Many of the prime principles summarised above are being actively investigated in several groups in Munich. The principal investigators of this proposal are already connected through participation in fours research networks (SFBs).
Protein DNA interactions that govern gene transcription are studied extensively in the groups of Becker, Imhof, Eick and Meisterernst. Becker and Meisterernst have developed powerful cell-free systems that allow revealing the mechanism of transcription initiation in a reconstituted chromatin environment. These are complemented by in vivo analyses in tissue culture cells and whole organisms.
The laboratories of Eick and Meisterernst (both GSF) specialize on the sequence-specific DNA binding proteins and their interactions with the RNA polymerase machinery. Meisterernst focuses on mediator proteins that connect transcription factors to the machinery. Eick studies the function and regulation of the cellular proto-oncogene c-Myc, which plays a central role in the development of multiple forms of cancer. The group has shown that c-Myc can induce a cellular growth program uncoupled from cell cycle activation by switching on ribosome biogenesis. The c-Myc protein activates and represses a large variety of genes. In a second project the group studies regulatory mechanisms of c-Myc gene expression involving the carboxy-terminal domain (CTD) of RNA polymerase II.
The Cremer group studies the three-dimensional chromatin organization and nuclear arrangements from the level of chromosome territories down to the level of single genes in cell cultures (e.g. mouse embryonic stem cells and human hematopoietic stem cells, differentating cells and terminally differentiated cell types) and tissue sections (e.g. mouse retina and cerebellum). Emphasis is laid on comparative studies of eukaryote architecture in evolutionary distant species (human and other primates, mouse, chicken, plathelminthes, hydra and ciliates). The goal of these studies is the elucidation of evolutionary conserved rules of nuclear architecture in metazoans. A further goal is to understand the functional relevance of topological aspects for gene regulation and DNA repair. For this purpose the Cremer group has joined forces with Leonhardt to analyze dynamic interactions of genes with nuclear speckles and bodies and with Eick to study the topography of a fully functional GFP-tagged RNA pol II in live cell experiments. Within a European program “Spatio-Temporal Organization of Genome Surveillance in Live Cells” the Cremer group studies topological aspects of DNA double strand break repair. A strong interaction exists with the Carell group (area E) amined to decipher the repair process of damaged DNA strands within the nucleus.
The Leonhardt group is interested in the role and regulation of DNA methylation in development and disease. DNA methyltransferases play a central role in the regulation of gene expression, chromatin condensation and genome stability. The regulation of DNA methylation in mammalian cells is still far from being understood, mostly owing to the fact that multiple methyltransferases are involved that interact with each other as well as with numerous nuclear factors. They are studying this extremely complex process with a combination of biochemical, genetic, cell biological and biophysical methods, which could be best described as „in vivo biochemistry“, that is, the study of enzymes in their natural environment, meaning in living cells. Towards this goal they developed a number of assays employing live cell microscopy with photobleaching and photoactivation techniques in combination with specific inhibitors and genetically engineered cell lines and transgenic animals. In collaboration with Cramer (area C) they wish to obtain at least partial crystal structure data. In collaboration with Carell (area E) they try to develop specific inhibitors. In collaboration with Becker they investigate the interaction with chromatin. A long-term goal of these studies is the development of highly specific inhibitors and strategies for epigenetic reprogramming in cancer therapy and tissue regeneration.
Becker and Imhof laboratories (BioMedical Center) study the changes of chromatin structure that determine whether a locus is permissive or repressive for function. Increasingly, they make use of the Drosophila system to characterise the changing chromatin organisation during development, from oogenesis through early embryonic development to somatic cell differentiation. Of interest are the principles that allow fast and finely tuned switches in activity as well as those that lead to stable, heritable states that constitute epigenetic memory. At the molecular level, the dynamics of chromatin structure is implemented by the action of ATP-consuming nucleosome remodelling machines. The post-translational modifications of histones that modulate the folding of the nucleosome fibre into more or less rigid states are being studied both in the Becker and Imhof labs. Together they are concerned about structure and function of close to ten nucleosome modification complexes.
Imhof mainly focuses on the isolation and the characterisation of histone methyltransferases from Drosophila melanogaster and the establishment and maintenance of histone modification patterns during embryonic development and cellular differentiation. Especially their finding that members of the trithorax group and the polycomb group possess methyltransferase activity, which is essential for its in vivo function, suggest that the specific methylation of histones plays a fundamental role in the establishment and maintenance of epigenetic states. Considering that the histone modifications can act as a stable indexing system for the genome it is also of great interest for us to understand the basic mechanisms that maintain the modification patterns through several cell generations. Finally, the disturbance of the indexing system was shown to play a causal role in the development of many human malignancies. Therefore the development of small molecules that interfere with such epigenetic modifiers is of high interest for the treatment of common diseases, the analysis of stem cell differentiation and the study of epigenetic gene regulation.
One recent technical development in the Becker and Meisterernst labs is the combination of chromatin immunoprecipitation (ChIP) with microarray analysis (chip), which allows mapping the interactions of any protein of interest, including epigenetic regulators or the modified chromatin marks within the intact cell along entire chromosomes at a resolution of 100 base pairs. The combination of molecular approaches of this kind with cutting edge high-resolution microscopy should allow determining the folding of the chromosomal fibre and their dynamic changes as a function of cell identity.
Jansen (Gene Center) focuses on the role of mRNA localization during asymmetric cell divisions. They employ two model systems, budding yeast and Drosophila neuroblast cells, to understand the intracellular dynamics of RNP transport. This involves the packaging of individual mRNAs into RNP particles, but whether this occurs individually or in larger assemblies is unknown. In collaboration with structural biologists they aim at getting an in-depth understanding of the assembly of localized RNPs at different stages of the assembly line. Additionally, they want to develop a system to track the trafficking of different individual RNA species in combination with their target organelles in living single cells. In a parallel line of research they want to understand the role of localized mRNAs in neuronal development in Drosophila. To date they have identified several new candidates for localized mRNAs that segregate asymmetrically during the stem cell like division of neuroblasts into the presumptive neuron but are absent from the stem cell like neuroblast cell. A major research aim will be to identify the machinery that localizes these mRNAs and the function of the proteins encoded by these mRNAs for proper neuronal development. The expertise of the Jansen lab in the area of RNA-Protein interaction is most valuable for the analysis of the role for non-coding RNA on chromatin structure and function, which is currently a focus of the Becker lab.
In 2003 the ZfP (Zentrallabor für Proteinanalytik), a core facility for protein identification and characterisation, headed by Axel Imhof was founded at the BioMedical Center (BMC). The ZfP has a strong track record in analysing nucleic acid interacting proteins such as transcription regulators and chromatin structure components and regulators and provides service and cooperation for several SFBs and beyond.