Work Package 5

Identification and characterization of in vivo interacting signaling molecules and transcription factors by biotin tagging

Introduction
Identification of in vivo interacting molecules from small amounts of tissues is limited by the available biochemical separation and analytical techniques. We have demonstrated that in vivo biotinylation of the GATA-1 transcription factor in cultured cells by the bacterial BirA biotin ligase provides a novel and powerful strategy allowing isolation of protein complexes containing the biotinylated protein through a single purification step. We will explore the possibilities of this approach to identify transcription factor complexes in the developing nervous system and hematopoietic system of the mouse. The POU domain transcription factor Oct-6 plays a pivotal role in peripheral nerve development and regeneration through timing and orchestration of the Schwann cell myelination program. The biological function of Oct-6 critically depends on physical interactions with other nuclear proteins and its target DNA sequence, most of which are still unknown. Similarly, we will identify TR2/TR4 interacting factors in erythroid differentiation as it occurs in the developing and adult animal. TR2 and TR4 are putative g-globin suppressors and thus prime molecular targets for pharmaceutical intervention in thalassaemias.

Objective
The key objectives of this project are: 1) to develop a generic tool to identify protein-protein interactions for any protein in any organ system of the developing and full-grown organism to develop a generic strategy and tools to isolate large protein complexes from complex biological samples. 2) to validate this approach by obtaining a full understanding of Oct-6 in Schwann cell biology through identification of Oct-6 interacting proteins and their regulatory targets in the peripheral nervous system of the developing organism. 3) to analyse TR2/TR4 associated proteins in vivo. 4) generate ""cross-hybridisation" libraries and databases of these sequences (and their target genes) and to cross-analyse libraries to uncover developmental and differentiation pathways.

Approach
As an example, the Oct-6 studies are described here. We will develop tools to isolate and identify Oct-6 interacting proteins through a single purification step, based on the high affinity of streptavidin for in vivo biotinylated Oct-6 protein, and identification of interacting proteins through liquid chromatography followed by mass spectrometry (LCMS). In vivo biotinylation of Oct-6 is dependent on expression of the bacterial biotin ligase (BirA) and a short target peptide engineered at the carboxy terminus of the Oct-6 polypeptide. We will generate mice that express the BirA gene from the endogenous ROSA26 locus. As this locus is active in every cell type examined, the resulting mouse will express the BirA gene in all its cells, making this mouse of great value for all studies involving in vivo biotinylation of target peptides. Proteins will be isolated from sciatic nerves dissected from neonatal mice carrying the ROSA26/BirA and tagged Oct-6 alleles, purified using streptavidin conjugated para-magnetic beads and identified through LCMS. In addition, paraformaldehyde cross-linked Oct-6 containing chromatin will be isolated and the DNA sequences will be cloned to generate target libraries for Oct-6.

Keywords: transcription factor, cell differentiation, Biotinylation, affinity chromatography

Links with other parts of the program: This research line is related to WP9 and 10 in which the role of developmental regulators in stem cells is investigated. There is also a link with WP6 as we will generate single chain antibodies against a number of proteins identified in this research line to further investigate their biological role.

Participants: Meijer, Grosveld