Transcriptional Activation of Heterochromatin by Recruitment of dCas9 Activators.
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.
['Frank|Lukas|L|', 'Weinmann|Robin|R|', 'Erdel|Fabian|F|', 'Trojanowski|Jorge|J|', 'Rippe|Karsten|K|']
D000818:Animals D000076987:CRISPR-Associated Protein 9 D002843:Chromatin D005347:Fibroblasts D005455:Fluorescent Antibody Technique D015870:Gene Expression D005786:Gene Expression Regulation D006570:Heterochromatin D007091:Image Processing, Computer-Assisted D051379:Mice D008856:Microscopy, Fluorescence D011485:Protein Binding D015533:Transcriptional Activation D014162:Transfection D057188:Workflow
dCas9 Pericentric heterochromatin Decondensation CRISPR Chromocenter Fluorescence microscopy Image quantification Mouse embryonic fibroblasts