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

DNA methylation repels binding of hypoxia-inducible transcription factors to maintain tumor immunotolerance.

BACKGROUND: Hypoxia is pervasive in cancer and other diseases. Cells sense and adapt to hypoxia by activating hypoxia-inducible transcription factors (HIFs), but it is still an outstanding question why cell types differ in their transcriptional response to hypoxia. RESULTS: We report that HIFs fail to bind CpG dinucleotides that are methylated in their consensus binding sequence, both in in vitro biochemical binding assays and in vivo studies of differentially methylated isogenic cell lines. Based on in silico structural modeling, we show that 5-methylcytosine indeed causes steric hindrance in the HIF binding pocket. A model wherein cell-type-specific methylation landscapes, as laid down by the differential expression and binding of other transcription factors under normoxia, control cell-type-specific hypoxia responses is observed. We also discover ectopic HIF binding sites in repeat regions which are normally methylated. Genetic and pharmacological DNA demethylation, but also cancer-associated DNA hypomethylation, expose these binding sites, inducing HIF-dependent expression of cryptic transcripts. In line with such cryptic transcripts being more prone to cause double-stranded RNA and viral mimicry, we observe low DNA methylation and high cryptic transcript expression in tumors with high immune checkpoint expression, but not in tumors with low immune checkpoint expression, where they would compromise tumor immunotolerance. In a low-immunogenic tumor model, DNA demethylation upregulates cryptic transcript expression in a HIF-dependent manner, causing immune activation and reducing tumor growth. CONCLUSIONS: Our data elucidate the mechanism underlying cell-type-specific responses to hypoxia and suggest DNA methylation and hypoxia to underlie tumor immunotolerance

Retinoid X receptor and its partners in the nuclear receptor family

Retinoid X receptor (RXR) and its dimerization partners in the nuclear receptor family recognize DNA response elements in which two AGGTCA binding sites are arranged in tandem. Target site selection by these complexes requires the spacing between the binding sites to act as the identity element. With the recent determination of three-dimensional structures of several different DNA-binding complexes of RXR, together with studies of protein conformational changes, it is clear how the interactions of RXR with its partners are precisely tuned to match the spacing between their DNA binding sites.status: publishe

Gene expression: Protein-DNA interactions probed by fluorine-19 NMR

All life processes are based on ordered, specific interactions among macromolecules. Complete three-dimensional structure determination is tedious and dependent on fortuitous crystallization or small molecular size. There is a need for bridging methods to obtain real space information in structures, to keep pace with the rapid accumulation of one-dimensional sequence information. The large size and complexity of most protein-DNA complexes provides a challenge for conventional structural methods. This thesis demonstrates that fluorine-19 incorporation in nucleic acids can be combined with \sp{19}F-NMR to detect specific protein-nucleic acid interactions to meet this challenge. 5-fluorouridine (5-FU) can be substituted for thymidine in synthetic DNA molecules. Coliphage T7 RNA polymerase (T7-Pol) can utilize 5-FU containing promoter DNA for RNA transcription. \sp{19}F-NMR spectroscopy allows probing of interactions at the DNA major groove. Several new interactions of T7-Pol were seen by \sp{19}F-NMR. NMR results combined with familiar chemical studies produce a more detailed picture of the promoter DNA. T7-Pol perturbs major groove and minor groove regions along both sides of T7 promoter. The nucleoside triphosphate (NTP) analog, 5-FUTP, allows incorporation of fluorine-19 into T7 transcripts for \sp{19}F-NMR observations at the RNA. To do this, ternary transcriptional complexes of T7-Pol bearing RNA transcripts positioned at defined template sequences are constructed. Addition of NTP results in processive elongation of transcript. \sp{19}F-NMR was also used to examine the prototypic gene-regulatory interaction between E. coli lac repressor and operator DNA. Although lac operator DNA contains a pseudo-two-fold axis, genetic and chemical data suggest asymmetric interactions with lac repressor. The interaction of lac repressor N-terminal-DNA-binding domain at symmetrically related operator sequences was probed using 5-FU substitutions. These domains produced \sp{19}F-NMR shifts consistent with non-equivalent protein interactions at DNA half-sites. However, altering right side DNA sequences to match the left side symmetrized the interactions. The results indicate that intrinsic variations in DNA structure, and not a mismatch between protein and DNA dimensions, cause the asymmetry of interaction. Two-dimensional heteronuclear \sp{19}F-\{\sp1H$\}$ Overhauser spectroscopy of the lac headpiece-operator complex revealed specific protein interactions at the DNA fluorines

Visualizing the Architectures and Interactions of Nuclear Receptors

Nuclear receptors (NRs) are master regulators of broad genetic programs in metazoans. These programs are regulated in part by the small-molecule ligands that bind NRs and modulate their interactions with transcriptional coregulatory factors. X-ray crystallography is now delivering more complete pictures of how the multidomain architectures of NR homo- and heterodimers are physically arranged on their DNA elements and how ligands and coactivator peptides act through these complexes. Complementary studies are also pointing to a variety of novel mechanisms by which NRs access their DNA-response elements within chromatin. Here, we review the new structural advances together with proteomic discoveries that shape our understanding of how NRs form a variety of functional interactions with collaborating factors in chromatin.status: publishe

Physical structure of nuclear receptor-DNA complexes

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Bacteriophage T7 RNA polymerase. 19F-nuclear magnetic resonance observations at 5-fluorouracil-substituted promoter DNA and RNA transcript

We have substituted 5-fluorodeoxyuridine (5-FdU) in place of thymidine in defined positions along synthetic bacteriophage T7 promoter DNA sequences. None of the fluoro-substitutions in the promoter DNA sequence reduced transcription yields with T7 RNA polymerase significantly. Substitutions on the coding template strand reduced transcription yields when placed at +3, but not at +4. 19F-n.m.r. spectra from transcription reactions and gel analysis of transcription products show that T7 RNA polymerase correctly and efficiently utilizes 5-FUTP as a RNA substrate analog. The fluorine atom provides a sensitive probe for monitoring the local environment, base sequence and solvent exposure at the DNA major groove through its 19F-n.m.r. resonance. Buffer dependencies of the fluorine chemical shift and digestion patterns with DNase I suggest that the T7 promoter base-pairs near the transcription start site are distorted with a more open minor groove and less solvent accessible major groove. Previous chemical footprinting data of promoter-polymerase complexes yield a picture that T7 RNA polymerase recognizes major groove features in the region from positions -7 to -11 and minor groove features on the same side of DNA flanking both sides of this region. Consistent with this, 19F-n.m.r. observations identify two additional positions, -8 and -17, involved in promoter recognition on this side of the DNA helix. On the other hand, our observations also implicate the opposite side of the DNA helix, primarily at positions -14 and -15, as major groove recognition sites for T7 RNA polymerase. In addition, n.m.r. spectra from 5-FdU-substituted base-pairs -2 and -3, suggest either additional interactions on the same side of the DNA helix as -14 and -15, or distortions in the DNA structure.status: publishe

Structural characterization of mammalian bHLH-PAS transcription factors

The mammalian basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) transcription factors share common architectural features that include a bHLH DNA-binding domain and tandemly positioned PAS domains. The sixteen members of this family include the hypoxia-inducible factors (HIF-1α and HIF-2α), ARNT (also known as HIF-1β), CLOCK and BMAL1. Most bHLH-PAS proteins have been genetically linked to variety of diseases in humans, including cancers, metabolic syndromes and psychiatric conditions. To function as transcription factors, the bHLH-PAS proteins must form heterodimeric complexes. Recent crystallographic studies of HIF-α-ARNT and CLOCK-BMAL1 complexes have unveiled the organization of their multi-domain bHLH-PAS-A-PAS-B segments, revealing how these architectures can give rise to unique patterns of heterodimerization. As our structural understanding becomes better integrated with ligand-discovery and target gene identification, a more comprehensive picture of their architectural and functional properties will emerge.status: publishe

Nuclear-receptor interactions on DNA-response elements

Nuclear receptors regulate transcription by binding to DNA-response elements using their conserved DNA-binding domains. These response elements contain conserved hexameric sequences that can be arranged in various bipartite configurations, including inverted and direct repeats. A series of structural studies on receptor--DNA binding complexes illustrate the strategies used by receptors to recognize the symmetry of their binding site as well as its sequence. These structures also indicate how cooperation between receptors enhances their joint affinity and selectivity for correctly configured sites.status: publishe

Large scale production of nuclear receptor ligand-binding domains

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