Title: Dissecting the biophysical properties and DNA-binding specificity of the ETV6 transcription factor
Abstract: Transcription factors bind to specific DNA sequences and regulate the expression of associated genes. In this thesis, I used several biophysical methods to investigate the structure and DNA-binding specificity profile of the eukaryotic transcription factor ETV6. I investigated the mechanisms by which ETV6 selectively binds its cognate DNA targets. ETV6 prefers DNAs containing a core GGAA motif, unlike other family members where GGA(A/T) is accepted. This specificity toward the fourth adenine is mediated by a single histidine, His396, contrasted with a tyrosine in other family members. Through NMR-monitored pH titrations, I found His396 adopts the neutral Nε2H tautomeric state when ETV6 is both free and DNA-bound. Both structural and surface plasmon resonance binding studies revealed the mutation of His396 to a tyrosine increased its affinity for GGAT-containing DNA, while not diminishing its affinity for DNAs with a GGAA core. Thus I propose that His396 does not serve to enable binding of DNA containing a GGAA core, but rather to disfavour association toward DNAs containing bases other than an adenine at this fourth position. This thereby restricts the transcriptional profile of ETV6 relative to other ETS factors. In a related study I investigated conformational flexibility within the DNA-binding domain of ETV6. All ETS family proteins share a structurally conserved ETS domain, allowing association toward many redundant DNA sequences. These proteins can also recognize subtly different DNA regions to enable transcriptional specificity. I proposed a contributing factor toward the profile of DNAs each family member recognized was domain flexibility. Testing this, I introduced cavity-forming mutations in the ETV6 ETS domain, resulting in increase flexibility determined through relaxation-dispersion NMR experiments. Then, using microarrays, I showed the increase in flexibility weakened selection of near-cognate DNAs, and thereby increased the specificity for cognate DNAs. These flexibility changes predominantly affected selection of DNA bases contacted solely via their phosphodiester backbone, indicating a link between protein flexibility and specificity for the sequence-dependent shape of DNA. Collectively my thesis uncovered many biophysical properties of the ETV6 ETS domain and illustrated how these features contribute to its DNA binding specificity at a molecular level.
Monday, August 29, 2022 at 2:30 pm – 3:30 pm via Zoom
Hosted by: Dr. Lawrence McIntosh