The EuTRACC consortium will use genomics, proteomics and in vivo phenotyping to describe the network of TFs that regulates embryonic stem cell differentiation into neuronal and haematopoietic lineages. Computational analyses of these data will allow major progress towards understanding the mechanisms of transcriptional control of lineage commitment, ultimately leading to the modelling of these processes. The project will consist of cycles of overlapping phases that will be conducted over 4 years. The project will be closely coordinated with the International Regulome Consortium.
Objectives of EuTRACC can be classified as:
- general objectives
- scientific objectives
- technical objectives
- coomericial objectives
- training objectives
General objectives for this project are to:
- Ensure a substantial contribution of EU based science to the field of transcriptional regulation of differentiation and development.
- Provide strong input from the EU to the International Regulome Consortium (IRC), a worldwide consortium that represents a third generation genomics project addressing the regulation of genome function at a higher level by mapping the genetic regulatory nodes and networks.
Scientific objectives for this project are to:
- Identify protein complexes of basic, general and tissue specific TFs and interacting partners expressed in neuronal and haematopoietic cell types.
- Use bioinformatic methods to correctly annotate mouse genes that encode bona fide TFs and interacting proteins, microarray data will be analysed to identify TF complexes expressed in the selected cell types. Validation of interactions by IPs and/or BiFC. Determine intracellular localisation of TFs and/or associated proteins.
- Identify target genes of the different TF complexes.
- Functionally analyse TFs and selected interacting proteins by morpholino injections in zebrafish and Xenopus embryos.
- Derive computational procedures and models that describe the mechanism of regulation of gene transcription to differentiate embryonic stem cells to neuronal or haematopoietic cells. Specific databases and tools will be developed to facilitate the collection, curation and analysis of the data, as well as for the public dissemination of findings.
Technical objectives for this project are to:
- Generate 100-200 knock-ins of protein tags for affinity purification of TF complexes and concurrently generate conditional TF KOs in ES cells.
- Generate homozygous null mutations in key TFs in ES cells and mice.
- Culture and differentiate TF tagged ES cells in vitro and isolate the relevant tissues from ES generated mice for genomic and proteomic analyses.
- Characterize the protein components of transcriptional complexes containing the tagged TFs in selected cell and tissue types.
- Purify and identify TF binding sites in selected cell types by two approaches: chromatin affinity purification, followed by DNA amplification, and hybridization to genome wide microarrays (Affymetrix, Agilent or Nimblegen). Fine mapping, when required, will be done by in vivo footprinting.
- Repeat cycles of tagging knock-ins for affinity purification etc. by tagging selected interaction partners from the previous screen (biochemistry & morpholinos).
Commercial objectives of this project are to:
- Innovations arising from project will be identified; intellectual property (IP) will be protected by individual partners or combinations of partners and technical feasibility will be assessed.
- Develop improved module libraries based on experimental results and develop new modeling approaches towards differentiation related processes. This will crucially depend on the new experimental results obtained during this project.
- Develop improved stem cell culture and differentiation practice towards neuronal and haematopoietic cell types for both small and large scale applications. The identification of key regulatory nodes in lineage commitment pathways can be applied using Stem Cell Sciences IP for regenerative therapy.
Training objectives for this project are to:
- Provide excellent training facilities for young researchers in the basic sciences, including data capture, data analysis, and the protection and exploitation of inventiveness.
- Dissemination of state-of-the-art genomic and proteomic technologies amongst consortium members.