CRISPR-based reagents to study the influence of the epigenome on gene expression

Abstract

The use of epigenome editing is set to expand our knowledge of how epigenetic landscapes facilitate gene expression capacity within a given cell. As epigenetic landscape profiling in health and disease becomes more commonplace, so does the requirement to assess the functional impact that particular regulatory domains and DNA methylation profiles have upon gene expression capacity. That functional assessment is particularly pertinent when analysing epigenomes in disease states where the reversible nature of histone and DNA modification might yield plausible therapeutic targets. In this review we discuss first the nature of the epigenetic landscape, secondly the types of factors that deposit and erase the various modifications, consider how modifications transduce their signals, and lastly address current tools for experimental epigenome editing with particular emphasis on the immune system.

Keywords: gene regulation; molecular biology; transcription factors.

Figure 1

Chromatin domains. Chromatin is organized into lamina‐associated domains (LADs) and topologically associating domains (TADs). LADs are enriched in genes that are either not expressed or are expressed at low levels. DNA sequences within individual TADs interact with one another physically with a greater frequency than they do with sequences outside the TAD suggesting that regulatory domains influencing expression of an individual gene will be constrained to a particular TAD.

Figure 2

Use of dCas9 fusion proteins in epigenetics. (a) dCas9 is cloned in frame with a Kruppel‐associated box (KRAB) domain which, upon delivery to cells along with specific gRNAs, can mediate gene silencing. Silencing is facilitated by the recruitment of KRAB‐associated protein‐1 (Kap1) to KRAB. Kap1 contains a heterochromatin protein1 (HP1) binding domain which is required for transcriptional repression, and a carboxy‐terminal tandem plant homeodomain (PHD) and bromodomain which can recruit the H3K9 methyl transferase SETDB1 to implement H3K9 methylation. (b) dCas9 is cloned in frame with the catalytic domain of the histone acetyl transferase p300. The histone acetyl transferase (HAT) is able to mediate the acetylation of H3K27 in a gRNA‐dependent manner. H3K27acetylation can be recognized by bromodomain‐containing proteins. (c) dCas9 is cloned in frame with an epitope such as FLAG, which can be used with specific gRNAs to immunoprecipitate fragmented chromatin and proteins bound to it.