Cyclic AMP Response Element Sites Research Articles

NFκB and Cyclic AMP Response Element Sites Mediate the Valproic Acid and UL138 Responsiveness of the Human Cytomegalovirus Major Immediate Early Enhancer and Promoter.

The major immediate early enhancer and promoter (MIEP) of human cytomegalovirus (HCMV) drives the transcription of the immediate early one (IE1) and IE2 genes, whose encoded proteins stimulate productive, lytic replication. The MIEP is activated by the virally encoded and tegument-delivered pp71 protein at the start of de novo lytic infections of fully differentiated cells. Conversely, the MIEP is silenced at the start of de novo latent infections within incompletely differentiated myeloid cells in part because tegument-delivered pp71 is sequestered in the cytoplasm in these cells, but also by viral factors that repress transcription from this locus, including the UL138 protein. During both modes of infection, MIEP activity can be increased by the histone deacetylase inhibitor valproic acid (VPA); however, UL138 inhibits the VPA-responsiveness of the MIEP. Here, we show that two families of cellular transcription factors, NF-κB and cAMP response element-binding protein (CREB), together control the VPA-mediated activation and UL138-mediated repression of the HCMV MIEP. IMPORTANCE Artificial regulation of the HCMV MIEP, either activation or repression, is an attractive potential means to target the latent reservoirs of virus for which there is currently no available intervention. The MIEP could be repressed to prevent latency reactivation or induced to drive the virus into the lytic stage that is visible to the immune system and inhibited by multiple small-molecule antiviral drugs. Understanding how the MIEP is regulated is a critical part of designing and implementing either strategy. Our revelation here that NF-κB and CREB control the responsiveness of the MIEP to the viral UL138 protein and the FDA-approved drug VPA could help in the formulation and execution of promoter regulatory strategies against latent HCMV.

Role of cyclic AMP response element binding protein (CREB) in angiotensin II-induced responses in vascular smooth muscle cells.

Cyclic AMP response element (CRE) binding protein (CREB) is a nuclear transcription factor that regulates the transcription of several genes containing the CRE sites on their promoters. CREB is activated by phosphorylation on a key serine residue, Ser311, in response to a wide variety of extracellular stimuli including angiotensin II (Ang II). Ang II is an important vasoactive peptide and mitogen for vascular smooth muscle cells (VSMC) that in addition to regulating the contractile response in VSMC also plays an important role in phenotypic switch of VSMC from contractile to a synthetic state. The synthetic VSMC are known to exhibit proliferative and migratory properties due to hyperactivation of Ang II-induced signaling events. Ang II has been shown to induce CREB phosphorylation/activation and transcription of genes implicated in proliferation, growth, and migration. Here, we have highlighted some key studies that have demonstrated an important role of CREB in Ang II-mediated gene transcription, proliferation, hypertrophy, and migration of VSMC.

Differential transcription factor use by the KIR2DL4 promoter under constitutive and IL-2/15-treated conditions.

KIR2DL4 is unique among human KIR genes in expression, cellular localization, structure, and function, yet the transcription factors required for its expression have not been identified. Using mutagenesis, EMSA, and cotransfection assays, we identified two redundant Runx binding sites in the 2DL4 promoter as essential for constitutive 2DL4 transcription, with contributions by a cyclic AMP response element (CRE) and initiator elements. IL-2- and IL-15-stimulated human NK cell lines increased 2DL4 promoter activity, which required functional Runx, CRE, and Ets sites. Chromatin immunoprecipitation experiments show that Runx3 and Ets1 bind the 2DL4 promoter in situ. 2DL4 promoter activity had similar transcription factor requirements in T cells. Runx, CRE, and Ets binding motifs are present in 2DL4 promoters from across primate species, but other postulated transcription factor binding sites are not preserved. Differences between 2DL4 and clonally restricted KIR promoters suggest a model that explains the unique 2DL4 expression pattern in human NK cells.

Activating transcription factor 2 targets c‐Fos, but not c‐Jun, in growth plate chondrocytes

Activating transcription factor 2 (ATF-2), c-Fos, and c-Jun belong to the bZIP family of transcription factors. Promoters of c-Fos, c-Jun, cyclin D1, and cyclin A are targets of ATF-2 in primary mouse chondrocytes. An ATF-2 expression vector was co-transfected with either c-Fos or c-Jun promoters in mutant ATF-2 chondrocytes in order to show by luciferase assay that ATF-2 increased promoter activity of c-Fos, but not c-Jun. Chromatin immunoprecipitation (ChIP) assays revealed that ATF-2 bound with the c-Fos promoter at the -294 cyclic AMP response element (CRE) site, but did not bind to the TPA responsive element (TRE) or activating protein-1 (AP1) sites of the c-Jun promoter. Dominant-negative (dn) c-Fos inhibited cyclin D1 promoter activity. However, dn c-Jun had minimal effect on this same promoter activity. c-Fos was capable of interactions with both the cyclin D1 CRE and AP1 sites, while c-Jun co-operated specifically with the cyclin D1 CRE site. Neither c-Fos nor c-Jun had any effect on cyclin A promoter activity. c-Fos was unable to bind to the cyclin A AP1 or CRE sites. In contrast c-Jun was competent in interactions with cyclin A AP1-2 as well as the CRE.

Upregulation of Cyclooxygenase-2 by 4-Nonylphenol is Mediated Through the Cyclic Amp Response Element Activation Pathway

The organic compound nonylphenol (NP) belongs to the family of alkylphenols and is a product of industrial synthesis formed during phenol alkylation. Nonylphenol is considered to be an endocrine disruptor due to weak ability to mimic estrogen and subsequently to disrupt the natural balance of hormones in a given organism. Since the endocrine and immune systems share portions of common signaling pathways, it is conceivable that NP may also affect immune system functions. However, the influence of NP on inflammation and macrophages responsiveness to NP is unclear. Thus, the effects of NP were investigated on cyclooxygenase (COX)-2 expression in cultured macrophages. NP induced COX-2 protein and gene expression in murine macrophage RAW264.7 cells and enhanced COX-2 promoter activity and prostaglandin E2 production. Transfection of RAW264.7 cells with hCOX-2 or various deletion and mutation promoter constructs revealed that the cyclic AMP response element (CRE) was the predominant mediator responsive to NP-induced effects. Moreover, transfection with pCRE-Luc plasmid followed by immunoblotting demonstrated that NP activated CRE sites and CRE binding protein (CREB) phosphorylation. NP also increased nuclear CREB accumulation and CREB binding to the COX-2 promoter. Phosphatidylinositol 3 (PI3)-kinase, Akt, and the mitogen-activated protein kinases (MAP kinases) p38 and JNK were also significantly activated by NP. Our data demonstrate that NP induces COX-2 expression through the PI3-kinase/Akt/MAP kinases/CRE pathway. These findings provide insight into the signal transduction pathways involved in the inflammatory responses induced by NP in macrophages.

Human Arm protein lost in epithelial cancers, on chromosome X 1 (ALEX1) gene is transcriptionally regulated by CREB and Wnt/β‐catenin signaling

The aberrant activation of Wnt signaling is a key process in colorectal tumorigenesis. Canonical Wnt signaling controls transcription of target genes via beta-catenin and T-cell factor/lymphoid enhancer factor family transcription factor complex. Arm protein lost in epithelial cancers, on chromosome X 1 (ALEX1) is a novel member of the Armadillo family which has two Armadillo repeats as opposed to more than six repeats in the classical Armadillo family members. Here we examine cis-regulatory elements and trans-acting factors involved in the transcriptional regulation of the ALEX1 gene. Site-directed mutations of a cyclic AMP response element (CRE) and an E-box impaired the basal activity of human ALEX1 promoter in colorectal and pancreatic cancer cell lines. Moreover, overexpression of CRE-binding protein (CREB) increased the ALEX1 promoter activity in these cell lines, whereas knockdown of CREB expression decreased the expression level of ALEX1 mRNA. Interestingly, luciferase reporter analysis and quantitative real-time RT-PCR demonstrated that the ALEX1 promoter was up-regulated in a CRE-dependent manner by continuous activation of Wnt/beta-catenin signaling induced by a glycogen synthase kinase-3 inhibitor and overexpression of beta-catenin. These results indicate that the CRE and E-box sites are essential cis-regulatory elements for ALEX1 promoter activity, and ALEX1 expression is regulated by CREB and Wntk/beta-catenin signaling.

Frequency-Dependent Regulation of Follicle-Stimulating Hormone β by Pulsatile Gonadotropin-Releasing Hormone Is Mediated by Functional Antagonism of bZIP Transcription Factors

Oscillatory synthesis and secretion of the gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), under the control of pulsatile hypothalamic gonadotropin-releasing hormone (GnRH), is essential for normal reproductive development and fertility. The molecular mechanisms by which various patterns of pulsatile GnRH regulate gonadotrope responsiveness remain poorly understood. In contrast to the alpha and LH beta subunit genes, FSH beta subunit transcription is preferentially stimulated at low rather than high frequencies of pulsatile GnRH. In this study, mutation of a cyclic AMP response element (CRE) within the FSH beta promoter resulted in the loss of preferential GnRH stimulation at low pulse frequencies. We hypothesized that high GnRH pulse frequencies might stimulate a transcriptional repressor(s) to attenuate the action of CRE binding protein (CREB) and show that inducible cAMP early repressor (ICER) fulfills such a role. ICER was not detected under basal conditions, but pulsatile GnRH stimulated ICER to a greater extent at high than at low pulse frequencies. ICER binds to the FSH beta CRE site to reduce CREB occupation and abrogates both maximal GnRH stimulation and GnRH pulse frequency-dependent effects on FSH beta transcription. These data suggest that ICER production antagonizes the stimulatory action of CREB to attenuate FSH beta transcription at high GnRH pulse frequencies, thereby playing a critical role in regulating cyclic reproductive function.

Prostaglandin E 2 activates cAMP response element-binding protein in glioma cells via a signaling pathway involving PKA-dependent inhibition of ERK

Prostaglandin E 2 (PGE 2) plays a critical role in influencing the biological behavior of tumor cells. We previously demonstrated that PGE 2 stimulates human glioma cell growth via activation of protein kinase A (PKA) type II. This study was undertaken to further elucidate the intracellular pathways activated by PGE 2 downstream to PKA. Stimulation of U87-MG glioma cells with PGE 2 increased phosphorylation of the cyclic-AMP response element (CRE) binding protein CREB at Ser-133 and CREB-driven transcription in a dose- and time-dependent manner. Expression of dominant CREB constructs that interfere with CREB phosphorylation at Ser-133 or with its binding to the CRE site markedly decreased PGE 2-induced CREB activation. Inhibition of PKA by H-89 or expression of a dominant negative PKA construct attenuated PGE 2-induced CREB activation. Moreover, inhibition of PKA type II decreased PGE 2-induced CREB-dependent transcription by 45% compared to vehicle-treated cells. To investigate the involvement of additional signaling pathways, U87-MG cells were pretreated with wortmannin or LY294002 to inhibit the PI3-kinase/AKT pathway. Both inhibitors had no effect on PGE 2-induced CREB phosphorylation and transcriptional activity, suggesting that PGE 2 activates CREB in a PI3-kinase/AKT independent manner. Challenge of U87-MG cells with PGE 2, at concentrations that induced maximal CREB activation, or with forskolin inhibited extracellular signal-regulated kinase (ERK) phosphorylation. Pretreatment of U87-MG cells with the ERK inhibitor PD98059, accentuated ERK inhibition and increased CREB phosphorylation at Ser-133 and CREB-driven transcription stimulated by PGE 2, suggesting that inhibition of ERK contributes to PGE 2-induced CREB activation. Inhibition of ERK by PGE 2 or by forskolin was rescued by treatment of cells with H-89 or by the dominant negative PKA construct. Moreover, PGE 2 or forskolin inhibited phosphorylation of Raf-1 phosphorylation at Ser-338. Challenge of U87-MG cells with 11-deoxy-PGE 1 increased CREB-driven transcription and stimulated cell growth, while other PGE 2 analogues had no effect. Together our results reveal a novel signaling pathway whereby PGE 2 signals through PKA to inhibit ERK and increase CREB transcriptional activity.

Tyrosine nitration of L‐type Ca channels prevents activation of the cyclic AMP Response Element (CRE)

Calcium influx through Cav1.2 has been implicated in signal transduction and gene expression. We have recently shown that colonic inflammation results in decreased Ca currents due to nitration of c‐terminal tyrosine residues that prevents src‐kinase mediated phosphorylation. In hCav1.2b transfected cells, depolarization by KCl and/or treatement with the Ca channel agonist BayK8644 induced phosphorylation of the cAMP response binding protein (CREB). Phospho‐CREB binds to CRE sites on promoter regions of several genes. A dual luciferase assay was used to determine activation of CRE in hCav1.2b transfected cells. CRE activation was increased by 2.4‐fold by KCl‐induced depolarization that was blocked by nifedipine. Nitration of hCav1.2b by peroxynitrite (150 uM) prevented KCl induced CRE activation. Both Forskolin and Ionomycin enhanced CRE activation by 2.8‐fold. Mutation of c‐terminal tyrosine residues (Y1837‐1861‐2134F) prevented CRE activation. These data suggest that tyrosine phosphorylation of the COOH terminus of Cav1.2 plays an important role in the downstream signaling for the activation of transcription factors. Supported by NIDDK046367.

Activity of the LMP1 gene promoter in Epstein–Barr virus-transformed cell lines is modulated by sequence variations in the promoter-proximal CRE site

The Epstein-Barr virus (EBV)-encoded tumour-associated latent membrane protein 1 (LMP1) gene expression is transactivated by EBV nuclear antigen 2 (EBNA2) in human B cells. We have previously identified a cyclic AMP-responsive element (CRE) in the B95-8 LMP1 promoter that is essential for transcription activation. Sequencing of LMP1 promoter in the P3HR1-derived EREB2.5 cell line revealed 25 single base pair substitutions in comparison to the B95-8 virus, one of them localized in the CRE element. Sequence variations in this element have been identified in several EBV isolates of both African and Asian origins. The effect of the P3HR1 CRE site variation on binding of factors to the LMP1 promoter sequence (LRS) and promoter activation was investigated with electrophoretic mobility-shift assays and reporter gene transfection assays. ATF1 and CREB1 transcription factors bound with reduced efficiency to the P3HR1 variant and below the detection level to the other tested variants. Accordingly, reporter plasmids carrying the P3HR1 CRE sequence in a B95-8 LRS context displayed 50 % lower activity in all tested cell lines. The impaired ability to activate transcription caused by the C to A substitution in CRE was not apparent when the mutated site was placed in a P3HR1 LRS context and the reporter transfected into Jijoye cells, most likely as a consequence of the other base pair substitutions in P3HR1 LRS. Overall, our results suggest that the mutations in the LRS CRE site have been conserved to adjust LMP1 expression to levels that favour cell survival in certain cellular and environmental contexts.

BBF2H7, a Novel Transmembrane bZIP Transcription Factor, Is a New Type of Endoplasmic Reticulum Stress Transducer

Endoplasmic reticulum (ER) stress transducers IRE1 (inositol requiring 1), PERK (PKR-like endoplasmic reticulum kinase), and ATF6 (activating transcription factor 6) are well known to transduce signals from the ER to the cytoplasm and nucleus when unfolded proteins accumulate in the ER. Recently, we identified OASIS (old astrocyte specifically induced substance) as a novel ER stress transducer expressed in astrocytes. We report here that BBF2H7 (BBF2 human homolog on chromosome 7), an ER-resident transmembrane protein with the bZIP domain in the cytoplasmic portion and structurally homologous to OASIS, is cleaved at the membrane in response to ER stress. The cleaved fragments of BBF2H7 translocate into the nucleus and can bind directly to cyclic AMP-responsive element sites to activate transcription of target genes. Interestingly, although BBF2H7 protein is not expressed under normal conditions, it is markedly induced at the translational level during ER stress, suggesting that BBF2H7 might contribute to only the late phase of unfolded protein response signaling. In a mouse model of focal brain ischemia, BBF2H7 protein is prominently induced in neurons in the peri-infarction region. Furthermore, in a neuroblastoma cell line, BBF2H7 overexpression suppresses ER stress-induced cell death, while small interfering RNA knockdown of BBF2H7 promotes ER stress-induced cell death. Taken together, our results suggest that BBF2H7 is a novel ER stress transducer and could play important roles in preventing accumulation of unfolded proteins in damaged neurons.

ATF-2 stimulates the human insulin promoter through the conserved CRE2 sequence

The insulin promoter contains a number of dissimilar cis-acting regulatory elements that bind a range of tissue specific and ubiquitous transcription factors. Of the regulatory elements within the insulin promoter, the cyclic AMP responsive element (CRE) binds by far the most diverse array of transcription factors. Rodent insulin promoters have a single CRE site, whereas there are four CREs within the human insulin gene, of which CRE2 is the only one conserved between species. The aim of this study was to characterise the human CRE2 site and to investigate the effects of the two principal CRE-associated transcription factors; CREB-1 and ATF-2. Co-transfection of INS-1 pancreatic β-cells with promoter constructs containing the human insulin gene promoter placed upstream of the firefly luciferase reporter gene and expression plasmids for ATF-2 or CREB-1 showed that ATF-2 stimulated transcriptional activity while CREB-1 elicited an inhibitory effect. Mutagenesis of CRE2 diminished the effect of ATF-2 but not that of CREB-1. ATF-2 was shown to bind to the CRE2 site by electrophoretic mobility shift assay and by chromatin immunoprecipitation, while siRNA mediated knockdown of ATF-2 diminished the stimulatory effects of cAMP related signalling on promoter activity. These results suggest that ATF-2 may be a key regulator of the human insulin promoter possibly stimulating activity in response to extracellular signals.

Involvement of Cyclic AMP Response Element (CRE) in Exendin‐4 Induction of Rat Egr‐1 Gene

We recently observed that exendin-4 (EX-4), a potent agonist of GLP-1, induced cyclin D1 expression through the activation of putative cAMP-responsive element (CRE) site and Egr-1 binding site on cyclin D1 promoter (J Endocrinol, 2006; Diabetologia, 2006, both in press). In the present study, induction of Egr-1 expression by EX-4 and the cis-acting elements involved in the transcriptional activation of Egr-1 gene were investigated in INS-cells. EX-4 (10 nM) rapidly and transiently induced the expressions of Egr-1 mRNA and protein. These inductions were completely inhibited by a specific PKA inhibitor, H-89, suggesting the involvement of cAMP/PKA signaling pathway. In promoter assay using serial-deletion constructs of rat Egr-1 promoter, EX-4 induced the highest promoter activity with full-length constructs (−462 to +27) and the activity of similar degree with the construct deleted up to −124. The construct deleted to −73, which was without serum response elements (SREs) site known to be involved in regulating Egr-1 transcription, still responded to EX-4 moderately. However, further deletion to −46 failed to respond to EX −4. Thus, in examining the role of the nucleotide region from −73 to −46, mutation of the putative CRE site located at −69 resulted in 52% reduction in promoter activity in response to EX-4. This study indicates that the putative CRE site as well as the SRE site is required for the induction of Egr-1 gene by EX-4. This research was grant-supported by Korea Science and Engineering Foundation (R-2004-000-10127-0), Republic of Korea.

Functional role of NF-IL6 and its sumoylation and acetylation modifications in promoter activation of cyclooxygenase 2 gene

NF-IL6β regulates gene expression and plays function roles in many tissues. The EGF-regulated cyclooxygenase-2 (cox-2) expression is mediated through p38MAPK signaling pathway and positively correlates with NF-IL6β expression in A431 cells. NF-IL6β coordinated with c-Jun on cox-2 transcriptional activation by reporter and small interfering RNA assays. NF-IL6β could directly bind to CCAAT/enhancer-binding protein (C/EBP) and cyclic AMP-response element (CRE) sites of the cox-2 promoter by in vitro-DNA binding assay. The C/EBP site was important for basal and, to a lesser extent, for EGF-regulated cox-2 transcription, while the CRE site was a more specific response to EGF inducibility of cox-2 gene. SUMO1 expression attenuated EGF- and NF-IL6β-induced cox-2 promoter activities. NF-IL6β was found to be sumoylated by in vivo- and in vitro-sumoylation assays, and the SUMO1-NF-IL6β (suNF-IL6β) lost its ability to interact with p300 in in vitro-binding assay. NF-IL6β was also acetylated by p300, and acetylation of NF-IL6β enhanced the cox-2 promoter activity stimulated by NF-IL6β itself. In vivo-DNA binding assay demonstrated that EGF stimulated the recruitment of p300 and NF-IL6β to the cox-2 promoter, yet promoted the dissociation of SUMO1-modificated proteins from the promoter. These results indicated that NF-IL6β plays a pivotal role in the regulation of basal and EGF-induced cox-2 transcription.

(2E,6Z,10E)-7-hydroxymethyl-3,11,15-trimethyl-2,6,10,14-hexadecatetraen-1-ol (Plaunotol) increases cyclooxygenase-2 expression via nuclear factor κB and cyclic AMP response element in rat gastric epithelial cells

Plaunotol, [(2E,6Z,10E)-7-hydroxymethyl-3,11,15-trimethyl-2,6,10,14-hexadecatetraen-1-ol], a gastroprotective agent, increases the prostaglandin production in the gastric mucosa and accelerates ulcer healing. The precise mechanisms underlying the gastroprotective actions by plaunotol are not known. On the other hand, cyclooxygenase (COX)-2 is a key enzyme in PGE 2 production and its induction is thought to have an important role in ulcer healing. We investigated the mechanism of plaunotol-mediated COX-2 induction in rat gastric epithelial (RGM1) cells. We used a PGE 2 enzyme-linked immunoassay kit and Western blot analysis to measure PGE 2 production and COX-2 induction with plaunotol treatment in serum-starved RGM1 cells. In addition, gel-shift assay, Western blot analysis and a reporter assay were performed to observe which Cox-2 promoter was involved in plaunotol-induced Cox-2 expression. The findings indicated that plaunotol treatment dose-dependently increased COX-2 expression and PGE 2 production. The nuclear factor κB (NF-κB) and cyclic AMP response element (CRE) sites of the COX-2 gene promoter were critical to plaunotol-mediated COX-2 expression. In conclusion, plaunotol induced COX-2 expression and increased PGE 2 production in serum-starved RGM1 cells via activation of the NF-κB and CRE sites of Cox-2 gene promoters.

Inhibition of MuSK Expression by CREB Interacting with a CRE-Like Element and MyoD

The type I receptor-like protein tyrosine kinase MuSK is essential for the neuromuscular junction formation. MuSK expression is tightly regulated during development, but the underlying mechanisms were unclear. Here we identified a novel mechanism by which MuSK expression may be regulated. A cyclic AMP response element (CRE)-like element in the 5'-flanking region of the MuSK gene binds to CREB1 (CRE-binding protein 1). Mutation of this element increases the MuSK promoter activity, suggesting a role for CREB1 in attenuation of MuSK expression. Interestingly, CREB mutants unable to bind to DNA also inhibit MuSK promoter activity, suggesting a CRE-independent inhibitory mechanism. In agreement, CREB1 could inhibit a mutant MuSK transgene reporter whose CRE site was mutated. We provide evidence that CREB interacts directly with MyoD, a myogenic factor essential for MuSK expression in muscle cells. Suppression of CREB expression by small interfering RNA increases MuSK promoter activity. These results demonstrate an important role for CREB1 in the regulation of MuSK expression.

Hypermethylation of the reelin (RELN) promoter in the brain of schizophrenic patients: A preliminary report

DNA methylation changes could provide a mechanism for DNA plasticity and dynamism for short-term adaptation, enabling a type of cell memory to register cellular history under different environmental conditions. Some environmental insults may also result in pathological methylation with corresponding alteration of gene expression patterns. Evidence from several studies has suggested that in schizophrenia and bipolar disorder, mRNA of the reelin gene (RELN), which encodes a protein necessary for neuronal migration, axonal branching, synaptogenesis, and cell signaling, is severely reduced in post-mortem brains. Therefore, we investigated the methylation status of the RELN promoter region in schizophrenic patients and normal controls as a potential mechanism for down regulation of its expression. Ten post-mortem frontal lobe brain samples from male schizophrenic patients and normal controls were obtained from the Harvard Brain Tissue Resources Center. DNA was extracted using a standard phenol-chloroform DNA extraction protocol. To evaluate differences between patients and controls, we applied methylation specific PCR (MSP) using primers localized to CpG islands flanking a potential cyclic AMP response element (CRE) and a stimulating protein-1 (SP1) binding site located in the promoter region. For each sample, DNA extraction, bisulfite treatment, and MSP were independently repeated at least four times to accurately determine the methylation status of the target region. Forty-three PCR trials were performed on the test and control samples. MSP analysis of the RELN promoter revealed an unmethylated signal in all reactions (43 of 43) using DNA from the frontal brain tissue, derived from either the schizophrenic patients or normal controls indicating that this region of the RELN promoter is predominantly unmethylated. However, we observed a distinct methylated signal in 73% of the trials (16 of 22) in schizophrenic patients compared with 24% (5 of 21) of controls. Thus, the hypermethylation of the CpG islands flanking a CRE and SP1 binding site observed at a significantly higher level (t = -5.07, P = 0.001) may provide a mechanism for the decreased RELN expression, frequently observed in post-mortem brains of schizophrenic patients. We also found an inverse relationship between the level of DNA methylation using MSP analysis and the expression of the RELN gene using semi-quantitative RT-PCR. Despite the small sample size, these studies indicate that promoter hypermethylation of the RELN gene could be a significant contributor in effecting epigenetic alterations and provides a molecular basis for the RELN gene hypoactivity in schizophrenia. Further studies with a larger sample set would be required to validate these preliminary observations.

CREB binds to multiple loci on human chromosome 22.

The cyclic AMP-responsive element-binding protein (CREB) is an important transcription factor that can be activated by hormonal stimulation and regulates neuronal function and development. An unbiased, global analysis of where CREB binds has not been performed. We have mapped for the first time the binding distribution of CREB along an entire human chromosome. Chromatin immunoprecipitation of CREB-associated DNA and subsequent hybridization of the associated DNA to a genomic DNA microarray containing all of the nonrepetitive DNA of human chromosome 22 revealed 215 binding sites corresponding to 192 different loci and 100 annotated potential gene targets. We found binding near or within many genes involved in signal transduction and neuronal function. We also found that only a small fraction of CREB binding sites lay near well-defined 5' ends of genes; the majority of sites were found elsewhere, including introns and unannotated regions. Several of the latter lay near novel unannotated transcriptionally active regions. Few CREB targets were found near full-length cyclic AMP response element sites; the majority contained shorter versions or close matches to this sequence. Several of the CREB targets were altered in their expression by treatment with forskolin; interestingly, both induced and repressed genes were found. Our results provide novel molecular insights into how CREB mediates its functions in humans.

Characterization of the prothrombin gene expression during nerve differentiation

The transcriptional regulation of the prothrombin gene expression in neuroblastomas was investigated because of the interest in non-hepatic thrombin expression and function in the nervous system. The data indicated that the murine prothrombin gene was distinctively transcribed in proliferating murine N2a cells and that the transcripts were decreased during the differentiation of N2a cells. The gene transcription in proliferating N2a cells was due to the C-I nuclear complex formation in the promoter region, −248/−140. Mutation analyses indicated that nucleotides from −237 to −231 are the core C-I binding site while the longer sequence −248/−140 is needed for the C-I binding. The C-I binding to the promoter −248/−140 could be inhibited by the presence of competitor probe −187/−166, and the mutation in nucleotides from −186 to −179 significantly diminished not only the formation of C-I binding in the promoter region but also the promoter activity in proliferating neuroblastoma cells. Cyclic AMP response element (CRE) modulator, CREM, appeared to selectively bind to the sequence encompassing −186/−179. Taken together, the results indicate that the prothrombin gene transcription in proliferating N2a cells was critically dependent on the cooperative interaction between the factor(s) binding to the C-1 cis-acting element (−237/−231) and the putative CRE site (−186/−179) in the prothrombin promoter, and that the lack of prothrombin expression that coincided with nerve differentiation was mainly due to the lack of C-I complex formation in the promoter.

Cytokines increase CRE binding but decrease CRE-mediated reporter activity in rat hepatocytes by increasing c-Jun

The cyclic AMP response element (CRE) has been implicated in the regulation of the expression of many genes and cellular processes important in hepatocyte function. CRE sites exist in the promoter regions of several genes expressed during inflammation. Numerous studies on the role of CRE in hepatocyte gene expression have been performed in resting hepatocytes, but the role of CRE during inflammation is unknown. To evaluate the regulation of CRE-mediated transcription during sepsis, cultured hepatocytes were exposed to proinflammatory cytokines and lipopolysaccharide (LPS) was injected into rats. Nuclear proteins were collected and CRE binding activity measured by electromobility shift assay (EMSA) using a consensus CRE oligonucleotide. CRE binding activity was increased in vitro by cytokines and in vivo by LPS administration but CRE-dependent reporter activity was decreased by cytokine stimulation. A c-jun N-terminal kinase (JNK) inhibitor reversed the cytokine-induced increase in CRE binding and increased CRE-dependent reporter activity. Supershift assays indicated that cyclic AMP response element binding protein (CREB) and c-Jun proteins were included in the CRE binding complex. CREB induced and c-Jun suppressed reporter activity using a CRE-dependent construct transfected into cultured primary hepatocytes. In conclusion, these data demonstrate that proinflammatory cytokines regulate CRE binding and activity in cultured hepatocytes and suggest that sepsis-induced changes in CRE binding may participate in the cellular response to inflammation.