The robust regenerative capacity of muscle stem cells (MuSCs) and their progenitors depends on their ability to undergo rapid and vast changes to their transcriptome during cell state changes. While transcription factors and epigenetic remodelling proteins are critical to render genes permissive for transcription, often these genes are found to have paused promoter proximal RNA Polymerase II (Pol II) which remains in a rate-limiting poised state. Indeed, while prior studies have shown poised Pol II is often regulated by the Negative Elongation Factor (NELF) to induce rapid changes in gene expression, the specific need for NELF in somatic stem cell populations has not been previously examined. In this thesis, we identify a specific requirement for NELF-dependent promoter proximal Pol II pausing in proliferating myogenic progenitors. Here, NELF stabilizes nascent transcripts associated with the paused RNA Pol II at genes required to maintain muscle progenitors in cell cycle. This promotes expansion of the pool of myogenic progenitors required to adequately repair damaged skeletal muscle. Our molecular analysis suggests that in proliferating progenitors, NELF-bound Pol II ensures the stabilization of transcripts, and continued expression of genes that prevent p53-mediated cell cycle withdrawal and terminal differentiation. Unexpectedly, this work revealed a previously unappreciated contribution of proliferating myogenic progenitors to replenish the stem cell niche in support of MuSC self-renewal during skeletal muscle regeneration. Based on our results, new therapeutic avenues which could treat muscle wasting disease are also discussed.

Advancements of antiretroviral therapies (ART) have made significant strides in reducing human immunodeficiency virus (HIV) viral loads in patients to undetectable levels. Upon interruption of ART, viral load rebounds and AIDS symptoms return. Latent reservoirs of virus are responsible for this phenomenon because they contain integrated provirus, which is transcriptionally silent, thus unaffected by ART and hidden from host immune surveillance. A commonly proposed mechanism for HIV latency is the presence of host cell transcription factors that lead to transcriptional silencing. CD4+ T cells and other immune cells, whether due to their subset phenotype, activation state, or stage in development, will vary in their battery of transcription factors. Of particular interest is Bcl11b, a critical transcription factor involved in the commitment to a T-cell fate during thymocyte development that has recently been shown to play a role in silencing HIV-1 transcription. Bcl11b is required for inhibiting the development of natural killer cell-like traits during the early development of T cells. The repressive role of this zinc-finger transcription factor has recently been shown to inhibit HIV-1 transcription in the context of microglial cells via recruitment of chromatin remodeling factors. Also, Bcl11b has been shown to interact with other HIV-1 transcriptional silencing factors such as NuRD and NCoR. Preliminary mass spectrophotometry results have pointed to a physical interaction of Bcl11b with NELF, another proven repressive factor of HIV transcription. We hypothesize that Bcl11b represses HIV transcription and is recruited to the HIV-1 long terminal repeat (LTR) through a paused RNA polymerase II complex, contributing to the establishment and maintenance of latency. Our studies confirm Bcl11b's repressive role in T cells, and investigate the mechanism with NELF. Transfection of HEK293T cells with HIV-LUC shows nearly 50% reduction in HIV transcription in the presence of Bcl11b, and analysis of viral protein output by p24 ELISA confirms this result. Furthermore, when co-transfected with NELF-B, the two transcription factors lead to nearly 90% reduction in HIV transcription. Results suggest that these factors cooperate to repress HIV transcriptional elongation. Protein and chromatin immunoprecipitations (ChIP) were also performed to see a direct interaction between the two transcription factors and the HIV LTR. Physical interaction of the two factors was not witnessed, while ChIP analysis shows enrichment of RNA polymerase II at the transcriptional start site suggesting Bcl11b increasing RNA polymerase II pausing. We conclude that Bcl11b plays a repressive role in HIV transcription through promoter-proximal pausing with a synergistic effect with NELF, but a yet to be identified factor is responsible for the coordination of the two factors. As an important T-cell commitment factor, Bcl11b may play an important role in establishing and maintaining cellular latency through transcriptional repression via a complex with NELF. Confirming Bcl11b's role as a repressive transcription factor and providing further support for a synergistic role with NELF, could highlight a new target for therapeutic strategies against the elusive latent reservoir.

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Koordinierte Phosphorylierung der C-terminale Domäne von RNA Polymerase II (RNAPII) ist essentiell für eine effiziente Kupplung von naszierender RNA Synthese und co-transkriptionalem RNA Prozessierens. Zirkuläre RNAs (circRNAs) sind eine neue Klasse von RNA Molekülen mit hoher Prävalenz in neuronalen Zelltypen. Die Biogenese von circRNAs ist noch ungeklärt, insbesondere die Frage warum das Intron upstream der circRNA während der Transkription des circRNA Exons zurückbehalten wird um Rück-Spleißen zu ermöglichen. Verschiede Belege suggerieren, dass unzulängliche Rekrutierung des Spleiceosoms zur circRNA Formation führen kann. In dieser Arbeit untersuche ich die Mechanismen die zu Defekten in der Erkennung und des Spleißens des Introns upstream der circRNA führen. Mit diesem Ziel erfasste ich die genomweite Verteilung von chromatinassoziierter RNAPII mit verschiedenen Phosphorylierungen, sowie Spleißfaktoren und Transkriptionsreglern mittels ChIP-seq in neuronaler Differenzierung von murinen embryonalen Stammzellen zu dopaminergen und Motoneuronen. Während der gesamten Differenzierung, aber insbesondere in den differenzieren Neuronen, konnten circRNAs detektiert werden. In meiner Arbeit finde ich, dass circRNAs detektiert werden, wenn Gene hohe Levels an mRNA exprimieren und, dass die Produktion von circRNA mit einer Dysbalance zwischen dem Laden der RNA-Polymerase II auf die DNA und dem Rekruitieren der Splice-Maschinerie zusammen hängt. Um funktionell mit den Pausier-Mechanismen der RNA-Polymerase II zu interferieren, habe ich einen ''promotor-proximal-pausing'' Faktor depletiert. Dabei stellte ich fest, dass diese Depletion genügt, um die circRNA Levels in embryonalen Stamzellen zu erhöhen. Die Ergebnisse die in dieser Arbeit gezeigt werden, beschreiben die Beteiligung des Pausierens der RNA-Polymerase II and der Formierung von circRNAs. / Coordinated phosphorylation of RNA polymerase II (RNAPII) C-terminal domain is essential for efficient coupling of nascent RNA synthesis with co-transcriptional RNA processing events. Circular RNAs (circRNAs) are a novel class of RNAs whose biogenesis remains ill understood, namely why the upstream intron is not spliced before the circRNA-exon is fully transcribed. Indirect evidence suggests that altered spliceosome recruitment can lead to circRNA formation. To investigate the mechanisms that may be involved in deficient recognition and splicing of introns upstream of exons included in circRNAs, I mapped the chromatin occupancy of RNAPII phosphorylated forms, splicing factors, and transcription regulators by ChIP-seq during mouse ESC differentiation to dopaminergic and spinal motor neurons. CircRNAs are detected throughout differentiation, peaking in differentiated neurons, as expected. I found that circRNAs are detected when genes express high levels of mRNA, and that circRNA production is associated with an imbalance between RNAPII loading and recruitment of the splicing machinery. To mechanistically interfere with pausing mechanisms, I depleted an RNAPII promoter-proximal pausing factor, and found that it was sufficient to increase the formation of circRNAs in stem cells. Results shown in this work implicate RNAPII regulation mechanisms in the formation of circRNAs.