The role of chromatin organization and structure in neuronal differentiation

Abstract

Chromatin structure is more than just a simple packaging scaffold for DNA. It organizes and coordinates the precise spatio-temporal transcription processes a cell needs for a properly orchestrated development. Epigenetics is the study of this type of regulation. It describes the processes that lead to chromatin decondensation or compaction and involves a multitude of different protein complexes and enzyme families. They carry out various functions like DNA-methylation, histone modification or energy-dependent chromatin remodeling and work together to ensure correct transcription of the DNA template. A dynamic transcription is most important during development. Here, the cell needs to execute lineage restriction while staying responsive to outside cues in order to differentiate properly. Neuronal development is of particular interest because it produces long-lived cell types and early mistakes in regulation can lead to severe disease phenotypes also later in life. However, once differentiation is completed and the neuronal cells have reached a mature postmitotic state they need to maintain a certain transcriptional balance over a long period of time. Recently, there have been a lot of studies linking epigenetic transcriptional regulation in neurons to processes that are also seen in neurodegenerative diseases. This thesis described the characterization of epigenetic modifiers in neurons and their possible implication in neurodegenerative disease development.First, we provided a comparative transcriptional profiling of an epigenetic modifier gene set in five neuronal and three non-neuronal cell types. With this sensitive qPCR-based approach we were able to find a cell-type-specific regulation of subunits in remodelers like the SWI/SNF-complex. We also observed a neuron-specific expression of modifiers such as PRMT8, CHD5 and HDAC9. We continued by describing a form of repressive chromatin that is localized at the nuclear periphery of neurons and characteristic for postmitotic cells. This area is defined by a differentiation-dependent relocalization of the heterochromatin marks H3K27me3 and H3K9me3. For this part of the thesis we used the well-established LUHMES model cell line that produces mature postmitottic neurons within 6 days. Upon differentiation LUHMES cells stop to proliferate, exit the cell cycle and develop a functional neuronal network. LUHMES cells are widely used as model system to study neurodegeneration in dopaminergic neurons. In an attempt to combine the well-studied neurodegenerative aspects of this system with epigenetic research we restimulated cell cycle after differentiation. While this led to an upregulation of cell cycle markers, a condition also seen in early stages of neurodegeneration, the peripheral localization of H3K27me3 did not change. In addition, the restimulation of proliferation indicated activation but not progression of cell cycle; again a condition that is also found in neurodegenerative disease models. Simultaneously, we could observe the expression of replication stress markers in those cells. We hypothesized that the death of cell-cycle-stimulated neurons is due to the restrictive nature of the “heterochromatin barrier” we observed. This structure appears block DNA-synthesis, cause replicative stress and eventually lead to apoptosis.In summary, we developed a qPCR-based array for the characterization of epigenetic modifier genes and we were able to link H3K27me3-relocalization in postmitotic neurons to a replication-stress-phenotype that is also seen in many neurodegenerative diseases. This work also confirms the usefulness of the LUHMES system as tool to study mechanistic and biochemical details of neurodegeneration.