Investigation into the role of heterochromatin protein 1 gamma (HP1[gamma]) in gene regulation in mammals

Abstract

Heterochromatin protein 1 (HP1) was identified as a key component of the condensed DNA surrounding centromeres in most eukaryotic cells. Genes placed in close proximity to such constitutively dense heterochromatin regions were found to be stochastically silenced in a proportion of the cells leading to variegation of expression, a classical epigenetic phenomenon known as position effect variegation (PEV). Mutagenesis screens identified HP1 and SUV39H as powerful suppressors of variegation which were necessary for heterochromatin-mediated PEV. SUV39H was found to methylate the histone H3 tail at lysine 9 at heterochromatin providing a binding site for HP1. This discovery provided the first direct evidence for a histone or epigenetic code in which the ‘writer’ of the code on chromatin would be the SUV39H and the ‘reader’ HP1. This mechanism was subsequently found to be conserved from S. pombe to humans. A mechanism which shares features with PEV has also been implicated in the pathogenesis of repeat-expansion diseases, such as Friedreich’s ataxia, in which the affected genes are aberrantly silenced. In mice there are 3 highly conserved HP1 isoforms, HP1α, HP1β and HP1γ. HP1α and β are found by microscopy to be localised to constitutive heterochromatic condensed regions in the nucleus whereas HP1γ has a pan-nuclear distribution and is therefore implicating in regulating euchromatic genes. There is a paucity of data examining how and where HP1γ regulates gene expression in vivo at the genome-wide level. This thesis employed knockout and knockdown strategies to investigate this. In view of the early lethality of mice in which HP1γ has been deleted by homologous recombination, the function of HP1γ was studied here by establishing mouse embryonic fibroblast cell lines. Immunoflourescent microscopy revealed for the first time that HP1γ was necessary for the localisation of HP1β at constitutive heterochromatic regions in the nucleus in most cells. This delocalisation of HP1β was associated with aberrant upregulation of the repetitive major satellite DNA associated with pericentromeric heterochromatin implying for the first time that HP1γ plays an important role in maintaining centromeric heterochromatin in a silenced state thought to be important for the maintenance of genome integrity. Strikingly, analysis of the transcriptome revealed a large number of genes (4293) to be dysregulated in male cells compared to females (1186) where the effect of HP1γ deficiency resulted in aberrant expression of immune related genes that would normally be repressed. This sexually dimorphic effect was investigated further by studying the effect of HP1γ deficiency on the subset of 176 genes found to differ in expression between normal males and females. Moreover, HP1γ in males was found essential for maintaining the relative repression of 114 genes in males compared to females, suggesting that the Y chromosome interacts with HP1γ to reduce the expression of these genes. In summary, a novel function for HP1γ in repressing pericentromeric DNA and in maintaining sexually dimorphic gene expression was discovered.