The Gasser laboratory “Functional organization of the nucleus”
 
The Gasser laboratory studies how nuclear organization impinges on mechanisms of repair and replication fork stability, and on epigenetic inheritance of cell fate decisions. We combine genome-wide mapping, synthetic lethal screens, quantitative live fluorescence imaging, biochemical reconstitution and standard yeast molecular genetics to address these questions at a molecular and cellular level. In respect to the questions of stem cell determination and epigenetic inheritance, we are working increasingly with the nematode C. elegans as it provides the opportunity to study the effects of nuclear organization on gene expression during well characterized cellular differentiation events. In C. elegans, we have developed means to study the position of genes in living, differentiating cells. We show that tissue specific genes shift from an inactive positive at the nuclear lamin to a lumenal position during differentiation. In contrast to differentiation specific genes, heat-shock promoters shift to the nuclear periphery when active. We have also demonstrated that by introducing into C elegans a mutant lamin that causes muscular dystrophy in humans – then a muscle specific promoter does not shift inwards in muscle. This impairs its tissue-specific activation and impairs muscle function in worms (papers: Meister et al., submitted; Mattout, Pike et al., in preparation). In general we are studying how the non-random organization of genes in the interphase nucleus contributes to cell type determination and inheritance of a determined cell lineage specific expression pattern. In order to study the subnuclear distribution of genes throughout development we have tagged loci in the C. elegans genome with lacO repeats and GFP-lacI binding. Integrated low-copy transgenes containing arrays of lacO sites and developmentally regulated promoters are visualized by the expression of GFP-lacI from a ubiquitously expressed promoter. These transgenes maintain their proper expression patterns and are detected as fluorescent foci. In early embryonic cells, transgenes like many endogenous loci, are randomly distributed in the nucleus. Over the course of differentiation, transgenes shift either inwards (when induced) or to the NE (when silenced). Large transgene arrays are at the NE in early embryos, and remain there until activated in a tissue-specific manner. The perinuclear domains bear epigenetic marks of heterochromatin. Our results suggest that the trigger for localization to the nuclear rim may be structural components of heterochromatin and rather than the binding of sequence specific factors. We will use genetics, biochemistry and genomic approaches to identify the mechanism and importance of these interactions for stress response and for cell differentiation generally.

Cell plasticity in Health and Disease: SystemsX node

Susan Gasser also leads the SystemX Research and Technology development project called Cell Plasticity, which is a network of 8 laboratories along with the computational biology platform of FMI and the deep sequencing platform of D-BSSE. The goal of “Cell Plasticity in Health and Disease”  is to develop a systems-level understanding of the gene regulatory networks that are responsible for cellular differentiation in mammals. We will focus in particular on elucidating and modeling the mechanisms by which the sequence-specific binding of transcription factors interacts with the dynamics of the “epigenetic code” along the genome, i.e. the local status of chromatin as determined by histone and DNA modifications. In order to derive general regulatory principles we propose to study a panel of mouse cellular differentiation systems including four “normal” differentiation processes and two cancer-related transformation events. All these systems have well-defined time courses of differentiation with clearly defined cellular states that allow collection of homogeneous populations of cells. To study this panel of mouse differentiation systems we will apply uniform measurement protocols and data processing procedures to obtain genome-wide time courses of mRNA expression, miRNA expression, histone modifications, DNA methylation and selected transcription factor binding, for each model system. The data from all systems will be analyzed by common novel mathematical and computational methods. In particular, we will use sophisticated methods for genome-wide annotation of transcription factor binding sites in combination with the time course data to develop quantitative models for the genome-wide interactions between chromatin state and the actions of sequence-specific transcription factors. Predictions from the computational models will be followed-up and validated by targeted perturbation experiments using mutant animals and cells, as well as knock-down or over-expression of transcription factors and chromatin modifying enzymes. Our final goal is to develop novel models of the transcriptional and epigenetic regulatory networks acting in these differentiation systems to the point that we can engineer them. That is, we aim to predict and confirm particular perturbations that reliably trans-differentiate a system from a given start state to a desired target state.