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Identification and manipulation of chromatin barriers in transcriptional reprogramming
Identification and manipulation of chromatin barriers in transcriptional reprogramming
Master transcription factors are cell fate determining factors that can force new identities onto cells by evoking distinct gene transcription patterns. This potential can be detrimental for an organism when activated erroneously, which is why genes of master transcription factors have to be regulated tightly. Chromatin features like DNA methylation have been implied in transcriptional regulation, the direct causalities and the underlying mechanisms are however still largely unclear. Here I investigated the role of the neurogenic transcription factor Sexdetermining-region-y-box 1 (Sox1) in directing neural stem cell identity. I employed a targeted trans-activating domain (dCas9-VP64) to induce Sox1 expression in vitro in neural progenitor cells (NPCs) and characterized the invoked phenotypic changes. Inducing Sox1 expression in NPCs restored their neuronal differentiation potential, and transcriptome analysis revealed a shift in cell identity towards neural stem cells (NSCs), underlining the role of Sox1 as cell fate determining factor. Analysis on single cell basis however revealed that only a small subset of NPCs responded to the targeted gene induction with Sox1 upregulation. Using a GFP knock in as reporter, I separated responsive from unresponsive cells and investigated differences in chromatin features at the Sox1 promoter. I identified DNA methylation as a strong barrier against trans-activation by combining transcriptional engineering and epigenome editing via dCas9-Tet1. Furthermore, I found similar barriers at the promoters of other master transcription factor genes, including Oct4 and Nkx2-2. Lastly, I employed a screening approach to identify potential regulatory regions distal of the Sox1 gene. By transducing NPCs with a gRNA library of high complexity, I was able to identify targeting sites for dCas9-VP64 in the locus of Sox1 that have the potency to induce gene transcription even outside of the promoter. In conclusion, I have confirmed Sox1 as a neurogenic master transcription factor and identified mechanisms that control expression of this gene. These findings could serve to optimize future trans-activation approaches and underline the importance of chromatin features in the regulation of cell fate determining factors.
Chromatin, CRISPR/Cas9, cell identity, reprogramming, Sox1
Baumann, Valentin
2019
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Baumann, Valentin (2019): Identification and manipulation of chromatin barriers in transcriptional reprogramming. Dissertation, LMU München: Graduate School of Systemic Neurosciences (GSN)
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Abstract

Master transcription factors are cell fate determining factors that can force new identities onto cells by evoking distinct gene transcription patterns. This potential can be detrimental for an organism when activated erroneously, which is why genes of master transcription factors have to be regulated tightly. Chromatin features like DNA methylation have been implied in transcriptional regulation, the direct causalities and the underlying mechanisms are however still largely unclear. Here I investigated the role of the neurogenic transcription factor Sexdetermining-region-y-box 1 (Sox1) in directing neural stem cell identity. I employed a targeted trans-activating domain (dCas9-VP64) to induce Sox1 expression in vitro in neural progenitor cells (NPCs) and characterized the invoked phenotypic changes. Inducing Sox1 expression in NPCs restored their neuronal differentiation potential, and transcriptome analysis revealed a shift in cell identity towards neural stem cells (NSCs), underlining the role of Sox1 as cell fate determining factor. Analysis on single cell basis however revealed that only a small subset of NPCs responded to the targeted gene induction with Sox1 upregulation. Using a GFP knock in as reporter, I separated responsive from unresponsive cells and investigated differences in chromatin features at the Sox1 promoter. I identified DNA methylation as a strong barrier against trans-activation by combining transcriptional engineering and epigenome editing via dCas9-Tet1. Furthermore, I found similar barriers at the promoters of other master transcription factor genes, including Oct4 and Nkx2-2. Lastly, I employed a screening approach to identify potential regulatory regions distal of the Sox1 gene. By transducing NPCs with a gRNA library of high complexity, I was able to identify targeting sites for dCas9-VP64 in the locus of Sox1 that have the potency to induce gene transcription even outside of the promoter. In conclusion, I have confirmed Sox1 as a neurogenic master transcription factor and identified mechanisms that control expression of this gene. These findings could serve to optimize future trans-activation approaches and underline the importance of chromatin features in the regulation of cell fate determining factors.