Presentation description

ZBTB33, ZBTB38, and ZBTB4 constitute a methyl-CpG binding protein (MBP) family who share a conserved set of three zinc fingers (ZFs) that play crucial roles in binding methylated DNA and mediating downstream transcriptional processes. ZBTB4 is comprised of two sets of ZF domains, an N-terminal (N-term) four ZF domain, and a unique C-terminal (C-term) two ZF domain. We have determined the ZBTB4 C-term ZFs exhibit broad-range nucleic acid binding. Interestingly, a subset of the genomic sites bound by ZBTB4 contain guanine (G) rich sequences linked to the potential formation of G-quadruplex (G4) non-canonical DNA structures, which are not recognized by the N-term ZF domain. Our lab has discovered that ZBTB4 can bind G4s through the C-term ZF domain, and given that G4s regulate transcriptional outcomes in disease states such as cancer, these findings potentially expand the role of ZBTB4 in modulating gene expression and cellular processes. We aim to shed light to the molecular nature of the novel binding interaction between ZBTB4 and G4s. To assist in this investigation, I successfully implemented a Palindrome-Nicking Dependent Amplification (PaNDA) protocol, a new enzymatic method that produces high-quality single-stranded DNA (ssDNA) oligonucleotides that will be used to carry out ZBTB4:G4 binding studies. I aim to characterize and validate the quality of the reaction products for further troubleshooting of a purification scheme. In parallel, I produced and purified different ZBTB4 C-term ZF constructs and began investigating their binding with a G4 DNA target. My efforts in ssDNA synthesis and purification, and in the production and isolation of ZBTB4 C-term ZFs constructs, will aid in uncovering the mechanisms by which ZBTB4 recognition of G4s may regulate transcriptional regulation.