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The transmission of extracellular signals into the nucleus involves inducible transcription factors, but how different signalling pathways act in a cell type-specific fashion is poorly understood. Here, we studied the regulatory role of the AP-1 transcription factor family in blood development using embryonic stem cell differentiation coupled with genome-wide transcription factor binding and gene expression analyses. AP-1 factors respond to MAP kinase signalling and comprise dimers of FOS, ATF and JUN proteins. To examine genes regulated by AP-1 and to examine how it interacts with other inducible transcription factors, we abrogated its global DNA-binding activity using a dominant-negative FOS peptide. We show that FOS and JUN bind to and activate a specific set of vascular genes and that AP-1 inhibition shifts the balance between smooth muscle and hematopoietic differentiation towards blood. Furthermore, AP-1 is required for de novo binding of TEAD4, a transcription factor connected to Hippo signalling. Our bottom-up approach demonstrates that AP-1- and TEAD4-associated cis-regulatory elements form hubs for multiple signalling-responsive transcription factors and define the cistrome that regulates vascular and hematopoietic development by extrinsic signals., Highlighted article: AP-1 activates the expression of vascular genes in hemogenic endothelial cells and is required for de novo binding of the Hippo signaling transcription factor TEAD4.

Stammzellen mit ihrer besonderen Fähigkeit sich selbst zu erneuern und zu differenzieren stellen einen faszinierenden Zelltyp für Grundlagenforschung und angewandte Wissenschaften dar. Pluripotente embryonale Stammzellen (ES Zellen), die aus Zellen der inneren Zellmasse von Präimplantationsembryonen etabliert werden, können ekto-, meso- und endodermale Zelltypen sowie Keimzellen hervorbringen. Im Gegensatz dazu sind multipotente adulte Stammzellen in ihrem Entwicklungspotential eingeschränkt, sie differenzieren sich zu allen Zelltypen ihres Gewebes. Zum Beispiel hämatopoetische Stammzellen (HSZs), die sich in Blut-bildenden Geweben wie dem Knochenmark befinden, vermögen sich in alle Blutzellen zu differenzieren. Während der Differenzierung von Stammzellen ändert sich nicht deren Genom, sondern ihre epigenetische Regulation. Durch epigenetische Mechanismen werden Zelltypen mit verschiedensten Phänotypen und Funktionen generiert. Für Stammzelltherapien ist ein tieferes Verständnis des Zusammenhangs von Epigenom und zellulärer Funktion wichtig. Im Rahmen dieser Dissertation war es mein Ziel, differenzierende Stammzellkulturen auf ihre Genexpression, ihre Chromatinregulation und ihr Differenzierungspotiential hin zu analysieren. Um Histonmodifikationen, die einen möglichen Mechanismus epigenetischer Regulation darstellen, global untersuchen zu können, sind zunächst, durchusszytometrische Protokolle etabliert worden, die die Analyse einzelner Zellen ermöglichen sollten. Mit dieser Methode konnten reduzierte Levels von Histonazetylierung in differenzierten ES Zellen gezeigt werden. Im Gegensatz dazu beobachtete ich vergleichbare Levels von Histonazetylierung in unreifen und reifen Knochenmarkzellen. Zusätzlich untersuchte ich die Wirkung des Histondeazetylase-Inhibitors (HDI) Trichostatin A (TSA) auf Knochenmarkzellkulturen, in denen auch HSZs enhalten sind. Nach Behandlung mit TSA erhöhte sich der Anteil von Zellen mit in vitro und in vivo hämatopoetischer Aktivität, während vor allem differenzierte Zellen in Apoptose gingen. Außerdem wurde der Verlust der Pluripotenz in differenzierenden ES Zellkulturen untersucht. Marker-basierte Analysen und funktionelle Tests wurden mit ES Zellen durchgeführt, die kurzfristig in vitro differenziert wurden. Es stellte sich heraus, dass nach funktionellen Gesichtspunkten die Pluripotenz bereits nach 2 Tagen Differenzierung deutlich reduziert war, beurteilt anhand der Fähigkeit Kolonien zu bilden, embryoide Körperchen (EK) zu formieren und zu kontrahierenden Herzmuskelzelltypen zu differenzieren. Im Gegensatz dazu verringerte sich die Expression von Pluripotenzmarkern erst zu späteren Zeitpunkten. Ich habe weiterhin beobachten können, dass die Wahl des Differenzierungssystems (Aggregations-EK, klonale EKs oder als adhärente Einzelzellschicht) einen Einfluss auf den Fortschritt und die Homogenität der Differenzierung hatte. Um das Ende der Pluripotenz genauer zu untersuchen, wurden differenzierte ES Zellen zurück in ES Zellkulturbedingungen gebracht. Die Ergebnisse deuten an, dass 3 Tage differenzierte ES Zellen einen Punkt überschritten haben, an dem eine Rückkehr zur Pluripotenz allein durch Kulturbedingungen noch möglich ist. Durch die Behandlung mit HDIs starben selektiv differenzierte ES Zellen. Des Weiteren war es Ziel dieser Arbeit, den Einuss von EED - einer essentiellen Untereinheit des Histon-methylierenden Polycomb repressive complex 2 (PRC2) - auf das Chromatin und die Funktion von ES Zellen hin zu analysieren. ES Zellen ohne EED wiesen neben dem bereits bekannten Verlust der Trimethylierung von Histon 3 an Lysin 27 (H3K27me3), global reduzierte H3K9me3 Levels sowie erhöhte Histonazetylierung auf. Trotz typischer ES Zell-Morphologie und normaler Expression von Pluripotenzgenen, besaßen EED knockout (KO)ES Zellen eine veränderte Organisation der Heterochromatinstruktur im Zellkern, eine verlangsamte Chromatinmobilität und Probleme bei der Differenzierung. Zusammenfassend gewähren meine Daten Einblick in die epigenetische Regulation von Stammzellen. Im Besonderen konnte ich zeigen, dass die Behandlung mit HDIs für differenzierende Knochenmarkzellen und differenzierende ES Zellen nachteilig war und zu deren selektivem Zelltod führte. Die hier durchgeführten Analysen ergaben, dass ES Zellen nach 3 Tagen Differenzierung das Ende der Pluripotenz erreicht hatten. Schließlich zeigten die Versuche mit EED KO ES Zellen, dass sie sich zwar selbst erneuerten und morphologisch identisch mit wildtypischen ES Zellen waren, jedoch Defekte bei der Differenzierung besaßen. Dies deutet darauf hin, dass EED nicht nur für undifferenzierte ES Zellen wichtig ist, sondern auch während der Differenzierung von Bedeutung ist., Stem cells with the particular potential to self renew and to differentiate into multiple cell lineages are fascinating cell types for basic and applied research. Pluripotent embryonic stem (ES) cells are derived from the inner cell mass (ICM) of preimplantation embryos. Upon differentiation ES cells can give rise to cells of ecto-, meso- and endoderm including germ cells. In contrast, multipotent adult stem cells are more restricted in their differentiation outcomes,they differentiate into cells of their tissue of origin. For example, hematopoietic stem cells (HSCs) that reside in hemogenic tissues such as the bone marrow (BM) differentiate into hemato-/lymphoid cell lineages. Upon differentiation of stem cells not the genome, but the epigenetic regulation changes. Differentiation-associated epigenetic changes generate cell types with distinct phenotypes and functions. For stem cell-based therapies it is important to deeper understand the relation between epigenome and cellular function. In the scope of this thesis I aimed to analyze cultures of differentiating stem cells with respect to gene expression, chromatin regulation and differentiation potential. For the analysis of global histone modification levels, which represent one mechanism for epigenetic regulation, fow cytometric protocols were established that allow single cell measurements. By applying this methodology decreased histone acetylation levels were shown in differentiated ES cell populations. In contrast, comparable histone acetylation levels were observed in differentiated and undifferentiated BM cells. In addition, I investigated effects of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) on murine BM cells, comprising also HSCs. Upon TSA treatment the frequency of cells with in vitro and in vivo hematopoietic activity was increased, while lineage committed cells underwent apoptosis. Next, the loss of pluripotency was assessed in differentiating ES cell cultures. Using short-term in vitro differentiation protocols marker-based analyses and functional assays were performed.Functionally pluripotency was diminished after 2 days of differentiation as assessed by colony formation, embryoid body (EB) formation and cardiomyogenic differentiation approaches. In contrast, pluripotency marker expression was reduced at later time points. Further, the application of distinct differentiation systems (aggregation EB, clonal EB or monolayer (ML) culture) had an impact on the progression and homogeneity of differentiation cultures. To further study the end of pluripotency, differentiated ES cells were placed under ES cell culture conditions. The data suggest that 3 days differentiated ES cells had passed a point of no return and failed to regain Oct4-eGFP expression and that HDAC inhibitor treatment selectively killed differentiated ES cells. Finally, I aimed to study the effect of EED - a core subunit of the histone methylating Polycomb repressive complex 2 (PRC2) - on ES cell chromatin and function. ES cells lacking EED showed loss of histone H3 lysine 27 trimethylation (H3K27me3) accompanied by increased histone acetylation and reduced H3K9me3 levels. Despite typical ES cell morphology and pluripotency marker expression, EED knockout (KO) ES cells exhibited altered nuclear heterochromatin organization, delayed chromatin mobility and a failure in proper differentiation. Conclusively, my data provide insights into the epigenetic regulation of stem cells. Particularly, the results suggest that HDAC inhibitor treatment was detrimental for differentiated BM as well as for differentiated ES cells and that ES cells after 3 days of differentiation had lost pluripotency. Further, the data demonstrate that EED KO ES cells self renewed, exhibited morphology and pluripotency marker expression similar to wild type ES cells, but failed to differentiate. This indicates an important role of EED not only for undifferentiated but also for differentiating ES cells.

Funder: RCUK | Medical Research Council (MRC); doi: https://doi.org/10.13039/501100000265, Funder: Max-Planck-Gesellschaft (Max Planck Society); doi: https://doi.org/10.13039/501100004189, Haematopoietic stem cells (HSCs) are normally quiescent, but have evolved mechanisms to respond to stress. Here, we evaluate haematopoietic regeneration induced by chemotherapy. We detect robust chromatin reorganization followed by increased transcription of transposable elements (TEs) during early recovery. TE transcripts bind to and activate the innate immune receptor melanoma differentiation-associated protein 5 (MDA5) that generates an inflammatory response that is necessary for HSCs to exit quiescence. HSCs that lack MDA5 exhibit an impaired inflammatory response after chemotherapy and retain their quiescence, with consequent better long-term repopulation capacity. We show that the overexpression of ERV and LINE superfamily TE copies in wild-type HSCs, but not in Mda5-/- HSCs, results in their cycling. By contrast, after knockdown of LINE1 family copies, HSCs retain their quiescence. Our results show that TE transcripts act as ligands that activate MDA5 during haematopoietic regeneration, thereby enabling HSCs to mount an inflammatory response necessary for their exit from quiescence.

Funder: RCUK | Medical Research Council (MRC); doi: https://doi.org/10.13039/501100000265, Funder: Max-Planck-Gesellschaft (Max Planck Society); doi: https://doi.org/10.13039/501100004189, Haematopoietic stem cells (HSCs) are normally quiescent, but have evolved mechanisms to respond to stress. Here, we evaluate haematopoietic regeneration induced by chemotherapy. We detect robust chromatin reorganization followed by increased transcription of transposable elements (TEs) during early recovery. TE transcripts bind to and activate the innate immune receptor melanoma differentiation-associated protein 5 (MDA5) that generates an inflammatory response that is necessary for HSCs to exit quiescence. HSCs that lack MDA5 exhibit an impaired inflammatory response after chemotherapy and retain their quiescence, with consequent better long-term repopulation capacity. We show that the overexpression of ERV and LINE superfamily TE copies in wild-type HSCs, but not in Mda5−/− HSCs, results in their cycling. By contrast, after knockdown of LINE1 family copies, HSCs retain their quiescence. Our results show that TE transcripts act as ligands that activate MDA5 during haematopoietic regeneration, thereby enabling HSCs to mount an inflammatory response necessary for their exit from quiescence.

Successful elimination of bacteria in phagocytes occurs in the phago-lysosomal system, but also depends on mitochondrial pathways. Yet, how these two organelle systems communicate is largely unknown. Here we identify the lysosomal biogenesis factor transcription factor EB (TFEB) as regulator for phago-lysosome-mitochondria crosstalk in macrophages. By combining cellular imaging and metabolic profiling, we find that TFEB activation, in response to bacterial stimuli, promotes the transcription of aconitate decarboxylase (Acod1, Irg1) and synthesis of its product itaconate, a mitochondrial metabolite with antimicrobial activity. Activation of the TFEB–Irg1–itaconate signalling axis reduces the survival of the intravacuolar pathogen Salmonella enterica serovar Typhimurium. TFEB-driven itaconate is subsequently transferred via the Irg1-Rab32–BLOC3 system into the Salmonella-containing vacuole, thereby exposing the pathogen to elevated itaconate levels. By activating itaconate production, TFEB selectively restricts proliferating Salmonella, a bacterial subpopulation that normally escapes macrophage control, which contrasts TFEB’s role in autophagy-mediated pathogen degradation. Together, our data define a TFEB-driven metabolic pathway between phago-lysosomes and mitochondria that restrains Salmonella Typhimurium burden in macrophages in vitro and in vivo.