Human gut bacteria produce ΤΗ17-modulating bile acid metabolites.

The microbiota modulates gut immune homeostasis. Bacteria influence the development and function of host immune cells, including T helper cells expressing interleukin-17A (TH17 cells). We previously reported that the bile acid metabolite 3-oxolithocholic acid (3-oxoLCA) inhibits TH17 cell differentiation1. Although it was suggested that gut-residing bacteria produce 3-oxoLCA, the identity of such bacteria was unknown, and it was unclear whether 3-oxoLCA and other immunomodulatory bile acids are associated with inflammatory pathologies in humans. Here we identify human gut bacteria and corresponding enzymes that convert the secondary bile acid lithocholic acid into 3-oxoLCA as well as the abundant gut metabolite isolithocholic acid (isoLCA). Similar to 3-oxoLCA, isoLCA suppressed TH17 cell differentiation by inhibiting retinoic acid receptor-related orphan nuclear receptor-γt, a key TH17-cell-promoting transcription factor. The levels of both 3-oxoLCA and isoLCA and the 3α-hydroxysteroid dehydrogenase genes that are required for their biosynthesis were significantly reduced in patients with inflammatory bowel disease. Moreover, the levels of these bile acids were inversely correlated with the expression of TH17-cell-associated genes. Overall, our data suggest that bacterially produced bile acids inhibit TH17 cell function, an activity that may be relevant to the pathophysiology of inflammatory disorders such as inflammatory bowel disease.

The Nuclear Receptor PPARγ Controls Progressive Macrophage Polarization as a Ligand-Insensitive Epigenomic Ratchet of Transcriptional Memory.

Macrophages polarize into distinct phenotypes in response to complex environmental cues. We found that the nuclear receptor PPARγ drove robust phenotypic changes in macrophages upon repeated stimulation with interleukin (IL)-4. The functions of PPARγ on macrophage polarization in this setting were independent of ligand binding. Ligand-insensitive PPARγ bound DNA and recruited the coactivator P300 and the architectural protein RAD21. This established a permissive chromatin environment that conferred transcriptional memory by facilitating the binding of the transcriptional regulator STAT6 and RNA polymerase II, leading to robust production of enhancer and mRNAs upon IL-4 re-stimulation. Ligand-insensitive PPARγ binding controlled the expression of an extracellular matrix remodeling-related gene network in macrophages. Expression of these genes increased during muscle regeneration in a mouse model of injury, and this increase coincided with the detection of IL-4 and PPARγ in the affected tissue. Thus, a predominantly ligand-insensitive PPARγ:RXR cistrome regulates progressive and/or reinforcing macrophage polarization.

Author Correction: Bile acid metabolites control TH17 and Treg cell differentiation.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Bile acid metabolites control TH17 and Treg cell differentiation.

Bile acids are abundant in the mammalian gut, where they undergo bacteria-mediated transformation to generate a large pool of bioactive molecules. Although bile acids are known to affect host metabolism, cancer progression and innate immunity, it is unknown whether they affect adaptive immune cells such as T helper cells that express IL-17a (TH17 cells) or regulatory T cells (Treg cells). Here we screen a library of bile acid metabolites and identify two distinct derivatives of lithocholic acid (LCA), 3-oxoLCA and isoalloLCA, as T cell regulators in mice. 3-OxoLCA inhibited the differentiation of TH17 cells by directly binding to the key transcription factor retinoid-related orphan receptor-γt (RORγt) and isoalloLCA increased the differentiation of Treg cells through the production of mitochondrial reactive oxygen species (mitoROS), which led to increased expression of FOXP3. The isoalloLCA-mediated enhancement of Treg cell differentiation required an intronic Foxp3 enhancer, the conserved noncoding sequence (CNS) 3; this represents a mode of action distinct from that of previously identified metabolites that increase Treg cell differentiation, which require CNS1. The administration of 3-oxoLCA and isoalloLCA to mice reduced TH17 cell differentiation and increased Treg cell differentiation, respectively, in the intestinal lamina propria. Our data suggest mechanisms through which bile acid metabolites control host immune responses, by directly modulating the balance of TH17 and Treg cells.

Structures of NPAS4-ARNT and NPAS4-ARNT2 heterodimers reveal new dimerization modalities in the bHLH-PAS transcription factor family.

Neuronal PER-ARNT-SIM (PAS) domain protein 4 (NPAS4) is a protective transcriptional regulator whose dysfunction has been linked to a variety of neuropsychiatric and metabolic diseases. As a member of the basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) transcription factor family, NPAS4 is distinguished by an ability to form functional heterodimers with aryl hydrocarbon receptor nuclear translocator (ARNT) and ARNT2, both of which are also bHLH-PAS family members. Here, we describe the quaternary architectures of NPAS4-ARNT and NPAS4-ARNT2 heterodimers in complexes involving DNA response elements. Our crystallographic studies reveal a uniquely interconnected domain conformation for the NPAS4 protein itself, as well as its differentially configured heterodimeric arrangements with both ARNT and ARNT2. Notably, the PAS-A domains of ARNT and ARNT2 exhibit variable conformations within these two heterodimers. The ARNT PAS-A domain also forms a set of interfaces with the PAS-A and PAS-B domains of NPAS4, different from those previously noted in ARNT heterodimers formed with other class I bHLH-PAS family proteins. Our structural observations together with biochemical and cell-based interrogations of these NPAS4 heterodimers provide molecular glimpses of the NPAS4 protein architecture and extend the known repertoire of heterodimerization patterns within the bHLH-PAS family. The PAS-B domains of NPAS4, ARNT, and ARNT2 all contain ligand-accessible pockets with appropriate volumes required for small-molecule binding. Given NPAS4's linkage to human diseases, the direct visualization of these PAS domains and the further understanding of their relative positioning and interconnections within the NPAS4-ARNT and NPAS4-ARNT2 heterodimers may provide a road map for therapeutic discovery targeting these complexes.

Retraction Note: DDX5 and its associated lncRNA Rmrp modulate TH17 cell effector functions.

Change History: This Article has been retracted; see accompanying Retraction. Corrected online 20 January: In this Article, author Frank Rigo was incorrectly listed with a middle initial; this has been corrected in the online versions of the paper.

Bidirectional modulation of HIF-2 activity through chemical ligands.

Hypoxia-inducible factor-2 (HIF-2) is a heterodimeric transcription factor formed through dimerization between an oxygen-sensitive HIF-2α subunit and its obligate partner subunit ARNT. Enhanced HIF-2 activity drives some cancers, whereas reduced activity causes anemia in chronic kidney disease. Therefore, modulation of HIF-2 activity via direct-binding ligands could provide many new therapeutic benefits. Here, we explored HIF-2α chemical ligands using combined crystallographic, biophysical, and cell-based functional studies. We found chemically unrelated antagonists to employ the same mechanism of action. Their binding displaced residue M252 from inside the HIF-2α PAS-B pocket toward the ARNT subunit to weaken heterodimerization. We also identified first-in-class HIF-2α agonists and found that they significantly displaced pocket residue Y281. Its dramatic side chain movement increases heterodimerization stability and transcriptional activity. Our findings show that despite binding to the same HIF-2α PAS-B pocket, ligands can manifest as inhibitors versus activators by mobilizing different pocket residues to allosterically alter HIF-2α-ARNT heterodimerization.

Structural integration in hypoxia-inducible factors.

The hypoxia-inducible factors (HIFs) coordinate cellular adaptations to low oxygen stress by regulating transcriptional programs in erythropoiesis, angiogenesis and metabolism. These programs promote the growth and progression of many tumours, making HIFs attractive anticancer targets. Transcriptionally active HIFs consist of HIF-α and ARNT (also called HIF-1ß) subunits. Here we describe crystal structures for each of mouse HIF-2α-ARNT and HIF-1α-ARNT heterodimers in states that include bound small molecules and their hypoxia response element. A highly integrated quaternary architecture is shared by HIF-2α-ARNT and HIF-1α-ARNT, wherein ARNT spirals around the outside of each HIF-α subunit. Five distinct pockets are observed that permit small-molecule binding, including PAS domain encapsulated sites and an interfacial cavity formed through subunit heterodimerization. The DNA-reading head rotates, extends and cooperates with a distal PAS domain to bind hypoxia response elements. HIF-α mutations linked to human cancers map to sensitive sites that establish DNA binding and the stability of PAS domains and pockets.

DDX5 and its associated lncRNA Rmrp modulate TH17 cell effector functions.

T helper 17 (TH17) lymphocytes protect mucosal barriers from infections, but also contribute to multiple chronic inflammatory diseases. Their differentiation is controlled by RORγt, a ligand-regulated nuclear receptor. Here we identify the RNA helicase DEAD-box protein 5 (DDX5) as a RORγt partner that coordinates transcription of selective TH17 genes, and is required for TH17-mediated inflammatory pathologies. Surprisingly, the ability of DDX5 to interact with RORγt and coactivate its targets depends on intrinsic RNA helicase activity and binding of a conserved nuclear long noncoding RNA (lncRNA), Rmrp, which is mutated in patients with cartilage-hair hypoplasia. A targeted Rmrp gene mutation in mice, corresponding to a gene mutation in cartilage-hair hypoplasia patients, altered lncRNA chromatin occupancy, and reduced the DDX5-RORγt interaction and RORγt target gene transcription. Elucidation of the link between Rmrp and the DDX5-RORγt complex reveals a role for RNA helicases and lncRNAs in tissue-specific transcriptional regulation, and provides new opportunities for therapeutic intervention in TH17-dependent diseases.

Chemical Proteomics and Phenotypic Profiling Identifies the Aryl Hydrocarbon Receptor as a Molecular Target of the Utrophin Modulator Ezutromid.

Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease arising from mutations in the dystrophin gene. Upregulation of utrophin to compensate for the missing dystrophin offers a potential therapy independent of patient genotype. The first-in-class utrophin modulator ezutromid/SMT C1100 was developed from a phenotypic screen through to a Phase 2 clinical trial. Promising efficacy and evidence of target engagement was observed in DMD patients after 24 weeks of treatment, however trial endpoints were not met after 48 weeks. The objective of this study was to understand the mechanism of action of ezutromid which could explain the lack of sustained efficacy and help development of new generations of utrophin modulators. Using chemical proteomics and phenotypic profiling we show that the aryl hydrocarbon receptor (AhR) is a target of ezutromid. Several lines of evidence demonstrate that ezutromid binds AhR with an apparent KD of 50 nm and behaves as an AhR antagonist. Furthermore, other reported AhR antagonists also upregulate utrophin, showing that this pathway, which is currently being explored in other clinical applications including oncology and rheumatoid arthritis, could also be exploited in future DMD therapies.

Nuclear receptor full-length architectures: confronting myth and illusion with high resolution.

The crystal structures of three nuclear receptor (NR) complexes have emerged to reveal their multidomain architectures on DNA. These pictures provide unprecedented views of interfacial couplings between the DNA-binding domains (DBDs) and ligand-binding domains (LBDs). The detailed pictures contrast with previous interpretations of low-resolution electron microscopy (EM) and small angle X-ray scattering (SAXS) data, which had suggested a common architecture with noninteracting DBDs and LBDs. Revisiting both historical and recent interpretations of NR architecture, we invoke new principles underlying higher-order quaternary organization and the allosteric transmission of signals between domains. We also discuss how NR architectures are being probed in living cells to understand dimerization and DNA-binding events in real time.

Response to Moras et al.

We recently reviewed full-length nuclear receptor (NR) structures in an Opinion article wherein we carefully evaluated a large body of literature. As heads of three separate laboratories working on NR architectures, we expressed our shared insights and critical comments. One group (Moras et al.) has declined to accept our strong concerns about several of their published reports. We comment on their letter.

Multidomain integration in the structure of the HNF-4α nuclear receptor complex.

The hepatocyte nuclear factor 4α (HNF-4α; also known as NR2A1) is a member of the nuclear receptor (NR) family of transcription factors, which have conserved DNA-binding domains and ligand-binding domains. HNF-4α is the most abundant DNA-binding protein in the liver, where some 40% of the actively transcribed genes have a HNF-4α response element. These regulated genes are largely involved in the hepatic gluconeogenic program and lipid metabolism. In the pancreas HNF-4α is also a master regulator, controlling an estimated 11% of islet genes. HNF-4α protein mutations are linked to maturity-onset diabetes of the young, type 1 (MODY1) and hyperinsulinaemic hypoglycaemia. Previous structural analyses of NRs, although productive in elucidating the structure of individual domains, have lagged behind in revealing the connectivity patterns of NR domains. Here we describe the 2.9 Å crystal structure of the multidomain human HNF-4α homodimer bound to its DNA response element and coactivator-derived peptides. A convergence zone connects multiple receptor domains in an asymmetric fashion, joining distinct elements from each monomer. An arginine target of PRMT1 methylation protrudes directly into this convergence zone and sustains its integrity. A serine target of protein kinase C is also responsible for maintaining domain-domain interactions. These post-translational modifications lead to changes in DNA binding by communicating through the tightly connected surfaces of the quaternary fold. We find that some MODY1 mutations, positioned on the ligand-binding domain and hinge regions of the receptor, compromise DNA binding at a distance by communicating through the interjunctional surfaces of the complex. The overall domain representation of the HNF-4α homodimer is different from that of the PPAR-γ-RXR-α heterodimer, even when both NR complexes are assembled on the same DNA element. Our findings suggest that unique quaternary folds and interdomain connections in NRs could be exploited by small-molecule allosteric modulators that affect distal functions in these polypeptides.

Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORγt activity.

CD4(+) T helper lymphocytes that express interleukin-17 (T(H)17 cells) have critical roles in mouse models of autoimmunity, and there is mounting evidence that they also influence inflammatory processes in humans. Genome-wide association studies in humans have linked genes involved in T(H)17 cell differentiation and function with susceptibility to Crohn's disease, rheumatoid arthritis and psoriasis. Thus, the pathway towards differentiation of T(H)17 cells and, perhaps, of related innate lymphoid cells with similar effector functions, is an attractive target for therapeutic applications. Mouse and human T(H)17 cells are distinguished by expression of the retinoic acid receptor-related orphan nuclear receptor RORγt, which is required for induction of IL-17 transcription and for the manifestation of T(H)17-dependent autoimmune disease in mice. By performing a chemical screen with an insect cell-based reporter system, we identified the cardiac glycoside digoxin as a specific inhibitor of RORγt transcriptional activity. Digoxin inhibited murine T(H)17 cell differentiation without affecting differentiation of other T cell lineages and was effective in delaying the onset and reducing the severity of autoimmune disease in mice. At high concentrations, digoxin is toxic for human cells, but non-toxic synthetic derivatives 20,22-dihydrodigoxin-21,23-diol and digoxin-21-salicylidene specifically inhibited induction of IL-17 in human CD4(+) T cells. Using these small-molecule compounds, we demonstrate that RORγt is important for the maintenance of IL-17 expression in mouse and human effector T cells. These data indicate that derivatives of digoxin can be used as chemical templates for the development of RORγt-targeted therapeutic agents that attenuate inflammatory lymphocyte function and autoimmune disease.

Structure of the intact PPAR-gamma-RXR- nuclear receptor complex on DNA.

Nuclear receptors are multi-domain transcription factors that bind to DNA elements from which they regulate gene expression. The peroxisome proliferator-activated receptors (PPARs) form heterodimers with the retinoid X receptor (RXR), and PPAR-gamma has been intensively studied as a drug target because of its link to insulin sensitization. Previous structural studies have focused on isolated DNA or ligand-binding segments, with no demonstration of how multiple domains cooperate to modulate receptor properties. Here we present structures of intact PPAR-gamma and RXR-alpha as a heterodimer bound to DNA, ligands and coactivator peptides. PPAR-gamma and RXR-alpha form a non-symmetric complex, allowing the ligand-binding domain (LBD) of PPAR-gamma to contact multiple domains in both proteins. Three interfaces link PPAR-gamma and RXR-alpha, including some that are DNA dependent. The PPAR-gamma LBD cooperates with both DNA-binding domains (DBDs) to enhance response-element binding. The A/B segments are highly dynamic, lacking folded substructures despite their gene-activation properties.

Structural overview of the nuclear receptor superfamily: insights into physiology and therapeutics.

As ligand-regulated transcription factors, the nuclear hormone receptors are nearly ideal drug targets, with internal pockets that bind to hydrophobic, drug-like molecules and well-characterized ligand-induced conformational changes that recruit transcriptional coregulators to promoter elements. Yet, due to the multitude of genes under the control of a single receptor, the major challenge has been the identification of ligands with gene-selective actions, impacting disease outcomes through a narrow subset of target genes and not across their entire gene-regulatory repertoire. Here, we summarize the concepts and work to date underlying the development of steroidal and nonsteroidal receptor ligands, including the use of crystal structures, high-throughput screens, and rational design approaches for finding useful therapeutic molecules. Difficulties in finding selective receptor modulators require a more complete understanding of receptor interdomain communications, posttranslational modifications, and receptor-protein interactions that could be exploited for target gene selectivity.

Decoding Allosteric Control in Hypoxia-Inducible Factors.

The mammalian family of basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) transcription factors possess the ability to sense and respond to diverse environmental and physiological cues. These proteins all share a common structural framework, comprising a bHLH domain, two PAS domains, and transcriptional activation or repression domain. To function effectively as transcription factors, members of the family must form dimers, bringing together bHLH segments to create a functional unit that allows for DNA response element binding. The significance of bHLH-PAS family is underscored by their involvement in many major human diseases, offering potential avenues for therapeutic intervention. Notably, the clear identification of ligand-binding cavities within their PAS domains enables the development of targeted small molecules. Two examples are Belzutifan, targeting hypoxia-inducible factor (HIF)-2α, and Tapinarof, targeting the aryl hydrocarbon receptor (AHR), both of which have gained regulatory approval recently. Here, we focus on the HIF subfamily. The crystal structures of all three HIF-α proteins have been elucidated, revealing their bHLH and tandem PAS domains are used to engage their dimerization partner aryl hydrocarbon receptor nuclear translocator (ARNT, also called HIF-1ß). A broad range of recent findings point to a shared allosteric modulation mechanism among these proteins, whereby small-molecules at the PAS-B domains exert direct influence over the HIF-α transcriptional functions. As our understanding of the architectural and allosteric mechanisms of bHLH-PAS proteins continues to advance, the possibility of discovering new therapeutic drugs becomes increasingly promising.

The protein architecture and allosteric landscape of HNF4α.

Hepatocyte nuclear factor 4 alpha (HNF4α) is a multi-faceted nuclear receptor responsible for governing the development and proper functioning of liver and pancreatic islet cells. Its transcriptional functions encompass the regulation of vital metabolic processes including cholesterol and fatty acid metabolism, and glucose sensing and control. Various genetic mutations and alterations in HNF4α are associated with diabetes, metabolic disorders, and cancers. From a structural perspective, HNF4α is one of the most comprehensively understood nuclear receptors due to its crystallographically observed architecture revealing interconnected DNA binding domains (DBDs) and ligand binding domains (LBDs). This review discusses key properties of HNF4α, including its mode of homodimerization, its binding to fatty acid ligands, the importance of post-translational modifications, and the mechanistic basis for allosteric functions. The surfaces linking HNF4α's DBDs and LBDs create a convergence zone that allows signals originating from any one domain to influence distant domains. The HNF4α-DNA complex serves as a prime illustration of how nuclear receptors utilize individual domains for specific functions, while also integrating these domains to create cohesive higher-order architectures that allow signal responsive functions.

Monoterpenoid aryl hydrocarbon receptor allosteric antagonists protect against ultraviolet skin damage in female mice.

The human aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is a pivotal regulator of human physiology and pathophysiology. Allosteric inhibition of AhR was previously thought to be untenable. Here, we identify carvones as noncompetitive, insurmountable antagonists of AhR and characterize the structural and functional consequences of their binding. Carvones do not displace radiolabeled ligands from binding to AhR but instead bind allosterically within the bHLH/PAS-A region of AhR. Carvones do not influence the translocation of ligand-activated AhR into the nucleus but inhibit the heterodimerization of AhR with its canonical partner ARNT and subsequent binding of AhR to the promoter of CYP1A1. As a proof of concept, we demonstrate physiologically relevant Ahr-antagonism by carvones in vivo in female mice. These substances establish the molecular basis for selective targeting of AhR regardless of the type of ligand(s) present and provide opportunities for the treatment of disease processes modified by AhR.