CREB Binding Sites Research Articles

Endoplasmic reticulum stress impedes regulated secretion by governing key exocytotic and granulogenic molecular switches.

Dense core vesicles (DCVs) and synaptic vesicles are specialised secretory vesicles in neurons and neuroendocrine cells, and abnormal release of their cargo is associated with various pathophysiologies. Endoplasmic reticulum (ER) stress and inter-organellar communication are also associated with disease biology. To investigate the functional status of regulated exocytosis arising from the crosstalk of a stressed ER and DCVs, ER stress was modelled in PC12 neuroendocrine cells using thapsigargin. DCV exocytosis was severely compromised in ER-stressed PC12 cells and was reversed to varying magnitudes by ER stress attenuators. Experiments with tunicamycin, an independent ER stressor, yielded similar results. Concurrently, ER stress also caused impaired DCV exocytosis in insulin-secreting INS-1 cells. Molecular analysis revealed blunted SNAP25 expression, potentially attributed to augmented levels of ATF4, an inhibitor of CREB that binds to the CREB-binding site. The effects of loss of function of ATF4 in ER-stressed cells substantiated this attribution. Our studies revealed severe defects in DCV exocytosis in ER-stressed cells for the first time, mediated by reduced levels of key exocytotic and granulogenic switches regulated via the eIF2α (EIF2A)-ATF4 axis.

Activation of TGR5 Increases Urine Concentration by Inducing AQP2 and AQP3 Expression in Renal Medullary Collecting Ducts

Introduction: G protein-coupled bile acid receptor (TGR5), the first G protein-coupled receptor for bile acids identified, is capable of activating a variety of intracellular signaling pathways after interacting with bile acids. TGR5 plays an important role in multiple physiological processes and is considered to be a potential target for the treatment of various metabolic diseases, including type 2 diabetes. Evidence has emerged that genetic deletion of TGR5 results in an increase in basal urine output, suggesting that it may play a critical role in renal water and salt reabsorption. The present study aims to elucidate the effect and mechanism of TGR5 activation on urine concentration. Methods: Mice were treated with TGR5 agonists (LCA and INT-777) for 3 days. The 24-h urine of mice was collected and analyzed for urine biochemical parameters. The mRNA expressions were detected by real-time PCR, and the protein expressions were detected by western blot. Immunohistochemistry and immunofluorescence were performed to examine the cellular location of proteins. The cultured primary medullary collecting duct cells were pretreated with H89 (a PKA inhibitor) for 1 h, followed by 12-h treatment of LCA and INT-777. Luciferase reporter assays were used to detect the effect of CREB on the gene transcription of AQPs. Gel electrophoretic mobility shift assays were used to analyze DNA–protein interactions. Results: Treatment of mice with the TGR5 agonist LCA and INT-777 markedly reduced urine output and increased urine osmolality, accompanied by a marked increase in AQP2 and AQP3 protein expression and membrane translocation. In cultured primary medullary collecting duct cells, LCA and INT-777 dose-dependently upregulated AQP2 and AQP3 expression in a cAMP/PKA-dependent manner. Mechanistically, both AQP2 and AQP3 gene promoter contains a putative CREB-binding site, which can be bound and activated by CREB as assessed by both gene promoter-driven luciferase and gel shift assays. Conclusion: Collectively, our findings demonstrate that activation of TGR5 can promote urine concentration by upregulation of AQP2 and AQP3 expression in renal collecting ducts. TGR5 may represent an attractive target for the treatment of patients with urine concentration defect.

DNA methylation of the promoter region at the CREB1 binding site is a mechanism for the epigenetic regulation of brain-specific PKMζ

Protein kinase M zeta, PKMζ, is a brain enriched kinase with a well characterized role in Long-Term Potentiation (LTP), the activity-dependent strengthening of synapses involved in long-term memory formation. However, little is known about the molecular mechanisms that maintain the tissue specificity of this kinase. Here, we characterized the epigenetic factors, mainly DNA methylation, regulating PKMζ expression in the human brain. The PRKCZ gene has an upstream promoter regulating Protein kinase C ζ (PKCζ), and an internal promoter driving PKMζ expression. A demethylated region, including a canonical CREB binding site, situated at the internal promoter was only observed in human CNS tissues. The induction of site-specific hypermethylation of this region resulted in decreased CREB1 binding and downregulation of PKMζ expression. Noteworthy, CREB binding sites were absent in the upstream promoter of PRKCZ locus, suggesting a specific mechanism for regulating PKMζ expression. These observations were validated using a system of human neuronal differentiation from induced pluripotent stem cells (iPSCs). CREB1 binding at the internal promoter was detected only in differentiated neurons, where PKMζ is expressed. The same epigenetic mechanism in the context of CREB binding site was identified in other genes involved in neuronal differentiation and LTP. Additionally, aberrant DNA hypermethylation at the internal promoter was observed in cases of Alzheimer's disease, correlating with decreased expression of PKMζ in patient brains. Altogether, we present a conserved epigenetic mechanism regulating PKMζ expression and other genes enhanced in the CNS with possible implications in neuronal differentiation and Alzheimer's disease.

IL-33 induces granzyme C expression in murine mast cells via an MSK1/2-CREB-dependent pathway.

Granzymes comprise a group of proteases involved in the killing of infected or cancerous cells by the immune system. Although best studied in T cells and natural killer (NK) cells, they are also expressed in some innate immune cells. Granzymes B and C are encoded in the mouse chymase locus that also encodes a number of mast cell-specific proteases. In line with this, mast cells can express granzyme B, although how this is regulated and their ability to express other granzymes is less well studied. We therefore examined how IL-33, a cytokine able to activate mast cells but not induce degranulation, regulated granzyme B and C levels in mast cells. Granzyme C, but not B, mRNA was strongly up-regulated in bone marrow-derived mast cells following IL-33 stimulation and there was a corresponding increase in granzyme C protein. These increases in both granzyme C mRNA and protein were blocked by a combination of the p38α/β MAPK inhibitor VX745 and the MEK1/2 inhibitor PD184352, which blocks the activation of ERK1/2. ERK1/2 and p38α activate the downstream kinases, mitogen and stress-activated kinases (MSK) 1 and 2, and IL-33 stimulated the phosphorylation of MSK1 and its substrate CREB in an ERK1/2 and p38-dependent manner. The promoter for granzyme C contains a potential CREB-binding site. Bone marrow-derived mast cells from either MSK1/2 double knockout or CREB Ser133Ala knockin mice were unable to up-regulate granzyme C. Together these results indicate that IL-33-induced granzyme C expression in mast cells is regulated by an MSK1/2-CREB-dependent pathway.

Analysis of the DNA methylation pattern of the promoter region of calcitonin gene-related peptide 1 gene in patients with episodic migraine: An exploratory case-control study

Recent studies suggested that epigenetic mechanisms, including DNA methylation, may be involved in migraine pathogenesis. The calcitonin gene-related peptide (CGRP), encoded by calcitonin gene-related peptide 1 (CALCA) gene, plays a key role in the disease. The aim of the study was to evaluate DNA methylation of CALCA gene in patients with episodic migraine. 22 patients with episodic migraine (F/M 15/7, mean age 39.7 ± 13.4 years) and 20 controls (F/M 12/8, mean age 40.5 ± 14.8 years) were recruited. Genomic DNA was extracted from peripheral blood. Cytosine-to-thymine conversion was obtained with sodium bisulfite. The methylation pattern of two CpG islands in the promoter region of CALCA gene was analyzed. No difference of methylation of the 30 CpG sites at the distal region of CALCA promoter was observed between migraineurs and controls. Interestingly, in patients with episodic migraine the methylation level was lower in 2 CpG sites at the proximal promoter region (CpG −1461, p = 0.037, and −1415, p = 0.035, respectively). Furthermore, DNA methylation level at different CpG sites correlates with several clinical characteristics of the disease, as age at onset, presence of nausea/vomiting, depression and anxiety (p < 0.05). In conclusion, we found that DNA methylation profile in two CpG sites at the proximal promoter region of CALCA is lower in migraineurs when compared to controls. Intriguingly, the −1415 hypomethylated unit is located at the CREB binding site, a nuclear transcription factor. In addition, we found a correlation between the level of CALCA methylation and several clinical features of migraine. Further studies with larger sample size are needed to confirm these results.

Selective suppression of IL-10 transcription by calcineurin in dendritic cells through inactivation of CREB.

Myeloid cells play a pivotal role in immune responses against bacterial and fungal infection. Among innate immune receptors, C-type lectin receptors (CLRs) can induce a wide spectrum of cytokines through immunoreceptor tyrosine-based activation motifs (ITAMs)-mediated signaling pathways. Dendritic cells (DCs) produce IL-10 through CLR stimulation; however, the regulatory mechanism of IL-10 expression has not been elucidated. In the current study, we report that calcium (Ca2+) signaling-deficient DCs produced more IL-10 than wild-type DCs. Mechanistically, Ca2+-dependent phosphatase calcineurin directly inactivates cAMP response element-binding protein (CREB), a transcription factor of Il10 in DCs, through dephosphorylating CREB at serine 133. In calcineurin-deficient DCs, CREB was highly phosphorylated and increased its binding to the Il10 promoter. Elimination of mitogen-activated protein kinase (MAPK) signaling that phosphorylates CREB, deficiency of CREB, as well as deletion of a CREB-binding site in the Il10 promoter could diminish IL-10 production in DCs. Our findings identified a novel substrate of calcineurin as well as a mechanism through which Ca2+ signaling regulates IL-10 expression downstream of CLRs. As IL-10 is a crucial immunosuppressive cytokine, this mechanism may counteract the over-activated IL-10-producing signals induced by CARD9 and MAPK pathways, preventing the ineffectiveness of the immune system during bacterial and fungal infection.

Elevation of Hyaluronan Synthase by Magnesium Supplementation Mediated through the Activation of GSK3 and CREB in Human Keratinocyte-Derived HaCaT Cells.

Skin barrier damage is present in the patients with hereditary disorders of the magnesium channel, but the molecular mechanism has not been fully understood. We found that the expressions of hyaluronan synthase (HAS), HAS2 and HAS3 are influenced by MgCl2 concentration in human keratinocyte-derived HaCaT cells. The exposure of cells to a high concentration (5.8 mM) of MgCl2 induced the elevation of HAS2/3 expression, which was inhibited by mRNA knockdown of nonimprinted in Prader-Willi/Angelman syndrome-like domain containing 4 (NIPAL4). Similarly, the content of hyaluronic acid (HA) was changed according to MgCl2 concentration and the expression of NIPAL4. The MgCl2 supplementation increased the reporter activities of HAS2/3, which were inhibited by NIPAL4 knockdown, indicating that the expressions of HAS2/3 are up-regulated at the transcriptional level. The reporter activities and mRNA levels of HAS2/3, and the production of HA were inhibited by CHIR-99021, a glycogen synthase kinase-3 (GSK3) inhibitor, and naphthol AS-E, a cyclic AMP-response element binding protein (CREB) inhibitor. Furthermore, the mutation in putative CREB-binding sites of promoter region in HAS2/3 genes inhibited the MgCl2 supplementation-induced elevation of promoter activity. Our results indicate that the expressions of HAS2/3 are up-regulated by MgCl2 supplementation in HaCaT cells mediated through the activation of GSK3 and CREB. Magnesium may play a pivotal role in maintaining the skin barrier function and magnesium supplementation may be useful to enhance moisturization and wound repair in the skin.

Abstract MP39: FHL5 , A Novel Cofactor Associated With Coronary Artery Disease, Regulates Smooth Muscle Cell Function Through A Transcriptional Network Linking Multiple Risk Loci

Introduction: Coronary artery disease (CAD) is the leading cause of death worldwide with an estimated heritability of ~50%. The most recent genome wide association study (GWAS) for CAD and myocardial infarction (MI) identified over 200 loci. One such novel CAD/MI locus, UFL1-FHL5 (P = 1.1E-8), is associated with additional vascular pathologies, including hypertension, migraines, and coronary calcification. We previously showed through statistical fine-mapping that FHL5 is the top candidate causal gene underlying each of these vascular trait associations and highly enriched in the contractile mural cell populations in the artery. These preliminary studies motivate our hypothesis that FHL5 functions as a transcriptional regulator of SMC contractility to affect vascular disease risk. Methods and Results: Given the reported role of FHL5 as a cofactor, we mapped 17,201 candidate FHL5 binding sites in coronary artery SMCs using the Cleavage Under Targets and Release Using Nuclease (CUT&amp;RUN) method. Binding sites were enriched for AP-1 family motifs and strongly overlapped CREB binding sites and H3K27ac enhancer marks. FHL5 target genes were functionally enriched in well characterized CAD pathways, such as extracellular matrix organization (P=5.2E-15, OR=2.1) and TGF-beta signaling (P=8.0E-5, OR=2.0). Interestingly, FHL5 binding sites were also enriched for CAD (P=1.8E-7, OR=2.3) and blood pressure (BP) risk variants (P=0.04, OR=1.2), thereby linking FHL5 with the regulation of multiple downstream CAD/BP loci. Weighted gene co-expression network and key driver analyses of human coronary artery transcriptomic data further support this regulatory role. Lastly, we validated these findings by performing confocal based calcium imaging and collagen gel contraction assays. Consistent with our functional genomic analyses, overexpression of FHL5 elevated intracellular calcium levels 2.5X and increased SMC contractility by ~45%. Conclusion: Taken together, our results provide evidence that FHL5 may impact CAD risk by regulating a network of disease-associated genes mediating SMC functions. These findings further contribute to our understanding of the heritable risk for multiple vascular diseases.

A Positive Feedback Loop Between DICER1 and Differentiation Transcription Factors Is Important for Thyroid Tumorigenesis.

Background: DICER1 plays a central role in microRNA biogenesis and functions as a tumor suppressor in thyroid cancer, which is the most frequent endocrine malignancy with a rapidly increasing incidence. Thyroid cancer progression is associated with loss of cell differentiation and reduced expression of thyroid differentiation genes and response to thyrotropin (TSH). Here we investigated whether a molecular link exists between DICER1 and thyroid differentiation pathways.Methods: We used bioinformatic tools to search for transcription factor binding sites in the DICER1 promoter. DICER1, NKX2-1, PAX8, and CREB expression levels were evaluated by gene and protein expression in vitro and by interrogation of The Cancer Genome Atlas (TCGA) thyroid cancer data. Transcription factor binding and activity were assayed by chromatin immunoprecipitation, band-shift analysis, and promoter–reporter gene activity. Gene-silencing and overexpression approaches were used to elucidate the functional link between DICER1 and differentiation.Results: We identified binding sites for NKX2-1 and CREB within the DICER1 promoter and found that both transcription factors are functional in thyroid cells. TSH induced DICER1 expression in differentiated thyroid cells, at least in part, through the cAMP/PKA/CREB pathway. TCGA analysis revealed a significant positive correlation between CREB and DICER1 expression in human thyroid tumors. NKX2-1 overexpression increased DICER1 promoter activity and expression in vitro, and this was significantly greater in the presence of CREB and/or PAX8. Gain- and loss-of-function assays revealed that DICER1 regulates NKX2-1 expression in thyroid tumor cells and vice versa, thus establishing a positive feedback loop between both proteins. We also found a positive correlation between NKX2-1 and DICER1 expression in human thyroid tumors. DICER1 silencing decreased PAX8 expression and, importantly, the expression and activity of the sodium iodide symporter, which is essential for the diagnostic and therapeutic use of radioiodine in thyroid cancer.Conclusions: The differentiation transcription factors NKX2.1, PAX8, and CREB act in a positive feedback loop with DICER1. As the expression of these transcription factors is markedly diminished in thyroid cancer, our findings suggest that DICER1 downregulation in this cancer is mediated, at least partly, through impairment of its transcription.

Crtc modulates fasting programs associated with 1-C metabolism and inhibition of insulin signaling

Fasting in mammals promotes increases in circulating glucagon and decreases in circulating insulin that stimulate catabolic programs and facilitate a transition from glucose to lipid burning. The second messenger cAMP mediates effects of glucagon on fasting metabolism, in part by promoting the phosphorylation of CREB and the dephosphorylation of the cAMP-regulated transcriptional coactivators (CRTCs) in hepatocytes. In Drosophila, fasting also triggers activation of the single Crtc homolog in neurons, via the PKA-mediated phosphorylation and inhibition of salt-inducible kinases. Crtc mutant flies are more sensitive to starvation and oxidative stress, although the underlying mechanism remains unclear. Here we use RNA sequencing to identify Crtc target genes that are up-regulated in response to starvation. We found that Crtc stimulates a subset of fasting-inducible genes that have conserved CREB binding sites. In keeping with its role in the starvation response, Crtc was found to induce the expression of genes that inhibit insulin secretion (Lst) and insulin signaling (Impl2). In parallel, Crtc also promoted the expression of genes involved in one-carbon (1-C) metabolism. Within the 1-C pathway, Crtc stimulated the expression of enzymes that encode modulators of S-adenosyl-methionine metabolism (Gnmt and Sardh) and purine synthesis (ade2 and AdSl) Collectively, our results point to an important role for the CREB/CRTC pathway in promoting energy balance in the context of nutrient stress.

Characterization of the G protein-coupled receptor kinase 6 promoter reveals a functional CREB binding site.

BackgroundG protein-coupled receptor kinase 6 (GRK6) is part of the G protein-coupled receptor kinase family, whose members act as key regulators of seven-transmembrane receptor signalling. GRK6 seems to play a role in regulation of inflammatory processes, but mechanisms of transcriptional regulation of GRK6 expression in inflammatory cell lines have not been characterized. Protein kinase C (PKC) signalling is also involved in inflammatory regulation and an impact of PKC activation on GRK6 protein expression was described previously. Thus, the aim of this study was to 1) characterize the GRK6 promoter, and 2) investigate a potential influence of PKC on GRK6 expression.MethodsFive deletion constructs of the GRK6 promoter were cloned. After transient transfection into a human T cell line, promoter activity was assessed using luciferase reporter gene assays. Putative transcription factor binding sites were identified, mutated, and binding was investigated using electrophoretic mobility shift assays (EMSA). Following stimulation with a PKC activator, GRK6 expression on mRNA and protein levels was assessed by reverse transcriptase qPCR and Western blots.ResultsInvestigation of the GRK6 promoter revealed a putative cAMP responsive element (CRE), whose mutation led to decreased promoter activity (p = 0.0006). Functionality of the CRE binding protein (CREB) binding site was verified in EMSA blots. Stimulation with a PKC activator resulted in decreased GRK6 promoter activity (p = 0.0027), mRNA (p = 0.04) and protein expression.ConclusionWe characterized the human GRK6 promoter and identified promoter activity to be influenced by a CREB binding site. PKC might be one determinant contributing to altered GRK6 expression.

Characterization of the expression and immunological impact of the transcriptional activator CREB in renal cell carcinoma

BackgroundThe non-classical human leukocyte antigen (HLA)-G is a strong immunomodulatory molecule. Under physiological conditions, HLA-G induces immunological tolerance in immune privileged tissues, while under pathophysiological situations it contributes to immune escape mechanisms. Therefore, HLA-G could act as a potential immune checkpoint for future anti-cancer immunotherapies. Recent data suggest an aberrant expression of the cAMP response element binding protein (CREB) in clear cell renal cell carcinoma (ccRCC), which is correlated with tumor grade and stage. Furthermore, preliminary reports demonstrated a connection of CREB as a control variable of HLA-G transcription due to CREB binding sites in the HLA-G promoter region. This study investigates the interaction between CREB and HLA-G in different renal cell carcinoma (RCC) subtypes and its correlation to clinical parameters.MethodsThe direct interaction of CREB with the HLA-G promoter was investigated by chromatin immunoprecipitation in RCC cell systems. Furthermore, the expression of CREB and HLA-G was determined by immunohistochemistry using a tissue microarray (TMA) consisting of 453 RCC samples of distinct subtypes. Staining results were assessed for correlations to clinical parameters as well as to the composition of the immune cell infiltrate.ResultsThere exists a distinct expression pattern of HLA-G and CREB in the three main RCC subtypes. HLA-G and CREB expression were the lowest in chromophobe RCC lesions. However, the clinical relevance of CREB and HLA-G expression differed. Unlike HLA-G, high levels of CREB expression were positively associated to the overall survival of RCC patients. A slightly, but significantly elevated number of tumor infiltrating regulatory T cells was observed in tumors of high CREB expression. Whether this small increase is of clinical relevance has to be further investigated.ConclusionsAn interaction of CREB with the HLA-G promoter could be validated in RCC cell lines. Thus, for the first time the expression of CREB and its interaction with the HLA-G in human RCCs has been shown, which might be of clinical relevance.

Nfil3, a target of the NACA transcriptional coregulator, affects osteoblast and osteocyte gene expression differentially.

Intermittent administration of PTH(1-34) has a profound osteoanabolic effect on the skeleton. At the cellular level, osteoblasts and osteocytes are two crucial cell types that respond to PTH stimulation in bone. The transcriptional cofactor Nascent polypeptide Associated Complex and coregulator alpha (NACA) is a downstream target of the PTH-Gαs-PKA axis in osteoblasts. NACA functions as a transcriptional cofactor affecting bZIP factor-mediated transcription of target promoters in osteoblasts, such as Osteocalcin (Bglap2). Here, we used RNA-Seq and ChIP-Seq against NACA in PTH-treated MC3T3-E1 osteoblastic cells to identify novel targets of the PTH-activated NACA. Our approach identified Nuclear factor interleukin-3-regulated (Nfil3) as a target promoter of this pathway. Knockdown of Naca reduced the response of Nfil3 to PTH(1-34) stimulation. In silico analysis of the Nfil3 promoter revealed potential binding sites for NACA (located within the ChIP fragment) and CREB. We show that following PTH stimulation, phosphorylated-CREB binds the proximal promoter of Nfil3 in osteoblasts. The activity of the Nfil3 promoter (-818/+182bp) is regulated by CREB and this activation relies on the presence of NACA. In addition, we show that knockdown of Nfil3 enhances the expression of osteoblastic differentiation markers in MC3T3-E1 cells while it represses osteocytic marker gene expression in IDG-SW3 cells. These results show that the PTH-induced NACA axis regulates Nfil3 expression and suggest that NFIL3 acts as a transcriptional repressor in osteoblasts while it exhibits differential activity as an activator in osteocytes.

Genetic and Epigenetic Regulation of the Smoothened Gene (SMO) in Cancer Cells.

(1) Background: The hedgehog (HH) signaling pathway is a key regulator of embryonic patterning, tissue regeneration, stem cell renewal, and cancer growth. The smoothened (SMO) protein regulates the HH signaling pathway and has demonstrated oncogenic activity. (2) Methods: To clarify the role of the HH signaling pathway in tumorigenesis, the expression profile of key HH signaling molecules, including SMO, PTCH1, GLI1, GLI2, and GLI3, were determined in 33 cancer cell lines and normal prostate cells and tissues. We performed a computational analysis of the upstream region of the SMO gene to identify the regulatory elements. (3) Results: Three potential CpG islands and several putative SMO promoter elements were identified. Luciferase reporter assays mapped key SMO promoter elements, and functional binding sites for SP1, AP1, CREB, and AP-2α transcription factors in the core SMO promoter region were confirmed. A hypermethylated SMO promoter was identified in several cancer cell lines suggesting an important role for epigenetic silencing of SMO expression in certain cancer cells. (4) Discussion: These results have important implications for our understanding of regulatory mechanisms controlling HH pathway activity and the molecular basis of SMO gene function. Moreover, this study may prove valuable for future research aimed at producing therapeutic downregulation of SMO expression in cancer cells.

Anti-Müllerian Hormone Regulates Stem Cell Factor via cAMP/PKA Signaling Pathway in Human Granulosa Cells by Inhibiting the Phosphorylation of CREB.

Anti-Müllerian hormone (AMH) downregulates the level of stem cell factor (SCF) via the cAMP/PKA signaling pathway in human granulosa cells (GCs). Little information is available on the molecular mechanism underlying the interaction. This study is aimed at determining whether AMH regulates expression of SCF via the cAMP-PKA-CREB signaling pathway in human GCs. In the present study, we verified the binding of cAMP-response element-binding protein (CREB) to promoter of SCF in human GCs. Furthermore, the effect of CREB was tested on the SCF promoter, and the site of CREB binding to SCF promoter was identified using truncations as well as assays of SCF-promoted mutation and CREB mutation. To investigate the correlation among AMH, SCF promoter, and CREB, pGL-Basic-SCF+CREB was transfected into overexpressed AMH GCs (AMH-high GCs), low expressed AMH GCs (AMH-low GCs), and normal GCs (GCs), respectively. Finally, immunofluorescence, double immunostaining, and Western blot were carried out in AMH-high and AMH-low GCs to confirm the AMH-mediated regulation of SCF expression by inhibiting the phosphorylation of CREB (pCREB) in GCs. Results indicated CREB interacted with SCF promoter and significantly enhanced the transcription level of SCF. The CREB binding site was localized at 318-321bp of SCF gene promote. AMH inhibits the expression of SCF by phosphorylation of CREB via the PKA signaling pathway in GCs. These findings provide an in-depth understanding of the molecular mechanism underlying AMH suppressing the follicle growth, which would aid in the development of a novel therapy.

Transcriptional regulation of human cyclic GMP-AMP synthase gene

Cyclic GMP-AMP synthase (cGAS, cGAMP synthase) plays crucial roles in autoimmune disease, anti-tumor response, anti-senescence and anti-inflammatory response. Many studies have focused on cGAS-mediated signaling pathway. However, transcriptional mechanisms of cGAS gene have remained largely unknown. Here, we cloned the cGAS promoter region and characterized the molecular mechanisms controlling the cGAS transcriptional activity. By a series of 5′ deletion and promoter constructions, we showed that the region (−414 to +76 relatives to the transcription start site) was sufficient for promoter activity. Mutation of Sp1 and CREB binding sites in this promoter region led to an apparent reduction of the cGAS promoter activity. Overexpression of Sp1 and CREB could obviously enhance promoter activity, whereas knocking-down of endogenous Sp1 and CREB markedly restrained the cGAS promoter activity. Sp1 and CREB binding to the cGAS promoter region in vivo was verified by Chromatin immunoprecipitation assay. These results pointed out that transcription factors Sp1 and CREB regulate the transcription of the cGAS gene.

Transcriptional repression of the tyrosinase-related protein 2 gene by transforming growth factor-β and the Kruppel-like transcription factor GLI2

BackgroundTyrosinase-Related Protein 2 (TRP2) is an enzyme involved in melanogenesis, that also exerts proliferative, anti-apoptotic and immunogenic functions in melanoma cells. TRP2 transcription is regulated by the melanocytic master transcription factor MITF. GLI2, a transcription factor that acts downstream of Hedgehog signaling, is also a direct transcriptional target of the TGF-β/SMAD pathway that contributes to melanoma progression and exerts transcriptional antagonistic activities against MITF. ObjectivesTo characterize the molecular events responsible for TGF-β and GLI2 repression of TRP2 expression. MethodsIn silico promoter analysis, transient cell transfection experiments with 5′-end TRP2 promoter deletion constructs, chromatin immuno-precipitation, and site-directed promoter mutagenesis were used to dissect the molecular mechanisms of TRP2 gene regulation by TGF-β and GLI2. ResultsWe demonstrate that TGF-β and GLI2-specific TRP2 repression involves direct mechanisms that occur in addition to MITF downregulation by TGF-β and GLI2. We identify two functional GLI2 binding sites within the TRP2 promoter that are critical for TGF-β and GLI2 responsiveness, one of them overlapping a CREB binding site. GLI2 and CREB competing for the same cis-element is associated with opposite transcriptional outcome. ConclusionOur results further refine the understanding of how TGF-β and GLI2 control the phenotypic plasticity of melanoma cells. In particular, we identify critical GLI2-binding cis-elements within the TRP2 promoter region that allow for its transcriptional repression independently from MITF concomitant downregulation.

CREB element is essential for unfolded protein response (UPR) mediating the Cu-induced changes of hepatic lipogenic metabolism in Chinese yellow catfish (Pelteobagrus fulvidraco)

The present study was conducted to explore the underlying mechanism of unfolded protein response (UPR) mediating the Cu-induced changes of hepatic lipogenic metabolism in a low vertebrate, freshwater teleost yellow catfish Pelteobagrus fulvidraco. To this end, three experiments were conducted. In Exp. 1, we cloned the regions of grp78, perk, ire-1α and atf-6α promoters, and found that multiple cAMP-response element binding protein (CREB) binding sites were identified in their promoter regions. Furthermore, these CREB binding sites played crucial role in transcriptional regulation of UPR. In Exp. 2, the involvement of perk, ire-1α and atf-6α in Cu-induced changes of hepatic lipid metabolism was confirmed by specific miRNA. In Exp. 3, the regulatory mechanism of CREB underlying UPR mediating Cu-induced hepatic lipogenic metabolism were investigated. Cu induced UPR via the activation of CREB binding sites in the promoter regions of grp78, perk, ire-1α and atf-6α. In addition, the inhibition of CREB markedly attenuated the Cu-induced up-regulation of hepatic lipogenic metabolism in hepatocytes. This conclusion was further supported by the results from the trial of CREB over-expression. Taken together, the present study indicated that CREB was essential for UPR mediating Cu-induced lipogenic metabolism, supporting a mechanistic link among CREB, UPR and Cu-induced changes of lipid metabolism.

Differential Gene Expression Profile Induced by Valproic Acid (VPA) in Pediatric Epileptic Patients.

Epilepsy is a neuronal disease that affects up to 70 million people worldwide. The development of effective therapies to combat childhood epilepsy requires early biomarkers. Here, we performed a whole-genome microarray analysis in blood cells to identify genes differentially expressed between epileptic and epileptic valproic acid (VPA)-treated children versus normal children to obtain information about the gene expression to help us to understand genetic aspects of this disease. We found that the most significant differentially expressed genes were related to the transcriptional factor cAMP-response element binding protein (CREB) that is overexpressed in children with epilepsy compared with normal children, and 6 and 12 months of VPA treatment reversed several of these changes. Interestingly, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), a type I transmembrane glycoprotein that binds collagen proteins and contains CREB binding sites, was one of the more up-regulated genes in epileptic patients, and treatment with VPA strongly reversed its up-regulation. CREB up-regulates genes related to epilepsy; here, we suggest that LAIR1 could activate CREB, and together, they trigger epilepsy. After VPA treatment, LAIR1 repressed genes by disrupting the functional LAIR1–CREB complex, resulting in successful treatment. A functional microarray analysis offers new information that could open novel avenues of research in biomarker discovery, which may be useful for the early identification of children with a predisposition to epilepsy.

Genome-Wide Mapping of DNA Accessibility and Binding Sites for CREB and C/EBPβ in Vasopressin-Sensitive Collecting Duct Cells.

Background Renal water excretion is controlled by vasopressin, in part through regulation of the transcription of the aquaporin-2 gene (Aqp2).Methods To identify enhancer regions likely to be involved in the regulation of Aqp2 and other principal cell-specific genes, we used several next generation DNA-sequencing techniques in a well characterized cultured cell model of collecting duct principal cells (mpkCCD). To locate enhancers, we performed the assay for transposase-accessible chromatin using sequencing (ATAC-Seq) to identify accessible regions of DNA and integrated the data with data generated by chromatin immunoprecipitation followed by next generation DNA-sequencing (ChIP-Seq) for CCCTC binding factor (CTCF) binding, histone H3 lysine-27 acetylation, and RNA polymerase II.Results We identified two high-probability enhancers centered 81 kb upstream and 5.8 kb downstream from the Aqp2 transcriptional start site. Motif analysis of these regions and the Aqp2 promoter identified several potential transcription factor binding sites, including sites for two b-ZIP transcription factors: CCAAT/enhancer binding protein-β (C/EBPβ) and cAMP-responsive element binding protein (CREB). To identify genomic binding sites for both, we conducted ChIP-Seq using well characterized antibodies. In the presence of vasopressin, C/EBPβ, a pioneer transcription factor critical to cell-specific gene expression, bound strongly at the identified enhancer downstream from Aqp2 However, over multiple replicates, we found no detectable CREB binding sites within 390 kb of Aqp2 Thus, any role for CREB in the regulation of Aqp2 gene transcription is likely to be indirect.Conclusions The analysis identified two enhancer regions pertinent to transcriptional regulation of the Aqp2 gene and showed C/EBPβ (but not CREB) binding.