Service Communautaire d'Information sur la Recherche et le Développement - CORDIS

FP6

TRANS-REG Résumé de rapport

Project ID: 502950
Financé au titre de: FP6-LIFESCIHEALTH
Pays: Greece

Final Report Summary - TRANS-REG (Transcription complex dynamics controlling specific gene expression programs)

The central objective of the project was the investigation of the connection between the dynamics of assembly and the spatial distribution of different complexes involved in RNA polymerase-II transcription in the nucleus of living cells. To obtain a comprehensive view of the transcription mechanism our focus was on the most important multi-subunit transcription complexes. We have studied their biochemical characteristics, gene specificity and cellular dynamics using model genes activated during cell differentiation, cell proliferation and signal transduction. To this end a multilateral course of action was planned, which involved fluorescence imaging applications together with genetic, proteomic and biochemical approaches.

During the first year the network has produced and quality assessed a large number of research tools (antibodies, epitope-tagged expression vectors). Several novel algorithms and statistical methods for the evaluation of FRET and FRAP experimental results have been developed. Drosophila and yeast strains with mutations on specific subunits of transcription complexes have been developed and used in assessing the functional roles of the individual proteins in the process of transcription. In addition, new improved chromatin immunoprecipitation protocols have been developed and used for the initial analyses of transcription factor recruitment and nucleosome modifications.

In the second year, the project identified new components of the general transcription machinery and developed a further set of antibodies and epitope-tagged vectors for the newly identified ones. More sophisticated algorithms and statistical methods for FRAP, and novel mouse and drosophila genetic models have been developed. Most importantly, however, by using the tools, which were developed in the first and second year, in specific scientific research activities, a number of important discoveries were made related to chromatin function and mechanism of transcription regulation. We have established an initial fairly complete map of in situ interactions between the components of the basic transcription factor machinery and obtained a large number of information about the molecular events involved in the mechanism of transcriptional regulation of several model genes. In addition, several novel posttranslational modifications of GTFs have been discovered, and studies on their biological role have been initiated.

In the second year and onward, there was a much more intense collaboration between the participants, which was fostered by the fact that we entered into the application stage of most of new technologies developed in the first year.

During the third year of the project, the consortiums' efforts were concentrated mainly on the in vivo functioning of general transcription factor complexes. Most of the activities were natural continuations of previous years' work aimed at the completion of the program deliverables. Fluorescence resonance energy transfer (FRET) based techniques were used to characterise the newly identified Tumour angiogenic factor (TAF) pairs within the Transcription factor IID (TFIID) complex and to complement the previous year's results.

We now have a complete interaction map of the components of the general transcription machinery, validated by several independent approaches. The building up of a detailed interaction map was a major and most challenging goal of the consortium three years ago. We have not only succeeded in this task, but also obtained several important results proving the functional importance of most of the identified interactions.

In situ imaging technologies were used to evaluate the topology of genes in nuclear territories. These studies revealed a clear correlation between gene activity and relocation of genes in specific nuclear regions. Together with the other in situ imaging results the above findings suggest an additional layer of regulation of gene expression. Of particular interest are the results obtained from genome-wide ChIP on ChIP studies. RNA pol-II and TBP were found together more often on promoters of highly expressed genes. These studies have also shed light into a novel function of NC2, playing role in the commitment of the promoters for transcription.

Our work also led to the discovery of distinct complexes with different TAF subunit composition and the presence of distinct CNOT subcomplexes. We have indications that except their role in cell proliferation, TAFs could also be involved in apoptotic events. Apoptosis and proliferation are key-processes in cell homeostasis and cancer and it is known that apoptotic and proliferative extracellular signals can utilise the same pathways to fulfil their opposite action. In this respect, our finding of the regulated shuttling of TAFs between the cytoplasm and nucleus in response to Ras signalling pathway, provides an important mechanistic evidence. The scenario of the TAFs, through modifying the activity of the complexes they belong, being involved in this cell decision is an intriguing finding. Finally, our work led to the significant improvement, standardization, quality assessment of chromatin immunoprecipitation technologies and algorithms.

Overall, the activities led to the successful completion of all the planned deliverables of the program. We have established the roles of the dynamic association of RNA polymerase-II with general transcription factor complexes within the nucleus, the trafficking of the components between nuclear structures, the forces governing complex-complex interactions and the pacemaker of their intrinsic dynamics at the DNA-protein surfaces. We have now a clearly more comprehensive view of the processes regulating gene expression. Our efforts provided novel critical insights into the process by the discovery of novel functions of a diverse array of factors and sophisticated processes that control the activation of genes in a spatially and temporally regulated manner. These include multiple levels of regulatory pathways operating in living cells that determine whether a gene is turned on or off in a given cell at a given time.

The success of the program is also demonstrated by the large number of publications. During the entire period of the program the consortium has published 6 joint and 76 individual scientific papers in peer-reviewed journals. Besides of scientific publications the results were also disseminated in several scientific conferences, workshops, seminar series and the network's web site: http://www.imbb.forth.gr/people/talianidis/strep.htm

Informations connexes

Reported by

FOUNDATION FOR RESEARCH AND TECHNOLOGY HELLAS
Nikolaou Plastira 100
HERAKLION
Greece

Thèmes

Life Sciences