The multivalency of the glucocorticoid receptor ligand-binding domain explains its manifold physiological activities.

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, 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 suggests different dimeric conformations within cells. In addition, we unveil pathophysiologically relevant quaternary assemblies of the receptor with important implications for glucocorticoid action and drug design.

Corticosterone pattern-dependent glucocorticoid receptor binding and transcriptional regulation within the liver.

Ultradian glucocorticoid rhythms are highly conserved across mammalian species, however, their functional significance is not yet fully understood. Here we demonstrate that pulsatile corticosterone replacement in adrenalectomised rats induces a dynamic pattern of glucocorticoid receptor (GR) binding at ~3,000 genomic sites in liver at the pulse peak, subsequently not found during the pulse nadir. In contrast, constant corticosterone replacement induced prolonged binding at the majority of these sites. Additionally, each pattern further induced markedly different transcriptional responses. During pulsatile treatment, intragenic occupancy by active RNA polymerase II exhibited pulsatile dynamics with transient changes in enrichment, either decreased or increased depending on the gene, which mostly returned to baseline during the inter-pulse interval. In contrast, constant corticosterone exposure induced prolonged effects on RNA polymerase II occupancy at the majority of gene targets, thus acting as a sustained regulatory signal for both transactivation and repression of glucocorticoid target genes. The nett effect of these differences were consequently seen in the liver transcriptome as RNA-seq analysis indicated that despite the same overall amount of corticosterone infused, twice the number of transcripts were regulated by constant corticosterone infusion, when compared to pulsatile. Target genes that were found to be differentially regulated in a pattern-dependent manner were enriched in functional pathways including carbohydrate, cholesterol, glucose and fat metabolism as well as inflammation, suggesting a functional role for dysregulated glucocorticoid rhythms in the development of metabolic dysfunction.

Targeted H3R26 deimination specifically facilitates estrogen receptor binding by modifying nucleosome structure.

Transcription factor binding to DNA in vivo causes the recruitment of chromatin modifiers that can cause changes in chromatin structure, including the modification of histone tails. We previously showed that estrogen receptor (ER) target gene activation is facilitated by peptidylarginine deiminase 2 (PAD2)-catalyzed histone H3R26 deimination (H3R26Cit). Here we report that the genomic distributions of ER and H3R26Cit in breast cancer cells are strikingly coincident, linearly correlated, and observed as early as 2 minutes following estradiol treatment. The H3R26Cit profile is unlike that of previously described histone modifications and is characterized by sharp, narrow peaks. Paired-end MNase ChIP-seq indicates that the charge-neutral H3R26Cit modification facilitates ER binding to DNA by altering the fine structure of the nucleosome. Clinically, we find that PAD2 and H3R26Cit levels correlate with ER expression in breast tumors and that high PAD2 expression is associated with increased survival in ER+ breast cancer patients. These findings provide insight into how transcription factors gain access to nucleosomal DNA and implicate PAD2 as a novel therapeutic target for ER+ breast cancer.

Smyd3-mediated immuno-modulation in HPV-negative head and neck squamous cell carcinoma mouse models.

SET and MYND-domain containing protein 3 (SMYD3) mediates epigenetic repression of type I IFN response genes in human papillomavirus (HPV)-negative HNSCC cells, and Smyd3 depletion using anti-sense oligonucleotides (ASOs) increases the sensitivity of syngeneic mouse oral carcinoma (MOC1) models to anti-PD-1 therapy. In this study, we utilized single-cell RNA-seq of MOC1 tumors treated with Smyd3 ASOs and found enrichment of type I IFN response pathways in cancer cells, a shift of CD8+ T-cells toward an activated/memory phenotype, and a shift of neutrophils toward an anti-tumorigenic phenotype. Mechanisms of resistance to the Smyd3 ASO and anti-PD-1 combination were derived from cancer cells, macrophages, and CD8+ T-cells, including neutrophil enrichment through the upregulation of Cxcl2, repression of Cxcl9, and defective antigen presentation. This study sheds light on the immunomodulatory functions of Smyd3 in vivo and provides insight into actionable mechanisms of resistance to improve the efficacy of Smyd3 ASOs and anti-PD-1 combination.

Short-term ocean acidification decreases pulsation and growth of the widespread soft coral Xenia umbellata.

Coral reefs may experience lower pH values as a result of ocean acidification (OA), which has negative consequences, particularly for calcifying organisms. Thus far, the effects of this global factor have been mainly investigated on hard corals, while the effects on soft corals remain relatively understudied. We therefore carried out a manipulative aquarium experiment for 21 days to study the response of the widespread pulsating soft coral Xenia umbellata to simulated OA conditions. We gradually decreased the pH from ambient (~8.3) to three consecutive 7-day long pH treatments of 8.0, 7.8, and 7.6, using a CO2 dosing system. Monitored response variables included pulsation rate, specific growth rate, visual coloration, survival, Symbiodiniaceae cell densities and chlorophyll a content, photosynthesis and respiration, and finally stable isotopes of carbon (C) and nitrogen (N) as well as CN content. Pulsation decreased compared to controls with each consecutive lowering of the pH, i.e., 17% at pH 8.0, 26% at pH 7.8 and 32% at pH 7.6, accompanied by an initial decrease in growth rates of ~60% at pH 8.0, not decreasing further at lower pH. An 8.3 ‰ decrease of δ13C confirmed that OA exposed colonies had a higher uptake and availability of atmospheric CO2. Coral productivity, i.e., photosynthesis, was not affected by higher dissolved inorganic C availability and none of the remaining response variables showed any significant differences. Our findings suggest that pulsation is a phenotypically plastic mechanism for X. umbellata to adjust to different pH values, resulting in reduced growth rates only, while maintaining high productivity. Consequently, pulsation may allow X. umbellata to inhabit a broad pH range with minimal effects on its overall health. This resilience may contribute to the competitive advantage that soft corals, particularly X. umbellata, have over hard corals.

HIV vaccine candidate efficacy in female macaques mediated by cAMP-dependent efferocytosis and V2-specific ADCC.

The development of an effective vaccine to protect against HIV acquisition will be greatly bolstered by in-depth understanding of the innate and adaptive responses to vaccination. We report here that the efficacy of DNA/ALVAC/gp120/alum vaccines, based on V2-specific antibodies mediating apoptosis of infected cells (V2-ADCC), is complemented by efferocytosis, a cyclic AMP (cAMP)-dependent antiphlogistic engulfment of apoptotic cells by CD14+ monocytes. Central to vaccine efficacy is the engagement of the CCL2/CCR2 axis and tolerogenic dendritic cells producing IL-10 (DC-10). Epigenetic reprogramming in CD14+ cells of the cyclic AMP/CREB pathway and increased systemic levels of miRNA-139-5p, a negative regulator of expression of the cAMP-specific phosphodiesterase PDE4D, correlated with vaccine efficacy. These data posit that efferocytosis, through the prompt and effective removal of apoptotic infected cells, contributes to vaccine efficacy by decreasing inflammation and maintaining tissue homeostasis.

SMYD3 represses tumor-intrinsic interferon response in HPV-negative squamous cell carcinoma of the head and neck.

Cancers often display immune escape, but the mechanisms are incompletely understood. Herein, we identify SMYD3 as a mediator of immune escape in human papilloma virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC), an aggressive disease with poor response to immunotherapy with pembrolizumab. SMYD3 depletion induces upregulation of multiple type I interferon (IFN) response and antigen presentation machinery genes in HNSCC cells. Mechanistically, SMYD3 binds to and regulates the transcription of UHRF1, encoding for a reader of H3K9me3, which binds to H3K9me3-enriched promoters of key immune-related genes, recruits DNMT1, and silences their expression. SMYD3 further maintains the repression of immune-related genes through intragenic deposition of H4K20me3. In vivo, Smyd3 depletion induces influx of CD8+ T cells and increases sensitivity to anti-programmed death 1 (PD-1) therapy. SMYD3 overexpression is associated with decreased CD8 T cell infiltration and poor response to neoadjuvant pembrolizumab. These data support combining SMYD3 depletion strategies with checkpoint blockade to overcome anti-PD-1 resistance in HPV-negative HNSCC.

Effective asexual reproduction of a widespread soft coral: comparative assessment of four different fragmentation methods.

BACKGROUND: Many coral reefs worldwide are experiencing declines in hard corals, resulting in other benthic organisms, e.g., soft corals, becoming more dominant. As such, more studies on the ecophysiology of soft corals are needed. Despite many methods for asexual reproduction of hard corals, effective methods for soft corals, i.e., without a hard skeleton, are scarce. This study, thus, assessed four fragmentation methods, the glue, rubber band, tunnel mesh, and plug mesh method for the pulsating soft coral Xenia umbellata that is widely distributed in the tropical Indo-Pacific. METHODS: Methods were comparatively assessed by determining the required time and labor for the fragmentation plus the health status of the fragmented corals by measuring their oxygen fluxes and pulsation rates, i.e., a special feature of this soft coral that can be used as a proxy for its health. RESULTS: There were no significant health status differences between methods. This was indicated by similar gross photosynthesis (between 7.4 to 9.7 µg O2 polyp-1 h-1) and pulsating rates (between 35 and 44 pulses min-1) among methods. In terms of time/labor intensity and success rates, i.e., the percentage of fragments attached to the desired surface, the plug mesh method was the most efficient method with a significantly higher success rate (95 ± 5%), while the others had a success rate between 5 ± 5 and 45 ± 15%. The time needed for fragmentation, though not significant, was also the shortest (78 ± 11 s fragment-1), while other methods required between 84 ± 14 and 126 ± 8 s frag-1. The plug mesh method may thus be a valuable tool related to the reproduction of soft corals for use in subsequent experimental work.

The glucocorticoid receptor associates with the cohesin loader NIPBL to promote long-range gene regulation.

The cohesin complex is central to chromatin looping, but mechanisms by which these long-range chromatin interactions are formed and persist remain unclear. We demonstrate that interactions between a transcription factor (TF) and the cohesin loader NIPBL regulate enhancer-dependent gene activity. Using mass spectrometry, genome mapping, and single-molecule tracking methods, we demonstrate that the glucocorticoid (GC) receptor (GR) interacts with NIPBL and the cohesin complex at the chromatin level, promoting loop extrusion and long-range gene regulation. Real-time single-molecule experiments show that loss of cohesin markedly diminishes the concentration of TF molecules at specific nuclear confinement sites, increasing TF local concentration and promoting gene regulation. Last, patient-derived acute myeloid leukemia cells harboring cohesin mutations exhibit a reduced response to GCs, suggesting that the GR-NIPBL-cohesin interaction is defective in these patients, resulting in poor response to GC treatment.

Predicted functions of MdmX in fine-tuning the response of p53 to DNA damage.

Tumor suppressor protein p53 is regulated by two structurally homologous proteins, Mdm2 and MdmX. In contrast to Mdm2, MdmX lacks ubiquitin ligase activity. Although the essential interactions of MdmX are known, it is not clear how they function to regulate p53. The regulation of tumor suppressor p53 by Mdm2 and MdmX in response to DNA damage was investigated by mathematical modeling of a simplified network. The simplified network model was derived from a detailed molecular interaction map (MIM) that exhibited four coherent DNA damage response pathways. The results suggest that MdmX may amplify or stabilize DNA damage-induced p53 responses via non-enzymatic interactions. Transient effects of MdmX are mediated by reservoirs of p53ratioMdmX and Mdm2ratioMdmX heterodimers, with MdmX buffering the concentrations of p53 and/or Mdm2. A survey of kinetic parameter space disclosed regions of switch-like behavior stemming from such reservoir-based transients. During an early response to DNA damage, MdmX positively or negatively regulated p53 activity, depending on the level of Mdm2; this led to amplification of p53 activity and switch-like response. During a late response to DNA damage, MdmX could dampen oscillations of p53 activity. A possible role of MdmX may be to dampen such oscillations that otherwise could produce erratic cell behavior. Our study suggests how MdmX may participate in the response of p53 to DNA damage either by increasing dependency of p53 on Mdm2 or by dampening oscillations of p53 activity and presents a model for experimental investigation.

Genome-wide binding potential and regulatory activity of the glucocorticoid receptor's monomeric and dimeric forms.

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 based on recovery of enriched half-site response elements suggest monomeric engagement on DNA. Here, we perform genome-wide studies on GRdim and a constitutively monomeric mutant. Our results show that impairing dimerization affects binding even to open chromatin. We also find that 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.

Nuclear pore protein NUP210 depletion suppresses metastasis through heterochromatin-mediated disruption of tumor cell mechanical response.

Mechanical signals from the extracellular microenvironment have been implicated in tumor and metastatic progression. Here, we identify nucleoporin NUP210 as a metastasis susceptibility gene for human estrogen receptor positive (ER+) breast cancer and a cellular mechanosensor. Nup210 depletion suppresses lung metastasis in mouse models of breast cancer. Mechanistically, NUP210 interacts with LINC complex protein SUN2 which connects the nucleus to the cytoskeleton. In addition, the NUP210/SUN2 complex interacts with chromatin via the short isoform of BRD4 and histone H3.1/H3.2 at the nuclear periphery. In Nup210 knockout cells, mechanosensitive genes accumulate H3K27me3 heterochromatin modification, mediated by the polycomb repressive complex 2 and differentially reposition within the nucleus. Transcriptional repression in Nup210 knockout cells results in defective mechanotransduction and focal adhesion necessary for their metastatic capacity. Our study provides an important role of nuclear pore protein in cellular mechanosensation and metastasis.

WHSC1 monomethylates histone H1 and induces stem-cell like features in squamous cell carcinoma of the head and neck.

Squamous cell carcinoma of the head and neck (SCCHN) is a malignancy with poor outcomes, thus novel therapies are urgently needed. We recently showed that WHSC1 is necessary for the viability of SCCHN cells through H3K36 di-methylation. Here, we report the identification of its novel substrate, histone H1, and that WHSC1-mediated H1.4K85 mono-methylation may enhance stemness features in SCCHN cells. To identify proteins interacting with WHSC1 in SCCHN cells, WHSC1 immunoprecipitation and mass spectrometry identified H1 as a WHSC1-interacting candidate. In vitro methyltransferase assays showed that WHSC1 mono-methylates H1 at K85. We generated an H1K85 mono-methylation-specific antibody and confirmed that this methylation occurs in vivo. Sphere formation assays using SCC-35 cells stably expressing either wild-type (FLAG-H1.4-WT) or mutated (FLAG-H1.4K85A) vector with lysine 85 to alanine substitution which is not methylated, indicated a higher number of spheres in SCC-35 cells expressing the wild type than those with the mutant vector. SCC-35 cells expressing the wild type H1.4 proliferated faster than those expressing the mutated vector. RNA sequencing, RT-PCR and Western blotting of the FLAG-H1.4-WT or FLAG-H1.4K85A SCC-35 cells revealed that OCT4 levels were higher in wild type compared to mutant cells. These results were reproduced in SCC-35 cells genetically modified with CRISPR to express H1.4K85R. Chromatin immunoprecipitation showed that FLAG-H1.4K85A had decreased occupancy in the OCT4 gene compared to FLAG-H1.4-WT. This study supports that WHSC1 mono-methylates H1.4 at K85, it induces transcriptional activation of OCT4 and stemness features in SCCHN cells, providing rationale to target H1.4K85 mono-methylation through WHSC1 in SCCHN.

High Quality ATAC-Seq Data Recovered from Cryopreserved Breast Cell Lines and Tissue.

DNA accessibility to transcription regulators varies between cells and modulates gene expression patterns. Several "open" chromatin profiling methods that provide valuable insight into the activity of these regulatory regions have been developed. However, their application to clinical samples has been limited despite the discovery that the Analysis of Transposase-Accessible Chromatin followed by sequencing (ATAC-seq) method can be performed using fewer cells than other techniques. Obtaining fresh rather than stored samples and a lack of adequate optimization and quality controls are major barriers to ATAC's clinical implementation. Here, we describe an optimized ATAC protocol in which we varied nuclear preparation conditions and transposase concentrations and applied rigorous quality control measures before testing fresh, flash frozen, and cryopreserved breast cells and tissue. We obtained high quality data from small cell number. Furthermore, the genomic distribution of sequencing reads, their enrichment at transcription start sites, and transcription factor footprint analyses were similar between cryopreserved and fresh samples. This updated method is applicable to clinical samples, including cells from fine needle aspiration and tissues obtained via core needle biopsy or surgery. Chromatin accessibility analysis using patient samples will greatly expand the range of translational research and personalized medicine by identification of clinically-relevant epigenetic features.

Detailed DNA methylation profiles of the E-cadherin promoter in the NCI-60 cancer cells.

E-cadherin (E-cad) is a transmembrane adhesion glycoprotein, the expression of which is often reduced in invasive or metastatic tumors. To assess E-cad's distribution among different types of cancer cells, we used bisulfite-sequencing for detailed, base-by-base measurement of CpG methylation in E-cad's promoter region in the NCI-60 cell lines. The mean methylation levels of the cell lines were distributed bimodally, with values pushed toward either the high or low end of the methylation scale. The 38 epithelial cell lines showed substantially lower (28%) mean methylation levels compared with the nonepithelial cell lines (58%). The CpG site at -143 with respect to the transcriptional start was commonly methylated at intermediate levels, even in cell lines with low overall DNA methylation. We also profiled the NCI-60 cell lines using Affymetrix U133 microarrays and found E-cad expression to be correlated with E-cad methylation at highly statistically significant levels. Above a threshold of approximately 20% to 30% mean methylation, the expression of E-cad was effectively silenced. Overall, this study provides a type of detailed analysis of methylation that can also be applied to other cancer-related genes. As has been shown in recent years, DNA methylation status can serve as a biomarker for use in choosing therapy.

Depicting combinatorial complexity with the molecular interaction map notation.

To help us understand how bioregulatory networks operate, we need a standard notation for diagrams analogous to electronic circuit diagrams. Such diagrams must surmount the difficulties posed by complex patterns of protein modifications and multiprotein complexes. To meet that challenge, we have designed the molecular interaction map (MIM) notation (http://discover.nci.nih.gov/mim/). Here we show the advantages of the MIM notation for three important types of diagrams: (1) explicit diagrams that define specific pathway models for computer simulation; (2) heuristic maps that organize the available information about molecular interactions and encompass the possible processes or pathways; and (3) diagrams of combinatorially complex models. We focus on signaling from the epidermal growth factor receptor family (EGFR, ErbB), a network that reflects the major challenges of representing in a compact manner the combinatorial complexity of multimolecular complexes. By comparing MIMs with other diagrams of this network that have recently been published, we show the utility of the MIM notation. These comparisons may help cell and systems biologists adopt a graphical language that is unambiguous and generally understood.

An analysis of the class of gene regulatory functions implied by a biochemical model.

Understanding the integrated behavior of genetic regulatory networks, in which genes regulate one another's activities via RNA and protein products, is emerging as a dominant problem in systems biology. One widely studied class of models of such networks includes genes whose expression values assume Boolean values (i.e., on or off). Design decisions in the development of Boolean network models of gene regulatory systems include the topology of the network (including the distribution of input- and output-connectivity) and the class of Boolean functions used by each gene (e.g., canalizing functions, post functions, etc.). For example, evidence from simulations suggests that biologically realistic dynamics can be produced by scale-free network topologies with canalizing Boolean functions. This work seeks further insights into the design of Boolean network models through the construction and analysis of a class of models that include more concrete biochemical mechanisms than the usual abstract model, including genes and gene products, dimerization, cis-binding sites, promoters and repressors. In this model, it is assumed that the system consists of N genes, with each gene producing one protein product. Proteins may form complexes such as dimers, trimers, etc. The model also includes cis-binding sites to which proteins may bind to form activators or repressors. Binding affinities are based on structural complementarity between proteins and binding sites, with molecular binding sites modeled by bit-strings. Biochemically plausible gene expression rules are used to derive a Boolean regulatory function for each gene in the system. The result is a network model in which both topological features and Boolean functions arise as emergent properties of the interactions of components at the biochemical level. A highly biased set of Boolean functions is observed in simulations of networks of various sizes, suggesting a new characterization of the subset of Boolean functions that are likely to appear in gene regulatory networks.

Diagnostic markers that distinguish colon and ovarian adenocarcinomas: identification by genomic, proteomic, and tissue array profiling.

Colon and ovarian cancers can be difficult to distinguish in the abdomen, and the distinction is important because it determines which drugs will be used for therapy. To identify molecular markers for that differential diagnosis, we developed a multistep protocol starting with the 60 human cancer cell lines used by the National Cancer Institute to screen for new anticancer agents. The steps included: (a) identification of candidate markers using cDNA microarrays; (b) verification of clone identities by resequencing; (c) corroboration of transcript levels using Affymetrix oligonucleotide chips; (d) quantitation of protein expression by "reverse-phase" protein microarray; and (e) prospective validation of candidate markers on clinical tumor sections in tissue microarrays. The two best candidates identified were villin for colon cancer cells and moesin for ovarian cancer cells. Because moesin stained stromal elements in both types of cancer, it would probably not have been identified as a marker if we had started with mRNA or protein profiling of bulk tumors. Villin appears at least as useful as the currently used colon cancer marker cytokeratin 20, and moesin also appears to have utility. The multistep process introduced here has the potential to produce additional markers for cancer diagnosis, prognosis, and therapy.

The Systems Biology Graphical Notation.

Circuit diagrams and Unified Modeling Language diagrams are just two examples of standard visual languages that help accelerate work by promoting regularity, removing ambiguity and enabling software tool support for communication of complex information. Ironically, despite having one of the highest ratios of graphical to textual information, biology still lacks standard graphical notations. The recent deluge of biological knowledge makes addressing this deficit a pressing concern. Toward this goal, we present the Systems Biology Graphical Notation (SBGN), a visual language developed by a community of biochemists, modelers and computer scientists. SBGN consists of three complementary languages: process diagram, entity relationship diagram and activity flow diagram. Together they enable scientists to represent networks of biochemical interactions in a standard, unambiguous way. We believe that SBGN will foster efficient and accurate representation, visualization, storage, exchange and reuse of information on all kinds of biological knowledge, from gene regulation, to metabolism, to cellular signaling.