The EuTRACC consortium proposes to determine the regulation of the genome by mapping the regulatory pathways and networks of transcription factors that control cellular functions. EuTRACC will be part of and work in close collaboration with the International Regulome Consortium (IRC), a worldwide network that will address the regulation of genome function at a higher level by mapping the genetic regulatory nodes and networks that control the activity of embryonic stem cells and the process of differentiation to specific cell types. This Integrated Project will focus on mapping the genetic circuitry that controls the formation of neural tissues and the blood system. The project will utilize genetics, proteomics and genomics tools in the mouse, zebrafish and Xenopus model organisms.
We will use our experience and novel methodology for in vivo protein tagging, protein affinity purification, and mass spectrometry, as well as mouse embryonic stem (mES) cell modification, genetics and bioinformatics to:
- Develop and apply high throughput protein tagging technology for transcription factors (TFs) to generate mES cell lines with bispecific “knocked-in” affinity tags at the N- and/or C-terminals. 100-200 TFs will be converted into null mutations in the same TFs in mES cells.
- Characterize the function of selected, pivotal factors by conditional mutagenesis in mice.
- Affinity purify the tagged proteins from undifferentiated (where appropriate) and differentiated ES cells and various adult mouse tissues from the haematopoietic and neural systems and identify interacting proteins by MALDI-TOF MS, and LC-MS/MS.
- Identify TF binding sites by chromatin affinity purification (ChIP) and hybridisations to whole genome arrays (chips) and/or in vivo footprinting.
- Validate interactions by immunoprecipitations, intracellullar co-localization by immunocytochemistry and/or bi-molecular fluorescence complementation (BiFC) and perform a functional analysis of novel TF interacting proteins by morpholino injections in zebrafish or Xenopus embryos.
- Format a database, which will be interfaced with ENSEMBL and other similar data sets.
- Build models and simulations of the TF networks.
These approaches will allow the characterisation of transcription factor complexes and the genome-wide identification of binding sites for these TFs in undifferentiated and differentiated ES cells or differentiated tissues (blood and neuronal system). By employing teams of scientists that apply these technologies in several cycles, we will create a “pipeline” that systematically identifies TF complexes and TF binding sites in vertebrate genomes required for the differentiation into haematopoietic and neural cells. The bio-informaticians will develop novel algorithms to extract and interpret the data and viewers to integrate the information in the ENSEMBL database. Data will be made available publicly by web based platforms and tools. Databases will be constructed that will include annotated TFs, TF-DNA interactions, protein- and RNA TF interactions and cellullar regulation (gene, effect, cell type, cell state). The project databases will be interfaced with existing micro-array databases as well as other public databases.
The project will provide new insights in understanding the regulation of cell function that stimulate translational research that is critical to develop novel therapies, particularly in the areas of stem cell transplantation and tissue engineering. The commercial implications of the project will be translated by the specialist enterprise for its therapeutic potential. Commercial partners on the reagent side will be asked to bid for participation.