Defining the functional roles of post-SET domain basic stretch for K36 methyltransferases

• Main Author: Szczepski, Kacper
• Other Authors: Jaremko, Lukasz
• Language: en
• Published: 2021
• Subjects: Methyltransferase; PKMT; NMR
• Online Access: Szczepski, K. (2021). Defining the functional roles of post-SET domain basic stretch for K36 methyltransferases. KAUST Research Repository. https://doi.org/10.25781/KAUST-6H3ZS; http://hdl.handle.net/10754/668950

Posttranslational modifications of nucleosomes play a crucial role for the proper functioning of the cell. One of the modifications called methylation is conducted by a family of SET-domain containing proteins called NSD1, NSD2, and NSD3. Recently, more evidence about the involvement of NSD proteins and their mutations in the oncogenesis has emerged. Various studies have found that post-SET domain and basic post-SET extension of NSD proteins are crucial for nucleosome interactions and for conducting enzymatic reactions. In this thesis, I attempt to define the role of post-SET domain basic extension on DNA binding, using nuclear magnetic resonance and isothermal titration calorimetry. Additionally, I have attempted to establish a methodology for obtaining nucleosomes, which could be used in future studies. NMR results showed that the post-SET domain extension is required for DNA to bind to NSD2 and NSD3 methyltransferases. The mutant form of NSD2, E1099K, exhibits stronger binding to DNA than does the wild type of NSD2. NMR results also show that the transplanted version of NSD3 containing the post-SET extension of NSD2 have an affinity similar to that of the NSD2 wild type. The NSD2 transplanted with a post-SET extension of NSD3 has close to 4 times less affinity towards DNA than does NSD2 wild type. The affinity of NSD3 T1232A and wild type could not be obtained, as the proteins could not be expressed in a sufficient amount for ITC experiments. However, the present literature confirms lower affinity of NSD3 (around 4 times less than NSD2) towards nucleosomes. Based on the empirical data and literature-based information, it can be assumed that post-SET domain basic extension determines the binding affinity of a NSD protein towards DNA. Additionally, a successful methodology for obtaining nucleosomes was established for future studies.