Nuclear architecture in differentiating embryonic stem cells

UoM administered thesis: Phd

  • Authors:
  • Fanni Kleinert

Abstract

Gene expression is regulated at various levels, such as transcription, RNA transport and translation. Additionally, it has been shown that chromatin structure, location and dynamics also have an important role in gene expression control. While active gene regions are strongly associated with an open chromatin structure at the surface of the chromosome territory (CT) and a location in the nuclear interior, inactive gene regions seem to be related with a closed structure within the CT and a position at the nuclear periphery. However, it is still unclear how these features are regulated. Importantly, malfunction of gene regulation can impact on health and longevity. Therefore, the aim of this project was to investigate the correlation of gene expression and chromatin organisation both in single gene loci and the MHC gene cluster. The MHC locus has the highest gene density in mammalian cells and contains genes that can be reprogrammed by pro-inflammatory cytokines. The original goal of this project was to label the MHC locus by the Lac operator/repressor (LacO/LacI) approach in order to study chromatin dynamics in living cells using labelled CTs as reference for genome mobility. The thymidine analogue EdU, that can be used to label CTs, was analysed for its effects on cell cycle progression and survival, and revealed to have a strong negative impact on the cells' well-being. In the end, the LacO/LacI-recognition system for live-cell imaging did not succeed, thus FISH analyses were carried out to study chromatin dynamics in snap-shots. The location and structure of the hybridised gene regions were analysed in response to gene activation and inactivation during ESC differentiation to neuroepithelial progenitors (NPs). Single-gene focused experiments were performed using the cell line specific genes, Oct4 and Sox1, together with Gapdh as a housekeeping gene. Even though, the results showed less changes between the days of differentiation on the Gapdh locus, the gene expression profiles for the cell line specific genes did not match with the hypothesised chromatin organisation (see above). However, investigations on the gene-dense MHC locus showed structural chromatin changes that correlated with the activation of genes in this region. Interestingly, ESC treated with TNFalpha were unable to activate NF-kappaB signalling, probably due to the lack of a functional IKK complex.In summary, this project was focussing on the regulation of gene expression by the chromatin architecture and revealed complex chromatin dynamics that are likely to be affected by the sum of genes in a genome region, rather than a single gene.

Details

Original languageEnglish
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Supervisors/Advisors
Award date1 Aug 2015