Expanding the repertoire of glucocorticoid receptor target genes by engineering genomic response elements.

Verena Thormann,Melissa Bothe,Maika C Rothkegel,Marina Borschiwer,Laura V Glaser,Sebastiaan H Meijsing,Alisa Fuchs

doi:10.26508/lsa.201800283

Verena Thormann, Melissa Bothe + Show 5 more

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https://doi.org/10.26508/lsa.201800283

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Abstract

The glucocorticoid receptor (GR), a hormone-activated transcription factor, binds to a myriad of genomic binding sites yet seems to regulate a much smaller number of genes. Genome-wide analysis of GR binding and gene regulation has shown that the likelihood of GR-dependent regulation increases with decreased distance of its binding to the transcriptional start site of a gene. To test if we can adopt this knowledge to expand the repertoire of GR target genes, we used CRISPR/Cas-mediated homology-directed repair to add a single GR-binding site directly upstream of the transcriptional start site of each of four genes. To our surprise, we found that the addition of a single GR-binding site can be enough to convert a gene into a GR target. The gain of GR-dependent regulation was observed for two of four genes analyzed and coincided with acquired GR binding at the introduced binding site. However, the gene-specific gain of GR-dependent regulation could not be explained by obvious differences in chromatin accessibility between converted genes and their non-converted counterparts. Furthermore, by introducing GR-binding sequences with different nucleotide compositions, we show that activation can be facilitated by distinct sequences without obvious differences in activity between the GR-binding sequence variants we tested. The approach to use genome engineering to build genomic response elements facilitates the generation of cell lines with tailored repertoires of GR-responsive genes and a framework to test and refine our understanding of the cis-regulatory logic of gene regulation by testing if engineered response elements behave as predicted.

Highlights

Cis-regulatory elements embedded in the genome encode the information to relay environmental and developmental cues into specific patterns of gene expression
Either no recruitment or a less robust acquired recruitment of glucocorticoid receptor (GR) was found for the genes that could not be converted (VSIG1 and GYPC, Fig 2) indicating that GR binding is required for the acquired GR-dependent regulation
Because GR almost exclusively binds to regions of open chromatin [7], differences in chromatin accessibility would provide a straightforward explanation for the gene-specific acquired activation observed

Summary

Introduction

Cis-regulatory elements embedded in the genome encode the information to relay environmental and developmental cues into specific patterns of gene expression. The information is decoded by transcription factors (TFs), which bind to cis-regulatory elements and set in motion a cascade of events to change the expression level of genes. An appealing feature of GR to study gene regulation is that its activity can be turned on or off by the addition or removal of its ligand (e.g., dexamethasone, a synthetic glucocorticoid ligand). This on/off switch facilitates the relatively straightforward identification of target genes by comparing gene expression levels between untreated cells and cells treated with hormone. GR binds as a dimer to typically imperfect half sites separated by a 3-bp spacer, and the exact sequence of the half site, the spacer, and of the nucleotides flanking the GR-binding sequence (GBS) can modulate GR’s activity towards target genes [10, 11]

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