TY - THES A1 - Baluapuri, Apoorva T1 - Molecular Mechanisms of MYC’s impact on Transcription Elongation T1 - Molekulare Mechanismen des Einflusses von MYC auf die Transkriptionselongation N2 - Expression of the MYC oncoprotein, which binds the DNA at promoters of most transcribed genes, is controlled by growth factors in non-tumor cells, thus stimulating cell growth and proliferation. Here in this thesis, it is shown that MYC interacts with SPT5, a subunit of the RNA polymerase II (Pol II) elongation factor DSIF. MYC recruits SPT5 to promoters of genes and is required for its association with Pol II. The transfer of SPT5 is mediated by CDK7 activity on TFIIE, which evicts it from Pol II and allows SPT5 to bind Pol II. MYC is required for fast and processive transcription elongation, consistent with known functions of SPT5 in yeast. In addition, MYC increases the directionality of promoters by stimulating sense transcription and by suppressing the synthesis of antisense transcripts. The results presented in this thesis suggest that MYC globally controls the productive assembly of Pol II with general elongation factors to form processive elongation complexes in response to growth-factor stimulation of non-tumour cells. However, MYC is found to be overexpressed in many tumours, and is required for their development and progression. In this thesis it was found that, unexpectedly, such overexpression of MYC does not further enhance transcription but rather brings about squelching of SPT5. This reduces the processivity of Pol II on selected set of genes that are known to be repressed by MYC, leading to a decrease in growth-suppressive gene transcription and uncontrolled tumour growth N2 - Die Expression des MYC-Onkoproteins, das die Promotoren der meisten exprimierten Gene bindet, wird in gesunden, nicht transformierten Zellen durch Wachstumsfaktoren reguliert und fördert das Zellwachstum und die Zellteilung. In dieser Arbeit wurde die Interaktion zwischen MYC und SPT5, einer Untereinheit des RNA-Polymerase (Pol II) Elongationsfaktors DSIF gezeigt. MYC ist für die Rekrutierung von SPT5 an Promotoren und die Assoziation mit Pol II notwendig. Der Transfer von SPT5 auf Pol II setzt die Aktivität der Proteinkinase CDK7 voraus, die TFIIE aus dem Pol II Komplex entfernt und es so SPT5 ermöglicht, an Pol II zu binden. MYC wird für eine schnelle und prozessive Transkriptionselongation benötigt, was mit bekannten Funktionen von SPT5 in Hefe übereinstimmt. Zusätzlich erhöht MYC die Direktionalität von Promotoren, indem es die Sense-Transkription stimuliert und die Synthese der Antisense-Transkripte unterdrückt. Die in dieser Arbeit vorgestellten Ergebnisse legen nahe, dass MYC in normalen, nicht-transformierten Zellen die produktive Assemblierung von Pol II mit allgemeinen Elongationsfaktoren global steigert, um prozessive Elongationskomplexe als Reaktion auf die Wachstumsfaktorstimulation zu bilden. Die meisten humanen Tumore exprimieren jedoch deutlich erhöhte Mengen des MYC Proteins, das für Tumorentstehung und Progression benötigt wird. In dieser Arbeit wurde festgestellt, dass eine solche Überexpression von MYC unerwarteterweise keine weitere Steigerung der Expression mit sich bringt, sondern zur Sequestrierung von SPT5 führt. Dies reduziert die Prozessivität von Pol II an ausgewählten Genen, welche durch MYC bekannterweise supprimiert werden, was zu einer Abnahme der wachstumsunterdrückenden Gentranskription und zu einem unkontrollierten Wachstum führt. KW - Transcription KW - MYC Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-243806 ER - TY - JOUR A1 - Hartmann, Oliver A1 - Reissland, Michaela A1 - Maier, Carina R. A1 - Fischer, Thomas A1 - Prieto-Garcia, Cristian A1 - Baluapuri, Apoorva A1 - Schwarz, Jessica A1 - Schmitz, Werner A1 - Garrido-Rodriguez, Martin A1 - Pahor, Nikolett A1 - Davies, Clare C. A1 - Bassermann, Florian A1 - Orian, Amir A1 - Wolf, Elmar A1 - Schulze, Almut A1 - Calzado, Marco A. A1 - Rosenfeldt, Mathias T. A1 - Diefenbacher, Markus E. T1 - Implementation of CRISPR/Cas9 Genome Editing to Generate Murine Lung Cancer Models That Depict the Mutational Landscape of Human Disease JF - Frontiers in Cell and Developmental Biology N2 - Lung cancer is the most common cancer worldwide and the leading cause of cancer-related deaths in both men and women. Despite the development of novel therapeutic interventions, the 5-year survival rate for non-small cell lung cancer (NSCLC) patients remains low, demonstrating the necessity for novel treatments. One strategy to improve translational research is the development of surrogate models reflecting somatic mutations identified in lung cancer patients as these impact treatment responses. With the advent of CRISPR-mediated genome editing, gene deletion as well as site-directed integration of point mutations enabled us to model human malignancies in more detail than ever before. Here, we report that by using CRISPR/Cas9-mediated targeting of Trp53 and KRas, we recapitulated the classic murine NSCLC model Trp53fl/fl:lsl-KRasG12D/wt. Developing tumors were indistinguishable from Trp53fl/fl:lsl-KRasG12D/wt-derived tumors with regard to morphology, marker expression, and transcriptional profiles. We demonstrate the applicability of CRISPR for tumor modeling in vivo and ameliorating the need to use conventional genetically engineered mouse models. Furthermore, tumor onset was not only achieved in constitutive Cas9 expression but also in wild-type animals via infection of lung epithelial cells with two discrete AAVs encoding different parts of the CRISPR machinery. While conventional mouse models require extensive husbandry to integrate new genetic features allowing for gene targeting, basic molecular methods suffice to inflict the desired genetic alterations in vivo. Utilizing the CRISPR toolbox, in vivo cancer research and modeling is rapidly evolving and enables researchers to swiftly develop new, clinically relevant surrogate models for translational research. KW - non-small cell lung cancer KW - CRISPR-Cas9 KW - mouse model KW - lung cancer KW - MYC KW - JUN Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230949 SN - 2296-634X VL - 9 ER -