22455 research outputs found
Sort by
Functional characterization of ARID1A mutations in follicular lymphoma
Get PDFBackground: Follicular lymphoma (FL) is one of the most common malignant lymphomas worldwide and the most common form of indolent lymphoma. FL is highly heterogeneous from both the clinical and molecular point of view. It remains a clinical challenge since advanced-stage disease is still considered incurable, and patients ultimately present with relapsed or re-fractory disease. Molecularly, FL is characterized by highly recurrent genetic mutations in genes coding for epigenetic modifiers. ARID1A mutations are among the most frequent mutations in FL (~10-20 % at the time of diagnosis). ARID1A mutations are a component of the prognostic clinic-genetic risk model m7-FLIPI (Pastore, Jurinovic et al. 2015). These mutations are primari-ly disruptive and result in protein haplodeficiency. Functionally, ARID1A is part of a SWI/SNF complex, which controls chromatin accessibility and is involved in numerous processes, includ-ing gene expression.
Aim: Functionally characterize ARID1A mutations in representative human FL model systems.
Methods: I used established and primary FL-like cell lines that harbor the hallmark t(14;18) translocation with or without heterozygous or homozygous ARID1A mutations (introduced by CRISPR/Cas9) or knock-down (by shRNA). I applied complementary omics approaches (RNA-Seq and ATAC-Seq) and functional assays to untangle the consequences of ARID1A loss in these FL model systems.
Results: ARID1A loss profoundly altered gene expression. Across three cell lines, we observed consistent down-regulation of genes involved in cell cycle regulation and apoptosis pathways upon ARID1A loss. In functional experiments, I could show that ARID1A mutant clones are characterized by significantly slower cell proliferation and increased formation of anaphase bridges.
Next, I demonstrated that ARID1A loss results in decreased FAS levels and lower sensitivity to FASLG-induced apoptosis. We discovered the underlying molecular mechanism through ad-vanced bioinformatics analyses and functional experiments. Briefly, ARID1A loss does not di-rectly affect FAS expression. Still, it results in reduced DNA accessibility and expression of the co-transcription factor RUNX3, thereby hindering RUNX3-ETS1 cooperativity and ETS1-induced FAS expression, which promotes a functionally and clinically relevant immune-evasive pheno-type.
Finally, RNA-Seq analysis indicated that ARID1A loss alters the plasma membrane and cyto-skeleton functions, as well as the overall abundance of ligands and receptors. Ex vivo co-cultures of FL-like cells with T cells suggested impaired immune synapse formation with CD4 T lymphocytes upon ARID1A loss.
Discussion and conclusion: Overall, our analyses provide novel insights into the functional consequences of ARID1A mutations in FL, most notably promoting immune evasion. A better understanding of mutation-specific biology, including its impact on interactions within the tumor microenvironment, holds promise for improved patient stratification and the development of personalized treatment approaches
Viral analysis of acute SARS-CoV-2 infections in the prospective COVID-19 cohort, Munich (KoCo19)
Get PDF
Warum ist die pathohistologische Diagnostik von Tumoren kleiner Speicheldrüsen viel schwieriger und damit gefährlicher als die von Tumoren großer Speicheldrüsen?
Get PDFIn-vivo-Verschleißverhalten von Restaurationen aus CAD/CAM- Komposit versus Lithium-Disilikat-Keramik bei Patienten nach prothetischer Gesamtrehabilitation
Get PDFHeterochromatic regulation of endogenous retroviruses in mouse embryonic stem cells
Get PDFDer Großteil des Säugetiergenoms liegt in Form von Heterochromatin vor, einer Struktur aus Desoxyribonukleinsäure (DNS) und Proteinen, die sich durch begrenzte Zugänglichkeit der Erbinformation, geringe Transkriptionsaktivität und Anreicherung von repetitiven Sequenzen auszeichnet. Die spezifische Regulierung und dichte Verpackung bestimmter DNS-Abschnitte ist entscheidend für die Entwicklung von Säugetieren, während Defekte zu Krebs und Zelltod führen können.
Die Unterdrückung von Retrotransposons aus der Gruppe der endogenen Retroviren (ERVs) durch Heterochromatisierung ist von wesentlicher Bedeutung für die Gewährleistung genomischer Stabilität und transkriptioneller Integrität. Die Zielmechanismen sowie die Akteure, die an der Etablierung und Aufrechterhaltung des heterochromatischen Zustands beteiligt sind, sind jedoch nicht vollständig bekannt.
Diese Doktorarbeit umfasst zwei Veröffentlichungen, in denen neue Beteiligte der Regulierung des Heterochromatins identifiziert und charakterisiert wurden, wobei die Mechanismen der ERV-Stilllegung in embryonalen Stammzellen der Maus als Modellsystem verwendet wurden.
Es wurde ein 160 Basenpaar (bp) langes Sequenzelement von Intracisternal A-Partikel (IAP) Retrotransposons identifiziert, welches die Bildung, Ausbreitung und Erhaltung von Heterochromatin auslöst. Diese kurze Heterochromatin induzierende (SHIN) Sequenz führt zu einer von den Proteinen SETDB1 und TRIM28 abhängigen Ablagerung von H3K9me3 (Trimethylierung von Lysin 9 des Histons 3), einer Histonmodifikation, die ein Kennzeichen des Heterochromatins darstellt. Ein SHIN Sequenz Reportersystem in Kombination mit genomweiten sh- und sgRNA-Screens identifizierte die neuen ERV Regulationsfaktoren ATRX und MORC3. Beide Proteine binden an IAP-Elemente und sind für eine effiziente Heterochromatinbildung und die Aufrechterhaltung von robustem Heterochromatin notwendig.
Als ein wesentliches Ergebnis konnte gezeigt werden, dass ein funktionsfähiger MORC3-ATPase-Zyklus und MORC3-SUMOylierung für die ERV-Chromatinregulation von besonderer Bedeutung sind. Proteomanalysen von mutierten MORC3-Proteinen zeigten eine beeinträchtigte Interaktion mit dem Histon-H3.3- Chaperon DAXX. Bedeutenderweise ist H3.3 an MORC3-Bindungsstellen in MORC3 ko und mutierten Zellen deutlich reduziert, was zeigt, dass MORC3 ein kritischer Regulator des DAXX vermittelten Histon H3.3-Einbaus in ERV-Regionen ist.
Zusammenfassend beschreibt diese Arbeit zwei neue Akteure der heterochromatischen ERV-Regulierung und gibt einen molekularen Einblick in den H3.3 abhängigen Stilllegungsmechanismus.The majority of the mammalian genome is present as heterochromatin, a structure composed of deoxyribonucleic acid (DNA) and proteins, characterized by limited accessibility of the heredity material, low transcriptional activity, and enrichment of repetitive sequences. The specific regulation and dense packaging of certain DNA segments are crucial for mammalian development whereas defects can lead to cancer and cell death. Repression of retrotransposons of the so-called endogenous retroviruses (ERVs) by heterochromatization is essential to ensure genomic stability and transcriptional integrity. Yet, the targeting mechanisms as well as players involved in both, establishing and maintaining the heterochromatic state, are not completely understood.
This thesis comprises two publications that identified and characterized novel players involved in heterochromatin regulation using the mechanisms of ERV silencing in mouse embryonic stem cells as a model system.
A 160 base pair (bp) sequence element of Intracisternal A particle (IAP) retrotransposons was identified to trigger the formation, spreading, and maintenance of heterochromatin. This short heterochromatin inducing (SHIN) sequence leads to deposition, of H3K9me3 (trimethylation of lysine 9 on Histone 3), a histone modification that represents a hallmark of heterochromatin, in a SETDB1 and TRIM28 dependent manner. A SHIN sequence-reporter system in combination with genome-wide sh- and sgRNA screens identified the novel players ATRX and MORC3. Both factors bind to IAP elements and are necessary for efficient heterochromatin formation and maintenance of robust heterochromatin.
Of particular significance, a functional MORC3 ATPase cycle and MORC3 SUMOylation are important for ERV chromatin regulation. Proteomic analyses of MORC3 mutant proteins revealed compromised interaction with the histone H3.3 chaperone DAXX. Importantly, H3.3 was strongly reduced on MORC3 binding sites in MORC3 ko and mutant cells, indicating that MORC3 is a critical regulator of DAXX-mediated histone H3.3 incorporation on ERV regions.
In summary, this work describes two novel players of heterochromatic ERV regulation and provides molecular insight into the H3.3-dependent silencing mechanism
Macrophage derived complement factor C3 impacts on cardiac remodeling
Get PDFMyocardial infarction (MI) is a common condition with strong impact on mortality and morbidity. Coronary occlusion induces myocardial necrosis triggering inflammatory responses of various cell types, leading to myocardial healing or scar formation. The majority of immune cells in the heart are myeloid cells, which play a role in the pathophysiology of MI. They are attracted by inflammatory mediators such as cytokines and chemokines, and recruited to ischemic myocardium. The notion that the complement system mediates immunological defense and is stimulated by MI is supported by prior research. Robust upregulation of the complement system contributes to the postinfarct inflammation process both directly, e.g. by chemoattracting immune cells, and indirectly, such as by inserting the membrane attack complex (MAC) into damaged cells within ischemic myocardium. Complement component 3 (C3) is a key factor in the complement cascade and massive C3 deposition is found in the ischemic region after MI. Blockage or genetic deficiency of C3 has been shown to reduce ischemic injury. However, the underlying mechanisms have been unclear. We utilized C3-tdTomato knock-in reporter mice to monitor C3 expression in immune cells in health and in response to MI, and characterized its role in postinfarct cardiac inflammation and remodeling. Using flow cytometry we identified profound expression of C3 by circulating myeloid cells already in steady state conditions. While presence of C3 was low in healthy mouse hearts, cardiac resident macrophages upregulated C3 within 48h after myocardial ischemia/reperfusion (I/R) injury. Transplantation of donor bone marrow (BM) cells to lethally irradiated mice demonstrated that BM-independent tissue macrophages, which originated from embryonic hematopoiesis, produce this complement factor whereas only minimal amounts of C3 are taken up from blood circulation. In line with this, circulating myeloid cells of bone marrow donors generate C3 in high abundance in recipient mice lacking serum-derived C3. While macrophage C3 had no significant impact on cardiac remodeling after short-term ischemia, tissue resident macrophage derived C3 impacted on infarct size and cardiac remodeling in mice undergoing chronic infarction. Taken together, we provide a comprehensive characterization of C3 expression in immune cells and decipher the upregulation of C3 in cardiac macrophages upon inflammation, shedding light on the importance of intracellularly generated C3 and paving the way for future investigations of post-ischemic inflammation
