Characterisation of novel regulators of polycombgroup function
Perera, Colombatantirige Pumi Mahika
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Although all cells in a multicellular organism contain the same set of genes, the spatiotemporal expression of these genes needs to be dynamically regulated for morphogenesis and life cycle transitions to take place. Polycomb-group (PcG) proteins are evolutionarily-conserved epigenetic regulators that function – via epigenetic marks such as H3K27me3 and modifications to chromatin structure – to maintain the repression of developmentally-important genes so that these genes are only expressed in the appropriate cells at the appropriate times. This repressive activity of the PcG is antagonised by the trithorax-group (trxG) of proteins. Although they maintain specific patterns of gene repression, PcG proteins are ubiquitously expressed. How their activity is regulated is largely unknown. To identify such regulatory pathways, a genetic screen for modifiers of PcG activity in Arabidopsis was carried out previously using the PcG mutant curly leaf (clf), which has moderately-severe developmental defects due to the ectopic or untimely expression of developmental regulators such as floral homeotic genes and the important flowering time regulator FLOWERING LOCUS T (FT). I characterised three novel potential regulators identified in this genetic screen: the chromatin-associated protein AT-HOOK MOTIF NUCLEAR LOCALISED PROTEIN 22 (AHL22), the 26S proteasome and the novel trithorax-group members ANTAGONIST OF LHP1 1 and 2 (ALP1 and ALP2). I found that the dominant sop-11D mutation is caused by over expression of AHL22 which suppresses the phenotype of clf by reducing FT expression. However, genetic analysis suggests that AHL22 may act in a parallel pathway to the PcG. I showed that mutations affecting diverse subunits of the 26S proteasome reduce the mis-expression of CLF targets and suppress the phenotypes of clf although they do not restore H3K27me3 levels at these targets. Pharmacological inhibition of the proteasome also alleviated the mis-expression of target genes found in clf mutants. Analysis of PcG protein levels following proteasome inhibition suggests that the 26S proteasome antagonises the PcG by degrading the key PcG member EMBRYONIC FLOWER 1 (EMF1), which is likely to be important for implementing target gene repression. Surprisingly, my proteomic analysis showed that the novel trxG members ALP1 and ALP2 are accessory components of a core PcG complex – the Polycomb Repressive Complex 2 (PRC2) – in vivo, suggesting that that ALP1 and ALP2 may antagonise PcG repression by preventing the association of core PRC2 components with accessory components EMF1, LIKE HETEROCHROMATIN PROTEIN 1 and the PHD finger proteins VERNALISATION5 and VIN3-LIKE 1. My results reveal a previously unknown role for 26S proteasomal degradation in the regulation of PcG activity during vegetative development and identify novel in vivo associators of the core PRC2 and point to their role in modulating PcG activity. These results thereby increase our understanding of how the PcG is regulated and serve as a starting point to discover how specificity is given to the PcG mediated repression, either by targeted degradation of EMF1 by various E3 ligases or by different combinations of PRC2 associators.