Transcriptional Activation of Heterochromatin by Recruitment of dCas9 Activators.

Autor:	Frank L; Division of Chromatin Networks, German Cancer Research Center (DKFZ) and Bioquant, Heidelberg, Germany., Weinmann R; Division of Chromatin Networks, German Cancer Research Center (DKFZ) and Bioquant, Heidelberg, Germany., Erdel F; LBME, Centre de Biologie Intégrative (CBI), CNRS, UPS, Toulouse, France., Trojanowski J; Division of Chromatin Networks, German Cancer Research Center (DKFZ) and Bioquant, Heidelberg, Germany., Rippe K; Division of Chromatin Networks, German Cancer Research Center (DKFZ) and Bioquant, Heidelberg, Germany. karsten.rippe@dkfz.de.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2021; Vol. 2351, pp. 307-320.
DOI:	10.1007/978-1-0716-1597-3_17
Abstrakt:	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. (© 2021. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze:	MEDLINE
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