Genome-wide Binding Potential and Regulatory Activity of the Glucocorticoid Receptor’s Monomeric and Dimeric Forms
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PROVIDER: PRJNA482932 | ENA |
REPOSITORIES: ENA
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Project description:A widely regarded model for glucocorticoid receptor (GR) action postulates that dimeric binding to DNA regulates unfavorable metabolic pathways while monomeric receptor binding promotes repressive gene responses related to its anti-inflammatory effects. This model has been built upon the characterization of the GRdim mutant, reported to be incapable of DNA binding and dimerization. Although quantitative live-cell imaging data shows GRdim as mostly dimeric, genomic studies, on the basis of recovery of enriched half-site response elements, suggest monomeric engagement on DNA. Here, we performed genome-wide studies on GRdim and a constitutively monomeric mutant. Our results show that impairing dimerization affects binding to even open chromatin. Also, GRdim does not exclusively bind half-response elements. Our results do not support a physiological role for monomeric GR and are consistent with a common mode of receptor binding via higher order structures that drives both the activating and repressive actions of glucocorticoids.
2021-01-19 | GSE117661 | GEO
Project description:The glucocorticoid receptor (GR) binds to DNA in at least three stoichiometric ratios to regulate gene expression. The dimeric binding of GR to a 15 base pair core sequence has been well studied, and is associated with activation of gene expression. Monomeric GR has been associated with repression. GR has also been shown to form tetramers on DNA in live cells. The role of DNA sequence in directing DNA-binding stoichiometry and subsequent gene regulation is not clear. We took an unbiased approach using SELEX-seq to calculate comprehensive monomeric- and dimeric-binding profiles for GR. The monomeric site is composed of a canonical half-site, but is distinguished from the dimer site by a downstream TTTg motif and the absence of a second half-site. To probe the mechanism of how this motif favors monomeric binding, we performed Spec-/Coop-seq. The TTTg increases monomeric affinity while decreasing cooperative dimeric binding. Crystal structures indicated that TTTg results in a narrowed minor groove bringing residues Y455 and K471 of GR into the proximity of DNA, increasing affinity. Interestingly, although monomeric binding is more often associated with repression of gene expression than dimeric binding, the trend is not significant. Consistent with live-cell imaging, a meta-analysis of GR:DNA structures reveals that, regardless of binding sequence, GR binds to DNA as a dimer of dimers using highly similar interfaces. This suggests that the functional stoichiometry of GR in the cell is tetrameric, and that sequence may modulate GR activity.
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Project description:Cone-rod homeobox (CRX) is a paired-like homeodomain transcription factor (TF) and a master regulator of photoreceptor development in vertebrates. The in vitro DNA binding preferences of CRX have been described in detail, but the degree to which in vitro binding affinity is correlated with in vivo enhancer activity is not known. In addition, paired-class homeodomain TFs can bind DNA cooperatively as both homodimers and heterodimers at inverted TAAT half-sites separated by two or three nucleotides. This dimeric configuration is thought to mediate target specificity, but whether monomeric and dimeric sites encode distinct levels of activity is not known. Here, we use a massively parallel reporter assay, CRE-seq, to determine how local sequence context shapes the regulatory activity of CRX binding sites in mouse photoreceptors. We assay inactivating mutations in >1700 TFBSs, and we find that dimeric CRX binding sites act as stronger enhancers than monomeric CRX binding sites. Furthermore, the activity of dimeric half-sites is cooperative, dependent on a strict three-base-pair spacing, and tuned by the identity of the spacer nucleotides. Saturating single-nucleotide mutagenesis of 195 CRX binding sites shows that, on average, changes in TFBS affinity are correlated with changes in regulatory activity, but this relationship is obscured when considering mutations across multiple CREs. Taken together, these results demonstrate that the activity of CRX binding sites is highly dependent on sequence context, providing insight into photoreceptor gene regulation and illustrating functional principles of homeodomain binding sites that may be conserved in other cell types.
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Project description:The glucocorticoid receptor (GR) is a ubiquitously expressed transcription factor that controls metabolic and homeostatic processes essential for life. Although numerous crystal structures of the GR ligand-binding domain (GR-LBD) have been reported, the functional oligomeric state of the full-length receptor (FL-GR), which is essential for its transcriptional activity, remains disputed. Here we present five new crystal structures of agonist-bound GR-LBD, along with a thorough analysis of previous structural work. We identify four distinct homodimerization interfaces on the GR-LBD surface, which can associate into 20 topologically different homodimers. Biologically relevant homodimers were identified by studying a battery of GR point mutants including crosslinking assays in solution, quantitative fluorescence microscopy in living cells, and transcriptomic analyses. Our results highlight the relevance of non-canonical dimerization modes for GR, especially of contacts made by loop L1-3 residues such as Tyr545. Our work illustrates the unique flexibility of GR’s LBD and suggest many dimeric conformations can coexist within cells. In addition, we unveil pathophysiologically relevant quaternary assemblies of the receptor with important implications for glucocorticoid action and drug design.
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2023-03-01 | GSE220962 | GEO
Project description:Cancer-related alterations of the p53 Tetramerization Domain (TD) abrogate wild-type (WT) p53 function. They result in a protein that preferentially forms monomers or dimers. These are also normal p53 states under basal cellular conditions. However, their physiological relevance is not well understood. We have established in vivo models for monomeric and dimeric p53 which model Li-Fraumeni Syndrome patients with germline p53 TD alterations. p53 monomers are inactive forms of the protein. Unexpectedly, p53 dimers conferred some tumor suppression that is not mediated by canonical WT p53 activities. p53 dimers upregulate the PPAR pathway. These activities are associated with lower prevalence of thymic lymphomas and inhibition of CD8+ T-cell accumulation. Lymphomas derived from dimeric p53 mice show cooperating alterations in the PPAR pathway, further implicating a role for these activities in tumor suppression. Our data reveal novel functions for p53 dimers and support the exploration of PPAR agonists as therapies.
2023-03-01 | GSE220961 | GEO
Project description:Cancer-related alterations of the p53 Tetramerization Domain (TD) abrogate wild-type (WT) p53 function. They result in a protein that preferentially forms monomers or dimers. These are also normal p53 states under basal cellular conditions. However, their physiological relevance is not well understood. We have established in vivo models for monomeric and dimeric p53 which model Li-Fraumeni Syndrome patients with germline p53 TD alterations. p53 monomers are inactive forms of the protein. Unexpectedly, p53 dimers conferred some tumor suppression that is not mediated by canonical WT p53 activities. p53 dimers upregulate the PPAR pathway. These activities are associated with lower prevalence of thymic lymphomas and inhibition of CD8+ T-cell accumulation. Lymphomas derived from dimeric p53 mice show cooperating alterations in the PPAR pathway, further implicating a role for these activities in tumor suppression. Our data reveal novel functions for p53 dimers and support the exploration of PPAR agonists as therapies.
2023-03-01 | GSE220964 | GEO
Project description:Cancer-related alterations of the p53 Tetramerization Domain (TD) abrogate wild-type (WT) p53 function. They result in a protein that preferentially forms monomers or dimers. These are also normal p53 states under basal cellular conditions. However, their physiological relevance is not well understood. We have established in vivo models for monomeric and dimeric p53 which model Li-Fraumeni Syndrome patients with germline p53 TD alterations. p53 monomers are inactive forms of the protein. Unexpectedly, p53 dimers conferred some tumor suppression that is not mediated by canonical WT p53 activities. p53 dimers upregulate the PPAR pathway. These activities are associated with lower prevalence of thymic lymphomas and inhibition of CD8+ T-cell accumulation. Lymphomas derived from dimeric p53 mice show cooperating alterations in the PPAR pathway, further implicating a role for these activities in tumor suppression. Our data reveal novel functions for p53 dimers and support the exploration of PPAR agonists as therapies.
2023-03-01 | GSE220963 | GEO
Project description:Cancer-related alterations of the p53 Tetramerization Domain (TD) abrogate wild-type (WT) p53 function. They result in a protein that preferentially forms monomers or dimers. These are also normal p53 states under basal cellular conditions. However, their physiological relevance is not well understood. We have established in vivo models for monomeric and dimeric p53 which model Li-Fraumeni Syndrome patients with germline p53 TD alterations. p53 monomers are inactive forms of the protein. Unexpectedly, p53 dimers conferred some tumor suppression that is not mediated by canonical WT p53 activities. p53 dimers upregulate the PPAR pathway. These activities are associated with lower prevalence of thymic lymphomas and inhibition of CD8+ T-cell accumulation. Lymphomas derived from dimeric p53 mice show cooperating alterations in the PPAR pathway, further implicating a role for these activities in tumor suppression. Our data reveal novel functions for p53 dimers and support the exploration of PPAR agonists as therapies.
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