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Proper development of the immune system is an intricate process dependent on many factors, including an intact DNA damage response. The DNA double-strand break signaling kinase ATM and its cofactor NBS1 are required during T cell development and for the maintenance of genomic stability. The role of a second ATM cofactor, ATMIN (also known as ASCIZ) in T cells is much less clear, and whether ATMIN and NBS1 function in synergy in T cells is unknown. Here, we investigate the roles of ATMIN and NBS1, either alone or in combination, using murine models. We show loss of NBS1 led to a developmental block at the double-positive stage of T cell development, as well as reduced TCRα recombination, that was unexpectedly neither exacerbated nor alleviated by concomitant loss of ATMIN. In contrast, loss of both ATMIN and NBS1 enhanced DNA damage that drove spontaneous peripheral T cell hyperactivation, proliferation as well as excessive production of proinflammatory cytokines and chemokines, leading to a highly inflammatory environment. Intriguingly, the disease causing T cells were largely proficient for both ATMIN and NBS1. In vivo this resulted in severe intestinal inflammation, colitis and premature death. Our findings reveal a novel model for an intestinal bowel disease phenotype that occurs upon combined loss of the DNA repair cofactors ATMIN and NBS1., Author Summary Defects in DNA repair pathways can lead to pathogenesis within the immune system, an example of which is inflammatory bowel disease (IBD). Yet the underlying genetic causes of IBD are often unclear. The DNA repair kinase ATM is crucial for the proper development and function of the immune system. ATM is regulated in a stimulus dependent manner by its cofactors, ATMIN and NBS1. These cofactors compete for ATM binding and in doing so regulate ATM kinase activity. Whereas both ATM and NBS1 function in T cell development and in the maintenance of genomic stability within such cells, the role of ATMIN (and the contribution of ATMIN and NBS1) in T cell function is unknown. Here, we show that whereas NBS1 has distinct ATMIN-independent functions during VDJ recombination, loss of both cofactors resulted in exacerbated DNA damage, T cell hyperactivation, inflammation and an IBD phenotype. The pathology was driven by T cells largely proficient for both ATMIN and NBS1. These data demonstrate additive effects revealed upon loss of both ATMIN and NBS1, thus illustrating the importance of these two DNA repair cofactors in proper T cell development and function.

Herzversagen stellt weltweit eine der häufigsten Todesursachen dar und ist hauptsächlich die Folge eines vorangegangenen Herzinfarkts. Die Tatsache, dass Herzgewebe nicht imstande ist, sich nach einem Infarkt zu regenerieren, führte zum allgemeinen Glauben, das Herz von Säugetieren sei ein ausdifferenziertes Organ. Kürzlich, jedoch, hat man im erwachsenen Herzen Zellen entdeckt, welche Stammzell-ähnlichen Charakter besitzen und die Fähigkeit haben könnten, geschädigtes Gewebe zu reparieren. Die Identifizierung von Faktoren, welche diese ruhenden Herzstammzellen aktivieren und zur Differenzierung zu funktionellen Herzzellen veranlassen können, wäre ein wichtiger Schritt in Richtung neuer Therapien von Herzkrankheiten. Ein Faktor, der für diese Tätigkeit in Frage kommt, ist der Leukämie Inhibitions Faktor (LIF). LIF ist ein polyfunktionelles Glykoprotein, welches mannigfaltige biologische Aktivitäten aufweist, wie unter anderem Wachstumsförderung und Differenzierung verschiedenster Zielzellen. In vorangegangenen Studien wurde gezeigt, dass speziell die Matrix-assoziierte Isoform (M-LIF) sich positiv auf die Kardiomyogenese auswirkt, im Unterschied zu ihrem diffundierbaren Gegenstück (D-LIF). Ziel dieser Diplomarbeit war es, die mögliche Rolle von LIF in Bezug auf Herzzellentwicklung zu untersuchen, wobei Unterschiede zwischen dem Effekt von M-LIF und D-LIF auf embryonale Stammzellen, „Embryoid bodies“, Herzvorläuferzellen und Kardiomyozyten analysiert wurden, sowie der Einfluss des Glykosylierungsgrades auf die biologische Aktivität. In den hier durchgeführten Experimenten konnte gezeigt werden, dass sowohl glykosyliertes M-LIF, als auch D-LIF, als Faktoren für die Expansion und gezielte Differenzierung von Herzvorläuferzellen eingesetzt werden können und sich damit in weiterer Folge als nützlich für die Zelltherapie von Herzkrankheiten erweisen könnten., Heart failure represents one of the leading causes of death in western civilization and is primarily the result of previous myocardial infarction. The fact that cardiac tissue is unable to regenerate after infarction led to the general belief that the heart of mammalian species is a terminally differentiated organ. Recently, however, evidence has emerged that the adult heart contains myocardial cells possessing stem cell-like properties which may have the potential to repair damaged tissue. The identification of factors which could activate these quiescent cardiac stem cells and cause them to differentiate into functional cardiomyocytes would be an important step towards finding new therapies for the treatment of heart diseases. One such factor has been proposed to be Leukemia Inhibitory Factor (LIF). LIF is a polyfunctional glycoprotein, showing a wide range of biological activities that include growth promotion and cell differentiation of various types of target cells. In previous studies, specifically the matrix-associated isoform of LIF (M-LIF) has been shown to positively influence cardiomyogenesis, in contrast to its diffusible counterpart (D-LIF). The aim of this diploma thesis was to investigate the potential role of LIF in cardiac differentiation, analyzing differences between the effect of M-LIF and D-LIF on embryonic stem cells, embryoid bodies, cardiac progenitor cells and cardiomyocytes, as well as the impact of glycosylation on biological activity. The conducted experiments reveal that both glycosylated M-LIF and D-LIF, respectively, can be utilized as factors to trigger expansion of cardiac stem cells and induce differentiation into functional heart cells in vitro which may ultimately prove useful for cell therapy of the diseased heart.

Maintenance of genome integrity via repair of DNA damage is a key biological process required to suppress diseases, including Fanconi anemia (FA). We generated loss-of-function human haploid cells for FA complementation group C (FANCC), a gene encoding a component of the FA core complex, and used genome-wide CRISPR libraries as well as insertional mutagenesis to identify synthetic viable (genetic suppressor) interactions for FA. Here we show that loss of the BLM helicase complex suppresses FANCC phenotypes and we confirm this interaction in cells deficient for FA complementation group I and D2 (FANCI and FANCD2) that function as part of the FA I-D2 complex, indicating that this interaction is not limited to the FA core complex, hence demonstrating that systematic genome-wide screening approaches can be used to reveal genetic viable interactions for DNA repair defects.

Defects in DNA repair can cause various genetic diseases with severe pathological phenotypes. Fanconi anemia (FA) is a rare disease characterized by bone marrow failure, developmental abnormalities and increased cancer risk that is caused by defective repair of DNA interstrand crosslinks (ICLs). By performing genome-wide loss-of-function screens across a panel of human haploid isogenic FA-defective cells (FANCA, FANCC, FANCG, FANCI, FANCD2), we identified the deubiquitylating enzyme USP48 as synthetic viable for FA gene deficiencies. Thus, as compared to FA-defective cells alone, FA-deficient cells additionally lacking USP48 are less sensitive to genotoxic stress induced by ICL agents and display enhanced, BRCA1-dependent, clearance of DNA damage. Consequently, USP48 inactivation reduces chromosomal instability of FA-defective cells. Our results highlight a role for USP48 in controlling DNA repair and suggest it as a potential target that could be therapeutically exploited for FA.