Nascent transcript analysis of glucocorticoid crosstalk with TNF defines primary and cooperative inflammatory repression

Abstract

The glucocorticoid receptor (GR) binds to specific DNA sequences and directly induces transcription of anti-inflammatory genes that contribute to cytokine repression, frequently in cooperation with NF-kB. Whether inflammatory repression also occurs through local interactions between GR and inflammatory gene regulatory elements remains controversial.Here, using Global Run-on Sequencing (GRO-seq) in human airway epithelial cells, we show that glucocorticoid signaling represses transcription within 10 minutes. Many repressed regulatory regions reside within 'hyper-ChIPable' genomic regions that are subject to nonspecific interactions with some antibodies. When this was accounted for, we determined that transcriptional repression occurs without local GR occupancy. Instead, widespread transcriptional induction through canonical GR binding sites is associated with reciprocal repression of distal TNF-regulated enhancers through a chromatin-dependent process, as evidenced by chromatin accessibility and enhancer-reporter assays. Simultaneously, transcriptional induction of key anti-inflammatory effectors is decoupled from primary repression through cooperation between GR and NF-kB at a subset of regulatory regions.Thus, glucocorticoids exert bimodal restraints on inflammation characterized by rapid primary transcriptional repression without local GR occupancy and secondary anti-inflammatory effects resulting from transcriptional cooperation between GR and NF-kB.3

Genomic determinants implicated in the glucocorticoid-mediated induction of KLF9 in pulmonary epithelial cells

“…Once bound to DNA, glucocorticoid-activated GR recruits cofactors that promote activation, or recruitment and activation, of the transcriptional machinery (17,18). This increases transcriptional activity at GREs, as evidenced by local RNA polymerase II (Pol II) recruitment and the production of bidirectional enhancer RNA (eRNA), plus leads to directional transcriptional activation across the body of target genes (19,20). Despite the large number of potential GRE sites throughout the genome, ligand-activated GR only binds to a small fraction of these sites in any given cell type or tissue (21).…”

Transcription factor enrichment analysis (TFEA): Quantifying the activity of hundreds of transcription factors from a single experiment

“…The simplest approach to this problem is to compare the MD-Scores between the set of differentially transcribed regions and regions whose transcription is unchanged, a method we refer to as the differential motif displacement analysis (MDD, see Supp. Fig.7 for full details) [52,24]. Unfortunately, the MDD method introduces an additional arbitrary threshold to classify regions as differentially transcribed or not and still uses the distance threshold set by the MD-Score approach.…”

“…To assess the effectiveness of the TFEA method, we first compared its performance to both the MD-Score [6] and MDD-Score [52,24] approaches. We examined a dataset in which a 1 hr Nutlin-3a treatment of HCT116 cells is used to activate TP53 [1].…”

“…More sophisticated approaches can take advantage of two additional factors: 1) positional information -where the motif is located relative to a region of interest [8,6] and 2) differential information -the amount of change occurring within that region of interest [45,12]. Relatively few techniques encode both types of information [39,52,24] and these currently provide no easily accessible software package or web-based application.…”

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

“…Next, we assessed whether differential occupancy contributes to the receptorspecific transcriptional regulation we observed. Given the small number of repressed genes for AR and the ambiguous link between GR binding and transcriptional repression (Sasse et al 2019), we decided to focus our analysis on upregulated genes. Genes were categorized as either non-regulated by either AR or GR, shared between AR and GR, GRspecific or AR-specific ( Fig.…”