Multiple Response Elements Research Articles

The transcriptional repressor Rev-erbα regulates circadian expression of the astrocyte Fabp7 mRNA.

The astrocyte brain-type fatty-acid binding protein (Fabp7) circadian gene expression is synchronized in the same temporal phase throughout mammalian brain. Cellular and molecular mechanisms that contribute to this coordinated expression are not completely understood, but likely involve the nuclear receptor Rev-erbα (NR1D1), a transcriptional repressor. We performed ChIP-seq on ventral tegmental area (VTA) and identified gene targets of Rev-erbα, including Fabp7. We confirmed that Rev-erbα binds to the Fabp7 promoter in multiple brain areas, including hippocampus, hypothalamus, and VTA, and showed that Fabp7 gene expression is upregulated in Rev-erbα knock-out mice. Compared to Fabp7 mRNA levels, Fabp3 and Fabp5 mRNA were unaffected by Rev-erbα depletion in hippocampus, suggesting that these effects are specific to Fabp7. To determine whether these effects of Rev-erbα depletion occur broadly throughout the brain, we also evaluated Fabp mRNA expression levels in multiple brain areas, including cerebellum, cortex, hypothalamus, striatum, and VTA in Rev-erbα knock-out mice. While small but significant changes in Fabp5 mRNA expression exist in some of these areas, the magnitude of these effects are minimal to that of Fabp7 mRNA expression, which was over 6-fold across all brain regions. These studies suggest that Rev-erbα is a transcriptional repressor of Fabp7 gene expression throughout mammalian brain.

Androgen/androgen receptor affects gentamicin-induced nephrotoxicity through regulation of megalin expression

AimInvestigation whether androgen/androgen receptor (AR) might regulate megalin expression and/or functionality and thus affecting Gentamicin-induced nephrotoxicity (GIN). Main methodsMale Wistar rats were treated with gentamicin with/out AR ligands (testosterone as agonist and flutamide as antagonist). Megalin expression in the kidney tissues was determined by real-time RT-PCR and western blot. Besides, megalin functionality was assessed using immunofluorescence imaging of fluorescein isothiocyanate (FITC) conjugated bovine serum albumin (BSA) (FITC-BSA). The effects of different treatments on the kidney were assessed at the structural level by histopathological evaluation and the biochemical level by colorimetric assay of blood urea nitrogen (BUN), serum creatinine (SCr) and urinary albumin/creatinine (A/C) ratio, besides, kidney expression of neutrophil gelatinase-associated lipocalin (NGAL) by immunoblotting. Key findingsOur results revealed that treatment with testosterone either alone or combined with gentamicin increased megalin expression at mRNA and protein levels as well as at the functional level. These effects were paralleled by increased GIN as manifested by increased SCr, BUN, A/C ratio, renal expression of NGAL or histopathological changes. On the other hand, treatment with flutamide ameliorated GIN and megalin expression and functionality. Computational analysis of megalin promotor revealed the presence of multiple response elements that mediate androgen response. SignificanceAndrogen/AR regulates megalin expression at the transcriptional level and consequently GIN. This may explain the sexual dimorphism in GIN and might represent a druggable target for treatment or prevention of GIN.

Sexual dimorphism in atrophic effects of topical glucocorticoids is driven by differential regulation of atrophogene REDD1 in male and female skin.

Topical glucocorticoids, well-known anti-inflammatory drugs, induce multiple adverse effects, including skin atrophy. The sex-specific effects of systemic glucocorticoids are known, but sexual dimorphism of therapeutic and side effects of topical steroids has not been studied. We report here that female and male mice were equally sensitive to the anti-inflammatory effect of glucocorticoid fluocinolone acetonide (FA) in ear edema test. At the same time, females were more sensitive to FA-induced skin atrophy. We recently reported that REDD1 (regulated in development and DNA damage 1) plays central role in steroid atrophy. We found that REDD1 was more efficiently activated by FA in females, and that REDD1 knockout significantly protected female but not male mice from skin atrophy. Studies using human keratinocytes revealed that both estradiol and FA induced REDD1 mRNA/protein expression, and cooperated when they were combined at low doses. Chromatin immunoprecipitation analysis confirmed that REDD1 is an estrogen receptor (ER) target gene with multiple estrogen response elements in its promoter. Moreover, experiments with GR and ER inhibitors suggested that REDD1 induction by these hormones was interdependent on functional activity of both receptors. Overall, our results are important for the development of safer GR-targeted therapies suited for female and male dermatological patients.

Cloning and functional identification of a strigolactone receptor gene MdD14 in apple

Strigolactones (SLs) are phytohomones that regulate shoot branching and hypocotyl elongation. Here, the strigolactone receptor gene MdD14 in apple was cloned. MdD14 was localized to the nucleus and cytoplasm. GUS staining showed that MdD14 was expressed in various tissues. A promoter analysis revealed that MdD14 contained multiple response elements and its expression levels were induced by various abiotic stress treatment. The post-translational of MdD14 was regulated by the SL analog GR24. Ectopic expression of MdD14 in Arabidopsis is resulted in a phenotype that inhibited shoot branching and hypocotyl elongation, as well as increased tolerance to salt, drought and low temperature stresses. These findings reveal the functions of MdD14 and lay a foundation for studying SLs in apple. The apple MdD14 plays a key role in the SL signal transduction pathway and is involved in various abiotic stress resistance.

Unearthing Regulatory Axes of Breast Cancer circRNAs Networks to Find Novel Targets and Fathom Pivotal Mechanisms

Circular RNAs (circRNAs) possess valuable characteristics for both diagnosis and treatment of several human cancers including breast cancer (BC). In this study, we combined several systems, biology tools and approaches to identify influential BC circRNAs, miRNAs, and related mRNAs as the members of competing endogenous RNAs (ceRNAs) networks and related RNA binding proteins (RBPs) to study and decipher the BC-triggering biological processes and pathways. Rooting from the identified total of 25 co-differentially expressed circRNAs (DECs) between triple negative (TN) and luminal A subtypes of BC from microarray analysis, five hub DECs (hsa_circ_0003227, hsa_circ_0001955, hsa_circ_0020080, hsa_circ_0001666, and hsa_circ_0065173) and top eleven RBPs (AGO1, AGO2, EIF4A3, FMRP, HuR (ELAVL1), IGF2BP1, IGF2BP2, IGF2BP3, EWSR1, FUS, and PTB) were explored to form the upper stream regulatory elements. All the hub circRNAs were regarded as a super sponge having multiple miRNA response elements (MREs). Then, three BC leading miRNAs (hsa-miR-149, hsa-miR-182, and hsa-miR-383) were also introduced from merging several established ceRNAs networks. The predicted 7- and 8-mer MREs matches between hub circRNAs and leading miRNAs ensured their enduring regulatory capability. The mined downstream mRNAs of the circRNAs-miRNAs network then were presented to STRING database to form the PPI network and to decipher the issue from another point of view. The BC interconnected enriched pathways and processes guarantee the merits of the ceRNAs network's members as targetable therapeutic elements. This study suggested extensive panels of novel therapeutic targets that are in charge of BC progression, hence their impressive role cannot be excluded and needs deeper empirical laboratory designs.

Role of the Fusarium oxysporum metallothionein Mt1 in resistance to metal toxicity and virulence.

Soil organisms exhibit high tolerance to heavy metals, probably acquired through evolutionary adaptation to contaminated environments. Essentially, metal tolerance in fungi involves several specific and non-specific mechanisms that include metal efflux, metal binding to cell walls, extracellular and intracellular sequestration and complexation with proteins. However, fungi have adopted different strategies to detoxify heavy metals, although species differ in the mechanisms used. In this complex molecular framework, metallothioneins (MTs) are becoming increasingly relevant in metal homeostasis, even though little is known about their role in metal adaptation and virulence in fungal pathogens. With the aim to decipher the function of metallothioneins in the opportunistic fungus Fusarium oxysporum, we have carried out an in silico analysis that revealed the presence of a hypothetical metallothionein (mt1) that has multiple metal responsive elements in its promoter region and conserved cysteine motifs in its coding sequence. Characterization of strain Δmt1 deficient in the mt1 gene revealed higher sensitivity of this mutant to copper, cadmium and zinc compared to the wild type strain (wt). Expression analyses revealed that Zn specifically activates mt1, but the lack of this gene did not lead to a transcriptional up-regulation of genes gapdh and prx, associated with the oxidative stress response. The lack of mt1 did not alter the pathogenic capacity of the fungus, either in tomato plant or in a murine model of systemic infection. Nevertheless, Δmt1 displayed lower resistance to macrophage killing, suggesting a connection between the absence of mt1 and impaired defence capacity against copper and reactive oxygen species.

Epstein-Barr Virus Gene BARF1 Expression is Regulated by the Epithelial Differentiation Factor ΔNp63α in Undifferentiated Nasopharyngeal Carcinoma.

Epstein–Barr Virus (EBV) BamHI-A rightward frame 1 (BARF1) protein is considered a viral oncogene in epithelial cells and has immune-modulating properties. During viral lytic replication BARF1 is expressed as an early gene, regulated by the immediate early EBV protein R. However, in viral latency BARF1 is exclusively expressed in epithelial tumors such as nasopharyngeal (NPC) and gastric carcinoma (GC) but not in lymphomas, indicating that activation of the BARF1 promoter is cell type specific. Undifferentiated NPC is characterized by high expression of ΔNp63 isoforms of the epithelial differentiation marker p63, a member of the p53 family of transcription factors. Transcription factor binding site analysis indicated potential p53 family binding sites within the BARF1 promoter region. This study investigated ability of various p53 family members to transactivate the BARF1 promoter. Using BARF1 promoter luciferase reporter constructs we demonstrate that only p63 isoform ΔNp63α is capable of transactivating the BARF1 promoter, but not the TAp63 isoforms, p53 or p73. Direct promoter binding of ΔNp63α was confirmed by Chromatin Immune Precipitation (ChIP) analysis. Deletion mutants of the BARF1 promoter revealed multiple ΔNp63 response elements to be responsible for BARF1 promoter transactivation. However, ΔNp63α alone was not sufficient to induce BARF1 in tumor cells harboring full EBV genomes, indicating that additional cofactors might be required for full BARF1 regulation. In conclusion, in EBV positive NPC and GC, BARF1 expression might be induced by the epithelial differentiation marker ΔNp63α, explaining BARF1 expression in the absence of lytic reactivation.

The aryl hydrocarbon receptor regulates the expression of TIPARP and its cis long non-coding RNA, TIPARP-AS1

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor and member of the basic helix-loop-helix-PAS family. AHR is activated by numerous dietary and endogenous compounds that contribute to its regulation of genes in diverse signaling pathways including xenobiotic metabolism, vascular development, immune responses and cell cycle control. However, it is most widely studied for its role in mediating 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity. The AHR target gene and mono-ADP-ribosyltransferase, TCDD-inducible poly-ADP-ribose polymerase (TIPARP), was recently shown to be part of a novel negative feedback loop regulating AHR activity through mono-ADP-ribosylation. However, the molecular characterization of how AHR regulates TIPARP remains elusive. Here we show that activated AHR is recruited to the TIPARP promoter, through its binding to two genomic regions that each contain multiple AHR response elements (AHREs), AHR regulates the expression of both TIPARP but also TIPARP-AS1, a long non-coding RNA (lncRNA) which lies upstream of TIPARP exon 1 and is expressed in the opposite orientation. Reporter gene and deletion studies showed that the distal AHRE cluster predominantly regulated TIPARP expression while the proximal cluster regulated TIPARP-AS1. Moreover, time course and promoter activity assays suggest that TIPARP and TIPARP-AS1 work in concert to regulate AHR signaling. Collectively, these data show an added level of complexity in the AHR signaling cascade which involves lncRNAs, whose functions remain poorly understood.

The Long Noncoding RNA HOTAIR in Breast Cancer: Does Autophagy Play a Role?

HOTAIR (HOX transcript antisense RNA) plays a critical role in chromatin dynamics through the interaction with histone modifiers resulting in transcriptional gene silencing. The promoter of the HOTAIR gene contains multiple estrogen response elements (EREs) and is transcriptionally activated by estradiol in estrogen receptor-positive breast cancer cells. HOTAIR competes with BRCA1, a critical protein in breast cancer and is a critical regulator of genes involved in epithelial-to-mesenchymal transition. It mediates an oncogenic action of c-Myc, essential for breast carcinogenesis. The carcinogenic action of HOTAIR was confirmed in breast cancer stem-like cells, in which it was essential for self-renewal and proliferation. Several miRNAs regulate the expression of HOTAIR and HOTAIR interacts with many miRNAs to support cancer transformation. Many studies point at miR-34a as a major component of HOTAIR–miRNAs–cancer cross-talk. The most important role of HOTAIR can be attributed to cancer progression as its overexpression stimulates invasion and metastasis. HOTAIR can regulate autophagy, important for breast cancer cells survival, through the interaction with miRNAs specific for autophagy genes and directly with these genes. The role of HOTAIR-mediated autophagy in breast cancer progression can be underlined by its interaction with matrix metalloproteinases, essential for cancer invasion, and β-catenin can be important for this interaction. Therefore, there are several mechanisms of the interplay between HOTAIR and autophagy important for breast cancer, but further studies are needed to determine more details of this interplay.

Pregnane X receptor (PXR) signaling in seabream primary hepatocytes exposed to extracts of seawater samples collected from polycyclic aromatic hydrocarbons (PAHs)-contaminated coastal areas

Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants damaging to the marine environment and the wildlife. Herein, we investigated the effects of extracts from coastal seawaters (central Adriatic sea, Italy), showing high concentrations of PAHs, on pregnane X receptor (PXR)-transcriptional regulation of the cytochrome P450 3A (CYP3A) gene using seabream primary hepatocytes. The results show that concentrated extracts of seawater with original ΣPAH concentrations above the putative threshold of 30 ng L−1 increased expression of PXR and its main target gene, CYP3A. Similar results were observed for LXR and its target gene SREBP-1c suggesting pathway cross-talk. These data are further supported by the finding of multiple PXR and LXR response elements in the putative promoters of their target genes. Overall, our data indicate the capacity of seawater extracts, containing environmentally relevant levels of PAHs, to affect multiple pathways, including lipid and cholesterol metabolism.

Development of Novel Cell Lines for High-Throughput Screening to Detect Estrogen-Related Receptor Alpha Modulators

Estrogen-related receptor alpha (ERRα), the first orphan nuclear receptor discovered, is crucial for the control of cellular energy metabolism. ERRα and its coactivator, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), are required for rapid energy production in response to environmental challenges. They have been implicated in the etiology of metabolic disorders such as type 2 diabetes and metabolic syndrome. ERRα also plays a role in the pathogenesis of breast cancer. Identification of compounds that modulate ERRα signaling may elucidate environmental factors associated with these diseases. Therefore, we developed stable cell lines containing an intact ERRα signaling pathway, with and without the coactivator PGC-1α, to use as high-throughput screening tools to detect ERRα modulators. The lentiviral PGC-1α expression constructs and ERRα multiple hormone response element (MHRE) reporters were introduced into HEK293T cells that express endogenous ERRα. A cell line expressing the reporter alone was designated “ERR.” A second cell line expressing both reporter and PGC-1α was named “PGC/ERR.” Initial screenings of the Library of Pharmacologically Active Compounds (LOPAC) identified 33 ERR and 22 PGC/ERR agonists, and 54 ERR and 15 PGC/ERR antagonists. Several potent ERRα agonists were dietary plant compounds (e.g., genistein). In conclusion, these cell lines are suitable for high-throughput screens to identify environmental chemicals affecting metabolic pathways and breast cancer progression.

The salt-regulated element in the promoter of lycopene β-cyclase gene confers a salt regulatory pattern in carotenogenesis of Dunaliella bardawil.

In the carotenoid biosynthesis, lycopene β-cyclase (LCYb) is a key regulatory enzyme involved in the conversion of lycopene into β-carotene. Under stress conditions, such as high salinity, high light and nutrient deprivation, large amounts of β-carotene can be accumulated in Dunaliella bardawil. To study on the molecular responses of salt stress in D. bardawil is of great significance to reveal the mechanisms of salt tolerance and engineer crop plants to be salt-tolerant. In this study, the full-length coding sequence of lcyb from D. bardawil (Dblcyb, GenBank: KX218392) was isolated by transcriptome sequencing. Then, the genomic sequence, promoter and terminator regions of Dblcyb were isolated by genome walking. The Dblcyb promoter (GenBank: KX218393) contained several typical transcription boxes, multiple light response elements and a salt-regulated element (SRE, GT1GMSCAM4). Dbpsy and Dblcyb responsible for β-carotene biosynthesis in D. bardawil was shown to be up-regulated under salt stress and their promoters contained the common SRE. By element deletion analysis and using Ble-EGFP as the reporter, the salt-inducible SRE was confirmed to confer salt-induced expression of Dblcyb promoter. It was indicated that the salt-regulated expression of Dblcyb may be attributed to the salt-responsive element (GT1GMSCAM4) and the GT-rich region in its genomic sequence.

Role of nuclear factor of activated T-cells 5 in regulating hypertonic-mediated secretin receptor expression in kidney collecting duct cells

A growing body of evidence suggests that secretin (SCT) is an important element in the osmoregulatory pathway. It is interesting to note that both SCT and its receptor (SCTR) gene are activated upon hyperosmolality in the kidney. However, the precise molecular mechanisms underlying the induction of the SCTR gene expression in response to changes in osmolality have yet to be clarified. Detailed DNA sequence analysis of the promoter regions of the SCTR gene reveals the presence of multiple osmotic response elements (ORE). The ORE is the binding site of a key osmosensitive transactivator, namely, the nuclear factor of activated T-cells 5 (NFAT5). SCTR and NFAT5 are co-expressed in the kidney cortex and medulla collecting duct cells. We therefore hypothesize that NFAT5 is responsible for modulating SCTR expression in hypertonic environments. In this study, we found hypertonicity stimulates the promoter activities and endogenous gene expression of SCTR in mouse kidney cortex collecting duct cells (M1) and inner medulla collecting duct cells (mIMCD3). The overexpression and silencing of NFAT5 further confirmed it to be responsible for the up-regulation of the SCTR gene under hypertonic conditions. A significant increase in the interaction between NFAT5 and the SCTR promoter was also observed following chromatin immunoprecipitation assay. In vivo, osmotic stress up-regulates the SCTR gene in the kidney cortex and medulla of wild-type mice, but does not do so in NFAT5+/− animals. Hence, this study provides comprehensive information on how NFAT5 regulates SCTR expression in different osmotic environments.

Abstract PR02: The diverse roles of long noncoding RNAs in the p53 tumor suppressor pathway

Abstract The p53 tumor suppressor is a transcription factor, which plays a central role in the cellular response to DNA damage and oncogenic stress. Using genetic approaches in the mouse as well as antisense technology, we have previously shown that the p53-regulated lncRNA, lincRNA-p21 activates the expression of its neighboring gene, the critical G1/S checkpoint mediator, p21. Mechanistically, we found that lincRNA-p21 localizes at its site of transcription, where it acts in cis to stabilize the binding of p53 and its transcriptional co-activator hnRNP-K at the promoter of p21. Importantly, using CRISPR-based approaches to modify the endogenous lincRNA-p21 locus in primary human fibroblasts, we show that the role of lincRNA-p21 in promoting p21 levels is conserved between mouse and human. To expand our understanding of the contribution of lncRNAs to the p53 tumor suppressor pathway, we have performed p53 ChIPseq and RNAseq under conditions of oncogenic stress and identified multiple previously uncharacterized lncRNAs that are regulated in a p53-dependent manner. One of them, lincRNA-Dhx15 is a novel lncRNA expressed from a bidirectional promoter shared with the RNA helicase Dhx15. LincRNA-Dhx15 is a direct target of p53 by virtue of multiple p53 response elements located in its first intron and appears to be preferentially expressed under conditions of senescence. We have generated a conditional knockout of lincRNA-Dhx15 in the mouse and found that absence of lincRNA-Dhx15 leads to impaired induction of senescence following prolonged exposure to DNA damage. Moreover, by gene expression profiling, we observed a downregulation of a subset of p53 target genes, suggesting that lincRNA-Dhx15 plays a global role in the p53 pathway. Surprisingly, we found that lincRNA-Dhx15 is exported to the cytoplasm and appears to promote the translational of p53 itself. The mechanistic basis for lincRNA-Dhx15 function is under active investigation. Altogether, these studies reveal that p53-regulated lncRNAs act through diverse mechanisms to add important layers of regulation to the p53 transcriptional network. Citation Format: Jordan Bendor, Jesse Zamudio, Tyler Jacks, Nadva Dimitrova. The diverse roles of long noncoding RNAs in the p53 tumor suppressor pathway. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr PR02.

Abstract 156: LncRNA BC200 is induced by estradiol and regulates apoptosis in MCF-7 breast cancer cells

Abstract LncRNA BC200 is a small functional RNA which has been implicated in the regulation of protein synthesis in neurons. It is dysregulated in invasive carcinomas of the breast. In this study, we reported that BC200 is upregulated in breast cancer, as detected by RT-PCR analysis of breast cancer tissue scan, suggesting its possible role as an oncogene. We then demonstrated that BC200 is transcriptionally induced by estradiol (E2) treatment in ER positive breast cancer cell lines through multiple functional estrogen response elements (EREs), including the full ERE sequence ∼800bp from transcriptional start site. Luciferase reporter assay combined with mutation analysis indicated that the full ERE region is critical to the estradiol-induced promoter activity. Furthermore, BC200 is crucial for cell growth and viability; its knockdown induces apoptosis in MCF-7 cells. This may explain in part why estrogen can promote cell proliferation in ER positive breast cancer cells. To better study its role in breast cancer development and progression, we generated BC200 knockout MCF-7 cell cells using CRISPR/Cas9 technique. BC200 knockout leads to the release of cytochrome c from mitochondria and promotes the cleavage of PARP-1, both of which are an indicator of cell apoptosis. This was further confirmed by TUNEL assay. It has been reported that hnRNP A2/B1 alters the bcl-X pre-mRNA splicing in favor of anti-apoptotic bcl-XL. Of great interest, BC200 knockout enhances expression of pro-apoptotic bcl-XS form at both mRNA and protein level. RNA pulldown assays using biotin-labeled BC200 probe suggested that BC200 can bind to hnRNP Q and hnRNP A2/B1. On the other hand, RNA immunoprecipitation using hnRNP A2/B1 antibody in BC200 knockout MCF-7 cells showed a significant reduction in enrichment of bcl-X mRNA as compared to control cells. We propose that the regulation of bcl-X alternative splicing depends upon endogenous expression of BC200 that would help hnRNP A2/B1 to position itself on bcl-X mRNA and modulate splicing in favor of bcl-XL. Thus, loss of BC200 leads to generation of bcl-XS protein synthesis and to apoptosis. Together, these results suggest that BC200 functions as a negative regulator of apoptosis in breast cancer and thus, it may serve as a potential target for cancer therapies. Citation Format: Ramesh Singh, Yin-yuan Mo. LncRNA BC200 is induced by estradiol and regulates apoptosis in MCF-7 breast cancer cells. [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 156. doi:10.1158/1538-7445.AM2015-156

Abstract 4194: APC and DIAPH1 collaborate to suppress myelodysplastic syndrome (MDS) pathogenesis

Abstract Myelodysplastic syndromes (MDS) are characterized by complex cytogenetic abnormalities, including loss of all or part of the long arm of chromosome 5 (5q). Known 5q tumor suppressors include APC1 and EGR1. APC is associated with familial colon cancer where it functions to suppress canonical Wnt signaling. Egr1 is a transcription factor that governs expression of tumor suppressor genes ranging from the p53 family to PTEN1. Adjacent to EGR1 is the DIAPH1 gene for the Rho GTPase effector formin DIAPH1. Knockout of Diaph1, Egr, or Apc in mice creates a range of MDS phenotypes. Likewise, mice with the multiple intestinal neoplasia (min/Apcmin) allele develop anemia and MDS most similar to the Diaph1 knockout phenotype. DIAPH1 builds linear actin filaments and stabilizes microtubule dynamics to establish and maintain cell polarity during adhesion, migration, and asymmetric cell division when activated by Rho GTPases, such as RhoB. Combinatorial deficiencies in RhoB (also a known tumor susceptibility gene) with Diaph1 exacerbates the Diaph1 MDS phenotype but not the rate of pathogenesis. RhoB-activated DIAPHs also signal and activate new gene expression by the myocardin-related (MRTF) and Mcm10/Agamous deficiens/ [serum response factor (SRF)] (MADS)-box transcription factors. MRTF-MADS registers changes in actin dynamics downstream of DIAPH1 to activate EGR1 expression through multiple serum response elements (SREs; recognized by SRF) within the EGR1 promoter. DIAPH1 also collaborates with APC in cytoskeletal remodeling as both proteins physically associate with and stabilize microtubules. APC can also nucleate new actin filaments that become processively elongated by DIAPH1. We examined interactions between APC and DIAPH1 signaling to SRF and in suppressing MDS pathogenesis. First, we found that both DIAPH1 and APC activate SRF- regulated gene expression. Consistent with biochemical data showing that DIAPH1 elongates APC-nucleated filaments, activation of SRF by APC relies upon DIAPH1. We bred Apcmin mice to our Diaph1 knockout mice and found that the MDS phenotype is significantly accelerated in Apcmin/Diaph1 knockout mice when compared to either the Apcmin or Diaph1 mutant phenotype. Pathological defects (dysplasia and anemia in peripheral blood and myeloid organs (spleen/bone marrow) requires at least 200 days to develop in either single mutant; combined mutations create aberrant peripheral blood cell pathology by 100 days of age. Consistent with early disease onset, mortality is accelerated in Apcmin /Diaph1-deficient mice. These novel studies show that genetic defects in Diaph1 and Apc synergize to effectively mimic 5q- MDS. Further, the data supports the idea that RhoB - DIAPH1/APC and MRTF-MADS comprise a novel tumor suppression pathway that is essential for hematopoiesis. Apcmin/Diaph1-deficient mice may prove to be a useful pre-clinical model for the development of more effective theranostic tools to attack MDS. Citation Format: Julie D. Turner, Susan M. Kitchen-Goosen, Andrew M. Howard, Heather L. Schumacher, Arthur S. Alberts. APC and DIAPH1 collaborate to suppress myelodysplastic syndrome (MDS) pathogenesis. [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 4194. doi:10.1158/1538-7445.AM2015-4194

Intrauterine Growth Restriction Disrupts Nucleosome Positioning at a Growth Hormone Response Element in the Insulin‐like Growth Factor 1 Gene

Intrauterine growth restriction (IUGR) increases the risk of adult onset diseases such as obesity and type II diabetes. Persistent downregulation of insulin‐like growth factor 1 (IGF1) has been implicated in these diseases. IUGR downregulates liver IGF1 expression in newborn and in day 21 (d21) mice. IGF1 expression at d21 is dependent on growth hormone (GH)‐mediated upregulation at multiple GH response elements (GHRE) including one containing tandem STAT5b binding sites within intron 2 (i2). We hypothesized that IUGR would disrupt prenatal epigenetic programming of the i2 GHRE and prevent postnatal i2 GHRE activation. IUGR mice were generated by thromboxane A2 analog infusion from gestation day 12.5 to term to elicit maternal hypertension. The GHRE chromatin state at the i2 GHRE was assessed by Na‐bisulfite sequencing for DNA methylation and GpC methyltransferase mapping for nucleosome positioning. In normal mice, the GHRE remains hypermethylated throughout gestation and begins to demethylate at d17. By d21 a single nucleosome is displaced. IUGR animals undergo similar DNA demethylation. However, we found two distinct nucleosome patterns at the d21 hepatic i2 GHRE. Some IUGR animals showed no nucleosome displacement, while in others, the nucleosome free region (NFR) was expanded on the 3′ side suggesting the NFR was misaligned. Evidence suggests different GHRE in IGF1 have unique functions and when tandem STAT5b sites are used, both must be functional for the enhancer to work. We speculate the altered NFR spacing could disrupt proper enhanceosome assembly and result in loss/altered GHRE activity.

Evolution of Novel Response Element Specificity in the Glucocorticoid Receptor

Specific recognition of DNA response elements by transcription factors is a key step in the regulation of gene expression. Some transcription factors recognize multiple, distinct response elements to mediate transrepression of transactivation of target genes. However, the mechanism by which a single transcription factor can evolve to recognize multiple response elements is unclear. Here, we study the evolution of the glucocorticoid receptor, which recognizes distinct response elements to mediate activation or repression its target genes. The glucocorticoid receptor binds to activating glucocorticoid response elements, or (+)GREs, as a dimer to activate transcription. Alternatively, GR binds to negative glucocorticoid response elements (nGREs) in a monomeric fashion to repress transcription. We demonstrate that, of the extant 3-keto steroid receptors, only the glucocorticoid receptor can bind to and repress transcription in a nGRE-dependent manner, despite the ability of all 3-keto steroid receptors to bind to and activate gene transcription from (+)GREs. Surprisingly, using ancestral gene reconstruction, we find that nGRE binding and gene repression was a feature of the ancestor of all extant 3-keto steroid receptors. nGRE binding and repression originated as a subfunction of the ancestral 3-keto steroid receptor coincident with (+)GRE binding, and this subfunction was optimized in the evolutionary lineage of the glucocorticoid receptor but lost in the ancestors of the mineralocorticoid, progesterone, and androgen receptors. Further, using x-ray crystallography and other structural biology approaches, we define the structural mechanisms by which the modern-day DNA binding specificity evolved in the glucocorticoid receptor.

Aryl hydrocarbon receptor-dependent stanniocalcin 2 induction by cinnabarinic acid provides cytoprotection against endoplasmic reticulum and oxidative stress.

The aryl hydrocarbon receptor (AhR) is a cytosolic ligand-activated transcription factor historically known for its role in xenobiotic metabolism. Although AhR activity has previously been shown to play a cytoprotective role against intrinsic apoptotic stimuli, the underlying mechanism by which AhR confers cytoprotection against apoptosis is largely unknown. Here, we demonstrate that activation of AhR by the tryptophan catabolite cinnabarinic acid (CA) directly upregulates expression of stanniocalcin 2 (Stc2) to elicit cytoprotection against apoptosis induced by endoplasmic reticulum stress and oxidative stress. Chromatin immunoprecipitation studies demonstrated that CA treatment induces direct AhR binding to a region of the Stc2 promoter containing multiple xenobiotic response elements. Using isolated primary hepatocytes from AhR wild-type (AhR floxed) and liver-specific AhR conditional knockout mice, we showed that pretreatment with CA conferred cytoprotection against hydrogen peroxide (H(2)O(2))-, thapsigargin-, and ethanol-induced apoptosis in an AhR-dependent manner. Furthermore, suppressing Stc2 expression using RNA interference confirmed that the cytoprotective properties of CA against H(2)O(2), thapsigargin, and ethanol injury were absolutely dependent on Stc2. Immunochemistry revealed the presence of Stc2 in the endoplasmic reticulum and on the cell surface, consistent with Stc2 secretion and autocrine and/or paracrine signaling. Finally, in vivo data using a mouse model of acute alcohol hepatotoxicity demonstrated that CA provided cytoprotection against ethanol-induced apoptosis, hepatic microvesicular steatosis, and liver injury. Collectively, our data uncovered a novel mechanism for AhR-mediated cytoprotection in the liver that is dependent on CA-induced Stc2 activity.

Constitutively active FOXO1 diminishes activin induction of Fshb transcription in immortalized gonadotropes.

In the present study, we investigate whether the FOXO1 transcription factor modulates activin signaling in pituitary gonadotropes. Our studies show that overexpression of constitutively active FOXO1 decreases activin induction of murine Fshb gene expression in immortalized LβT2 cells. We demonstrate that FOXO1 suppression of activin induction maps to the −304/−95 region of the Fshb promoter containing multiple activin response elements and that the suppression requires the FOXO1 DNA-binding domain (DBD). FOXO1 binds weakly to the −125/−91 region of the Fshb promoter in a gel-shift assay. Since this region of the promoter contains a composite SMAD/FOXL2 binding element necessary for activin induction of Fshb transcription, it is possible that FOXO1 DNA binding interferes with SMAD and/or FOXL2 function. In addition, our studies demonstrate that FOXO1 directly interacts with SMAD3/4 but not SMAD2 in a FOXO1 DBD-dependent manner. Moreover, we show that SMAD3/4 induction of Fshb-luc and activin induction of a multimerized SMAD-binding element-luc are suppressed by FOXO1 in a DBD-dependent manner. These results suggest that FOXO1 binding to the proximal Fshb promoter as well as FOXO1 interaction with SMAD3/4 proteins may result in decreased activin induction of Fshb in gonadotropes.