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Characterization of signalling functions and genomic targets of SWI3C-associated chromatin remodelling complexes

Final Report Summary - SWI3C-CRCS (Characterization of signalling functions and genomic targets of SWI3C-associated chromatin remodelling complexes)

Chromatin-remodelling complexes (CRCs) play pivotal roles in the regulation of basic cellular processes, including transcription, DNA replication, repair and cell cycle in eukaryotes. Although several orthologues of CRC components are conserved between higher plants and other eukaryotes, our current knowledge on plant CRCs is rather limited. The project was dedicated to perform genetic and biochemical characterisation of regulatory roles of SWI/SNF CRC complexes that carry an ATSWI3C subunit.

Major aims of the project were:
i) Determination of transcription regulatory targets of ATSWI3C CRCs by transcript profiling;
ii) Chromatin cross-linking and fractionation of ATSWI3C-containing chromatin remodelling complexes;
iii)Proteomics analysis of subunit composition of ATSWI3C containing CRC complexes;
iv) Analysis of subcellular localisation and dynamics of ATSWI3C;
v) Study of genetic interactions between ATSWI3C and developmental and hormonal signalling pathways.

During his previous study, the fellow has identified in yeast two-hybrid screens numerous proteins that may represent auxiliary interacting partners of Arabidopsis SWI/SNF complexes. Part of these these two-hybrid interactions were confirmed by FRET confocal microscopy studies during the project. During his Marie Curie Intra-European Fellowship (IEF) at the MPIPZ, the fellow performed extensive Affymetrix transcript profiling studies. The transcript profiling data were confirmed by real-time PCR measurements studies and the fellow has identified numerous genes that show similarly changed transcript levels in the atswi3c and atbrm mutants.

It was also found that the BRM ATPase and ATSWI3C occur in common SWI/SNF complexes and that mutations in these genes cause very similar molecular and developmental defects. These data were summarised in the publication of Archacki R-., Sarnowski T. J.- Puzio J., Brzeska, K., Prymakowska-Bosak, M., Koncz, C.- and Jerzmanowski, A-. (2009) Genetic analysis of functional redundancy of BRM ATPase and ATSWI3C subunits of Arabidopsis SWI/SNF chromatin remodelling complexes. Planta, 229, 1281-1292.(--authors with equal contribution).

During his Marie Curie IEF project, the fellow has also characterised several genetic interactions of atswi3c mutations that cause characteristic changes in physiological and developmental responses to different hormones, biotic and abiotic stresses. The results indicate that ATSWI3C is implicated in the control of root, leaf, and stomata development. Analysis of double mutants also suggests that ATSWI3C is required for proper function of several key components of ethylene hormone signalling pathway. Moreover, the genetic studies indicated that ATSWI3C-containing CRCs play a role in the control of flowering time and leaf development, as well as in genetic interaction with Constans, regulate chromatin status and/or assembly. To accomplish a further task of the Marie Curie project, the fellow performed subcellular localisation of ATSWI3 proteins using in vivo imaging, and established a novel tandem affinity tag labelling system for purification of SWI/SNF complexes and subsequent analysis of their subunit composition by mass spectrometry. This approach is currently used for chromatin precipitation to demonstrate association of SWI3 proteins with transcriptional regulatory regions of candidate genes in the Arabidopsis genome.

During his Marie Curie IEF project, the fellow has strengthened collaborative research interactions between the Laboratory of Plant Molecular Biology at the Institute of Biochemistry and Biophysics PAS/Warsaw University in Warsaw, Poland (his former laboratory) and the Max-Planck Institute for Plant Breeding Research (his Marie Curie IEF host) in terms of transferring knowledge to Ph.D. students of the Polish Laboratory. The fellow took responsibility to organise several short scientific visits of Ph.D. students from Warsaw in Cologne that allowed them to receive an intensive training in new methods. These methods were subsequently successfully adapted in the Polish laboratory. This extended collaboration with the MPIPZ host laboratory is expected to result in future joint proposals for research grants and training programmes. The collaboration is also expected to yield joined scientific publications and reports, which will be presented during international conferences and meetings.

The Marie Curie IEF grant provided the fellow with a unique opportunity to increase his scientific skills. During his stay at the MPIPZ in Cologne, the fellow has undergone intensive training in advanced methods of transcriptomics, bioinformatics, chromatin immuno-precipitation (ChIP), proteomics, in vivo fluorescent imaging, functional genomics and genetics. The fellow received complementary training in writing of scientific manuscripts, research planning, scientific communication, grant preparation, as well as has contributed to training of Ph.D. students in the host laboratory. In addition, the fellow gained significant interdisciplinary experience and internationally significant competence in affinity purification and analysis of plant protein complexes. He improved his English and German knowledge, and was trained in public relation-related communication issues, such as scientific ethical questions, problems of GMOs, and the role of plant research in sustainable agricultural developments. The fellowship has stimulated the fellow to develop his skill comparable to those who completed their post-doctoral training in the best universities and research institutes of Europe and USA.

As the project also aimed at strengthening interregional European collaboration in the area of a highly competitive research field, the fellow was instrumental to promote and establish a strong collaborative link between the fellowship host and his former research group. This collaboration is planned to be extended by involving other European research partners of both institutes in frame of future joined research programs, in particular because both institutions contribute actively to the ongoing COST action FA605-INPAS (International Network of Plant Abiotic Stress).

Finally, the fellow has also benefited from that an excellent contact exists between the MPIPZ and the University of Cologne, which provided him with the opportunity to participate and contribute to a wide-scale of joined seminars, lectures and training courses. Given the fact that about half of MPIPZ collaborators are foreigners, the MPIPZ provided the fellow with a multicultural environment, which stimulated new research and personal contacts promoting future worldwide collaborations and training activities. After finishing the Marie Curie IEF the fellow will be re-employed in Poland in the Laboratory of Plant Molecular Biology Polish Academy of Sciences/Warsaw University, Pawinskiego 5A 02-106 Warsaw, Poland (E-mail address: tsarn@ibb.waw.pl) and will be supported by a Marie Curie FP7-Reintegration-Grant.