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Periodic Report Summary 1 - CHIP-ET (Chromatin in Plants – European Training and Mobility)

Chromatin in Plants – European Training and Mobility (CHIP-ET)
Marie Curie Initial Training Network FP7-PEOPLE-2013-ITN607880

The Research Training Network CHIP-ET focuses on the study of protein complexes that modulate chromatin structure to activate RNA polymerase II (RNAPolII)–mediated transcription. Research will address how developmental stimuli, light and circadian rhythm signal to these complexes to modulate transcription. The interactome of the complexes will be determined, as well as their genome-wide target genes and the common and specific components in their molecular networks. The impact of perturbation of complex components or regulators on plant development and stress tolerance is analyzed in the Arabidopsis plant model. The epigenome of crop epilines will identify novel epialleles for yield and plant breeding. Four research objectives have been defined: (1) Biochemistry and nuclear architecture of chromatin complexes (2) Chromatin complex components and regulator's function in plant development, circadian clock and stress tolerance (3) Chromatin complex target genes (4) Chromatin and crop improvement. The partners provide multi-disciplinary cutting-edge technologies and know-how in chromatin research that will create synergy and added value for Europe. Full participation of the industrial partner make trainees aware of exploitation of research results and the importance of molecular breeding for a sustainable agriculture.

In the first work package, subunits of the transcription activating complexes HUB1, TFIIS, FACT, SPT4, SDG8 and TCP14 pulled down a number of proteins through affinity purification and mass spectrometry technologies and revealed subunit composition and putative interactors. The same technologies revealed that the structure of condensin that plays a central role in chromosome assembly and segregation during mitosis and meiosis is conserved in plants. Common interactors suggest consecutive interactions during transcript elongation. Specific interactors link the chromatin complexes and transcript elongation to RNA related processes or upstream signaling pathways that cross-talk to environmental stimuli and might be plant-specific. The subcellular and nuclear distribution and co-localization of subunits or interactors is being determined by regular confocal or super-resolution microscopy.

In the second work package, the function for the chromatin complexes and their interactors identified in work package one was examined. Studies focused on in plant growth and development, such as germination in darkness and specific light qualities so-called skoto- and photomorphogenesis, seedling growth, flowering time, circadian rhythm and abiotic stress tolerance was identified. Mutant and overexpression lines, and introgressed molecular markers were instrumental to define specific stages in development or specific conditions in which a particular gene of interest was active and performed a function. Phenotypic information was furthered with the so-called molecular phenotype that was obtained by genome-wide transcriptome analyses of the perturbed lines at specific developmental stages or conditions. Double and triple mutant combinations provided additional functional information and positioned the complexes and their interactors in molecular signaling pathways.
In the third work package, the function for the chromatin complexes in aspects or stages of plant growth and development or upon stress studied in work package two, was further consolidated in mutant lines by chromatin immune precipitation using antibodies against a modified histone, coupled to quantification by PCR and revealed that several regulators in development, cell cycle, circadian clock and flowering time were direct targets of the chromatin complex enzymatic activities. Interactors either assisted the chromatin complexes in their histone modification activity or showed other activities such as in RNA-related biology and suggested exciting links between the chromatin complexes and mRNA processing mechanisms during RNAPolII-mediated transcript elongation.

Chromatin complexes activating RNAPolII-mediated gene expression are studied at protein, RNA and DNA level, linking upstream regulation to protein biochemistry and interactome to downstream molecular networks and function in aspects of plant growth and development in order to obtain a mechanistic insight into their function. The new insights into the cross-talk of the chromatin complexes with environmental factors such as light, circadian rhythm and abiotic stress that are essential for plant growth, development and yield will provide new possibilities for optimization of elite genotypes in plant breeding to broaden the geographical production area in terms of more adverse conditions, altered light qualities, day length or circadian rhythm.

In a fourth work package, transcriptome, cytosine methylation and chromatin modifications were analysed in canola and rice epilines and pathways and genes have been identified that underlay the selected phenotypes such as enhanced energy use efficiency, drought tolerance, nitrogen use efficiency, vitality upon SA treatment and yield in field trials. A number of genes susceptible for epigenetic modification, so-called epi-alleles, and their transgenerational inheritance and stability are under study. Putative epigenetic markers have been selected and their use in molecular breeding is being tested. No doubt the research on epilines will lead to the identification of molecular markers for molecular breeding and Intellectual Property within the time frame of the CHIP-ET ITN. The epigenetic variation responsible for epi-alleles in the selected epilines most likely originates during seed set and might account for unexpected variability in progeny and seed batches. Our research on epilines will provide a better insight into the types of epigenetic variation and their genomic distribution, and thus ultimately will be useful to adjust commercial seed production.

A next-gen-sequencing (NGS) profiling method for differential cytosine methylation in plants has been developed through optimized parameters such as plant DNA isolation, type of restriction enzymes, library construction, sequencing depth and bioinformatics workflow for large dataset analysis. The reproducibility of covered regions, sequence annotation and methylation levels of focused regions were assessed. The profiling method was validated in the rice epilines selected for improved energy use efficiency, transgenerational inheritance of cytosine methylation was observed using the newly-developed method. The NGS-based cytosine methylation profiling method will enable the plant science community and plant breeders to routinely detect cytosine methylation on nucleotide resolution in multiple samples.

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Life Sciences