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The transcription in the eukaryotic cells involves epigenetic regulatory mechanisms that control local and higher-order chromatin remodelling. In the skin, keratinocyte-specific genes are organized into distinct loci including Epidermal Differentiation Complex (EDC) and Keratin type I/II loci. This thesis introduces bioinformatics approaches to analyze multi-level regulatory mechanisms that control skin development and keratinocyte-specific differentiation. Firstly, integration of gene expression data with analyses of linear genome organization showed dramatic downregulation of the genes that comprise large genomic domains in the sweat glands including EDC locus, compared to ii hair follicles, suggesting substantial differences in global genome rearrangement during development of these two distinct skin appendages. Secondly, comparative analysis of the genetic programmes regulated in keratinocytes by Lhx2 transcription factor and chromatin remodeler Satb1 revealed that significant number of their target genes is clustered in the genome. Furthermore, it was shown in this study that Satb1 target genes are lineage-specific. Thirdly, analysis of the topological interactomes of Loricrin and Keratin 5 in hair follicle steam cells revealed presence of the cis- and trans-interactions and lineage specific genes (Wnt, TGF-beta/activin, Notch, etc.). Expression levels of the genes that comprise interactomes show correlation with their histone modification status. This study demonstrates the crucial role for integration of transcription factormediated and epigenetic regulatory mechanisms in establishing a proper balance of gene expression in keratinocytes during development and differentiation into distinct cell lineages and provides an integrated bioinformatics platform for further analyses of the changes in global organization of keratinocyte-specific genomic loci in normal and diseased skin.

Die Komposition der Skelettmuskulatur resultiert aus der fein abgestimmten Balance von Proteinauf- und Abbaumechanismen. Die Skelettmuskelatrophie kann in verschiedenen Situationen entstehen bzw. von diversen Krankheiten ausgelöst werden (Altern, Hunger, Krebs, Nervenschädigung, Kachexie) und ist meist die Folge von gesteigertem Proteinabbau, der die Proteinsynthese überwiegt. Der Muskelabbau ist physiologisch teilweise sinnvoll und dient der Notversorgung von lebenswichtigen Organen mit Lipiden, Aminosäuren und Glukose. Insgesamt ist eine funktionsfähige Muskulatur sehr wichtig, sowohl für Gesunde als auch Erkrankte, da bei Muskelatrophie auslösenden Erkrankungen das Gesamtüberleben wesentlich verringert ist und die Lebensqualität der Patienten enorm reduziert ist. Der Abbau von strukturellen Muskelproteinen wurde hauptsächlich dem Ubiquitin-Proteasom System zugeschrieben, dessen Regulation und von seinen einzelnen Enzymen muss genauestens verstanden sein, um in der Zukunft zielgerichtete Therapien entwickeln zu können. Eines der zentralen Enzyme in der Skelett- und Herzmuskelatrophie ist die E3 Ubiquitin Ligase MuRF1. In nahezu allen Modellen für Muskelatrophie wurde eine starke Zunahme der Expression von MuRF1 beschrieben. Betrachtet man die sehr zentrale Rolle von MuRF1 im UPS, dort vermittelt MuRF1 den Abbau von strukturellen Proteinen des Sarkomers, und der beobachteten starken Regulation bei diversen Atrophie-Modellen, wird klar, wie wichtig das Verständnis der transkriptionellen Regulation von MuRF1 selbst ist. In den letzten Jahren wurden bereits einige Transkriptionsfaktoren identifiziert, die an der Regulation von MuRF1 bei verschiedenen Atrophie-Modellen beteiligt sind, die Studien zeigten aber auch, dass noch nicht alle Modelle erklärt werden konnten. Um die verbleibenden Wissenslücken zu füllen, wurde in dieser Studie nach neuen transkriptionellen Regulatoren von MuRF1 gesucht und deren Beteiligung an bereits bekannten Signalwegen analysiert. / Skeletal muscle mass is permanently balanced as a result of fine tuned protein synthesis and degradation mechanisms. Skeletal muscle atrophy occurs when protein degradation exceeds protein synthesis, which happens in a variety of conditions, such as aging, starvation, cancer, cachexia or denervation. Degradation of muscle mass can sometimes be useful, e.g. as source for lipids, amino acids and glucose in case of critical malnutrition as well as several other physiological conditions. But a solid composition and thereby functional maintenance of muscles is necessary for healthy individuals as well as individuals suffering from atrophy releasing diseases as to retain their mobility and to preserve full heart functions. Since degradation of structural proteins in muscle tissue has been addressed mainly to the ubiquitin-proteasome-system, the regulation of the participating components needs to be understood in detail to develop constructive treatments and therapies for atrophy prevention. One of the key enzymes in skeletal and heart muscle atrophy is the E3 ubiquitin ligase MuRF1. Its expression levels and protein content was found to be elevated in almost every know atrophy model. MuRF1 is very critical for the muscles composition and thus their functional integrity, as it marks and initiates degradation of structural and contractile proteins via the UPS. Since MuRF1 plays a prominent role in muscle atrophy, its transcriptional regulation needs to be well understood to develop effective therapies for all the different atrophy models MuRF1 has been linked to. Several transcription factors have been identified to regulate MuRF1 at different ratios and in diverse atrophy models. Importantly, they do not explain all MuRF1 inducing events observed. To fill some of the remaining knowledge gaps, the studies aims were to find new transcriptional regulators for MuRF1 and to analyze potential involvements of the obtained candidates in pathways affecting skeletal muscle atrophy.

Transkription är stokastisk, där utbrottsmässiga episoder av RNA-transkription genererar RNA-molekyler. Trots att detta är en kärndel av eukaryotiskt liv, är lite känt om hur DNA-bindande transkriptionsfaktorer och transkriptionella kofaktorer formar gen-specifik transkriptionell utbrottskinetik. Syftet med detta examensarbete var att tyda rollerna hos kofaktorerna Med14 och P300/CBP inom transkriptionell utbrottskinetik. För detta ändamål användes Auxin inducible degron systemet för snabb nedbrytning av Med14 eller P300/CBP-proteiner i HCT116-celler, följt av Smart-seq3xpress single cell-RNA-sekvensering. Ett särskild fokus i denna avhandling var även att utvärdera förmågan att härleda direkta  genuttrycksförändringar genom analys av introniska reads – detta då introner ko-transkriptionellt splitsas och dess nyttjande skulle fånga effekter av mycket närliggande transkription. Resultaten visar en tidsberoende minskning av introniskt innehåll och en nedreglering av genuttryck för majoriteten av generna i de behandlade cellinjerna, medan opåverkade kontroller inte visar sådana trender. Utbrottskinetikresultaten indikerar att det inte finns någon korrelation mellan P300/CBP-pertuberade cellers geners ursprungliga utbrottsstorlek och några trender i genuttryckets relativa förändring, medan detsamma kan sägas för Med14-pertuberade cellers geners utbrottsfrekvens. Svaga trender från P300/CBP-påverkade cellers utbrottskinetik och uttrycksändring kan antyda att deras utbrottsfrekvens och inte utbrottsstorlek har påverkats. Resultaten antyder att perturbationen var framgångsrik och att P300/CBP inte påverkar utbrottsstorlek samt att Med14 kan reglera utbrottsfrekvensen för alla påverkade gener i lika hög grad. Vidare forskning behövs inom utbrottskinetikdata för att utöka vår förståelse av denna studies implikationer gällande Med14:s och P300/CBP:s reglerande roller på transkriptionella utbrott. / Transcription is stochastic with episodes of RNA transcription generating bursts of RNA molecules. Despite being a core part of eukaryotic life, little is known about how DNA-binding transcription factors and transcriptional co-factors shape gene-specific transcriptional bursting kinetics. The aim of this thesis was to decipher the roles of the co-factors Med14 and P300/CBP in transcriptional burst kinetics. To this end, the Auxin inducible degron system was used for rapid Med14 or P300/CBP protein degradation in HCT116 cells, followed by Smart-seq3xpress single-cell RNA-sequencing. A particular focus of this thesis was to evaluate the abilities to infer direct gene expression changes by analysis of intronic reads – since introns are co-transcriptionally spliced and would capture very recent transcription. Results show a time dependent decrease of intronic contents and a downregulation in gene expression for a majority of genes in the perturbed cell lines, while unperturbed controls show no such trends. Bursting kinetics results indicate that there is no correlation between P300/CBP perturbed cells’ gene’s original bursting size and any trends in gene expression fold change while the same can be said for Med14 perturbed cell’s gene’s burst frequency. Weak trends from P300/CBP perturbed cells’ bursting kinetics and expression fold change could imply that their bursting frequency and not bursting size has been affected. The results imply that the perturbation was successful and that P300/CBP does not affect bursting size as well as that Med14 could regulate bursting frequency for all affected genes to an equal degree. Further research is needed into the bursting kinetics data to expand our understanding of this study’s implications regarding regulatory roles of Med14 and P300/CBP on transcriptional bursting.