de Boer, J.; (2005) The role of nuclear protein HBP1 in differentiation and proliferation using conditionally deficient mice. Doctoral thesis , University of London.

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Abstract

HBP1 (HMG box containing protein 1) is a member of the high mobility group chromosomal proteins. The HMG box contained in this protein binds DNA in a sequence specific manner and other domains in the protein allow its participation in protein-protein interactions that can have profound effects in the regulation of chromatin structure and, as a consequence, gene expression, proliferation and differentiation programmes. The protein also contains a novel domain called AXH that is also present in Ataxin-1 (ATX1), a human protein responsible for spinocerebellar ataxia type 1. The function of the AXH domain is unknown. An initial study to define the function of the AXH domain was to measure its structural properties and stability. This was achieved by producing protein constructs spanning the AXH modules of ATX1 and HBP1 and comparing their properties. This led to the identification of the minimal region sufficient for forming independently folded units (domains). It was also shown that the AXH of ATX1 contains a dimerization domain whereas the AXH of HBP1 stays as a monomer. In order to define the subcellular localisation of HBP1 the protein was tagged with enhanced yellow fluorescent protein (EYFP) and expressed in HEK293 cells. Subcellular localization of the respective fusion proteins was analyzed by direct fluorescence microscopy. EYFP-tagged wild type HBP1 was located exclusively in nuclei, while EYFP alone was equally distributed throughout the cells. To identify the NLS, several HBP1 deletion mutants were tagged with EYFP. Mutant HBP1 that contained only the HMG box also localized exclusively in nuclei, while the deletion of the HMG box resulted in the loss of preference for nucleus. These results indicate that HBP1 is a nuclear protein and that the NLS is located within the HMG box of HBP1. To assess the function of HBP1, standard gene-targeting techniques were used to produce a mouse in which the exons coding for the HMG box, an essential region of HBP1, is flanked by loxP sites (floxed), so that this domain can be deleted specifically at will by directing the expression of Cre in a tissue specific manner. Using the above strategy two mouse lines were generated: one carrying HBP 1 alleles with floxed HMG box coding exons to be used in conditional deletion studies and one with a constitutive deletion of these exons (AHMG). Furthermore, we generated two Cre transgenic mice lines that allow the deletion of floxed gene segments either only in the lymphoid compartment (hCD2-iCre) or in the entire haematopoietic compartment (Vav-iCre). Crossing these Cre transgenic lines to mice carrying the floxed alleles will allow the inactivation of genes only in certain tissues so that biological questions can be answered with increased specificity. Mice homozygous for the gene deletion (HBP1AHMG/AHMG mice) were viable, fertile, and have normal numbers and percentages of haematopoietic lineages. However, T lymphocytes from HBP 1AHMG/AHMG animals proliferate at a significantly higher rate in response to anti-CD3 in vitro. Initial experiments indicate that the increased proliferation by HBP1AHMG/AHMGT lymphocytes might be due to the fact that the cells are in a "poised for activation" state and, thus, predisposed for immediate response to stimulation.

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