| Popis: |
Large-scale chromatin folding is a key mechanism in gene regulation leading to variable and dynamic gene activation in each cell. Multiple mechanisms contribute to gene regulation including the binding of regulatory factors to specific genomic sequences, chromatin interactions and repositioning of genomic regions relative to specific nuclear landmarks. The aim of this thesis was to explore genome architecture at single allele-resolution either in a gene-specific context in relation to its expression pattern or in a genome-wide context. Upon viral infection, Interferon ? (IFN?), a critical player in innate immunity, is expressed stochastically only in a fraction of cells. I used Sendai virus infected mouse embryonic fibroblasts (MEFs) as a model system to study the influence of nuclear positioning on the stochastic activity of the IFN? gene. A mouse fibroblast cell line (L929) overexpressing the IFN? transcription factor IRF7 was used as a model of constitutive expression, for comparison. IFN? was found to associate with the nuclear lamina in MEFs and L929 cells prior to infection and dissociate upon infection. Induced repositioning of the IFN? gene away from the lamina by TSA treatment, in MEFs, or by IRF7 overexpression, in L929 cells, lead to higher IFN? expression with retention of stochasticity in MEFs, but complete attenuation of stochasticity in L929 cells. Thus, the IFN? association with the lamina correlates with its on/off state and stochastic behaviour. Identification of long-range chromatin interactions by 3C approaches plays major roles in advancing our understanding of gene regulation. However, current methodologies have several limitations. I contributed to the development of a novel genome-wide mapping technology to measure chromatin contacts. In contrast with 3C technologies, this approach is compatible with mapping high multiplicity interactions at single allele and cell resolution, in complex tissues. The dynamic nature of gene regulation lies in diverse features of large-scale chromatin folding. The IFN? study highlights a role of lamina association in the mechanism of gene-specific stochastic transcriptional activation. The novel mapping technology will provide a genome-wide qualitative tool to study the dynamics of chromatin contacts in single cells. |
