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

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.

Androgen and glucocorticoid receptor direct distinct transcriptional programs by receptor-specific and shared DNA binding sites

“…ATAC-seq data for GR (1 μM Dex, 1.5 h or 0.1% EtOH as vehicle control) is from a previous study ( 30 ), ArrayExpress accession number E-MTAB-7746. For AR (5 nM R1881 or 0.1% dmso as a vehicle control, 4 h), ATAC-seq was done as described ( 30 ). ArrayExpress accession number for ATAC-seq data: E-MTAB-9606.…”

“…Processing of ATAC-seq data was done as previously described in ( 30 ), with the addition that reads of mapping quality <10 were filtered out using SAMtools v1.10 ( 40 ). BigWig tracks for genome browser visualization were generated with bamCoverage (options: –normalizeUsing RPKM; –binSize 20; –smoothLength 60) from deepTools v3.4.1 ( 41 ).…”

Fighting the Fire: Mechanisms of Inflammatory Gene Regulation by the Glucocorticoid Receptor

“…X-ray crystallography of GR DBD dimers bound to different GBSs showed that conformation of the lever arm in the DBD appeared to be influenced by the DNA sequence (24, 119). Furthermore, the addition of a single GR-binding site was sufficient to convert a gene, which was normally not regulated by GR, into a target gene, such as IL-1 β and IL1R2 in U2OS cells (120). The presence of classical GREs in GR-bound enhancers near both activated and repressed genes in murine bone marrow-derived macrophages (BMDM) stimulated with LPS and Dexamethasone (Dex) challenge these models.…”

Mechanisms and Clinical Applications of Glucocorticoid Steroids in Muscular Dystrophy

“…Because GRs function within complexes, GR binding to a GRE, on its own, is not a strong predictor of GR-dependent gene regulation. The likelihood of an occupied GRE driving transcriptional regulation of a gene increases the closer that the GRE is to the gene’s transcriptional start site [ 13, 14 ]. GR binding sites can also work concordantly, with clusters of GREs mediating GR-dependent transcription [ 14 ].…”