Project Details
Projekt
Revealing the contribution of phase separation for establishing heterochromatin domains
Applicants
Professor Dr. Axel Imhof; Professor Dr. Karsten Rippe
Subject Area
Biochemistry
Term
since 2019
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 419067076
Pericentric repeat sequences in Drosophila and mouse self-organize in the cell nucleus into distinct µm-size heterochromatin subcompartments called chromocenters. Chromocenters accumulate protein factors like heterochromatin protein 1 (HP1) and are marked by histone H3 lysine 9 trimethylation. Intact chromocenters are essential for silencing repeat transcription and genome stability. In the first funding period we studied liquid-liquid phase separation (LLPS) mechanisms for maintaining chromocenters. The results obtained with mouse fibroblasts argue against LLPS of HP1 being involved this process, in line with other studies that report the lack of an HP1 phenotype on mesoscale chromatin structure in differentiated cells. However, LLPS could be relevant for formation/disassembly of chromocenters during cellular differentiation and the cell cycle. Accordingly, we now focus on chromocenter transitions between the condensed heterochromatin state and the decondensed transcriptionally active state as well as loss of clustering of pericentric repeat sequences. This will allow us to distinguish between factors that govern chromocenter formation versus its maintenance. Furthermore, we will also broaden our analysis to include protein factors other than HP1 that are involved in this process. Since our super-resolution microscopy analysis of chromocenters points to a multi-layered granular structure with smaller subdomains we will characterize their functional properties. To reveal intermediates that form during chromocenter state transitions we will apply and further advance dCas9 based approaches that we have developed. A focus will be on the histone acetylase p300 that we found to spatially segregate into liquid-like droplets during chromocenter activation. Furthermore, we will assess if mechanisms inferred from studies of ectopically perturbed chromocenters also apply to endogenous transitions during Drosophila embryonic development and the cell cycle. Our findings from the last funding period suggest that components of the chromocenter that regulate its formation have differentially evolved in Drosophila species. We will now continue to study the influence of phase separation on hybrid viability. By comparing different chromocenter states with a combination of advanced fluorescence microscopy methods and mass spectrometry-based analysis of their proteome we will be able to address the question if the establishment/disassembly of chromocenters involves phase separated intermediates. Thus, our project with reveal how the dynamic molecular organization of chromocenters is linked to their function and shed light on the role of phase separation in this process.
DFG Programme
Priority Programmes
