Presentation description

ZBTB4 belongs to a family of methyl-CpG binding proteins (MBPs), which are specialized transcription factors (TFs) responsible for regulating gene expression of key cellular pathways through the readout of the 5-methylcytosine (5mC) epigenetic marker. ZBTB4 has two zinc finger (ZF) domains: the three N-terminal (N-term) ZFs conserved between other members in the ZBTB MBP family capable of epigenetic readout, and the C-terminal (C-term) ZFs which are unique to ZBTB4. While the DNA recognition by the N-term ZFs has been previously explored and characterized, the functions of the C-term ZF domain remain to be elucidated. Given that a subset of ZBTB4 gene targets does not correspond to the N-term ZFs DNA recognition, we hypothesize that the C-term ZFs may play a role in readout at these unique gene targets. Therefore, the binding scope of DNA-protein interactions by the C-term ZFs will provide insight into the molecular mechanism by which ZBTB4 exerts gene expression regulation. A combined biophysical approach was used to attribute functions and DNA binding partners to the C-term ZFs and will provide a mechanistic model for understanding the transcriptional readout by ZBTB4. Our preliminary findings using solution NMR and electrophoretic mobility shift assays (EMSAs) show that the C-term ZFs of ZBTB4 are multimodal nucleic acid binders. One key step in determining the DNA binding scope of the C-term ZF domain is the identification of an optimal construct capable of high affinity binding. Therefore, the focus of these studies has been the design, production, purification, and evaluation of ZBTB4 C-term ZF constructs to establish the required protein elements for binding and recognition. Combined, these results begin to illuminate the C-term ZF domain function in transcriptional regulation in addition to and independent from the epigenetic readout provided by the N-term ZFs.