Abstract
The transition from silenced heterochromatin to a biologically active state and vice versa is a fundamental part of the implementation of cell type-specific gene expression programs. To reveal structure–function relationships and dissect the underlying mechanisms, experiments that ectopically induce transcription are highly informative. In particular, the approach to perturb chromatin states by recruiting fusions of the catalytically inactive dCas9 protein in a sequence-specific manner to a locus of interest has been used in numerous applications. Here, we describe how this approach can be applied to activate pericentric heterochromatin (PCH) in mouse cells as a prototypic silenced state by providing protocols for the following workflow: (a) Recruitment of dCas9 fusion constructs with the strong transcriptional activator VPR to PCH. (b) Analysis of the resulting changes in chromatin compaction, epigenetic marks, and active transcription by fluorescence microscopy-based readouts. (c) Automated analysis of the resulting images with a set of scripts in the R programming language. Furthermore, we discuss how parameters for chromatin decondensation and active transcription are extracted from these experiments and can be combined with other readouts to gain insights into PCH activation.
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Acknowledgments
This work was funded by the Deutsche Forschungsgemeinschaft (DFG) Priority Program 2191 “Molecular Mechanisms of Functional Phase Separation” via grant RI1283/16-1 and the START-HD project of the HMLS program of the University of Heidelberg. Data storage at SDS@hd was supported by the Ministry of Science, Research and the Arts Baden-Württemberg (MWK) and the DFG through grants INST 35/1314-1 FUGG and INST 35/1503-1 FUGG.
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R image segmentation and quantification scripts and functions with associated sample data: The script SegmentCC.R with associated external functions makeNucMask() and makeChromocenterMask() (as .R files) is used for image segmentation with subsequent quantification. The scripts curateCC.R and plotCC.R enable semi-automated curation and plotting of results obtained by SegmentCC.R (see Fig. 1b–d for workflow). Furthermore, sample .tif images are provided, intended for use with the SegmentCC.R script. Images are z-maximum intensity projections of image stacks acquired for iMEFs that have been transfected with MSR-targeting sgRNA and dCas9-GFP-VPR constructs. All scripts and sample data are also available at https://github.com/RippeLab/Chromocenters.
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Frank, L., Weinmann, R., Erdel, F., Trojanowski, J., Rippe, K. (2021). Transcriptional Activation of Heterochromatin by Recruitment of dCas9 Activators. In: Borggrefe, T., Giaimo, B.D. (eds) Enhancers and Promoters. Methods in Molecular Biology, vol 2351. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1597-3_17
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DOI: https://doi.org/10.1007/978-1-0716-1597-3_17
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