Glucocorticoid Receptor Binding Elements Research Articles

Enhanced transgene expression from single-stranded AAV vectors in human cells in vitro and in murine hepatocytes in vivo

We identified that distal 10 nucleotides in the D-sequence in AAV2 inverted terminal repeat (ITR) share partial sequence homology to ½ binding site of glucocorticoid receptor-binding element (GRE). Here, we describe that (i) purified GR binds to AAV2 D-sequence, and the D-sequence competes with GR binding to its cognate binding site; (ii) dexamethasone-mediated activation of GR pathway significantly increases the transduction efficiency of AAV2 vectors in human cells; (iii) human osteosarcoma cells, U2OS, which lack expression of GR, are poorly transduced by AAV2 vectors, but stable transfection with a GR expression plasmid restores vector-mediated transgene expression; (iv) replacement of the distal 10 nucleotides in the D-sequence of the AAV2 ITR with a full-length GRE consensus sequence significantly enhances transgene expression in human cells in vitro and in murine hepatocytes in vivo, and (v) none of the ITRs in AAV1, AAV3, AAV4, AAV5, and AAV6 genomes contains the GRE ½ binding site, and insertion of with a full-length GRE consensus sequence in the AAV6-ITR also significantly enhances transgene expression from AAV6 vectors, both in vitro and in vivo. These novel vectors, termed generation Y (GenY) AAV vectors, which are serotype-, transgene-, or promoter-agnostic, should be useful in human gene therapy.

Mutant glucocorticoid receptor binding elements on the interleukin-6 promoter regulate dexamethasone effects

BackgroundGlucocorticoids (GCs) have been extensively used as essential modulators in clinical infectious and inflammatory diseases. The GC receptor (GR) is a transcription factor belonging to the nuclear receptor family that regulates anti-inflammatory processes and releases pro-inflammatory cytokines, such as interleukin (IL)-6.ResultsFive putative GR binding sites and other transcriptional factor binding sites were identified on theIL-6 promoter, and dexamethasone (DEX) was noted to reduce the lipopolysaccharide (LPS)-induced IL-6 production. Among mutant transcriptional factor binding sites, nuclear factor-kappa B (NF-κB), activator protein (AP)-1, and specificity protein (Sp)1–2 sites reduced basal and LPS-induced IL-6 promoter activities through various responses. The second GR binding site (GR2) was noted to play a crucial role in both basal and inducible promoter activities in LPS-induced inflammation.ConclusionsWe concluded that selective GR2 modulator might exert agonistic and antagonistic effects and could activate crucial signaling pathways during the LPS-stimulated inflammatory process.

Identification of Pathway-Specific Serum Biomarkers of Response to Glucocorticoid and Infliximab Treatment in Children with Inflammatory Bowel Disease.

Objective:Serum biomarkers may serve to predict early response to therapy, identify relapse, and facilitate drug development in inflammatory bowel disease (IBD). Biomarkers are particularly important in children, in whom achieving early remission and minimizing procedures are especially beneficial.Methods:We profiled protein and micro RNA (miRNA) in serum from patients pre- and post-therapy, to identify molecular markers of pharmacodynamic effect. Serum was obtained from children with IBD before and after treatment with either corticosteroids (prednisone; n=12) or anti-tumor necrosis factor-α biologic (infliximab; n=7). Over 1,100 serum proteins were assayed using aptamer-based SOMAscan proteomics, and 22 miRNAs analyzed by quantitative real time PCR. Concordance of longitudinal changes between the groups was used to identify markers responsive to treatment. Bioinformatic analysis was used to build insight into mechanisms of changes in response to treatment.Results:We identified 18 proteins and three miRNAs responsive to both prednisone and infliximab. Eight markers that decreased are associated with inflammation and have gene promoters regulated by nuclear factor (NF)-κB. Several that increased are associated with resolving inflammation and tissue damage. We also identified six markers that appear to be steroid-specific, three of which have glucocorticoid receptor binding elements in their promoter region.Conclusions:Serum markers regulated by the inflammatory transcription factor NF-κB are potential candidates for pharmacodynamic biomarkers that, if correlated with later outcomes like endoscopic or histologic healing, could be used to monitor treatment, optimize dosing, and enhance drug development. The pharmacodynamic biomarkers identified here hold potential to improve both clinical care and drug development. Further studies are warranted to investigate these markers as early predictors of response, or possibly surrogate outcomes.

11. The Role of Glucocorticoid Receptor Signaling in Adeno-Associated Virus 2 Infection

We previously observed that purified recombinant glucocorticoid receptor (GR) protein specifically binds to the D-sequence within the inverted terminal repeat of the AAV2 genome because the D-sequence shares partial homology to the consensus half-site of the glucocorticoid receptor-binding element (GRE). The half-site is an essential core element, which has been reported to be sufficient to mediate GR signaling. Substitution of the D-sequence with the authentic GRE further increased AAV2 vector-mediated transgene expression. Based on these observations, we systemically examined whether AAV2 infection involves the GR signaling pathway. Our results showed that following infection by wild-type (WT) AAV2, or transduction with recombinant AAV2 vectors in vitro, GR was activated. The serine phosphorylated form of GR in whole cell lysates was significantly increased in a dose-dependent manner 18 hours post viral infection/vector transduction. Translocation and accumulation of GR in the nucleus was also increased correspondingly. To further corroborate our findings in an in vivo model, C57BL/6 mice were injected via tail-vein with either WT-AAV2 (1×1012 vgs/mouse) or AAV2-EGFP vectors (5×1011 vgs/mouse) and sacrificed 16 hours post virus/vector administration. A significant increase in the serine phosphorylated form of GR was also observed in liver tissues. We also observed that in cell cultures, dexamethasone, a well-known activator of GR, significantly enhanced rAAV2 vector-mediated transgene expression in various GR-positive cell lines, but not in a GR-deficient human osteosarcoma cell line, U2OS. Furthermore, stable transfection of U2OS cells with a GR expression plasmid not only facilitated AAV2 vector transduction, but also restored the ability of dexamethasone to further increase AAV2 vector-mediated transgene expression. Together with our previous data, we propose a model for elucidating the role of glucocorticoid receptor signaling pathway in AAV2 infection/AAV2 vector transduction (Fig. 1Fig. 1). Upon viral transduction or stimulation with glucocorticoids, the cytoplasmic GR is phosphorylated and translocated into the nucleus. Following AAV2 entry into the nucleus, the viral genome is released. Upon viral second-strand DNA synthesis or annealing of the complementary DNA strands, the activated GR binds to the double-stranded D-sequence in the viral inverted terminal repeats and consequently, enhances transgene expression. The activated GR also regulates its downstream pathways, such as NF-kB, which we have previously shown to have a substantial effect on AAV2 vector-mediated transgene expression (Proc Natl Acad Sci USA, 108:3743-3738, 2011). A better understanding of the complex interaction between GR and NF-kB signaling pathways notwithstanding, the availability of the fully functional GRE site-substituted novel AAV vectors to achieve high-efficiency transgene expression has implications in the potential use of these vectors in human gene therapy.View Large Image | Download PowerPoint Slide

310. Development of Generation X Recombinant AAV Vectors for Human Gene Therapy

We have previously reported the development of capsid-modified next generation (NextGen) AAV serotype vectors that transduce cells and tissues more efficiently at reduced vector doses (Proc. Natl. Acad. Sci., USA, 105: 7827-7832, 2008). More recently, we have also described the development of genome-modified generation X (GenX) AAV vectors that also transduce cells and tissues more efficiently (J. Virol, 89: 952-961, 2015). The recombinant AAV genome contains inverted terminal repeats (ITRs) of 145 nucleotides at both ends. In these studies, a 20-nucleotide sequence, termed the D-sequence, was replaced with a substitute sequence, which led to enhanced transgene expression in human cell lines in vitro an in murine hepatocytes in vivo (J. Virol., 89: 952-961, 2015). In our present studies, we observed that a sequence, GGTTCCT, at the end of the D-sequence, shares partial homology to the consensus glucocorticoid receptor-binding element (GRE) site, 5’-GGTACANNNTGTT/CCT-3’. The TGTTCT half-site is an essential core element, which has been reported to be sufficient to relay glucocorticoid signaling. In electrophoretic mobility-shift assays (EMSAs), we documented that purified GR protein could specifically bind to double-stranded D-sequence oligonucleotides, suggesting that the D-sequence potentially functions as a ½ GRE site. Based on these results, we hypothesized that replacement of the D-sequence with a full GRE binding-site in the ITR might further increase transgene expression from these GenX AAV genomes. To this end, recombinant AAV vectors were generated in which the D-sequence was replaced with a fully functional GRE site. Insertion of a full GRE binding-site in the ITR significantly increased the transgene expression from these GenX AAV genomes following encapsidation in the wild-type (WT) AAV2 capsid vectors in human cell lines in vitro, and the extent of the transgene expression was further increased by dexamethasone-treatment. When 1×1010 vgs of recombinant AAV2 vectors containing the Gaussia luciferase (Gluc) reporter gene in the unmodified AAV genome, or those containing the GRE sequence, were administered via tail-vein into C57BL6/J mice, the transduction efficiency of the AAV-GRE vectors was ~8-fold higher in murine hepatocytes in vivo up to 9-weeks post-vector administration. More interestingly, when AAV-GRE genomes containing the enhanced green fluorescence protein (EGFP) reporter gene were encapsidated in the optimal NextGen AAV capsid-modified quadruple-mutant (Y444F+Y500F+Y730F+T491V) AAV2 vectors, the transduction efficiency of these vectors was further increased by ~8-fold in murine hepatocytes in vivo at a dose as low as 5×108 vgs/mouse. Taken together, the availability of these novel GenX AAV vectors containing the D-sequence substitution, and the fully functional GRE site insertion, to achieve high-efficiency transgene expression, has implications in the use of these vectors in human gene therapy.

Abstract 1075: Steroid receptor reprogramming of the chromatin landscape: crosstalk at the genomic level.

Abstract Crosstalk between the estrogen receptor (ER) and the glucocorticoid receptor (GR) plays an important role in controlling many cellular processes. Physiological interactions between ER and GR are not only important for the development of certain tissues, such as the uterus and bone, but also may play an important role in breast cancer. Recent studies have shown that the GR and ER status in breast cancer is a significant factor for determining the outcome of the disease. However, the mechanistic details defining the cellular interactions between ER and GR are poorly understood. Since the regulation of receptor binding to response elements controls the transcriptional output in response to hormones, it is logical to suspect that co-treatment of cells with corticosteroids and estradiol would have an effect on the genome-wide binding landscapes for GR and ER. Therefore, using chromatin immunoprecipitation followed by high-throughput sequencing we investigated the changes in genome-wide binding of ER and GR upon co-activation and revealed that there is global re-arrangement of steroid receptor binding. Furthermore, we unveiled a novel mechanism dictating the molecular interplay between ER and GR. Genome-wide sequencing of DnaseI hypersensitive sites upon induction of GR shows that GR facilitates selective access of ER to specific sites in the genome by maintaining an accessible configuration at these response elements. In addition, activation of ER can affect chromatin structure at novel GR binding sites, resulting in a new class of GR binding elements. Further studies show that at the sites where ER binding is facilitated by GR, binding to these newly accessible sites is not dependent on the direct binding of ER to its response element. Instead ER is brought to these sites through interactions with other factors, such as AP1. Overall, these studies define genome level interactions between ER and GR whereby the activation of multiple steroid receptors has a dramatic impact on controlling which regulatory elements are accessible to each receptor. The unveiling of this mechanism is important for understanding the molecular interplay between ER and GR in development and in cancer and may represent a general mechanism for crosstalk between receptors. Citation Format: Tina B. Miranda, Ty C. Voss, Myong-Hee Sung, Songjoon Baek, Sam John, Mary Hawkins, Lars Grontved, R. Louis Schiltz, Gordon L. Hager. Steroid receptor reprogramming of the chromatin landscape: crosstalk at the genomic level. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1075. doi:10.1158/1538-7445.AM2013-1075

TAZ is downregulated by dexamethasone during the differentiation of 3T3-L1 preadipocytes

TAZ (transcriptional co-activator with PDZ binding motif) is a transcriptional modulator of mesenchymal stem cell differentiation. We have found that TAZ was expressed in postconfluent 3T3-L1 preadipocytes and downregulated during differentiation. Downregulation of TAZ was specifically mediated by dexamethasone (DEX), one component of induction cocktails routinely used in adipocyte differentiation. DEX repressed the transcription of TAZ by direct binding of the glucocorticoid receptor (GR) to the GR binding element in its promoter. More importantly, overexpression of TAZ inhibited adipogenesis and promoted the trans-differentiation of preadipocytes into osteocytes. This establishes a new functional interaction between DEX and TAZ that contributes to the mechanism of adipogenesis.

The MuRF1 Promoter is Synergistically Activated by the Glucocorticoid Receptor and the Forkhead Family Member, FoxO1

Skeletal muscle wasting is observed in a wide range of physiological conditions. The recently discovered ubiquitin E3 ligases, MAFbx and MuRF1, have been implicated in the regulation of protein degradation during muscle atrophy. Analysis of the MAFbx promoter has led to the observation that the FoxO family of transcription factors plays a critical role in regulating expression during atrophic conditions. Interestingly, MuRF1 and MAFbx expression in vivo has been shown to increase in response to treatment with dexamethasone, however no functional glucocorticoid receptor binding element (GRE) has been found in the MAFbx proximal promoter. To understand the regulation of MuRF1 expression, a 5000 base pair fragment of the mouse MuRF1 promoter was cloned. Analysis of the sequence revealed a GRE that is conserved in the mouse, rat and human promoter, as well as, a conserved FoxO binding element (FBE) that lies immediately adjacent to the GRE. Transcriptional reporter assays performed in both HepG2 and C2C12 cells demonstrate that the MuRF1 promoter is highly responsive to dexamethasone-activated glucocorticoid receptor (GR) and FoxO1 individually, while co-overexpression of GR and FoxO1 leads to a dramatic synergistic increase in reporter activity. Furthermore, mutation of either the GRE or the FBE alone or in combination is sufficient to significantly impair GR and/or FoxO1-mediated transcriptional activation of the MuRF1 promoter. These initial findings strongly suggest that corticosteroid-activated GR and FoxO1 act synergistically to drive MuRF1 expression under atrophy-inducing conditions.

The Synergistic Effect of Dexamethasone and All-trans-retinoic Acid on Hepatic Phosphoenolpyruvate Carboxykinase Gene Expression Involves the Coactivator p300

Activation of phosphoenolpyruvate carboxykinase (PEPCK) gene transcription in response to all-trans-retinoic acid (RA) or a glucocorticoid such as dexamethasone (Dex) requires a distinct arrangement of DNA-response elements and their cognate transcription activators on the gene promoter. Two of the accessory factor-binding elements involved in the Dex response (gAF1 and gAF3) coincide with the DNA-response elements involved in the RA response. We demonstrate here that the combination of Dex/RA has a synergistic effect on endogenous PEPCK gene expression in rat hepatocytes and H4IIE hepatoma cells. Reporter gene studies show that the gAF3 element and one of the two glucocorticoid receptor-binding elements (GR1) are most important for this effect. Chromatin immunoprecipitation assays revealed that when H4IIE cells were treated with Dex/RA, ligand-activated retinoic acid receptors (retinoic acid receptor/retinoid X receptor) and glucocorticoid receptors are recruited to this gene promoter, as are the transcription coregulators p300, CREB-binding protein, p/CIP, and SRC-1. Notably, the recruitment of p300 and RNA polymerase II to the PEPCK promoter is increased by the combined Dex/RA treatment compared with Dex or RA treatment alone. The functional importance of p300 in the Dex/RA response is illustrated by the observation that selective reduction of this coactivator, but not that of CREB-binding protein, abolishes the synergistic effect in H4IIE cells.

Glucocorticoid-dependent action of neural crest factor AP-2: stimulation of phenylethanolamine N-methyltransferase gene expression.

AP-2 is a vertebrate transcription factor expressed in neural crest cells and their derivative tissues, including the adrenal medulla, where epinephrine is produced. AP-2 is shown to stimulate expression of the gene encoding the epinephrine biosynthetic enzyme phenylethanolamine N-methyltransferase (PNMT). However, stimulation of the PNMT gene by AP-2 requires glucocorticoids and appears to be mediated through the interaction of AP-2 with activated type II glucocorticoid receptors. Mutation of AP-2 and/or glucocorticoid receptor binding elements within the PNMT promoter disrupts the ability of AP-2 and glucocorticoids to induce PNMT promoter activity. These findings suggest, in the case of PNMT, that AP-2 stimulates gene expression through a novel glucocorticoid-dependent mechanism.

V + fibronectin mRNA is increased in the brains of aged rats: effect of food restriction

Food restriction increases life-span in rodents. With regard to the central nervous system, underfeeding has been shown to have beneficial effects on synaptic transmission in old rats. However, the molecular events underlying functional changes in the brains of food-restricted rats are largely unknown. In the present study the levels of fibronectin mRNA containing the alternatively spliced segment V (FN-V +) as well as the levels of FN mRNA containing the alternatively spliced segment EIIIB (FN-EIIIB +), were examined by RNA gel blot hybridization in the brains of 3-day-old, 24-day-old, 10-month-old, 18-month-old, 30-month-old ad libitum (AL) fed, 30-month-old food-restricted (FR), and 35–37-month-old, FR rats. The hybridization signal for the FN-EIIIB + mRNA was relatively abundant at early postnatal stages but very few transcripts were detected in the brains of adult, AL rats. The transcripts coding for FN-V + mRNA were moderately expressed in the brains of 3-day-old, 24-day-old, 10-month-old, and 18-month-old rats. However, the FN-V + mRNA signal was then prominently increased (approx. 3-fold) in the brains of the 30-month-old, AL rats vs. 10-month-old, AL rats, and further increased (approx. 2-fold) in the brains of 30-month-old, FR, as compared to 30-month-old, AL rats. However, the levels of FN-V + mRNA were slightly decreased in the brains of very old (35–37-month) FR rats vs. 30-month-old FR rats. The distribution of fibronectin messenger RNA and protein was also investigated by non-radioactive in situ hybridization and immunohistochemistry, respectively. The most prominent expression of FN-V + messenger RNA was seen in neurons of the hippocampus, including the granule cells of the dentate gyrus, and in layers III and V of the cortex of 30-month-old, FR rats. FN immunostaining closely paralleled the distribution of FN mRNA and was confined to the neuronal cell periphery. The upregulation of fibronectin gene expression upon exposure to glucocorticoids is well documented. Prolonged food restriction, acting as a stress factor, combined with decreased plasticity of glucocorticoid regulatory responses in the aged rats could cause an increase in the levels of FN mRNA and protein in the brains of old, FR rats. Since FN has been shown to provide an adhesive substrate for extending neurites, we conclude that food restriction may potentiate synaptic plasticity, via glucocorticoid receptor binding elements of the FN gene, in the brains of old rats.

V+ Fibronectin mRNA is increased in the brains of aged rats: effect of food restriction

Food restriction increases life-span in rodents. With regard to the central nervous system, underfeeding has been shown to have beneficial effects on synaptic transmission in old rats. However, the molecular events underlying functional changes in the brains of food-restricted rats are largely unknown. In the present study the levels of fibronectin mRNA containing the alternatively spliced segment V (FN-V+) as well as the levels of FN mRNA containing the alternatively spliced segment EIIIB (FN-EIIIB+), were examined by RNA gel blot hybridization in the brains of 3-day-old, 24-day-old, 10-month-old, 18-month-old, 30-month-old ad libitum (AL) fed, 30-month-old food-restricted (FR), and 35–37-month-old, FR rats. The hybridization signal for the FN-EIIIB+ mRNA was relatively abundant at early postnatal stages but very few transcripts were detected in the brains of adult, AL rats. The transcripts coding for FN-V+ mRNA were moderately expressed in the brains of 3-day-old, 24-day-old, 10-month-old, and 18-month-old rats. However, the FN-V+ mRNA signal was then prominently increased (approx. 3-fold) in the brains of the 30-month-old, AL rats vs. 10-month-old, AL rats, and further increased (approx. 2-fold) in the brains of 30-month-old, FR, as compared to 30-month-old, AL rats. However, the levels of FN-V+ mRNA were slightly decreased in the brains of very old (35–37-month) FR rats vs. 30-month-old FR rats. The distribution of fibronectin messenger RNA and protein was also investigated by non-radioactive in situ hybridization and immunohistochemistry, respectively. The most prominent expression of FN-V+ messenger RNA was seen in neurons of the hippocampus, including the granule cells of the dentate gyrus, and in layers III and V of the cortex of 30-month-old, FR rats. FN immunostaining closely paralleled the distribution of FN mRNA and was confined to the neuronal cell periphery. The upregulation of fibronectin gene expression upon exposure to glucocorticoids is well documented. Prolonged food restriction, acting as a stress factor, combined with decreased plasticity of glucocorticoid regulatory responses in the aged rats could cause an increase in the levels of FN mRNA and protein in the brains of old, FR rats. Since FN has been shown to provide an adhesive substrate for extending neurites, we conclude that food restriction may potentiate synaptic plasticity, via glucocorticoid receptor binding elements of the FN gene, in the brains of old rats.

Identification of multiple glucocorticoid receptor binding sites in the rat osteocalcin gene promoter.

The biosynthesis of osteocalcin (OC), a bone-specific, noncollagenous protein, is stringently regulated during differentiation of the osteoblast phenotype. Glucocorticoids, and also 1,25(OH)2D3, mediate the developmental regulation of OC gene transcription. In this study, we established that the -1097 to +23 promoter (pOCZCat) of the rat OC gene confers glucocorticoid responsiveness to both basal and vitamin D-induced OC expression. The presence of multiple glucocorticoid receptor (GR) binding sites in the proximal rat OC gene promoter was determined by the combined use of DNase I footprinting, dimethyl sulfate fingerprinting, and gel mobility shift analysis with glucocorticoid receptor protein. One glucocorticoid receptor binding element (GRE) resides immediately downstream of the TATA box (-16 to -1). In vivo activity was established by cotransfection of ROS 17/2.8 osteosarcoma cells with an OC-CAT construct in the presence of cloned GRE sequences (wild type or mutant) as competitors. A putative second, less protected GR binding site is located further upstream in the OC gene basal promoter within the region overlapping the TATA box. This is in direct contrast to the organization of GREs in the human OC proximal promoter wherein GR binding at the upstream GRE overlapping the TATA is stronger than at the downstream GRE. In addition, we detected sequence-specific binding of GR protein to another basal promoter element, the OC box (-99 to -76), which contains a central CCAAT motif. The presence of multiple GR binding sites in the rat OC gene proximal promoter indicates that regulation of basal and vitamin D-enhanced transcription by glucocorticoids may involve the integrated activities of multiple, independent GREs.

Regulatory elements in the human immunodeficiency virus type 1 long terminal repeat LTR (HIV-1) responsive to steroid hormone stimulation.

Within the long terminal repeat (LTR) of the human immunodeficiency virus type 1 (HIV-1) provirus there exists a steroid hormone-responsive element corresponding to the TGTTCT sequence identified as the glucocorticoid receptor binding element within the LTR of mouse mammary tumor virus. We have used an LTR(HIV-1)-chloramphenicol acetyl transferase (CAT) plasmid construct to transfect infected H9V3 and noninfected H9 cells. Four hours before harvest the cells were divided into two parts and half was treated with hydrocortisone (10(-7) M). The cells were harvested and washed, and the CAT activity was measured. In eight repeat experiments an increased expression of the CAT gene has consistently been observed in H9V3 cells in response to the glucocorticoid but no significant effect of the steroid was observed in noninfected cells. Double transfection of LTR(HIV-1)-TAT and LTR(HIV-1)-CAT into noninfected H9 cells results in a cell population in which the CAT gene was responsive to glucocorticoid stimulation. A time course and dose response for the steroid effect have been determined and the binding of steroid receptor fo the LTR-DNA characterized by gel retardation experiments.