Ottaviani, D.; (2009) The genomic anchors of the human major histocompatibility complex. Doctoral thesis , UCL (University College London).

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Abstract

Eukaryotic chromatin is organised into a hierarchy of topologically constrained loop structures. Matrix Attachment Regions (MARs) are genomic sequences that mediate the anchoring of chromatin to the insoluble proteinaceous fraction of the nucleus known as the nuclear matrix. Since only a few MARs have been characterised so far, their role in genomic structure and function is not well understood. The aim of this thesis is to use the human Major Histocompatibility Complex (MHC) as model region to provide novel insights into the relationship between chromatin folding mediated by MARs and gene expression. This large locus contains critical genes for immunity and is associated with more diseases than any other genomic region. Classical MHC genes are expressed in a cell type specific pattern, and can be induced by cytokines such as IFN-γ. MARs were identified across the entire MHC in uninduced fibroblasts, IFN-γ induced fibroblasts and B lymphoblastoid cells. Expression array analysis showed that these cell types exhibit different MHC expression profiles. MARs were first isolated treating nuclei with hypertonic buffers followed by nuclease digestion and then mapped by hybridizing them onto a novel tiling path array for the MHC. The suitability of this array platform to study DNA-protein interactions was verified using hybridisations of CIITA-enriched DNA and DNA enriched in H3-K9/K14 acetylation. The findings reveal that MARs are unevenly distributed across the MHC, and that they can be classified into three classes: constitutive, cell type specific and transcriptiondependent. These sequences are mainly positioned in intergenic regions and in close proximity to the MHC class boundaries, subdividing the locus into physical domains. By comparing the position of MARs in uninduced fibroblasts, IFN-γ induced fibroblasts and B lymphoblastoid cells, transcriptional activation of the MHC was found to be associated with a reconfiguration of chromatin architecture resulting from the formation of additional genomic anchors. These results suggest that the dynamic tethering of chromatin is linked to transcriptional regulation.

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