INTEGER · Integrated approaches to study gene regulation during cellular differentiation

Integrated approaches to study gene regulation during cellular differentiation

The Integer ITN (www.integer-itn.eu) started in January 2009 bringing together 8 partner institutions (7 academic and one industrial) coordinated by Dr. John Strouboulis at the BSRC "Alexander Fleming" Athens, Greece. The ITN aimed to employ state-of-the-art genomic approaches in an Integrated effort to investigate all layers of Gene Regulation from trans-acting factors through to nuclear structure during cellular differentiation. This aim translated into the following specific research objectives: 1. Transcription factor function as master regulators of differentiation 2. Chromatin structure and epigenetic modifications in cellular memory 3. Spatial nuclear organisation of gene interactions and gene activity. The ITN recruited a total of 12 Fellows (10 ESRs and 2 ERs) of eight different nationalities and with a very good gender balance (7 female and 5 male Fellows). Of the 10 ESRs recruited by the Network, 7 are expected to obtain PhDs within the next year. The scientific research and technological objectives of the network were, to a very large extent, achieved with the great majority of milestones and deliverables fulfilled. Several publications have been produced as a result of the research activities of the network, some of which were co-authored by the Fellows. Technology highlights of the network include the development of a number of cutting-edge genomic and proteomic methodologies. Of particular note are the development of: (i) a cost-effective methodology and computational analysis pipeline for the characterization of the physical organization around specific gene regulatory elements using chromosome conformation capture combined with high-throughput sequencing (4C-seq); (ii) the development of novel technology for mapping epigenetic non-coding (nc) RNA binding sites in chromatin by combining the chromatin immunoprecipitation (ChIP) of Polycomb-Group associated ncRNAs with massive parallel sequencing and bioinformatic analysis; (iii) the development of novel technology for the quantitative proteomic characterization by mass spectrometry of immunoprecipitated, formaldehyde crosslinked chromatin domains. The main achievements of the research activities of the network are many and include: (i) the identification of complex cross-regulatory mechanisms amongst the major transcriptional regulators in hepatic differentiation; (ii) the description of “bookmarking” functions for several hepatic transcription factors in keeping the chromatin of hepatic gene regulatory regions open in early development; (iii) the integration of transcription factor genome-wide occupancy data with epigenomic modifications during erythropoiesis, leading to the identification of distinct classes of genes sharing specific epigenetic and functional characteristics; (iv) the combination of in vitro protein-DNA binding assays with chromatin immunoprecipitation (ChIP) assays, both coupled to massive parallel sequencing for the characterization of DNA binding specificities for lineage specific transcription factors; (v) the demonstration that the macro H2A histone variant maintains “stemness” by promoting cellular differentiation at the expense of cell differentiation in mouse embryonic stem (ES) cells; (vi) the proteomic characterization of chromatin domains incorporating specific histone variants; (vii) the analysis of the 3-dimensional folding of the active and inactive X chromosomes during ES cell differentiation; (viii) the demonstration of a physical association between active (phosphorylated) RNA Polymerase II and Polycomb Repressor Complexes (PRCs) and the identification of a cohort of active PRC genes in mouse ES cells; (iX) using a novel quantitative proteomics-ChIP analysis of RNA polymerase II-bound chromatin, the identification of a large number of novel interacting proteins which are specifically associated with different stages of the transcription cycle. The InteGeR ITN also executed a very strong training program which included four advanced research workshops and a scientific conference, in addition to four workshops on bioethics and various transferable skills. Many external speakers that are leaders in their fields were invited to the workshops which, it should be noted, were highly successful as they were attended by many external participants. Overall, the successful execution of the research and training programs of the InteGeR ITN has made, and will continue to make, a significant impact in generating considerable new knowledge on basic transcriptional and epigenetic mechanisms in gene regulation in relation to chromosome and nuclear three-dimensional structures in cell differentiation. Also, the cross disciplinary nature of the research and training programs has ensured that Fellows will develop as highly skilled scientists prepared for careers in post-genomic biology and its applications in academia or industry. Lastly, a major benefit of a successful outcome of the network will be the integration of the existing scientific activities into a larger durable European partnership. Together, these aspects will help enhance Europe’s competitiveness in the cutting-edge sector of biomedical research and development. The InteGeR ITN logo is shown in Appendix 2. Group photos of the Network members are shown in Appendix 3. Contact details: John Strouboulis PhD Coordinator of InteGeR FP7 Marie Curie ITN Division of Molecular Oncology Biomedical Sciences Research Center "Alexander Fleming" P.O. Box 74145 GR-16602, Varkiza Greece

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