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  • br GR Is a Transcriptional

    2022-05-13


    GR Is a Transcriptional Activator How GR activates some genes while repressing others remains unsettled after decades of study. While significant controversy exists regarding GR-mediated repression, a consensus model for transcriptional activation has emerged: GR activates transcription through sequence-specific binding to the genome at palindromic motifs. This is strongly supported by genomic studies. Dimeric sites preferentially associate with ligand-activated gene expression on a genome scale 57, 58, 63, and become occupied by transcriptional cofactors, activating histone modifications and RNAPII in response to GR binding during cell differentiation 67, 68. While these data are correlative, self-transcribing active regulatory region sequencing (STARR-seq) has been used to directly examine the transcriptional regulatory properties of GBSs. The assay interrogates enhancer function in a direct, quantitative, and high-throughput manner by placing DNA fragments from any source downstream of a minimal promoter and introducing the reporter library into CH 223191 mg [69]. Analysis of GR ChIP DNA by STARR-seq revealed that 95% of the fragments conferring GC regulation increased reporter gene expression in response to exogenous GC [70]. Moreover, sequence analysis identified the GR palindromic motif as the sole predictor of GC regulation, and ChIP-exo revealed a characteristic dimeric profile at the GC-regulated regions, demonstrating that GC-induced enhancers encode dimeric sites for GR. Similar to ligands, DNA functions as an allosteric regulator of GR by modulating its activity downstream of genomic occupancy to induce a select fraction of the GR transcriptome 64, 71, 72, 73. More generally, GR activates gene expression by integrating signals to nucleate the assembly of cofactors and the general transcription machinery. Recruitment of the mediator complex 43, 68, a cofactor that facilitates long-range chromatin interactions between TFs and the transcription initiation machinery 74, 75, implicates GR in the formation of DNA loops that potentially connect GBSs with promoters to activate distant genes. Similarly, DNA looping was invoked to explain the observation that dimeric sites are frequently surrounded by additional GBSs that lack both the palindromic motif and the ability to confer transcriptional regulation in response to GC [70]. The model posits that many ChIP-seq peaks represent chromatin-bound GR tethered to remotely bound TFs via looping, but this requires testing by chromatin confirmation capture techniques. It is also possible that monomeric binding by GR helps to explain the clustered arrangement of GBSs. Monomeric sites are enriched near dimeric sites in liver, yet they outnumber dimeric sites by 5:1 [58]. In comparison with GR dimers, monomers are suboptimized for DNA binding 62, 64 and transcriptional activation 71, 76, 77. This suggests that they are intermediates in the evolution of dimeric sites. However, with the discovery that genomic recognition sequences are suboptimized for TF affinity to CH 223191 mg favor tissue specificity at the expense of activity [78], it is also possible that monomeric sites are important for the tissue-specific functions of GR. Consistent with this, chromatin-bound monomers colocalize more frequently with lineage-dependent TFs than do dimers 58, 59, which may be a general property of steroid nuclear receptors 60, 79.
    Glucocorticoid-Mediated Repression and GR Approximately half of the genes affected by GC treatment are downregulated independent of the experimental system under study. This is important given that the immunosuppressive properties of GCs are mediated, at least in part, by transcriptional inhibition of proinflammatory genes in immune cells [80]. Unlike transactivation, a clear mechanism for GR transrepression has not emerged from genomic data. However, what appears clear is that the two prominent models for repression, namely (i) tethering of GR monomers and (ii) binding of GR to repressive DNA motifs termed ‘negative glucocorticoid response elements’, or nGREs 81, 82, are not supported by an unbiased examination of GBSs. ChIP-seq in primary macrophages [83] and liver tissue [39] revealed similar enrichment of GR near both ligand-activated and ligand-repressed genes. Despite expectations, sequence analyses failed to find motifs distinguishing the putatively activating and repressing GBSs and, thus, failed to implicate potential TFs mediating GR-dependent repression through tethering. Furthermore, the nGRE was not enriched in either these studies or any other published GR ChIP-seq data set, challenging the idea that GR binds directly to this motif under physiological conditions. It is striking that STARR-seq failed to identify GBSs that repress reporter gene activity in response to exogenous GC [70]. A technical deficiency is unlikely given that STARR-seq found sequences conferring negative regulation by steroid hormone in flies [84]. In this case, the repression occurred independently of receptor binding to the regulatory sequence, suggesting an indirect cause. A potential concern of STARR-seq is that current iterations measure reporter activity from plasmids, and substantial differences exist between enhancer activity encoded on episomes versus chromosomes [85]. While it is formally possible that STARR-seq is unable to detect GR-mediated repression requiring a chromosomal context, this is unlikely for tethering, which was originally described using transiently transfected reporter plasmids.