Abstract
Ubiquitylation plays an essential role in nearly every process in eukaryotic cells, including being a key post-translational modification of histones. Ubiquitylation controls how compact chromatin is and therefore regulates gene transcription. This project focuses on the protein UBR7 (Ubiquitin Protein Ligase E3 Component N-Recognin 7), which has been reported to bind and ubiquitinate histones. UBR7 is a 50 kDa protein with two well-characterised domains, a UBR box and a PHD domain. PHD domains are known to recognize histones, and UBR7 has been shown to bind histone subunits H3 and H2B, with suggestions that the PHD domain is critical. However, it has also recently been reported that the PHD domain is essential for UBR7 to
ubiquitinate H2B’s C-terminal tail. This raises the question of whether the PHD domain is acting as a RING (Really Interesting New Gene) domain and facilitating Ub transfer, making UBR7 an E3 ligase. This project aimed to further establish the roles of the UBR and PHD domains in binding and E3 ligase activity, and to establish purification protocols for UBR7, the PHD domain and histones.
Several new constructs were designed based on sequence and structure analysis to improve the expression and solubility of UBR7. Based on previous work, two constructs with differing lengths of the PHD domain (PHD1, PHD2) were also made. Purification protocols were established and optimized for UBR7, PHD1, PHD2, and three forms of histones. Various other components were purified using established methods. These proteins were then utilized in activity assays designed to compare PHD and UBR7 activity. However, no evidence of UBR7s E3 ligase activity was found. Pulldown assays were also done to establish the role of the PHD domain in histone interaction. The pulldowns appear to confirm that UBR7 interacts with H2B and H3, but the results are inconclusive. While these results are overall unexpected, the findings suggest avenues for further study. There’s potential for research on the CTR1 region and its impact on activity, as well as examining the reason behind the insolubility of UBR7. The establishment of various purification protocols also makes ITC a possibility.