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The most common form of DNA methylation involves the addition of a methyl group to a cytosine base in the context of a cytosine-phosphate-guanine (CpG) dinucleotide. Genomes from more primitive organisms are more abundant in CpG sites that, through the process of methylation, deamination and subsequent mutation to thymine-phosphate-guanine (TpG) sites, can produce new transcription factor binding sites. Here, we examined the evolutionary history of the over 36 000 glucocorticoid receptor (GR) consensus binding motifs in the human genome and identified a subset of them in regulatory regions that arose via a deamination and subsequent mutation event. GR can bind to both unmodified and methylated pre-GR binding sequences (GBSs) that contain a CpG site. Our structural analyses show that CpG methylation in a pre-GBS generates a favorable interaction with Arg447 mimicking that made with a TpG in a GBS. This methyl-specific recognition arose 420 million years ago and was conserved during the evolution of GR and likely helps fix the methylation on the relevant cytosines. Our study provides the first genetic, biochemical and structural evidence of high-affinity binding for the likely evolutionary precursor of extant TpG-containing GBS., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nuclear receptors (NRs) are a family of transcription factors that regulate numerous physiological processes such as metabolism, reproduction, inflammation, as well as the circadian rhythm. NRs sense changes in lipid metabolite levels to drive differential gene expression, producing distinct physiologic effects. This is an allosteric process whereby binding a cognate ligand and specific DNA sequences drives the recruitment of diverse transcriptional co-regulators at chromatin and ultimately transactivation or transrepression of target genes. Dysregulation of NR signaling leads to various malignances, metabolic disorders, and inflammatory disease. Given their important role in physiology and ability to respond to small lipophilic ligands, NRs have emerged as valuable therapeutic targets. Here, we summarize and discuss the recent progress on understanding the complex mechanism of action of NRs, primarily from a structural perspective. Finally, we suggest future studies to improve our understanding of NR signaling and better design drugs by integrating multiple structural and biophysical approaches., (© 2018 The Protein Society.)

Glucocorticoids (GCs) are potent repressors of NF-κB activity, making them a preferred choice for treatment of inflammation-driven conditions. Despite the widespread use of GCs in the clinic, current models are inadequate to explain the role of the glucocorticoid receptor (GR) within this critical signaling pathway. GR binding directly to NF-κB itself-tethering in a DNA binding-independent manner-represents the standing model of how GCs inhibit NF-κB-driven transcription. We demonstrate that direct binding of GR to genomic NF-κB response elements (κBREs) mediates GR-driven repression of inflammatory gene expression. We report five crystal structures and solution NMR data of GR DBD-κBRE complexes, which reveal that GR recognizes a cryptic response element between the binding footprints of NF-κB subunits within κBREs. These cryptic sequences exhibit high sequence and functional conservation, suggesting that GR binding to κBREs is an evolutionarily conserved mechanism of controlling the inflammatory response.

Microtubules are highly dynamic tubulin polymers that are required for a variety of cellular functions. Despite the importance of a cellular population of tubulin dimers, we have incomplete information about the mechanisms involved in the biogenesis of αβ-tubulin heterodimers. In addition to prefoldin and the TCP-1 Ring Complex, five tubulin-specific chaperones, termed cofactors A-E (TBCA-E), and GTP are required for the folding of α- and β-tubulin subunits and assembly into heterodimers. We recently described the purification of a novel trimer, TBCD•ARL2•β-tubulin. Here, we employed hydrogen/deuterium exchange coupled with mass spectrometry to explore the dynamics of each of the proteins in the trimer. Addition of guanine nucleotides resulted in changes in the solvent accessibility of regions of each protein that led to predictions about each's role in tubulin folding. Initial testing of that model confirmed that it is ARL2, and not β-tubulin, that exchanges GTP in the trimer. Comparisons of the dynamics of ARL2 monomer to ARL2 in the trimer suggested that its protein interactions were comparable to those of a canonical GTPase with an effector. This was supported by the use of nucleotide-binding assays that revealed an increase in the affinity for GTP by ARL2 in the trimer. We conclude that the TBCD•ARL2•β-tubulin complex represents a functional intermediate in the β-tubulin folding pathway whose activity is regulated by the cycling of nucleotides on ARL2. The co-purification of guanine nucleotide on the β-tubulin in the trimer is also shown, with implications to modeling the pathway., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The glucocorticoid receptor (GR) is a ligand-regulated transcription factor that controls the expression of extensive gene networks, driving both up- and down-regulation. GR utilizes multiple DNA-binding-dependent and -independent mechanisms to achieve context-specific transcriptional outcomes. The DNA-binding-independent mechanism involves tethering of GR to the pro-inflammatory transcription factor activator protein-1 (AP-1) through protein-protein interactions. This mechanism has served as the predominant model of GR-mediated transrepression of inflammatory genes. However, ChIP-seq data have consistently shown GR to occupy AP-1 response elements (TREs), even in the absence of AP-1. Therefore, the current model is insufficient to explain GR action at these sites. Here, we show that GR regulates a subset of inflammatory genes in a DNA-binding-dependent manner. Using structural biology and biochemical approaches, we show that GR binds directly to TREs via sequence-specific contacts to a GR-binding sequence (GBS) half-site found embedded within the TRE motif. Furthermore, we show that GR-mediated transrepression observed at TRE sites to be DNA-binding-dependent. This represents a paradigm shift in the field, showing that GR uses multiple mechanisms to suppress inflammatory gene expression. This work further expands our understanding of this complex multifaceted transcription factor., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The synthetic glucocorticoids (GCs) dexamethasone, mometasone furoate, and triamcinolone acetonide are pharmaceutical mainstays to treat chronic inflammatory diseases. These drugs bind to the glucocorticoid receptor (GR), a ligand-activated transcription factor and member of the nuclear receptor superfamily. The GR is widely recognized as a therapeutic target for its ability to counter proinflammatory signaling. Despite the popularity of GCs in the clinic, long-term use leads to numerous side effects, driving the need for new and improved drugs with less off-target pharmacology. X-ray crystal structures have played an important role in the drug-design process, permitting the characterization of robust structure-function relationships. However, steroid receptor ligand-binding domains (LBDs) are inherently unstable, and their crystallization requires extensive mutagenesis to enhance expression and crystallization. Here, we use an ancestral variant of GR as a tool to generate a high-resolution crystal structure of GR in complex with the potent glucocorticoid triamcinolone acetonide (TA) and a fragment of the small heterodimer partner (SHP). Using structural analysis, molecular dynamics, and biochemistry, we show that TA increases intramolecular contacts within the LBD to drive affinity and enhance stability of the receptor-ligand complex. These data support the emerging theme that ligand-induced receptor conformational dynamics at the mouth of the pocket play a major role in steroid receptor activation. This work also represents the first GR structure in complex with SHP, which has been suggested to play a role in modulating hepatic GR function., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

The glucocorticoid receptor (GR) is a constitutively expressed transcriptional regulatory factor (TRF) that controls many distinct gene networks, each uniquely determined by particular cellular and physiological contexts. The precision of GR-mediated responses seems to depend on combinatorial, context-specific assembly of GR-nucleated transcription regulatory complexes at genomic response elements. In turn, evidence suggests that context-driven plasticity is conferred by the integration of multiple signals, each serving as an allosteric effector of GR conformation, a key determinant of regulatory complex composition and activity. This structural and mechanistic perspective on GR regulatory specificity is likely to extend to other eukaryotic TRFs.

Oct4 is a transcription factor required for maintaining pluripotency and self-renewal in stem cells. Prior to differentiation, Oct4 must be silenced to allow for the development of the three germ layers in the developing embryo. This fine-tuning is controlled by the nuclear receptors (NRs), liver receptor homolog-1 (LRH-1) and germ cell nuclear factor (GCNF). Liver receptor homolog-1 is responsible for driving the expression of Oct4 where GCNF represses its expression upon differentiation. Both receptors bind to a DR0 motif located within the Oct4 promoter. Here, we present the first structure of mouse GCNF DNA-binding domain in complex with the Oct4 DR0. The overall structure revealed two molecules bound in a head-to-tail fashion on opposite sides of the DNA. Additionally, we solved the structure of the human LRH-1 DNA-binding domain bound to the same element. We explore the structural elements that govern Oct4 recognition by these two NRs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Many genomes contain families of paralogs--proteins with divergent function that evolved from a common ancestral gene after a duplication event. To understand how paralogous transcription factors evolve divergent DNA specificities, we examined how the glucocorticoid receptor and its paralogs evolved to bind activating response elements [(+)GREs] and negative glucocorticoid response elements (nGREs). We show that binding to nGREs is a property of the glucocorticoid receptor (GR) DNA-binding domain (DBD) not shared by other members of the steroid receptor family. Using phylogenetic, structural, biochemical, and molecular dynamics techniques, we show that the ancestral DBD from which GR and its paralogs evolved was capable of binding both nGRE and (+)GRE sequences because of the ancestral DBD's ability to assume multiple DNA-bound conformations. Subsequent amino acid substitutions in duplicated daughter genes selectively restricted protein conformational space, causing this dual DNA-binding specificity to be selectively enhanced in the GR lineage and lost in all others. Key substitutions that determined the receptors' response element-binding specificity were far from the proteins' DNA-binding interface and interacted epistatically to change the DBD's function through DNA-induced allosteric mechanisms. These amino acid substitutions subdivided both the conformational and functional space of the ancestral DBD among the present-day receptors, allowing a paralogous family of transcription factors to control disparate transcriptional programs despite high sequence identity.