Exploring the interactome of the Drosophila SUMO E3 ligase PIAS

Drosophila Protein Inhibitor of Activated STAT (PIAS) is required for chromatin organisation. PIAS proteins are SUMO E3 ligases that provide target specificity for the addition of small ubiquitin like modifiers (SUMO) to proteins to alter their function. Previous experiments in the Heun laboratory, which used a PIAS overexpression system, identified a relationship between PIAS and XNP (X-linked nuclear protein). Both proteins play a role in silencing transposable elements and likely interact. XNP and ADD1 (ADD (ATRX-DNMT3-DNMT3L) domain-containing protein 1) together make up the two halves of their mammalian homologue, the chromatin remodeller ATRX. XNP and ADD1 have been shown by us and others to be SUMOylated. We hypothesise that the SUMOylation of XNP by PIAS could be essential for its role in chromatin organisation and aimed to study this interaction within a Drosophila model using S2 tissue culture cells and embryos. In this thesis, I defined the interactomes of PIAS, ADD1 and XNP using immunoprecipitation-mass spectrometry (IP-MS) in the presence and absence of SUMOylation. Using CRISPR/Cas9 epitope tagged PIAS in S2 cell lines and primary antibodies against endogenous PIAS, I could confirm an interaction between PIAS and XNP in a SUMO dependent manner. Interestingly, I found PIAS interacts with ADD1 and heterochromatin protein 1 (HP1) in a SUMO independent manner. These interactomes support the hypothesis that SUMOylation of XNP by PIAS is involved in the association of XNP with ADD1 to promote heterochromatin organisation.