title: Heterochromatin composition, organization and MeCP2 post-translational modifications
Creators: name: Schmidt, Annika
Publisher: name: UNSPECIFIED
Year: 2023

description: In the cell nucleus, the DNA together with several other factors is packaged into chromatin which is organized into active and inactive compartments called euchromatin and heterochromatin. The maintenance of the silenced and compacted heterochromatin state is highly important for the genome stability and spatial organization of nuclear compartments and its misfunction can cause severe deficits in cellular processes. Heterochromatin is comprised of facultative heterochromatin that becomes active in specific situations and constitutive heterochromatin that is mostly silenced and constantly compacted. Constitutive heterochromatin from (peri)centromeric regions of several chromosomes fuses during interphase in mouse cells making up heterochromatin clusters. As the mechanism of the formation of constitutive heterochromatin and the factors involved in the process are not fully understood, we investigated the composition of constitutive heterochromatin in mouse tissues. Therefore, we optimized a protocol for native and unbiased isolation of heterochromatin clusters from mouse tissues and combined it with a quantitative mass spectrometry approach. We identified and validated previously unknown proteins along with proteins known to be involved in constitutive heterochromatin cluster formation and function. We observed differing heterochromatin organization between tissues and identified heterochromatin proteins with distinct abundance between mouse brain and liver tissue. Especially MeCP2, ATRX, and histone H1 might play a role in the distinct heterochromatin organization between tissues, but also newly identified candidate heterochromatin proteins could be involved. We found the methyl-CpG binding protein (MeCP2) enriched in the mouse brain. It binds to methylated cytosines on the DNA, recruits numerous chromatin-associated factors, and was shown to induce dose-dependent heterochromatin reorganization during terminal differentiation. Mutations in the MECP2 gene were associated with the neurological disorder Rett syndrome and altered protein levels were reported to cause disease phenotypes. Thus, we quantified MeCP2 levels in mouse brain nuclei and a cellular system upon exogenous expression of Mecp2-GFP. We showed that the level of MeCP2 in mouse brains is similar to its level in low transfected cells in our cellular system. In addition, we estimated the MeCP2 protein concentrations in the cell nucleus and heterochromatin compartments. We established a system that enables us to adjust the MeCP2 level of in cellulo and in vitro experiments to the in vivo protein level, thus ensuring comparability of experimental results to the physiological situation. Heterochromatin organization and its function are not only regulated by the proteins involved and their levels, but also by post-translational modifications of chromatin proteins. Hence, we isolated MeCP2 from the adult mouse brain and analyzed its modifications by mass spectrometry. We identified various post-translational modifications on MeCP2 and confirmed the influence of arginine methylation and to a lesser extent serine phosphorylation on MeCP2-mediated heterochromatin organization in a cellular system. Coexpression of arginine methyltransferases 1 and 6 lead to a decrease in heterochromatin clustering. Interestingly, we identified the Rett syndrome mutation R106 as a possible dimethylation site, implicating a role of MeCP2 post-translational modifications in the pathology of Rett syndrome. In summary, our data contribute to a better understanding of the constitutive heterochromatin organization in cells and its role in disease development.