Investigating the protein-protein interactions of ATRX/Daxx in an ALT tumor suppressor complex
Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
This project investigates the interaction between the Death-domain asssociated protein 6 (Daxx) and the chromatin remodeler ATRX. These proteins form an important tumor suppressor somplex that specifically inhibits the homologous recombination-based Alternative Lengthening of Telomeres (ALT) pathway of tumorgenesis. It has been reported that a small, globular, domain of Daxx (the Daxx helical bundle, or DHB) interacts with ATRX, however, the interacting region/ s of ATRXare poorly defined. The aim of thos project was to identify ATRX-derived peptide sequences that bind to the DHB domain of Daxx, as well as prepare for future experiments aiming to repeat this experiment with more comprehensive Daxx xonstructs which include the hostine binding domain, HBDm in complex with a H4/H3.3 heterpdimer. To this end. a Daxx construct corresponding to the DHB domain (Daxx46-144) and histones H4 and H3.3 were expressed in Escherichia coli and purified. A randomized phage peptide library and phage display was then employed to generate a consensus sequence of peptides with hifh affinity for the Daxx DHB domain, to be aligned- and compared to the full-lenth sequence of ATRX in an attempt to identify the ATRX region/s housing the Daxx interaction.
Several high-affinity peptides were obtained with this approach; however, these were too few or disparate to generate a comprehensive consensus sequence. Instead, individual peptides were aligned- and compared to the full-length ATRX, yielding one candidate sequence, D10, which partly corresponds to a conserved motif in the c-terminal helicase domain of ATRX. This sequence may indicate the site of the Daxx interaction; however, more research is required tp investigate the significance of the peptide.
Place, publisher, year, edition, pages
Daxx, ATRX, chromatin, hetrochromatin, epigenetics, phage display
Engineering and Technology
IdentifiersURN: urn:nbn:se:kth:diva-150565OAI: oai:DiVA.org:kth-150565DiVA: diva2:744727