hintergrund ist eine balancierte Translokation t(8;12)(q13;p11.2). Der Bruchpunkt auf derivativem Chromosom der(8) liegt 86 kb strangaufwärts des chondrogenetisch essentiellen Kandidatengens PTHLH (Parathyroid hormone like hormone). PTHLH ist für die Differenzierungsrate von proliferativen Chondrozyten verantwortlich. Positiv oder negativ reguliertes Pthlh führen zu einer Dysbalance mit Brachydaktylie-ähnlichen Phänotypen in murinen Tiermodellen. Der Leserahmen des Kaliumkanals KCNB2 auf Chromosom 8 wurde durch die Translokation in Intron 2 getrennt. Chrondrogenetische KCNB2 Funktionen konnten durch in situ Hybridisierungen ausgeschlossen werden. Der Translokationsbruchpunkt auf der(8) liegt in einer in Mammalia hochkonservierten Region und beeinhaltet ein Bindungsmotiv für AP1 Transkriptionsfaktoren. Durch die Translokation befindet sich in unmittelbarer Nähe eine Kernkonsensussequenz für ETS Transkriptionsfaktoren. AP1 und ETS Transkriptionsfaktoren interagieren und wurden auf eine potentielle PTHLH Regulation untersucht. Epigenetische Histonmodifizierungen, charakteristisch für cis-regulatorische Elemente, sowie Reportergenassays mit AP1 und ETS1 Bindungsmotiven zeigten einen Bezug zur PTHLH Regulation. Bindungsassays mit AP1 und ETS1 Transkriptionsfaktoren an den Bruchpunktsequenzen, sowie funktionelle in vitro Experimente mit Chondrozyten verifizierten die Hypothese, dass der Translokationsbruchpunkt strangaufwärts von PTHLH regulatorische Eigenschaften besitzt. Die AP1 und ETS1 Transkriptionsfaktoren regulierten PTHLH positiv in ATDC5 und C28/I2 Chondrozyten. In chondrogeninduzierten Patientenfibroblasten war die PTHLH Expression inhibiert. Die molekulare Pathogenese der BDE wurde durch die bisher unbekannte chondrogene PTHLH Fehlregulation dargestellt. / We studied a 3-generation family with Brachydactyly Type E (BDE) and identified a t(8;12)(q13;p11.2) translocation. We identified PTHLH (Parathyroid hormone like hormone) on chromosome 12p11.2 and the ionchannel KCNB2 on chromosome 8q13 as candidate genes. KCNB2 was disrupted in intron 2, while the chromosome 12 breakpoint is localized 86 kb upstream of PTHLH; only the latter gene is involved in chondrogenesis. The 12p11.2 breakpoint is conserved and features an AP1 binding site 86 kb upstream of PTHLH. Due to the translocation, an ETS binding site from 8q13 resided near the AP1 site. Since both transcription factors interact, we tested if AP1 and ETS1 can activate PTHLH in ATDC5 and C28/I2 chondrocytes. We used the breakpoint sequences of the derivative chromosomes 8 and 12 and the nonaffected chromosome 8 and 12 allele sequences in reporter-gene assays. Reporter-gene constructs containing the der(8) breakpoint revealed activation in murine and human chondrocytes. The enrichment of histone modifications, implicating cis-regulatory effects were investigated in the breakpoint area. We found the enriched histone H3K4me1 modification at the chromosome 12 breakpoint position in murine and human chondrocytes, while affected fibroblasts showed higher H3K4me1 enrichment at the der(8) breakpoint compared to wt(12) allele. Furthermore, the breakpoint sequence bound to AP1 and C-ets-1 in EMSA. Western blotting after PMA-stimulated AP1 and ETS1 activation and overexpression of different AP1 and ETS1 combinations showed activated PTHrP expression in chondrocytes. In chondrogenic induced BDE fibroblasts PTHLH was inhibited, while IHH was upregulated. We suggest that PTHLH was dysregulated by the translocation in BDE chondrocytes. This could lead to BDE. We highlight the impact to characterize genomic breakpoints in detail and demonstrate a novel AP1- and ETS1-directed chondrogenic PTHLH regulation in wild-type chondrocytes and dysregulation in the pathogenesis of BDE.

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