UC San Diego

Protein interaction studies using chromatin immunoprecipitation (ChIP) to map transcription factor targets genome-wide has led to an increased understanding of the regulation and function of these transcription factors. However, the current methods are hindered by inefficient purification systems due to the lack of specific antibodies. In this thesis, we developed the BLRP -biotinylation system to mark two specific transcription factors that are critical for the transcriptional regulation of the hypothalamic-pituitary-gonadal (HPG) axis, SF-1 and Six6, to allow for ChIP or ChIP-seq analysis. We expressed the bacterial BirA biotin ligase in hypothalamic and pituitary cells, which allows the efficient biotinylation of our tagged proteins that contain a small biotin ligase recognition peptide (BLRP) tag. We showed that these BLRP-tagged proteins are expressed and retain functions such as DNA-binding and transcriptional regulation. Taking advantage of the strong noncovalent interaction between biotin and streptavidin, we showed that the biotinylated tagged proteins were efficiently pulled-down with this single step purification method in vitro. Lastly, we used the pulled-down fraction for ChIP analysis to study the binding of SF-1 and Six6 on gene targets in gonadotrope L[Beta]T2 and hypothalamic GT1 -7 cell lines. Therefore, the BLRP-biotinylation system provides a novel method to specifically label and purify proteins, and ultimately allows for the identification of complex protein-DNA and protein-protein interactions in vitro