Isolation and characterization of nanobodies that bind the btb domain of patz1 transcription factor

Official URL: https://risc01.sabanciuniv.edu/record=b2767435_(Table of contents)

PATZ1 is a transcription factor that belongs to the ZBTB protein family. It is found to be overexpressed in some human malignancies such as colorectal and testicular tumors, suggesting a proto-oncogenic role. However, this association remains poorly understood. The aim of this study was to generate potent and selective nanobodies against the PATZ1- BTB domain to assist the characterization of its structure and function. For this, a phage display nanobody library was screened for PATZ1-BTB domain binders. Candidate binders were analyzed by ELISA, expressed in E. coli and purified using affinity and size exclusion chromatography. Binding affinities were determined using surface plasmon resonance. Size-exclusion chromatography and a fluorescent two-hybrid assay were used for further assessment of specificity and affinity. The effect of the selected nanobodies on the expression levels of the known PATZ1 target genes (CTH, ETV1) was assessed using real-time PCR. Three nanobody clones with nanomolar affinities were produced as soluble proteins at high concentration. Co-elution analysis showed specific complex formation between these nanobodies and the PATZ1-BTB domain. In the fluorescent two- hybrid assay, these nanobodies showed colocalization with their target protein in the nuclei of live cells. Intracellular stable expression of PATZ1-BTB domain-targeting nanobodies resulted in significant increase of ETV1 gene expression and decrease of the CTH target gene. Our results suggest that the specific binding of nanobodies to the BTB domain of PATZ1 might regulate its gene transcriptional activity. Further investigation will clarify if these nanobodies might be used as PATZ1 modulators.