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.
| Date of Award | Mar 2021 |
|---|
| Original language | English (US) |
|---|
| Awarding Institution | - Biological, Environmental Sciences and Engineering
|
|---|
| Supervisor | Lukasz Jaremko (Supervisor) |
|---|