Molecular Epigenetics and Chromosome Biology
Out of approximately 30,000 genes in the human genome, less than one third is actively transcribed at any given time in any single cell type. In fact, it is the stringent regulation of gene expression that allows the body to be functionally divided into tissues and organs, all with individual patterns of proteins specially adapted to the tasks of these organs. Consequently, mistakes in the regulation of genes are the primary cause of the majority of human diseases.
It is the central motivation of my research to better understand the mechanisms of cellular regulation processes, and explore ways how this knowledge might eventually be exploited for therapeutic purposes.
Research in my laboratory is concerned with several independent but related aspects of the temporal and spatial regulation of gene expression and genome replication.
Functional Architecture and Dynamics of the Cell Nucleus
The first focus of our scientific work aims at understanding the functional organization of the living cell nucleus. The internal structure of the nucleus has long been neglected with regard to regulatory roles, until enormous efforts from many laboratories over the past years have demonstrated a fundamental link between nuclear "architecture" and the control of key genetic processes. It is now generally accepted that dynamic interactions of nuclear components make up functional subcompartments in the nuclear interior, such as chromosome territories, splicing speckles, and a growing number of nuclear “bodies”. This mosaic-like organization of the nuclear interior provides spatially separated, dedicated locations with different local environments sustaining different functions. Even though there are still critical deficiencies in our knowledge of the underlying molecular mechanisms, it is no longer disputed that nuclear architecture plays an important role in the regulation of gene expression, RNA maturation, DNA replication and repair. We now recognize that understanding how the nucleus is organized and how this affects fundamental genetic processes will enable the design of entirely new approaches for the cure of human diseases originating from cellular dysregulation.
Our work is concerned with "architectural" components in the nucleus, which act as integrators of nuclear structure and function. Using quantitative measurements of in vivo dynamics of proteins and nucleic acids, we demonstrated the presence of a stable nuclear substructure in live cells that is essential for DNA replication, and is also involved in locally constrained gene silencing during the inactivation of X chromosomes for dosage compensation.
Chromosome Territory photobleaching. In a HEK293 cell with two inactive X chromosomes, SAF-A in one Xi territory was photobleached (arrow) and recovery was investigated over 85 minutes. Note that the enrichment of SAF-A in the bleached Xi is not re-established even after very long time. (scale bar: 10µm.)
Components of a stable proteinaceous substructure of the nucleus are under study in our laboratory for over 15 years now, and our detailed molecular knowledge of their interactions allows us to experimentally modify this structure to investigate its functional implications. In fact, we have already shown that an architectural nuclear protein, Scaffold Attachment Factor A (SAF-A), is essential for the assembly and maintenance of the nuclear scaffold, as well as the spatial regulation of DNA replication in vivo. These results do not only add to the fundamental understanding of the cell nucleus in terms of basic science, but have already allowed - in collaboration with the group of Hans Lipps at the University of Witten - the rational design of a new class of episomally replicating vectors that will be useful for gene therapy in mammalian cells.
In a related project, we investigate the dynamics of individual chromatin domains in live cells. While chromatin is known to be mobile on a small scale (<1µm), it is characterized by a very low mobility on the global nuclear level. For measurements in live cells, we employ an in-vivo visualization technique originally devised in Andrew Belmont's laboratory, where a DNA vector with an array of lac-operators is stably integrated into cells, and is later visualized by expression of a fusion protein of the lac-repressor and green fluorescent protein. This allows detection of single integration sites of the vector as a bright green spot in the living nucleus, and enables us to determine its mobility by confocal time-lapse microscopy. Published results from this project confirmed earlier data that chromatin loci located close to the nuclear periphery are more constrained in their movement than loci in the nuclear interior. Interestingly, however, inhibition of transcription did not affect the mobility of either locus. This suggests that active transcription is not necessary to render chromatin more mobile on a small scale, as often suggested in the literature. In the next step, we now investigate the role of chromatin modifications on its mobility, to elucidate the mechanism behind the differences in constraint between peripheral and internal chromatin loci. In addition, we determine local chromatin dynamics after DNA damage by genotoxic agents and irradiation, where massive modifications of chromatin structure occur, and in transformed versus normal cells. These experiments will provide novel insights into intranuclear dynamics, and how the mobility of chromatin is regulated in vivo to affect the expression of genes.
Posttranslational Methylation of Arginine
The second focus of our research is placed on the functional role of a widespread, but not yet well-understood posttranslational modification of proteins, the methylation of arginine residues. In contrast to phosphorylation and acetylation, which are central to cellular signal transduction as molecular “switches” to modulate protein function in vivo, the role of arginine methylation is much less clear. Methylation of arginine residues mostly affects nuclear proteins, in line with recent results that suggest a role of methylation in regulating gene expression associated with differentiation processes, and roles in replication of certain virus genomes. In addition, there is evidence for crosstalk between arginine methylation and other posttranslational modifications, as, for example, methylation of Arg-3 in histone H4 facilitates H4 acetylation, which then enhances transcriptional activation by nuclear hormone receptors. However, a general concept of the physiological consequences of arginine methylation has not yet been formulated. With the ultimate goal to identify novel pathways involving arginine methylation that may be suitable targets for drug development, we take other protein modifications as a conceptual guide, and investigate the potential function of arginine methylation by focusing on the involved enzymes and their substrates. In contrast to phosphorylation that is catalyzed by several hundred different kinases, arginine methylation requires only a small family of enzymes, the Protein Arginine Methyl Transferases (PRMT), of which more than ten members have been described up to now in higher eukaryotes including plants.
Schematic representation of the eight canonical members of the human PRMT family. The highly conserved PRMT core region (grey), signature motifs I, post I, II, and III (black) and the conserved THW loop (red), present in all PRMTs, are indicated. Note that PRMT7 has a duplication of these motifs and PRMT2 and PRMT3 have an N-terminal SH3 domain (orange) or a zinc finger (green), respectively. The nuclear localization signal (NLS, blue) targets PRMT6 to the nucleus whereas N-terminal myristoylation (~) tethers PRMT8 to the plasma membrane. The size of individual PRMTs is indicated at the C terminus of each enzyme.
Individual enzymes of the PRMT family differ in activity, substrate specificity and subcellular localization, but may join forces to exert biological effects. For example, PRMT1 cooperates with PRMT4/CARM1 and the histone acetylase p300 to stimulate p53-mediated transcription, at least in vitro. It is now essential to characterize the individual members of the family, and their functional interactions, in the living cell. These investigations will shape our understanding of PRMT enzymatic function, will help to determine the physiological role of the individual family members and, most importantly, will clarify the significance of arginine methylation of proteins in health and disease.
In collaborations with leading laboratories in the field, we use our expertise in in vivo mobility measurements and delineating regulatory molecular interactions to gain unprecedented insight into arginine methylation pathways. Recent published results from these projects have, for the first time, provided a comprehensive comparison of all eight human members of the PRMT family with regard to intracellular localization, substrate specificity, in-vivo dynamics, and complex formation. We also demonstrated that the predominant arginine methyl transferase in human cells, PRMT1 reversibly relocates to the nucleus when methylation is inhibited, where it - but not the closely related enzyme PRMT6 - becomes trapped by unmethylated substrates such the core histones, until the methylation reaction was successfully executed. This finding was more recently confirmed in our laboratory with a catalytically inactive construct of the enzyme, showing that enzymatic activity of PRMT1 is required for shuttling the protein between cytoplasm and nucleus, and the release of the enzymes from histones and other substrates. These findings provide a solid basis for investigating epigenetic effects of PRMT1 (and other PRMTs which behave similarly) by identifying genes on which PRMT1 accumulates.
Further experiments along this line now specifically take into account that PRMT1 exists in at least seven different isoforms that result from alternative splicing, and which differ in their expression patterns from ubiquitous to highly cell-type specific, subcellular localization, catalytic activity and substrate specificity. Similar variations are also likely for other PRMT family members, and will add to the complexity of regulatory networks involving arginine methylation, including epigenetic modification of histones for the modulation of gene expression. These ongoing efforts in our laboratory will help understanding the physiological role of arginine methylation, and will ultimately reveal new ways to interfere with the involved cellular pathways for medical purposes.