Glucocorticoid Receptor Response Element Research Articles

Specificity protein 1 (Sp1) and glucocorticoid receptor (GR) stimulate bovine alphaherpesvirus 1 (BoHV-1) replication and cooperatively transactivate the immediate early transcription unit 1 promoter.

Bovine alphaherpesvirus 1 (BoHV-1) infections cause respiratory tract disorders and suppress immune responses, which can culminate in bacterial pneumonia. Following acute infection, BoHV-1 establishes lifelong latency in sensory neurons present in trigeminal ganglia (TG) and unknown cells in pharyngeal tonsil. Latently infected calves consistently reactivate from latency after an intravenous injection of the synthetic corticosteroid dexamethasone (DEX), which mimics the effects of stress. The immediate early transcription unit 1 (IEtu1) promoter drives expression of infected cell protein 0 (bICP0) and bICP4, two key viral transcriptional regulators. The IEtu1 promoter contains two functional glucocorticoid receptor (GR) response elements (GREs), and this promoter is transactivated by GR, DEX, and certain Krüppel transcription factors that interact with GC-rich motifs, including consensus specificity protein 1 (Sp1) binding sites. Based on these observations, we hypothesized that Sp1 stimulates productive infection and transactivates key BoHV-1 promoters. DEX treatment of latently infected calves increased the number of Sp1+ TG neurons and cells in pharyngeal tonsil indicating that Sp1 expression is induced by stress. Silencing Sp1 protein expression with siRNA or mithramycin A, a drug that preferentially binds GC-rich DNA, significantly reduced BoHV-1 replication. Moreover, BoHV-1 infection of permissive cells increased Sp1 steady-state protein levels. In transient transfection studies, GR and Sp1 cooperatively transactivate IEtu1 promoter activity unless both GREs are mutated. Co-immunoprecipitation studies revealed that GR and Sp1 interact in mouse neuroblastoma cells (Neuro-2A) suggesting this interaction stimulates IEtu1 promoter activity. Collectively, these studies suggested that the cellular transcription factor Sp1 enhances productive infection and stress-induced BoHV-1 reactivation from latency.IMPORTANCEFollowing acute infection, bovine alphaherpesvirus 1 (BoHV-1) establishes lifelong latency in sensory neurons in trigeminal ganglia (TG) and pharyngeal tonsil. The synthetic corticosteroid dexamethasone consistently induces BoHV-1 reactivation from latency. The number of TG neurons and cells in pharyngeal tonsil expressing the cellular transcription factor specificity protein 1 (Sp1) protein increases during early stages of dexamethasone-induced reactivation from latency. Silencing Sp1 expression impairs BoHV-1 replication in permissive cells. Interestingly, mithramycin A, a neuroprotective antibiotic that preferentially binds GC-rich DNA, impairs Sp1 functions and reduces BoHV-1 replication suggesting that it is a potential antiviral drug. The glucocorticoid receptor (GR) and Sp1 cooperatively transactivate the BoHV-1 immediate early transcript unit 1 (IEtu1) promoter, which drives expression of infected cell protein 0 (bICP0) and bICP4. Mithramycin A also reduced Sp1- and GR-mediated transactivation of the IEtu1 promoter. These studies revealed that GR and Sp1 trigger viral gene expression and replication following stressful stimuli.

Slug, a Stress-Induced Transcription Factor, Stimulates Herpes Simplex Virus 1 Replication and Transactivates a cis-Regulatory Module within the VP16 Promoter.

Stress-mediated activation of the glucocorticoid receptor (GR) and specific stress-induced transcription factors stimulate herpes simplex virus 1 (HSV-1) productive infection, explant-induced reactivation, and immediate early (IE) promoters that drive expression of infected cell protein 0 (ICP0), ICP4, and ICP27. Several published studies concluded the virion tegument protein VP16, ICP0, and/or ICP4 drives early steps of reactivation from latency. Notably, VP16 protein expression was induced in trigeminal ganglionic neurons of Swiss Webster or C57BL/6J mice during early stages of stress-induced reactivation. If VP16 mediates reactivation, we hypothesized stress-induced cellular transcription factors would stimulate its expression. To address this hypothesis, we tested whether stress-induced transcription factors transactivate a VP16 cis-regulatory module (CRM) located upstream of the VP16 TATA box (-249 to -30). Initial studies revealed the VP16 CRM cis-activated a minimal promoter more efficiently in mouse neuroblastoma cells (Neuro-2A) than mouse fibroblasts (NIH-3T3). GR and Slug, a stress-induced transcription factor that binds enhancer boxes (E-boxes), were the only stress-induced transcription factors examined that transactivated the VP16 CRM construct. GR- and Slug-mediated transactivation was reduced to basal levels when the E-box, two 1/2 GR response elements (GREs), or NF-κB binding site was mutated. Previous studies revealed GR and Slug cooperatively transactivated the ICP4 CRM, but not ICP0 or ICP27. Silencing of Slug expression in Neuro-2A cells significantly reduced viral replication, indicating Slug-mediated transactivation of ICP4 and VP16 CRM activity correlates with enhanced viral replication and reactivation from latency. IMPORTANCE Herpes simplex virus 1 (HSV-1) establishes lifelong latency in several types of neurons. Periodically cellular stressors trigger reactivation from latency. Viral regulatory proteins are not abundantly expressed during latency, indicating cellular transcription factors mediate early stages of reactivation. Notably, the glucocorticoid receptor (GR) and certain stress-induced transcription factors transactivate cis-regulatory modules (CRMs) essential for expression of infected cell protein 0 (ICP0) and ICP4, key viral transcriptional regulatory proteins linked to triggering reactivation from latency. Virion protein 16 (VP16) specifically transactivates IE promoter and was also reported to mediate early stages of reactivation from latency. GR and Slug, a stress-induced enhancer box (E-box) binding protein, transactivate a minimal promoter downstream of VP16 CRM, and these transcription factors occupy VP16 CRM sequences in transfected cells. Notably, Slug stimulates viral replication in mouse neuroblastoma cells suggesting Slug, by virtue of transactivating VP16 and ICP4 CRM sequences, can trigger reactivation in certain neurons.

OR07-5 PARP1 Contributes to the Glucocorticoid Receptor Transcriptional Response And Influences the Skeletal Muscle Phenotype

Abstract Background NAD+-dependent PARP1 (Poly-(ADP-ribosyl)polymerase 1) generates the post-translational modification ADP-Ribosylation (ADPR). Given PARP1-ADPR determines cellular NAD+ availability, which in turn impacts a range of hormonally governed actions, we sought to identify the molecular roles of PARP1 in relation to glucocorticoids in the endocrine sensitive tissue skeletal muscle. Importance: Activity of the glucocorticoid receptor (GR) is dictated by molecular partners whose actions skew the frequency with which GR produces meaningful transcriptional outcomes. As a result, our molecular understanding of this steroid hormone receptor remains incomplete. Hypothesis PARP1 through ADPR actions impacts skeletal muscle phenotype and co-regulates glucocorticoid-mediated transcriptional responses. Experimental Design: Murine muscle myoblasts (C2C12) or primary murine muscle cultures were treated with PARP inhibitors (PJ34(10uM) or BYK204165(1uM)) or transfected with silencing RNA targeting PARP1. Cells were treated with ± dexamethasone (1uM). Major Results Analysis of differentiating muscle revealed PARP1 and ADPR are dynamic at days 0-5 (measured by Western immunoblot. p<0.001; n=6). Chemical inhibition of PARP1 significantly decreased ADPR (Western immunoblot. p<0.001; n=9) but did not prevent myotube formation. To identify if initial ADPR dynamics held consequence for fully differentiated myotubes, cells were treated with a single dose of the PARP1 inhibitor BYK204165 at induction of differentiation. Myotube lysates were collected 5 days later and were subjected to unlabelled SWATH-MS. This measured the abundance of 180 proteins as significantly different in PARP inhibitor-treated cells (n=7). Analyses of these proteins revealed over-representation of pathways governing muscle development (FDR 8.99-08), contraction (FDR 9.47-08, myofiber assembly (FDR 8.06-05), and energy metabolism (FDR 2.93-06). Indicating PARP1-ADPR regulates myogenesis and plays a role in the striated muscle contractile response. ChIP assays for PARP1 binding in cells showed a (30-fold enrichment ±3.2SEM) at GR response elements and detectable ADPR was reduced when exposed to dexamethasone (Western immunoblot n=5). These data support ADPR as responsive to glucocorticoid exposure and PARP1 influences GR recruitment to the genome. RNAseq of siPARP1 (n=4) versus scrambled myoblasts (n=5) identified differential expression of 165 genes. Gene ontology revealed over-representation in siPARP1 cells of those pathways regulating muscle differentiation, including actin-binding, cytoskeletal structure, and NAD+ binding genes. SiRNA of PARP1 had no influence over the ability of glucocorticoids to activate archetype target genes (DUSP1, MURF1, ATROGIN1 & GILZ), but differentially impacted the expression of 434 other genes. Conclusions PARP1 mediated ADPR is critical to the 'early phase' events of muscle cell differentiation. PARP1 influences short-term transcriptional behaviors of the glucocorticoid receptor; in turn, glucocorticoids themselves impact PARP1-genome interaction events. These findings are important for the understanding of general GR mechanisms and have relevance for the widespread functional decline observed during tissue glucocorticoid excess. Presentation: Saturday, June 11, 2022 12:30 p.m. - 12:45 p.m.

Independent Cis-Regulatory Modules within the Herpes Simplex Virus 1 Infected Cell Protein 0 (ICP0) Promoter Are Transactivated by Krüppel-like Factor 15 and Glucocorticoid Receptor.

A corticosteroid antagonist impairs Herpes Simplex Virus 1 (HSV-1) productive infection and explant-induced reactivation from latency, suggesting corticosteroids and the glucocorticoid receptor (GR) mediate certain aspects of these complex virus–host interactions. GR-hormone complexes regulate transcription positively and negatively, in part, by binding GR response elements (GREs). Recent studies revealed infected cell protein 0 (ICP0), ICP4, and ICP27 promoter/cis-regulatory modules (CRMs) are cooperatively transactivated by GR and Krüppel-like factor 15 (KLF15), which forms a feed-forward transcription loop. We hypothesized the ICP0 promoter contains independent CRMs that are transactivated by GR, KLF15, and the synthetic corticosteroid dexamethasone (DEX). This hypothesis is based on the finding that the ICP0 promoter contains multiple transcription factor binding sites, and GR and KLF15 cooperatively transactivate the full-length ICP0 promoter. ICP0 promoter sequences spanning −800 to −635 (fragment A) were efficiently transactivated by GR, KLF15, and DEX in monkey kidney cells (Vero), whereas GR and DEX significantly enhanced promoter activity in mouse neuroblastoma cells (Neuro-2A). Furthermore, ICP0 fragment B (−458 to −635) was efficiently transactivated by GR, KLF15, and DEX in Vero cells, but not Neuro-2A cells. Finally, fragment D (−232 to −24) was transactivated significantly in Vero cells by GR, KLF15, and DEX, whereas KLF15 and DEX were sufficient for transactivation in Neuro-2A cells. Collectively, these studies revealed efficient transactivation of three independent CRMs within the ICP0 promoter by GR, KLF15, and/or DEX. Finally, GC-rich sequences containing specificity protein 1 (Sp1) binding sites were essential for transactivation.

TAZ inhibits glucocorticoid receptor and coordinates hepatic glucose homeostasis in normal physiological states.

The elucidation of the mechanisms whereby the liver maintains glucose homeostasis is crucial for the understanding of physiological and pathological states. Here, we show a novel role of hepatic transcriptional co-activator with PDZ-binding motif (TAZ) in the inhibition of glucocorticoid receptor (GR). TAZ is abundantly expressed in pericentral hepatocytes and its expression is markedly reduced by fasting. TAZ interacts via its WW domain with the ligand-binding domain of GR to limit the binding of GR to the GR response element in gluconeogenic gene promoters. Therefore, liver-specific TAZ knockout mice show increases in glucose production and blood glucose concentration. Conversely, the overexpression of TAZ in mouse liver reduces the binding of GR to gluconeogenic gene promoters and glucose production. Thus, our findings demonstrate that hepatic TAZ inhibits GR transactivation of gluconeogenic genes and coordinates gluconeogenesis in response to physiological fasting and feeding.

Regulation of neurotropic herpesvirus productive infection and latency-reactivation cycle by glucocorticoid receptor and stress-induced transcription factors.

Neurotropic α-herpesvirinae subfamily members, herpes simplex virus type 1 (HSV-1) and bovine herpesvirus 1 (BoHV-1), are important viral pathogens in their respective hosts. Following acute infection on mucosal surfaces, these viruses establish life-long latency in neurons within trigeminal ganglia (TG) and central nervous system. Chronic or acute stress (physiological or psychological) increases the frequency of reactivation from latency, which leads to virus shedding, virus transmission, and recurrent disease. While stress impairs immune responses and inflammatory signaling cascades, we predict stressful stimuli directly stimulate viral gene expression and productive infection during early stages of reactivation from latency. For example, BoHV-1 and HSV-1 productive infection is impaired by glucocorticoid receptor (GR) antagonists but is stimulated by the synthetic corticosteroid dexamethasone. Promoters that drive expression of key viral transcriptional regulatory proteins are cooperatively stimulated by GR and specific Krüppel like transcription factors (KLF) induced during stress induced reactivation from latency. The BoHV-1 immediate early transcription unit 1 promoter and contains two GR response elements (GRE) that are essential for cooperative transactivation by GR and KLF15. Conversely, the HSV-1 infected cell protein 0 (ICP0) and ICP4 promoter as well as the BoHV-1 ICP0 early promoter lack consensus GREs: however, these promoters are cooperatively transactivated by GR and KLF4 or KLF15. Hence, growing evidence suggests GR and stress-induced transcription factors directly stimulate viral gene expression and productive infection during early stages of reactivation from latency. We predict the immune inhibitory effects of stress enhance virus spread at late stages during reactivation from latency.

Progesterone increases the incidence of bovine herpesvirus 1 reactivation from latency and stimulates productive infection

Bovine herpesvirus 1 (BoHV-1), including modified live vaccines, can cause abortions in pregnant cows. Progesterone maintains pregnancy and promotes spermiogenesis and testosterone biosynthesis in males: furthermore, progesterone is a neuro-steroid. Recent published studies demonstrated progesterone stimulated the BoHV-1 immediate early transcription unit 1 (IEtu1) promoter, and two glucocorticoid receptor response elements within the promoter were required for progesterone mediated transactivation. In this study, we tested whether progesterone induces reactivation from latency in rabbits. As expected, the synthetic corticosteroid dexamethasone consistently induced reactivation from latency in males and females. While progesterone induced reactivation from latency in approximately one-half of male rabbits, virus shedding was sporadic compared to dexamethasone and less efficient in female rabbits. Progesterone significantly increased productive infection in rabbit skin cells, which correlated with stimulating reactivation. These studies suggest progesterone promotes BoHV-1 spread in cattle, in part, by increasing the frequency of reactivation from latency.

The Glucocorticoid Receptor (GR) Stimulates Herpes Simplex Virus 1 Productive Infection, in Part Because the Infected Cell Protein 0 (ICP0) Promoter Is Cooperatively Transactivated by the GR and Krüppel-Like Transcription Factor 15.

Following acute infection, herpes simplex virus 1 (HSV-1) establishes lifelong latency in neurons. Physical, emotional, and chemical stresses are linked to increasing the incidence of reactivation from latency, but the mechanism of action is not well understood. In general, stress increases corticosteroid levels, leading to activation of the glucocorticoid receptor (GR), a pioneer transcription factor. Consequently, we hypothesized that stress-mediated activation of the GR can stimulate productive infection and viral gene expression. New studies demonstrated that the GR-specific antagonist (CORT-108297) significantly reduced HSV-1 productive infection in mouse neuroblastoma cells (Neuro-2A). Additional studies demonstrated that the activated GR and Krüppel-like transcription factor 15 (KLF15) cooperatively transactivated the infected cell protein 0 (ICP0) promoter, a crucial viral regulatory protein. Interestingly, the synthetic corticosteroid dexamethasone and GR or KLF15 alone had little effect on ICP0 promoter activity in transfected Neuro-2A or Vero cells. Chromatin immunoprecipitation (ChIP) studies revealed that the GR and KLF15 occupied ICP0 promoter sequences important for transactivation at 2 and 4 h after infection; however, binding was not readily detected at 6 h after infection. Similar results were obtained for cells transfected with the full-length ICP0 promoter. ICP0 promoter sequences lack a consensus "whole" GR response element (GRE) but contain putative half-GREs that were important for dexamethasone induced promoter activity. The activated GR stimulates expression of, and interacts with, KLF15; consequently, these data suggest KLF15 and the GR form a feed-forward loop that activates viral gene expression and productive infection following stressful stimuli.IMPORTANCE The ability of herpes simplex virus 1 (HSV-1) to periodically reactivate from latency results in virus transmission and recurrent disease. The incidence of reactivation from latency is increased by chronic or acute stress. Stress increases the levels of corticosteroids, which bind and activate the glucocorticoid receptor (GR). Since GR activation is an immediate early response to stress, we tested whether the GR influences productive infection and the promoter that drives infected cell protein 0 (ICP0) expression. Pretreatment of cells with a GR-specific antagonist (CORT-108297) significantly reduced virus replication. Although the GR had little effect on ICP0 promoter activity alone, the Krüppel-like transcription factor 15 (KLF15) cooperated with the GR to stimulate promoter activity in transfected cells. In transfected or infected cells, the GR and KLF15 occupied ICP0 sequences important for transactivation. Collectively, these studies provide insight into how stress can directly stimulate productive infection and viral gene expression.

Synergistic Activation of Bovine Herpesvirus 1 Productive Infection and Viral Regulatory Promoters by the Progesterone Receptor and Krüppel-Like Transcription Factor 15.

Bovine herpesvirus 1 (BoHV-1), including modified live vaccines, readily infects the fetus and ovaries, which can lead to reproductive failure. The BoHV-1 latency reactivation cycle in sensory neurons may further complicate reproductive failure in pregnant cows. The immediate early transcription unit 1 (IEtu1) promoter drives expression of important viral transcriptional regulators (bICP0 and bICP4). This promoter contains two functional glucocorticoid receptor (GR) response elements (GREs) that have the potential to stimulate productive infection following stressful stimuli. Since progesterone and the progesterone receptor (PR) can activate many GREs, we hypothesized that the PR and/or progesterone regulates productive infection and viral transcription. New studies demonstrated that progesterone stimulated productive infection. Additional studies revealed the PR and Krüppel-like transcription factor 15 (KLF15) cooperated to stimulate productive infection and IEtu1 promoter activity. IEtu1 promoter activation required both GREs, which correlated with the ability of the PR to interact with wild-type (wt) GREs but not mutant GREs. KLF15 also cooperated with the PR to transactivate the bICP0 early promoter, a promoter that maintains bICP0 protein expression during productive infection. Intergenic viral DNA fragments (less than 400 bp) containing two GREs and putative KLF binding sites present within genes encoding unique long 52 (UL-52; component of DNA primase/helicase complex), Circ, bICP4, and IEtu2 were stimulated by KLF15 and the PR more than 10-fold, suggesting that additional viral promoters are activated by these transcription factors. Collectively, these studies suggest progesterone and the PR promote BoHV-1 spread to reproductive tissues, thus increasing the incidence of reproductive failure.IMPORTANCE Bovine herpesvirus 1 (BoHV-1) is the most frequently diagnosed cause of abortions in pregnant cows and can cause "abortion storms" in susceptible herds. Virulent field strains and even commercially available modified live vaccines can induce abortion, in part because BoHV-1 replicates efficiently in the ovary and corpus luteum. We now demonstrate that progesterone and the progesterone receptor (PR) stimulate productive infection. The BoHV-1 genome contains approximately 100 glucocorticoid receptor (GR) response elements (GREs). Interestingly, the PR can bind and activate many promoters that contain GREs. The PR and Krüppel-like transcription factor 15 (KLF15), which regulate key steps during embryo implantation, cooperate to stimulate productive infection and two viral promoters that drive expression of key viral transcriptional regulators. These studies suggest that the ability of progesterone and the PR to stimulate productive infection has the potential to promote virus spread in reproductive tissue and induce reproductive failure.

Abstract 45: Snai2 is a new target to mediate glucocorticoid signaling on breast cancer cell migration

Abstract Steroid hormone receptors such as estrogen and progesterone receptors are well studied in breast cancer pathology; they are also used as drug targets for breast cancer therapy in the clinic. In contrast, glucocorticoid (GC) as a ubiquitous stress activated steroid hormone is less investigated in breast cancer. However, GC is frequently used as a co-treatment for breast cancer chemotherapy that generates some controversial effects to even promote cancer progression or recurrence in certain subtypes of breast cancer. To address this clinical issue, we focused on investigating the specificity of GC signaling on breast cancer cell behaviors in this study. When breast cancer cells were treated by dexamethasone (Dex), they were more responsive to cell migration (measured by electric cell-substrate impedance sensing, ECIS) than the effects on cell proliferation and apoptosis, wherein MDA-231 (triple negative breast cancer) cell was more sensitive to Dex than MCF7 cell (luminal A subtype). Further gene expression analysis by qRT-PCR microarray revealed that the glucocorticoid receptor (GR) responsive gene patterns are different between these two subtypes of breast cancer cells. One of remarked changes was that Snai2 (a zinc finger transcriptional factor) was highly activated in both cell lines. Western blotting analysis confirmed that MDA-231 cells have much higher basal level of Snai2 but with less fold increase upon Dex treatment than MCF-7. GR response elements (GREs) were also identified in the promoter region of Snai2, thus confirming Snai2 as a new target gene of GR in breast cancer. When Snai2 was knocked out by CRISPR, it was found that the basal migration rate was decreased in both cell lines. More importantly, their response rates to Dex treatment were also decreased as compared to the wild type cells. These decreases in cell migration are similar to those treated by GR antagonist RU486, thus confirming that Snai2 is a mediator to regulate the effects of GC/GR signaling on breast cancer cell migration. In summary, this study identified Snai2 as a new target gene of GR to regulate cell migration and potentially metastasis in response to GC signaling. The differential regulation of different subtypes of breast cancer cells by GC signaling was also confirmed. (+ equal contribution; * Corresponding author). Citation Format: Jun Ling, Adit Singhal, Zenaida P. Lopez-Dee, Brittany Porreca, Trinity Sprague. Snai2 is a new target to mediate glucocorticoid signaling on breast cancer cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 45.

Combinatorial Effects of the Glucocorticoid Receptor and Krüppel-Like Transcription Factor 15 on Bovine Herpesvirus 1 Transcription and Productive Infection.

Bovine herpesvirus 1 (BoHV-1), an important bovine pathogen, establishes lifelong latency in sensory neurons. Latently infected calves consistently reactivate from latency following a single intravenous injection of the synthetic corticosteroid dexamethasone. The immediate early transcription unit 1 (IEtu1) promoter, which drives bovine ICP0 (bICP0) and bICP4 expression, is stimulated by dexamethasone because it contains two glucocorticoid receptor (GR) response elements (GREs). Several Krüppel-like transcription factors (KLF), including KLF15, are induced during reactivation from latency, and they stimulate certain viral promoters and productive infection. In this study, we demonstrate that the GR and KLF15 were frequently expressed in the same trigeminal ganglion (TG) neuron during reactivation and cooperatively stimulated productive infection and IEtu1 GREs in mouse neuroblastoma cells (Neuro-2A). We further hypothesized that additional regions in the BoHV-1 genome are transactivated by the GR or stress-induced transcription factors. To test this hypothesis, BoHV-1 DNA fragments (less than 400 bp) containing potential GR and KLF binding sites were identified and examined for transcriptional activation by stress-induced transcription factors. Intergenic regions within the unique long 52 gene (UL52; a component of the DNA primase/helicase complex), bICP4, IEtu2, and the unique short region were stimulated by KLF15 and the GR. Chromatin immunoprecipitation studies revealed that the GR and KLF15 interacted with sequences within IEtu1 GREs and the UL52 fragment. Coimmunoprecipitation studies demonstrated that KLF15 and the GR were associated with each other in transfected cells. Since the GR stimulates KLF15 expression, we suggest that these two transcription factors form a feed-forward loop that stimulates viral gene expression and productive infection following stressful stimuli.IMPORTANCE Bovine herpesvirus 1 (BoHV-1) is an important viral pathogen that causes respiratory disease and suppresses immune responses in cattle; consequently, life-threatening bacterial pneumonia can occur. Following acute infection, BoHV-1 establishes lifelong latency in sensory neurons. Reactivation from latency is initiated by the synthetic corticosteroid dexamethasone. Dexamethasone stimulates lytic cycle viral gene expression in sensory neurons of calves latently infected with BoHV-1, culminating in virus shedding and transmission. Two stress-induced cellular transcription factors, Krüppel-like transcription factor 15 (KLF15) and the glucocorticoid receptor (GR), cooperate to stimulate productive infection and viral transcription. Additional studies demonstrated that KLF15 and the GR form a stable complex and that these stress-induced transcription factors bind to viral DNA sequences, which correlates with transcriptional activation. The ability of the GR and KLF15 to synergistically stimulate viral gene expression and productive infection may be critical for the ability of BoHV-1 to reactivate from latency following stressful stimuli.

GR and ER Coactivation Alters the Expression of Differentiation Genes and Associates with Improved ER+ Breast Cancer Outcome.

In estrogen receptor (ER)-negative breast cancer, high tumor glucocorticoid receptor (GR) expression has been associated with a relatively poor outcome. In contrast, using a meta-analysis of several genomic datasets, here we find that tumor GR mRNA expression is associated with improved ER(+) relapse-free survival (RFS; independently of progesterone receptor expression). To understand the mechanism by which GR expression is associated with a better ER(+) breast cancer outcome, the global effect of GR-mediated transcriptional activation in ER(+) breast cancer cells was studied. Analysis of GR chromatin immunoprecipitation followed by high-throughput sequencing in ER(+)/GR(+) MCF-7 cells revealed that upon coactivation of GR and ER, GR chromatin association became enriched at proximal promoter regions. Furthermore, following ER activation, increased GR chromatin association was observed at ER, FOXO, and AP1 response elements. In addition, ER associated with GR response elements, suggesting that ER and GR interact in a complex. Coactivation of GR and ER resulted in increased expression (relative to ER activation alone) of transcripts that encode proteins promoting cellular differentiation (e.g., KDM4B, VDR) and inhibiting the Wnt signaling pathway (IGFBP4). Finally, expression of these individual prodifferentiation genes was associated with significantly improved RFS in ER(+) breast cancer patients. Together, these data suggest that the coexpression and subsequent activity of tumor cell GR and ER contribute to the less aggressive natural history of early-stage breast cancer by coordinating the altered expression of genes favoring differentiation. The interaction between ER and GR activity highlights the importance of context-dependent nuclear receptor function in cancer. Mol Cancer Res; 14(8); 707-19. ©2016 AACR.

High activity of the stress promoter contributes to susceptibility to stress in the tree shrew.

Stress is increasingly present in everyday life in our fast-paced society and involved in the pathogenesis of many psychiatric diseases. Corticotrophin-releasing-hormone (CRH) plays a pivotal role in regulating the stress responses. The tree shrews are highly vulnerable to stress which makes them the promising animal models for studying stress responses. However, the mechanisms underlying their high stress-susceptibility remained unknown. Here we confirmed that cortisol was the dominate corticosteroid in tree shrew and was significantly increased after acute stress. Our study showed that the function of tree shrew CRH - hypothalamic-pituitary-adrenal (HPA) axis was nearly identical to human that contributed little to their hyper-responsiveness to stress. Using CRH transcriptional regulation analysis we discovered a peculiar active glucocorticoid receptor response element (aGRE) site within the tree shrew CRH promoter, which continued to recruit co-activators including SRC-1 (steroid receptor co-activator-1) to promote CRH transcription under basal or forskolin/dexamethasone treatment conditions. Basal CRH mRNA increased when the aGRE was knocked into the CRH promoter in human HeLa cells using CAS9/CRISPR. The aGRE functioned critically to form the “Stress promoter” that contributed to the higher CRH expression and susceptibility to stress. These findings implicated novel molecular bases of the stress-related diseases in specific populations.

Gestational dietary betaine supplementation suppresses hepatic expression of lipogenic genes in neonatal piglets through epigenetic and glucocorticoid receptor-dependent mechanisms

Methyl donors play critical roles in nutritional programming through epigenetic regulation of gene expression. Here we fed gestational sows with control or betaine-supplemented diets (3g/kg) throughout the pregnancy to explore the effects of maternal methyl-donor nutrient on neonatal expression of hepatic lipogenic genes. Betaine-exposed piglets demonstrated significantly lower liver triglyceride content associated with down-regulated hepatic expression of lipogenic genes acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD) and sterol regulatory element-binding protein-1c. Moreover, s-adenosyl methionine to s-adenosyl homocysteine ratio was elevated in the liver of betaine-exposed piglets, which was accompanied by DNA hypermethylation on FAS and SCD gene promoters and more enriched repression histone mark H3K27me3 on SCD gene promoter. Furthermore, glucocorticoid receptor (GR) binding to SCD gene promoter was diminished along with reduced serum cortisol and liver GR protein content in betaine-exposed piglets. GR-mediated SCD gene regulation was confirmed in HepG2 cells in vitro. Dexamethasone (Dex) drastically increased the luciferase activity of porcine SCD promoter, while the deletion of GR response element on SCD promoter significantly attenuated Dex-mediated SCD transactivation. In addition, miR-let-7e, miR-1285 and miR-124a, which respectively target porcine SCD, ACC and GR, were significantly up-regulated in the liver of betaine-exposed piglets, being in accordance with decreased protein content of these three genes. Taken together, our results suggest that maternal dietary betaine supplementation during gestation attenuates hepatic lipogenesis in neonatal piglets via epigenetic and GR-mediated mechanisms.

Abstract 4675: GR activation modifies ER transcriptional activity and results in decreased estrogen-mediated breast cancer cell proliferation

Abstract Approximately 75% of breast cancers (BC) express estrogen receptor-alpha (ER). The proliferative and anti-apoptotic actions of estrogen signaling in ER+ BC are well-established. Our laboratory recently reported that approximately 40% of ER+ breast cancers express significant glucocorticoid receptor (GR) mRNA; a meta-analysis of early-stage ER+ BC gene expression unexpectedly found that patients with tumors expressing high GR had significantly improved relapse-free survival (p-value 7.8E-8). Consistent with the improved outcome observed in ER+/GR+ BC patients, our laboratory and others have reported that GR activation can alter ER-mediated transcription and also antagonize estradiol (E2)-mediated cell proliferation. We therefore hypothesized that GR activation directly antagonizes the expression of ER-mediated pro-proliferative genes. Recapitulating observations reported by others, we first performed cell cycle analysis and found that the synthetic glucocorticoid dexamethasone (Dex) decreased E2-mediated proliferation of MCF-7 BC cells. To understand how GR activation inhibits ER-mediated pro-proliferative activity in ER+ BC cells, we performed chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to examine global GR chromatin occupancy in MCF-7 cells in the presence or absence of ER activation (+/- E2 at 10 nM). Surprisingly, in GR ChIP experiments we observed that in the presence of Dex, E2 treatment resulted in significantly increased GR association with ER response elements (EREs). Likewise, ER ChIP experiments showed that in the presence of E2, Dex treatment significantly increased the association of ER with GR response elements (GREs). Using genome-wide gene expression array analyses of MCF-7 cells, the gene expression signatures resulting from Dex and E2 co-treatment compared to E2 treatment alone are being examined. In analyses done to date, we found that in the presence of E2, Dex treatment inhibits the induction of ER-mediated pro-proliferative genes related to the cell cycle such as CCND1 (Cyclin D1) and CDC6. Furthermore, microarray analysis suggested that E2-mediated ER activation represses pro-apoptotic CASP7 and BIK gene expression, while co-treatment with Dex inhibits this repression. Additionally, anti-GR ChIP-seq analysis showed GR enrichment at CASP7 regulatory regions upon co-treatment with Dex and E2. Together, these data suggest that GR expression and subsequent GR activation may inhibit ER-mediated pro-proliferative gene expression as well as inhibit ER-mediated repression of anti-apoptotic genes through direct GR-chromatin interaction. These experiments suggest that GR activation can be an important component modulating ER activity in ER+/GR+ BC. Citation Format: Diana C. West, Charles F. Pierce, Deng Pan, Kyle Hernandez, Masha Kocherginsky, Suzanne D. Conzen. GR activation modifies ER transcriptional activity and results in decreased estrogen-mediated breast cancer cell proliferation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4675. doi:10.1158/1538-7445.AM2015-4675

Bovine herpesvirus 1 productive infection and immediate early transcription unit 1 promoter are stimulated by the synthetic corticosteroid dexamethasone

The primary site for life-long latency of bovine herpesvirus 1 (BHV-1) is sensory neurons. The synthetic corticosteroid dexamethasone consistently induces reactivation from latency; however the mechanism by which corticosteroids mediate reactivation is unclear. In this study, we demonstrate for the first time that dexamethasone stimulates productive infection, in part, because the BHV-1 genome contains more than 100 potential glucocorticoid receptor (GR) response elements (GREs). Immediate early transcription unit 1 (IEtu1) promoter activity, but not IEtu2 or VP16 promoter activity, was stimulated by dexamethasone. Two near perfect consensus GREs located within the IEtu1 promoter were necessary for dexamethasone-mediated stimulation. Electrophoretic mobility shift assays and chromatin immunoprecipitation studies demonstrated that the GR interacts with IEtu1 promoter sequences containing the GREs. Although we hypothesize that DEX-mediated stimulation of IEtu1 promoter activity is important during productive infection and perhaps reactivation from latency, stress likely has pleiotropic effects on virus-infected cells.

Fibroblast Growth Factor (Fgf) 21 is a Novel Target Gene of Glucocorticoid Receptor

Fibroblast growth factor (Fgf) 21 is a metabolic regulator that plays an important role in the maintenance of glucose, lipid, and energy homeostasis. Recent studies also suggest that Fgf21 induction can attenuate chemical‐induced toxicities, such as of acetaminophen, alcohol and dioxin. Glucocorticoid receptor (GR) is indispensable for cell growth and development, drug metabolism as well as immune response. However, the impact of GR activation on regulation of Fgf21 is unknown. The present study was designed to investigate whether activation of GR by the synthetic glucocorticoid Dexamethasone (DEX), can alter Fgf21 expression. Our results showed that in mouse liver, both low (2mg/kg) and high (50mg/kg) doses of DEX up‐regulated Fgf21 mRNA and protein expression. In addition, DEX induced Fgf21 mRNA and protein expression in both mouse (Hepa1c1c7) and human (Hep3B) hepatoma cells in a concentration‐ and time‐dependent manner. Generally, DEX produces its effects via activation of GR and/or pregnane X receptor (PXR). By using PXR‐null mouse model, we showed that 50mg/kg of DEX induced Fgf21 mRNA expression similarly in both wild‐type and PXR‐null mouse liver, indicating that the induction of Fgf21 by DEX is PXR‐independent. The in silico DNA sequence analysis revealed the presence of several putative GR response elements (GREs) in the 3kb promoter of mouse and human Fgf21/FGF21 genes. Further, GR silencing using GR‐specific siRNA, attenuated DEX‐induced Fgf21 expression in both mouse (Hepa1c1c7) and human (Hep3B) hepatoma cells. In summary, activation of GR induced Fgf21 expression in mouse liver as well as in cultured mouse and human hepatocytes.

A functional study of proximal goat β-casein promoter and intron 1 in immortalized goat mammary epithelial cells

Goat β-casein (CSN2) promoter has been extensively used to derive expression of recombinant therapeutic protein in transgenic goats; however, little direct evidence exists for signaling molecules and the cis-elements of goat CSN2 promoter in response to lactogenic hormone stimulation in goat mammary epithelial cells. Here, we use an immortalized caprine mammary epithelial cell line (CMC) to search for evidence of the above. Serial 5′-flanking regions deleted of promoter and intron 1 in goat CSN2 (−4,047 to +2,054) driven by firefly luciferase reporter gene were constructed and applied to measure promoter activity in CMC. The intron 1 region (+393 to +501) significantly decreased basal activity of the promoter. This finding contradicts other studies of the role of intron 1. The signal transducer and activator of transcription (STAT)5a played a significant role in activating promoter activity by prolactin stimulation. Hydrocortisone enhanced and prolonged the activity of STAT5a and promoter in CMC, but was independent of the glucocorticoid receptor response element. The minimum length of the CSN2 promoter segment in response to lactogenic stimulation was confirmed by 5′ serial deletions. A cis-element located from −300 to −90 in proximal goat CSN2 promoter that is absent in bovine and human CSN2 promoter was newly identified. We demonstrated the presence of a STAT5a binding site (−102 to −82) and preservation of the guanosine nucleotide at position −90 based on responses to the presence of lactogenic hormone using internal deletions and point mutations of the predicted STAT5a binding site, and chromatin immunoprecipitation assay. Together, these findings demonstrate that the proximal −300 bp of goat CSN2 promoter containing the STAT5a binding site (−102 to −82) is the response element for lactogenic hormone stimulation. Additionally, intron 1 may be required for tissue or developmental stage-specific expression in mammary gland. The role of the far-distal regions of goat CSN2 promoter in high-level lactogenic hormone induction and specific expression require further examination.

FKBP5 allele-specific epigenetic modification in gene by environment interaction.

The likelihood to develop stress-related psychiatric disorders in response to childhood trauma exposure may be moderated by the individual's genetic predisposition (Manuck and McCaffery, 2014). One of the genetic variants reported to alter the risk for psychiatric disorders following childhood trauma is a functional variant in FKBP5, a gene encoding a co-chaperone of the glucocorticoid receptor (GR). FKBP5 is strongly induced following stress exposure via binding of activated GR to a number of intronic and promoter GR response elements (GREs). The protein itself then binds to the GR complex, reduces the affinity of GR to cortisol and decreases translocation of the GR to the nucleus, providing an ultrashort negative feedback for GR activation on the genomic and protein level (Zannas and Binder, 2014). We have identified a functional polymorphism in close proximity of a GRE in intron 2 of FKBP5, which alters the extent of mRNA and protein induction following GR activation, likely by an altered 3D conformation. This results in a variable interaction of the intron 2 GRE with the transcription start site, leading to increased or reduced mRNA induction, respectively (Klengel et al, 2013). Individuals carrying the allele associated with stronger FKBP5 mRNA induction show GR resistance, prolonged cortisol response following stress, altered activation of brain regions important for threat response, such as the amygdala, and increased risk to a number of psychiatric disorders including major depression and post-traumatic stress disorder when exposed to childhood trauma. Interestingly, while the genetic effects on the physiological stress response are seen in adults, no interaction of adult trauma with this genotype on psychiatric risk is observed, suggesting an additional mechanism that explains the FKBP5 × childhood trauma interaction. In fact, we could show that exposure to childhood trauma leads to allele-specific epigenetic changes with a decrease in DNA methylation in a second GRE located in intron 7 of the gene, but only in carriers of the risk allele. This demethylation further de-represses FKBP5 induction following GR exposure and is likely mediated by the genetically determined increase in cortisol response following stress (Klengel et al, 2013) (Figure 1). Indeed, direct GR activation with a selective agonist in a neuronal progenitor cell line leads to a demethylation in exactly the same CG dinucleotides (CpGs) that are shown to be less methylated in DNA from peripheral blood in trauma-exposed risk allele carriers. These CpGs are located either within or between GR consensus binding site sequences, while more proximal CpGs are unaffected. We thus speculate that the demethylation is an active demethylation, induced by GR binding. Such active demethylation at GREs has been described before (Kress et al, 2006) and, although not directly shown, hydroxymethylation could be an intermediate step in this active transcription factor binding-induced demethylation (Bhutani et al, 2011), a process that has been described for other transcription factors as well (Feldmann et al, 2013). On a more general level, any genetic variant that alters binding of stress-induced transcription factors may thus lead to local differences in subsequent epigenetic changes. Thereby, allele-specific epigenetic modifications can contribute to gene × environment interactions, leading to long-term effects of stress on endocrine levels, brain activity, and the risk to develop psychiatric disorders. Figure 1 Schematic representation of an allele-specific epigenetic modification in FKBP5. The single-nucleotide polymorphism rs1360780 close to a functional GRE in intron 2 constitutes the genetic predisposition to an increased FKBP5 transcriptional response to ...

Bifunctional Ligands Allow Deliberate Extrinsic Reprogramming of the Glucocorticoid Receptor

Therapies based on conventional nuclear receptor ligands are extremely powerful, yet their broad and long-term use is often hindered by undesired side effects that are often part of the receptor's biological function. Selective control of nuclear receptors such as the glucocorticoid receptor (GR) using conventional ligands has proven particularly challenging. Because they act solely in an allosteric manner, conventional ligands are constrained to act via cofactors that can intrinsically partner with the receptor. Furthermore, effective means to rationally encode a bias for specific coregulators are generally lacking. Using the (GR) as a framework, we demonstrate here a versatile approach, based on bifunctional ligands, that extends the regulatory repertoire of GR in a deliberate and controlled manner. By linking the macrolide FK506 to a conventional agonist (dexamethasone) or antagonist (RU-486), we demonstrate that it is possible to bridge the intact receptor to either positively or negatively acting coregulatory proteins bearing an FK506 binding protein domain. Using this strategy, we show that extrinsic recruitment of a strong activation function can enhance the efficacy of the full agonist dexamethasone and reverse the antagonist character of RU-486 at an endogenous locus. Notably, the extrinsic recruitment of histone deacetylase-1 reduces the ability of GR to activate transcription from a canonical GR response element while preserving ligand-mediated repression of nuclear factor-κB. By providing novel ways for the receptor to engage specific coregulators, this unique ligand design approach has the potential to yield both novel tools for GR study and more selective therapeutics.