Transcriptome analysis identifies a robust gene expression program in the mouse intestinal epithelium on aging

The intestinal epithelium undergoes constant regeneration driven by intestinal stem cells. How old age affects the transcriptome in this highly dynamic tissue is an important, but poorly explored question. Using transcriptomics on sorted intestinal stem cells and adult enterocytes, we identified candidate genes, which change expression on aging. Further validation of these on intestinal epithelium of multiple middle-aged versus old-aged mice highlighted the consistent up-regulation of the expression of the gene encoding chemokine receptor Ccr2, a mediator of inflammation and several disease processes. We observed also increased expression of Strc, coding for stereocilin, and dramatically decreased expression of Rps4l, coding for a ribosome subunit. Ccr2 and Rps4l are located close to the telomeric regions of chromosome 9 and 6, respectively. As only few genes were differentially expressed and we did not observe significant protein level changes of identified ageing markers, our analysis highlights the overall robustness of murine intestinal epithelium gene expression to old age

Smarcad1 mediates microbiota-induced inflammation in mouse and coordinates gene expression in the intestinal epithelium

Background How intestinal epithelial cells interact with the microbiota and how this is regulated at the gene expression level are critical questions. Smarcad1 is a conserved chromatin remodeling factor with a poorly understood tissue function. As this factor is highly expressed in the stem and proliferative zones of the intestinal epithelium, we explore its role in this tissue. Results Specific deletion of Smarcad1 in the mouse intestinal epithelium leads to colitis resistance and substantial changes in gene expression, including a striking increase of expression of several genes linked to innate immunity. Absence of Smarcad1 leads to changes in chromatin accessibility and significant changes in histone H3K9me3 over many sites, including genes that are differentially regulated upon Smarcad1 deletion. We identify candidate members of the gut microbiome that elicit a Smarcad1-dependent colitis response, including members of the poorly understood TM7 phylum. Conclusions Our study sheds light onto the role of the chromatin remodeling machinery in intestinal epithelial cells in the colitis response and shows how a highly conserved chromatin remodeling factor has a distinct role in anti-microbial defense. This work highlights the importance of the intestinal epithelium in the colitis response and the potential of microbial species as pharmacological and probiotic targets in the context of inflammatory diseases

Epigenetic chromatin states during embryonic development and adult homeostasis of the mammalian gut

The mammalian small intestine is a complex 3D-structure comprised of various specialized, proliferative and fully differentiated cell types. Although highly specialized in adults, all cellular lineages originate from a monolayered epithelial tube in the early embryo, which consists of a presumably homogenous population. As the fastest proliferating tissue, the murine small intestine is an established model to study embryonic development and adult homeostasis. These processes are mainly regulated in two ways: intracellular signaling, such as the proliferative Wnt-pathway in combination with Notch- Hedgehog- and BMP-signaling, and epigenetic mechanisms, on which this thesis focuses. Here, I assessed the epigenetic marks H3K27Ac, H3K27me3, H3K4me3, H2A.Z and DNA-methylation, which are involved in the regulation of gene activity, providing a cell type and a development specific expression pattern. The main goal of the projects described in this thesis was to elucidate epigenetic mark deposition and their interaction on the genome-wide scale and to assess their functional impact on gene activity at single genetic loci. Furthermore, single cell in situ analysis was employed to visualize tissue heterogeneity of gene expression and epigenetic patterning. Both approaches were used to deepen our understanding of epigenetic mechanisms involved in development and homeostasis as well as in cancer pathogenesis. Active and repressive epigenetic marks’ genomic distribution was analyzed throughout the intestinal development from embryonic to the fully differentiated adult cell types. In combination with RNA-seq data, these ChIP-seq and MBD-seq experiments provided a comprehensive dataset, which allows for the investigation of epigenetic marks’ interactions along the intestinal cell development timecourse. Hereby, H2A.Z, a histone variant previously annotated to active genes, was found to be positioned on responsive rather than on active promoters. Moreover, H2A.Z was found to be decoupled from gene activity regulation in fully differentiated cells. The well-known active promoter and enhancer mark H3K27Ac was additionally found to mark poised enhancers, long before the corresponding gene’s expression. This novel premarking effect, in combination with the available timecourse gene expression data, could be used for improved genome-wide enhancer prediction and target gene identification. Numerous potential developmental marker genes were identified through developmental clustering. Single locus analysis of these genes revealed strong heterogeneity of gene expression and epigenetic levels in adult intestinal tissue. Strikingly, similar gene expression patterns were also observed at embryonic stages, which previously were believed to be uniform. This heterogeneity might be the key to adult tissue homeostasis and functionality as well as to the early cell fate commitment and structural patterning in the embryonic epithelium.Der Dünndarm besitzt eine komplexe dreidimensionale Struktur, aufgebaut aus mehreren spezialisierten, proliferativen und voll ausdifferenzierten Zelltypen. Trotz des hohen Spezialisierungsgrads im adulten Gewebe stammen alle Zellen des Dünndarmepithels von einer homogen aussehenden embryonalen Population ab. Der sich alle 5 Tage erneuernde Dünndarm ist das sich am schnellsten teilende Gewebe in der Maus und damit ein gängiges Modell für Studien der Embryonalentwicklung sowie der adulten Homöostase. Diese Prozesse werden vor allem auf zwei Arten gesteuert: Intrazelluläre Signalwege und epigenetische Mechanismen. Letzteres steht im Fokus dieser Doktorarbeit. Hierfür wurden die epigenetischen Histonmarkierungen H3K27Ac/me3, H3K4me3 und H2A.Z, sowie die DNA-Methylierung untersucht, die zusammen an der Steuerung der entwicklungsspezifischen Genexpression beteiligt sind. Die Zielsetzung dieser Arbeit war es, die Verteilung und Wechselwirkung epigenetischer Markierungen genomweit zu bestimmen und ihre Auswirkung auf die Aktivität einzelner Gene zu überprüfen. Zusätzlich wurden bildgebende in situ Methoden verwendet, um die Heterogenität adulten und embryonalen Gewebes, insbesondere im Hinblick auf Genexpression und Histonmarkierungen, zu untersuchen. Beide Ansätze dienten dazu, unser Verständnis epigenetischer Mechanismen, die an der Embryonalentwicklung und Homöostase, aber nicht zuletzt auch an der Karzinogenese beteiligt sind, zu vertiefen. Die Verteilung aktiver und repressiver epigenetischer Markierungen, sowie die Genexpression wurde während der Differenzierung von embryonalen Stadien zum adulten Dünndarmepithelium mithilfe von ChIP- und RNA-seq analysiert. Der hieraus entstandene umfassende Datensatz ermöglicht eine detaillierte Untersuchung epigenetischer Wechselwirkungen während des gesamten Entwicklungsverlaufs. Hierbei wurde H2A.Z, eine Histonvariante, deren Positionierung bisher mit aktiven Promotern und Enhancern assoziiert wurde, als ein Marker von regulationsempfindlichen genetischen Elementen identifiziert und zwar unabhängig von der Genaktivität. Insbesondere in voll ausdifferenzierten Zellen wurde eine Abkopplung der H2A.Z-Positionierung von der Genregulation beobachtet. Die Histonmarkierung H3K27Ac, bekannt für ihre Positionierung auf aktiven Promotern und Enhancern, wurde auch auf bereitstehenden Enhancern lange vor Expression des dazugehörenden Gens gefunden. Dieser neue Vormarkierungseffekt könnte zur verbesserten Identifizierung von Enhancersequenzen sowie der dazugehörigen Zielgene verwendet werden. Zahlreiche Gene wurden mithilfe der epigenetischen Gruppierung als potentielle Entwicklungsmarker identifiziert. Ihre Einzelanalyse deckte in vielen Fällen eine starke Heterogenität der Genexpression und epigenetischer Markierungen im adulten Gewebe auf. Interessanterweise wurde dies oft auch in den bisher als größtenteils homogen beschriebenen, embryonalen Stadien beobachtet. Diese Heterogenität könnte der Schlüssel zur adulten Homöostase sowie der embryonalen Spezialisierung des Dünndarmepithels sein

Effect of protein phosphatase gene sppA deletion on hyphal growth in Streptomyces coelicolor when combined with different protein kinase gene afsK mutations

Streptomyces species are known for their bacteria-untypical growth and sporulation. Growth by tip extension building up a mycelium consisting of long branching hyphae is a highly polar and regulated process. An essential protein invalved in hyphal growth and branching regulation is DiviVA. In S. coelicolor branching and growth is regulated by phosphorylation of DiviVA by the serine/tlu•eonine lcinase AfsK. The phosphatase SppA dephosphorylates DiviVA and leads to less branching growth, an interaction to be further investigated in this project. The aim of this project was to combine a sppA knockout mutation with different afsK modifications, such as deletion and overexpression mutants. A cosmid carrying a new ilsppA::vph allele was constructed using the Å-Red system in E. coli. The allele was transfened to S. coelicolor and recombinant strains with replacement of the sppA gene were selected. Of special interest was the ilsppA ilafsK double lu1ockout to reveal possible additional DiviVA phosphorylation due to other kinases and to check if the wild type colony size phenotype (previously small colonies were observed in ilsppA strains) would be regained. The obtained strains were examined for growth phenotypes and phosphorylation patterns of DiviVA were visualized by Western blotting. sppA knockout mutants were tested for increased sensitivity to overexpression of afsK. The obtained results suppmi the previously raised hypothesis, that SppA and AfsK are direct counteractors on DiviVA phosphorylation. The sppA afsK double knockout mutant pmily regains the wild type colony size. Sensitivity to overexpression of ajsK seems to be strongly increased by an sppA knockout. As time was limited the results are preliminary and further experiments should be carried out with the generated strains to confirm and extend the results