The use of a promiscuous biotin ligase to identify partners of the HOXD4 transcription factor
Teo, Erica Mei Ling
Date of Issue2015
School of Biological Sciences
Hox genes specify the development of structures along the anterior-posterior axis of bilaterian embryos. Following their discovery, it was shown that they encode homeodomain transcription factors that control diverse developmental processes, embryo patterning, and physiological processes. Hox gene deregulation is involved in many diseases and current methods to identify Hox protein partners have their limitations. Further insight into Hox protein function would be gained by identifying novel interaction partners. In this study, I tested the feasibility of using the BioID method for identifying novel protein partners of the vertebrate HOXD4 protein. BioID makes use of a mutant "promiscuous" biotin ligase derived from the E.coli birA gene. By fusing the region coding for the mutant BirA (BirA*) in frame with that of the zebrafish hoxd4a gene and expressing the fusion construct in mammalian HEK293T cells, proteins in close association with the fusion protein are expected to be biotin labelled over a defined biotinylation period. Using this approach, potential partners of the HOXD4A protein could be pulled down. Known partners of Hox proteins such as Pbx are expressed in HEK293T cells, and biotinylated, overexpressed PBX was pulled down, thus validating my approach.
DRNTU::Science::Biological sciences::Molecular biology
Final Year Project (FYP)
Nanyang Technological University