Genomic targeting and function of polycomb repressive complex 2 and ISWI chromatin remodelers

Barišić, Darko. Genomic targeting and function of polycomb repressive complex 2 and ISWI chromatin remodelers. 2017, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_12459

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In eukaryotes, chromatin provides a way to compact the genetic material into the confined space of a nucleus. It is also a means to store the same genetic information in different chromatin states. Alteration of these states is enabled by chromatin modifying and remodeling machineries – enzymes that utilize a diverse range of structural changes to chromatin. Despite their apparent importance in gene regulation, it is unclear how they facilitate the transition between chromatin states. Within two distinct projects, we aimed to (1) decipher how chromatin modifying complexes, namely the Polycomb group proteins, are targeted to chromatin and (2) how chromatin remodelers, specifically the ISWI remodeling complexes, change chromatin structure.
Polycomb group proteins assemble as chromatin-modifying complexes that maintain the memory of the silent transcriptional state, in part through methylation of lysine 27 on histone H3. Despite their established importance during development, it is largely unclear how these complexes are recruited to specific target genes and how they impair transcription. In flies, Polycomb is recruited by Polycomb response elements that are abundant in various DNA-binding factor motifs. However, the contribution of individual motifs is not yet resolved. In mammals, equivalents of Polycomb response elements are not yet characterized. Here, we aimed to dissect Polycomb-mediated silencing in the mouse genome by identifying DNA determinants of Polycomb recruitment and investigating the role of Polycomb recruitment in transcriptional silencing. More specifically, we developed an assay to test many DNA sequences with various sequence properties for their ability to drive PRC2 recruitment in mouse embryonic stem cells. The assay enabled integration of hundreds of sequences into a defined genomic location in parallel. We found that high density of unmethylated CG motifs within a synthetic backbone sequence is sufficient to recruit PRC2. Furthermore, to link PRC2 recruitment with transcriptional repression, we used CRISPR/Cas9 technology to delete the core PRC2 (Eed) component and monitored the transcriptional response by RNA-seq. Upon depletion of global H3K27me3 levels, we observed no significant changes in gene expression in mouse embryonic stem cells but global deregulation of PRC2 targets during differentiation into neuronal progenitors. These results indicate that recruitment of PRC2 and subsequent H3K27 methylation is important for cell-fate transition, but not required for gene repression in mouse embryonic stem cells.
For the second project, we were interested in chromatin remodelers (ISWI) and their role in regulating chromatin structure. Chromatin remodelers are known to use the energy of ATP hydrolysis to evict, slide and reposition nucleosomes, yet we do not fully understand how nucleosome positioning and occupancy affects transcription factor binding. To this aim, we deleted Snf2h, the ATPase subunit of the ISWI chromatin remodeling family, in mouse embryonic stem cells. The Snf2h knockout mouse embryonic stem cells are viable with unchanged expression of pluripotency markers, which is exciting as this is the first viable knockout of an ATPase remodeler subunit. To determine global changes upon deletion of Snf2h, we monitored nucleosome positioning, chromatin accessibility and transcriptional response in Snf2h knockout cells using MNase, ATAC and RNA sequencing, respectively. Extensive data analysis revealed global changes in nucleosome positioning proximal to transcription start sites and transcription factor motifs. Analyzing nucleosome positioning and chromatin accessibility data, we identified transcription factors that require Snf2h to bind their target sites, such as CTCF. It seems that in the absence of Snf2h, nucleosomes cannot be evicted from CTCF motifs, which in turn results in loss of CTCF binding. Taken together, these results indicate that ISWI complexes enable transcription factor binding, at both promoters and distal regulatory regions, by sliding of motif-bound nucleosomes.
Advisors:Schübeler, Dirk and Wutz, Anton
Faculties and Departments:09 Associated Institutions > Friedrich Miescher Institut FMI > Epigenetics > Gene regulation in chromatin (Schübeler)
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:12459
Thesis status:Complete
Number of Pages:1 Online-Ressource (X, 139 Seiten)
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Last Modified:08 Feb 2020 14:47
Deposited On:22 Feb 2018 14:34

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