Characterization of Hematopoietic Transcription Factor Complexes in Erythroid Cells

textabstractEfficient tagging methodologies are an integral aspect of protein complex characterization by proteomic approaches. Due to biotin’s very high affinity for avidin and streptavidin, biotinylation tagging offers an attractive approach for the efficient purification of protein complexes. The very high affinity of the biotin/(strept)avidin system also offers the potential for the single-step capture of lower abundance protein complexes, such as transcription factor complexes. The identification of short peptide tags that are efficiently biotinylated by the bacterial BirA biotin ligase, led to an approach for the single-step purification of transcription factor complexes by specific in vivo biotinylation tagging. A short sequence tag fused Nterminally to the transcription factor of interest is very efficiently biotinylated by BirA co-expressed in the same cells, as was demonstrated by the tagging of the essential hematopoietic transcription factor GATA-1. The direct binding to streptavidin of biotinylated GATA-1 in nuclear extracts resulted in the single-step capture of the tagged factor and associated proteins, which were eluted and identified by mass spectrometry. This led to the characterization of several distinct GATA1 complexes with other transcription factors and chromatin remodeling co-factors, which are involved in activation and repression of gene targets. Thus, BirA-mediated tagging is an efficient approach for the direct capture and characterization of transcription factor complexes