ERC FUNDED PROJECTS

"Eukaryotic nuclei are organised into functional compartments, – local microenvironments that are enriched in certain molecules or biochemical activities and therefore specify localised functional outputs. Our study seeks to unveil fundamental principles of co-regulation of genes in a chromo¬somal compartment and the preconditions for homeostasis of such a compartment in the dynamic nuclear environment. The dosage-compensated X chromosome of male Drosophila flies satisfies the criteria for a functional com¬partment. It is rendered structurally distinct from all other chromosomes by association of a regulatory ribonucleoprotein ‘Dosage Compensation Complex’ (DCC), enrichment of histone modifications and global decondensation. As a result, most genes on the X chromosome are co-ordinately activated. Autosomal genes inserted into the X acquire X-chromosomal features and are subject to the X-specific regulation. We seek to uncover the molecular principles that initiate, establish and maintain the dosage-compensated chromosome. We will follow the kinetics of DCC assembly and the timing of association with different types of chromosomal targets in nuclei with high spatial resolution afforded by sub-wavelength microscopy and deep sequencing of DNA binding sites. We will characterise DCC sub-complexes with respect to their roles as kinetic assembly intermediates or as representations of local, functional heterogeneity. We will evaluate the roles of a DCC- novel ubiquitin ligase activity for homeostasis. Crucial to the recruitment of the DCC and its distribution to target genes are non-coding roX RNAs that are transcribed from the X. We will determine the secondary structure ‘signatures’ of roX RNAs in vitro and determine the binding sites of the protein subunits in vivo. By biochemical and cellular reconstitution will test the hypothesis that roX-encoded RNA aptamers orchestrate the assembly of the DCC and contribute to the exquisite targeting of the complex."

2 482 770 €

Start date: 2012-02-01, End date: 2017-01-31

The interaction of cells with the extracellular matrix or neighboring cells plays a crucial role in many cellular functions, such as motility, differentiation and controlled cell death. Expanding on pioneering studies on defined 2-D model systems, the role of the currently known determinants (geometry, topography, biochemical functionality and mechanical properties) is currently addressed in more relevant 3-D matrices. However, there is a clear lack in currently available approaches to fabricate well defined microenvironments, which are asymmetric or in which these factors can be varied independently. The central objective of ASMIDIAS is the development of a novel route to asymmetric microenvironments for cell-matrix interaction studies. Inspired by molecular self-assembly on the one hand and guided macroscale assembly on the other hand, directed assembly of highly defined microfabricated building blocks will be exploited to this end. In this modular design approach different building blocks position themselves during assembly on pre-structured surfaces to afford enclosed volumes that are restricted by the walls of the blocks. The project relies on two central elements. For the guided assembly, the balance of attractive and repulsive interactions between the building blocks (and its dependence on the object dimensions) and the structured surface shall be controlled by appropriate surface chemistry and suitable guiding structures. To afford the required functionality, new approaches to (i) topographically structure, (ii) biochemically functionalize and pattern selected sides of the microscale building blocks and (iii) to control their surface elastic properties via surface-attached polymers and hydrogels, will be developed.The resulting unique asymmetric environments will facilitate novel insight into cell-matrix interactions, which possess considerable relevance in the areas of tissue engineering, cell (de)differentiation, bacteria-surface interactions and beyond.

1 484 100 €

Start date: 2011-11-01, End date: 2016-10-31

Cardiovascular disease keeps the top spot in mortality statistics in Europe with 2 million deaths annually and although prevention and therapy have continuously been improved, the prevalence of heart failure continues to rise. While contractile (systolic) dysfunction is readily accessible to pharmacological treatment, there is a lack of therapeutic options for reduced ventricular filling (diastolic dysfunction). The diastolic properties of the heart are largely determined by the giant sarcomeric protein titin, which is alternatively spliced to adjust the elastic properties of the cardiomyocyte. We have recently identified a titin splice factor that plays a parallel role in cardiac disease and postnatal development. It targets a subset of genes that concertedly affect biomechanics, electrical activity, and signal transduction and suggests alternative splicing as a novel therapeutic target in heart disease. Here we will build on the titin splice factor to identify regulatory principles and cofactors that adjust cardiac isoform expression. In a complementary approach we will investigate titin mRNA binding proteins to provide a comprehensive analysis of factors governing titin’s differential splicing in cardiac development, health, and disease. Based on its distinctive role in ventricular filling we will evaluate titin splicing as a therapeutic target in diastolic heart failure and use a titin based reporter assay to identify small molecules to interfere with titin isoform expression. Finally, we will evaluate the effects of altered alternative splicing on diastolic dysfunction in vivo utilizing the splice deficient mutant and our available animal models for diastolic dysfunction. The overall scientific goal of the proposed work is to investigate the regulation of cardiac alternative splicing in development and disease and to evaluate if splice directed therapy can be used to improve diastolic function and specifically the elastic properties of the heart.

1 499 191 €

Start date: 2012-01-01, End date: 2017-06-30

Timing is everything in ecology, and because plants provide the foundation for most land-based food webs, the timing of their activities profoundly orchestrates the majority of ecological interactions. Most photosynthetic and growth processes are under circadian control, but many additional processes--approximately 30-40% of all genes—are under circadian control, and yet the Darwinian fitness impact of being “in synch” with the environment has not been systematically studied for any organism. We have developed a toolbox for a native tobacco plant, Nicotiana attenuata, that allows us to “ask the plant” which genes, proteins or metabolites are regulated in particular plant-mediated ecological interactions; identify “the genes that matter” for a given interaction; silence or ectopically express these genes, and conduct field releases with the transformed plants at a nature preserve in the Great Basin Desert to rigorously test hypotheses of gene function. By taking advantage of both our understanding of what it takes for this plant to survive in nature, and the procedures established to disentangle the skein of subtle interactions that determine its performance, we will systematically examine the importance of synchronous entrained endogenous rhythms at all life stages: longevity in the seed bank, germination, rosette growth, elongation, flowering and senescence. Specifically, we propose to silence a key components (starting with NaTOC1) of the plant’s endogenous clock to shorten the plant’s circadian rhythm, both constitutively and with strong dexamethasone-inducible promoters, at all life stages. With a combination of real-time phenotype imaging, metabolite and transcriptome analysis, and ecological know-how, the research will reveal how plants adjust their physiologies to the ever-changing panoply of environmental stresses with which they must cope; by creating arrhythmic plants, we will understand why so many processes are under circadian control.

2 496 002 €

Start date: 2012-04-01, End date: 2017-03-31