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  • Genomic studies have afforded new insight into the influence

    2022-01-03

    Genomic studies have afforded new insight into the influence of Oleamide structure on GR binding. An important clue came from genome-wide mapping of chromatin structure using DNase I hypersensitive site sequencing (DNase-seq), and comparing the locations of open chromatin to GBSs [10]. DNase-seq in the absence and presence of ligand revealed that up to 95% of GR occupancy is directed to pre-existing regions of accessible chromatin. Similar results were gathered in vivo with an adrenalectomized mouse model [39], suggesting that GR on its own cannot efficiently counteract histone-mediated repression to access DNA in nucleosomes. Given that this is a distinguishing feature of pioneer TFs [47], the ability of GR to serve a pioneering role on its own is limited [48]. Yet, GR binding further remodels chromatin to increase accessibility for other TFs through a mechanism termed ‘assisted loading’, originally advanced to describe how TFs targeting the same DNA motifs can stimulate, rather than inhibit, each other’s occupancy via dynamic TF–DNA interactions [49]. The model depends on the ability of TFs to trigger chromatin remodeling [50], and provides insight into the collaborative binding widely observed at neighboring cis-regulatory sequences 51, 52, 53, 54, 55 and, conversely, helps to explain why enhancers typically contain several closely spaced TF-binding sites. Along with open chromatin, it is important to emphasize that the palindromic GR motif is a critical determinant of genomic occupancy. Before the emergence of genomics, characterization of the recognition sequence of GR had been primarily determined by in vitro approaches. Thus, it was reassuring that motif analyses of GR ChIP-seq data revealed robust enrichment of the palindromic sequence in vivo. However, due to the limited resolution of ChIP-seq, strong enrichment of additional colocalized motifs was also observed, confounding a definitive interpretation of the genomic substrate of GR. This was resolved by ChIP with lambda exonuclease digestion and sequencing (ChIP-exo), a technique designed to unequivocally identify bound motifs through unbiased, high-resolution mapping of TFs to the genome [56]. With ChIP-exo, several groups demonstrated GR binding at the expected palindromic sequence in native chromatin 57, 58, 59.
    GR Monomers Occupy the Genome Sequence Specifically and via Tethering Binding sites lacking the expected motif are present as a minor fraction in most TF ChIP-seq data sets, but can still account for thousands of sites in some cistromes. While experimental artifacts, such as antibody cross-reactivity with chromatin-bound proteins other than GR, may partially explain these sites, it is possible that TF binding in the absence of a motif may be biologically relevant. Of interest to GR, bound sites lacking the motif may represent protein–protein interactions between GR and other DNA-bound TFs to form so-called ‘tethered sites’ 60, 61. Indirect DNA binding is thought to explain the effects of GR on the expression of reporter genes lacking GR motifs, but unambiguous determination of the interacting protein(s) is challenging because presumed tethered sites often reside at composite enhancers bound by multiple TFs. With its superior resolution, ChIP-exo has identified tethering partners for GR monomers 58, 59. GR monomers were also found to interact sequence specifically with the GR half-site motif, consistent with earlier in vitro studies 62, 63, 64. It is intriguing that tethered sites strongly colocalize with monomeric, genomic sites, suggesting that transient contacts between monomers and nearby TFs are promoted and/or stabilized by GR interaction with half-site motifs. This idea, termed ‘half-site-facilitated tethering’, designates DNA as the primary recruiter of monomeric GR (Figure 2A), and stably bound TF neighbors as targets for subsequent protein–protein interactions through GR tracking on chromatin 62, 65. As the evidence for TF tethering mounts in vivo[66], better understanding of its prevalence and role(s) is needed.