Mechanisms of gene positioning in the muscle cell nucleus

The genome inside the cell nucleus is structurally organized at different levels, involving compaction of DNA molecules by specific protein complexes into chromatin fibers and chromatin domains. Recent studies have shown that the positioning of genomic regions and genes within the three-dimensional space of the nucleus is an important pathway to regulate chromatin and to activate or repress specific genes during the formation of specific cell types. Long regions of the genome, which are not needed in specific cell types are tethered to the nuclear envelope and thereby densely compacted and silenced. Specific genes, which have to be repressed at specific stages of cell differentiation, but need to be expressed at other stages were shown to move from the nuclear periphery to the center of the nucleus upon their activation. Conversely, many genes that need to be de-activated during differentiation move from the center to the nuclear periphery. The molecular players mediating gene movement and their regulation during cell differentiation are poorly understood and are the main subject of this study. We will generate a novel muscle cell system to follow muscle cell differentiation in the laboratory and at the same time follow the movement of differentiation-specific genes away from or towards the nuclear envelope. To do this, we will introduce a tag into muscle genes by a novel genome editing technology, and microscopically follow this tag during differentiation. This gene track cell system will be used to identify molecular components of the nuclear envelope involved in tethering genes to the nuclear periphery during muscle differentiation. We will systematically delete over 30 nuclear envelope candidate proteins using the genome editing technology, and analyze the effect on gene movement during muscle differentiation. This approach will identify gene tethers in the nuclear envelope and determine at which stages of muscle differentiation they work. We will also address how gene tethering is regulated during muscle differentiation. What determines or initiate the release from or the attachment of specific genes to tethers at the nuclear envelope? We will test the possibility that the nuclear envelope tether changes localization or is differentially expressed at different stages of muscle differentiation.Alternatively, specific sequence motives and modifications in the moving genes may regulate gene attachment and release. We will identify these elements by genome-wide identification of chromatin regions bound to the tethers. Overall this study will provide important mechanistic insight into the regulation of gene localization during cell differentiation. This is particularly important because several genetic diseases, such as muscular dystrophies or premature ageing syndromes, are linked to mutations in nuclear envelope components and show chromatin defects that may cause some of the disease pathologies.

Mechanisms of gene positioning in the cell nucleus during muscle differentiation. The genome that harbors all our genes inside the cell nucleus is structurally organized into chromatin domains. The position of genomic regions and genes within the three-dimensional space of the nucleus contributes to genome regulation during embryonic development and the formation of different cell types in the body. For instance, whole regions of the genome, which are no longer needed in a specific cell type are tightly tethered to the nuclear envelope and thereby "silenced". Similarly, individual genes, which have to be shut down during the formation of specific cell types are tethered to the nuclear envelope. In contrast, genes that need to be activated during establishment of specific cell types move away from the nuclear envelope into the center of the nucleus. The molecular players mediating attachment to and release of genes from the nuclear envelope are poorly understood and were the main subject of this study. In this project we investigated the formation of muscle cells from isolated muscle precursor cells outside the body. During this process, muscle specific genes (MyoD) have to be activated while muscle-precursor cell genes (Pax7) have to be silenced. By genetically manipulating the cells we generated a reporter muscle cell system that allows visualizing MyoD and Pax7 genes inside the nucleus of living cells by microscopic techniques. In this way one can follow the specific localization of these genes within the three-dimensional space of the nucleus during the transition from a muscle-precursor cell to a muscle cell. As expected, we observed translocation of the muscle cell gene MyoD from the nuclear envelope to more central positions in the nucleus, while the precursor cell gene Pax7 showed the opposite behavior - translocation from the nuclear center to the nuclear envelope. Following the successful generation of this reporter cell system, we deleted several components of the nuclear envelope alone or in combination to test, which of these components, if any, are involved in tethering genes to the nuclear envelope during muscle cell formation. So far, we identified several nuclear envelope components that, when deleted, dramatically affect the position of whole genomic regions in the nucleus, but did not affect the anchorage of specific genes at the nuclear envelope. Overall, we developed a powerful experimental system to study new components and pathways involved in the regulation of gene positioning. Thus, our system will allow to substantially increase our knowledge on this important genetic pathway for gene regulation in future experiments. This knowledge is particularly important for a better understanding of the molecular cause of many nuclear envelope-linked diseases, such as muscular dystrophies or premature ageing syndromes, where chromatin organization is impaired.