The role of polyhomeotic in polycomb group-mediated regulation of the tumour suppressor P16INK4A

• Main Author: de Breed, L. A.
• Published: University College London (University of London) 2010
• Subjects: 570
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.564979

The INK4A tumour suppressor gene is negatively regulated by the Polycomb Group (PcG) complexes, which plays an important role in stem cell- and cancer biology. Polycomb Repressive Complex 1 (PRC1) members posterior sex comb protein BMI1 and polycomb proteins CBX7 and CBX8 have been shown to be directly involved in repression of INK4A and delay of senescence, whereas evidence from mouse genetics suggests that polyhomeotic protein Ph2 is also required. However, it is still unknown why the mammalian PRC1 complex has so many possible permutations compared to the basic system in Drosphila and how this relates to the regulation of a single target gene. The aim was therefore to define the requirement and function of the three human polyhomeotic members, HPH1, -2 and -3, for INK4A repression. It was found that depletion of all HPH proteins by shRNA-mediated knockdown in primary human fibroblasts results in derepression of INK4A and premature senescence. In contrast, overexpression of HPH proteins leads to activation of INK4A accompanied by loss of PRC1 binding and PcG-associated histone H3K27 trimethylation at the locus. These diametrically opposite findings suggest that a fine balance in HPH protein levels is required for PcG repression. Furthermore, biochemical investigation of the interaction between CBX and HPH proteins did not demonstrate any level of specificity, but emphasised the RNA binding capacity of both proteins, which was further investigated. In addition, by tandem affinity purification and mass spectrometry analysis it was found that HPH2 interacts with both PRC1 components and novel proteins, which were studied for involvement in INK4A repression. In summary, this thesis demonstrates an essential role for the HPH proteins in the regulation of INK4A and provides novel insights into their functional contribution to PcG repression.