RT Journal Article
SR Electronic
T1 Expanding the repertoire of glucocorticoid receptor target genes by engineering genomic response elements
JF Life Science Alliance
JO Life Sci. Alliance
FD Life Science Alliance LLC
SP e201800283
DO 10.26508/lsa.201800283
VO 2
IS 2
A1 Verena Thormann
A1 Laura V Glaser
A1 Maika C Rothkegel
A1 Marina Borschiwer
A1 Melissa Bothe
A1 Alisa Fuchs
A1 Sebastiaan H Meijsing
YR 2019
UL https://www.life-science-alliance.org/content/2/2/e201800283.abstract
AB 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.