Coordinated Regulation of DDIT4 Transcriptional Heterogeneity By a Cluster of Glucocorticoid Receptor Binding Sites

Synthetic glucocorticoids are widely prescribed drugs used to treat a variety of human diseases including auto-immune diseases, asthma, cancer, and COVID-19. The transcriptional response to glucocorticoids such as Dexamethasone (Dex) is elicited by the Glucocorticoid Receptor (GR). Upon Dex treatment, GR enters the nucleus and interacts with thousands of enhancer elements throughout the genome. We recently demonstrated that the Dex response in human breast cancer cells is highly heterogeneous and that individual cells have unique transcriptional responses to Dex. To examine whether this heterogeneity arises from differential utilization and activity of GR-bound enhancers, we focused on the Dex response at the GR target gene DNA Damage Inducible Transcript 4 (DDIT4). Using ChIP-seq, ATAC-seq, and RNA-seq we identify 4 potential enhancers with GR binding sites (GBSs) 18-30kb upstream of the DDIT4 TSS and differential patterns of chromatin accessibility, histone acetylation, SWI/SNF recruitment, and eRNA transcription. We generated homozygous deletions of each GBS with CRISPR/CAS9 and used single molecule FISH (smFISH), RT-PCR, and ChIP-seq to determine their unique requirements. While GR binding to these GBSs was independent, each GBS had unique effects on histone acetylation, DDIT4 transcription, and the activity of the other GBSs/enhancers. Three of the GBSs were required for the timing and/or degree of DDIT4 induction. Conversely, the fourth GBS acted as a suppressor and deletion resulted in significant up-regulation of both DDIT4 transcription and enhancer RNA transcription from the other GBSs. Strikingly, smFISH revealed that these enhancers contributed to cellular heterogeneity, as deleting GBSs altered the frequency and amplitude of DDIT4 transcription across cell populations. Taken together, these results demonstrate that individual GBSs uniquely contribute to cell-to-cell heterogeneity within the transcriptional response of DDIT4 to Dex. Furthermore, they underscore the possibility that targeted modification of individual GBSs could be utilized to tailor patient-specific strategies for treatment of human diseases.