Project description:Global chromatin epigentic profiles of human cancer and NPC cell lines treated with neurodevelopmental compounds, epigenetic compounds and kinase inhibitors.
Project description:The structure of chromatin is critical for many aspects of cellular physiology and is considered to be the primary medium to store epigenetic information. It is defined by the histone molecules that constitute the nucleosome, the positioning of the nucleosomes along the DNA and the non-histone proteins that associate with it. These factors help to establish and maintain a largely DNA sequence-independent but surprisingly stable structure. Chromatin is extensively disassembled and reassembled during DNA replication, repair, recombination or transcription in order to allow the necessary factors to gain access to their substrate. Despite such constant interference with chromatin structure, the epigenetic information is generally well maintained. Surprisingly, the mechanisms that coordinate chromatin assembly and ensure proper assembly are not particularly well understood. Here, we use SWATH-MS to describe the kinetics of in vitro assembled chromatin supported by an embryo extract prepared from preblastoderm Drosophila melanogaster embryos. This system allows easy manipulation of distinct aspects of chromatin assembly such as post-translational histone modifications, the levels of histone chaperones and the concentration of distinct DNA binding factors. Our findings support the idea that chromatin assembly factors and factors important for chromatin structure bind chromatin in an ordered manner, which is -at least in part- regulated by histone deacetylation. We are able to identify functional clusters of proteins based on their different binding kinetics. Whereas many proteins bind exclusively during the onset of chromatin assembly, a few proteins show a clear tendency towards matured chromatin.
Project description:Epigenetic-mediated gene regulation orchestrates brain cell-type gene expression programmes, and epigenetic dysregulation is a major driver of aging and disease-associated changes. Proteins that mediate gene regulation are mostly localised to the nucleus, however, nuclear-localised proteins are often under-represented in gene expression studies and have been understudied in the context of the brain. To address this challenge, we have optimised an approach for nuclei isolation that is compatible with proteomic analysis. This was coupled to a mass spectrometry protocol for detecting proteins in low-concentration samples. We have generated nuclear proteomes for neurons, microglia, and oligodendrocytes from the mouse brain cortex and identified cell-type nuclear proteins associated with chromatin structure and organisation, chromatin modifiers such as transcription factors, and RNA-binding proteins, among others. Our nuclear proteomics platform paves the way for assessing brain cell type changes in the nuclear proteome across health and disease, such as neurodevelopmental, aging, neurodegenerative, and neuroinflammatory conditions.
