Downstream Regulatory Element Antagonistic Modulator Research Articles

Characterization of the Role of Individual EF-Hands in Dream in Modulating Conformational Dynamics, Oligomeric States, and Interaction with DNA

Downstream Regulatory Element Antagonistic Modulator (DREAM) carries four calcium binding motifs (EF-hand) of which EF-3 and EF-4 bind to Ca2+ with high affinity, whereas EF-2 preferentially binds to Mg2+ and EF-1 is inactive. In neuronal cells, DREAM serves as a gene transcriptional repressor of dynorphin, an endogenous ligand, to regulate pain transmission by controlling kappa receptor activation. Recent findings have shown that tetrameric ApoDREAM binds to DNA with a high affinity, whereas association of Ca2+ abolishes the interaction with DNA. Despite mechanisms of interactions between DREAM and DNA have been proposed, it remains unclear which EF-hand in DREAM triggers tetramer dissociation and thus controls DNA affinity. In this study, a combination of fluorescence steady-state, time-resolved anisotropy, circular dichroism (CD), and isothermal titration calorimetry (ITC) techniques were employed to investigate the role of individual EF-hands. Here, we showed that Mg2+ binding to EF-2 does not affect the tertiary and secondary structures of DREAM. Ca2+ binding to EF-3 induces conformational changes in DREAM evidenced by a blue-shift and a decrease in emission intensity of the single tryptophan, whereas the association of Ca2+ to EF-4 does not disturb the DREAM structure. We also showed that elimination of Mg2+ binding to DREAM results in dimerization in Apo form and increase of the dimeric fraction in Ca2+ form. In contrast, abolishment of Ca2+ binding to EF-4 results in a high fraction of DREAM tetramer (60-75%) in Apo, Mg2+, Ca2+, and Ca2+Mg2+ bound forms. Finally, ITC results showed that ApoDREAM wild-type endothermically (ΔH=+32 kcal/mol) binds to prodynorphin DRE oligomer with a Kd of 0.5 µM, whereas a weak interaction was observed in the Ca2+ form (Kd∼200 µM). Interactions of DREAM mutants with DNA will also be discussed.

Evaluation of Downstream Regulatory Element Antagonistic Modulator Gene in Human Multinodular Goiter

BackgroundDREAM (Downstream Regulatory Element Antagonistic Modulator) is a neuronal calcium sensor that was suggested to modulate TSH receptor activity and whose overexpression provokes an enlargement of the thyroid gland in transgenic mice. The aim of this study was to investigate somatic mutations and DREAM gene expression in human multinodular goiter (MNG).Material/MethodsDNA and RNA samples were obtained from hyperplastic thyroid glands of 60 patients (54 females) with benign MNG. DREAM mutations were evaluated by PCR and direct automatic sequencing, whereas relative quantification of mRNA was performed by real-time PCR. Over- and under-expression were defined as a 2-fold increase and decrease in comparison to normal thyroid tissue, respectively. RQ M (relative quantification mean); SD (standard deviation).ResultsDREAM expression was detected in all nodules evaluated. DREAM mRNA was overexpressed in 31.7% of MNG (RQ M=6.26; SD=5.08), whereas 53.3% and 15% had either normal (RQ M=1.16; SD=0.46) or underexpression (RQ M=0.30; SD=0.10), respectively. Regarding DREAM mutations analysis, only previously described intronic polymorphisms were observed.ConclusionsWe report DREAM gene expression in the hyperplastic thyroid gland of MNG patients. However, DREAM expression did not vary significantly, and was somewhat underexpressed in most patients, suggesting that DREAM upregulation does not significantly affect nodular development in human goiter.

The N-Terminal Extension of KChIP3 is Responsible for KChIP3-Calmodulin Complex Formation

Potassium channel interacting proteins (KChIPs), belong to the family of neuronal calcium sensors that are expressed in brain, lung and heart tissue. KChIPs bind to Kv4 channels and regulate channel trafficking, membrane association, and current kinetics. Among them, KChIP-3, also known as Downstream Regulatory Element Antagonistic Modulator (DREAM) and calsenilin, interacts with other intracellular partners (presenilin, calmodulin, and DNA) and was implied in Alzheimer's disease and pain sensing, although a molecular mechanism of KChIP-3 interactions with distinct intracellular partners remains unknown. The objective of this study is to provide a molecular insight into the mechanism of KChIP-3 interactions with calmodulin and determine the role of the N-terminus of KChIP-3 in the formation of calmodulin:KChIP-3 complex. Full length KChIP-3 (residue 1-256) and ΔN KChIP-3 (residue 65-256) were over-expressed in E. coli and purified according to an established protocol. In addition, a peptide that corresponds to residues 29-44 in KChIP-3 N-terminus was synthesized. The equilibrium dissociation constants and rotational correlation times for calmodulin: KChIP-3 complexes were determined using fluorescence anisotropy. Molecular dynamic simulations were implemented to provide an insight into the potential molecular structure of the calmodulin:KChIP-3 protein complex. Calmodulin interaction with KChIP-3 shows a Kd=3.3 µM whereas the peptide analogous to KChIP-3(29-44) binds with Kd=150 nM, deletion of 64 residues from the KChIP-3 N-terminus abolishes the complex formation. The interactions between KChIP-3 and calmodulin are regulated by the presence of Ca2+. A rotational correlation time of 35ns was determined for the KChIP-3:calmodulin complex, in agreement with a heterodimer of ellipsoidal shape. These results show that interactions between calmodulin and KChIP-3 are controlled by the intracellular calcium concentration and that the N-terminal extension in KChIP-3 provides a binding interface for calmodulin.

DREAM, a Transcription Repressor, Is Critical for Calcium Signaling and Platelet Thrombus Formation Independently of Its Transcriptional Activity

DREAM (Downstream Regulatory Element Antagonistic Modulator) is a neuronal calcium sensor and acts as a transcriptional repressor for pain modulation. Recent studies showed that endothelial cell DREAM transcriptionally regulates NF-κB signaling during inflammation (Tiruppathi et al. Nat Immunol. 2014). However, it is unknown whether DREAM plays a role during thrombosis and hemostasis. Using real-time fluorescence intravital microscopy with DREAM knockout (KO) mice, we demonstrated that intravascular cell DREAM is required for platelet thrombus formation and fibrin generation at the site of laser-induced cremaster arteriolar injury. Studies of bone marrow chimeric mice further revealed that both hematopoietic and endothelial cell DREAM regulate thrombus formation following arteriolar injury. Using in vitro flow chamber assays, we found that DREAM is important for platelet thrombus formation on collagen-coated surfaces under shear conditions. Further, tail bleeding time and blood loss significantly increased in DREAM KO mice, compared with wild-type (WT) mice, suggesting the importance of DREAM for hemostasis as well as thrombus formation in vivo. We observed that DREAM KO platelets show markedly decreased aggregation, ATP secretion, P-selectin exposure, and αIIbβ3 integrin activation induced by thrombin, collagen-related peptide, ADP, and A23187 (calcium ionophore). Interestingly, DREAM deletion did not affect phorbol 12-myristate 13-acetate-induced platelet functions. Consistently, DREAM KO platelets exhibited decreased phosphorylation of phosphoinositide-3-kinase, AKT, and ERK, but not protein kinase C (PKC) isoforms during cell activation, suggesting that DREAM does not regulate protein kinase C signaling pathway. Electron microscopic analysis demonstrated that ultrastructural features of DREAM KO platelets are indistinguishable from those of WT platelets. Since DREAM is a transcription factor that binds to calcium in four EF-hand motifs, we investigated whether platelet DREAM regulates calcium signaling. Indeed, DREAM deletion in platelets resulted in significant reduction in both intracellular calcium release and store-operated calcium entry (SOCE) following agonist stimulation. When we transiently knocked down DREAM in a human megakaryoblastic leukemia cell line (MEG-01) using siRNA, we found that A23187-induced calcium release and SOCE are impaired by DREAM knockdown in MEG-01 cells without altering the expression of calcium signaling molecules, such as inositol trisphosphate receptor, stromal interaction molecule 1, and ORAI1. These defects were rescued by overexpression of siRNA-resistant WT but not EF-hand domain mutant DREAM. Therefore, our results provide novel evidence that platelet DREAM regulates calcium signaling independently of its transcriptional factor activity, thereby playing a critical role during thrombus formation and hemostasis in vivo. DisclosuresNo relevant conflicts of interest to declare.

Activation of DREAM (Downstream Regulatory Element Antagonistic Modulator), a Calcium-Binding Protein, Reduces L-DOPA-Induced Dyskinesias in Mice

BackgroundPrevious studies have implicated the cyclic adenosine monophosphate/protein kinase A pathway as well as FosB and dynorphin-B expression mediated by dopamine D1 receptor stimulation in the development of 3,4-dihydroxyphenyl-L-alanine (L-DOPA)–induced dyskinesia. The magnitude of these molecular changes correlates with the intensity of dyskinesias. The calcium-binding protein downstream regulatory element antagonistic modulator (DREAM) binds to regulatory element sites called DRE in the DNA and represses transcription of target genes such as c-fos, fos-related antigen-2 (fra-2), and prodynorphin. This repression is released by calcium and protein kinase A activation. Dominant-active DREAM transgenic mice (daDREAM) and DREAM knockout mice (DREAM−/−) were used to define the involvement of DREAM in dyskinesias. MethodsDyskinesias were evaluated twice a week in mice with 6-hydroxydopamine lesions during long-term L-DOPA (25 mg/kg) treatment. The impact of DREAM on L-DOPA efficacy was evaluated using the rotarod and the cylinder test after the establishment of dyskinesia and the molecular changes by immunohistochemistry and Western blot. ResultsIn daDREAM mice, L-DOPA-induced dyskinesia was decreased throughout the entire treatment. In correlation with these behavioral results, daDREAM mice showed a decrease in FosB, phosphoacetylated histone H3, dynorphin-B, and phosphorylated glutamate receptor subunit, type 1 expression. Conversely, genetic inactivation of DREAM potentiated the intensity of dyskinesia, and DREAM−/− mice exhibited an increase in expression of molecular markers associated with dyskinesias. The DREAM modifications did not affect the kinetic profile or antiparkinsonian efficacy of L-DOPA therapy. ConclusionsThe protein DREAM decreases development of L-DOPA-induced dyskinesia in mice and reduces L-DOPA-induced expression of FosB, phosphoacetylated histone H3, and dynorphin-B in the striatum. These data suggest that therapeutic approaches that activate DREAM may be useful to alleviate L-DOPA-induced dyskinesia without interfering with the therapeutic motor effects of L-DOPA.

The Potassium Channel Interacting Protein 3 (DREAM/KChIP3) Heterodimerizes with and Regulates Calmodulin Function

Downstream regulatory element antagonistic modulator (DREAM/KChIP3), a neuronal EF-hand protein, modulates pain, potassium channel activity, and binds presenilin 1. Using affinity capture of neuronal proteins by immobilized DREAM/KChIP3 in the presence and absence of calcium (Ca(2+)) followed by mass spectroscopic identification of interacting proteins, we demonstrate that in the presence of Ca(2+), DREAM/KChIP3 interacts with the EF-hand protein, calmodulin (CaM). The interaction of DREAM/KChIP3 with CaM does not occur in the absence of Ca(2+). In the absence of Ca(2+), DREAM/KChIP3 binds the EF-hand protein, calcineurin subunit-B. Ca(2+)-bound DREAM/KChIP3 binds CaM with a dissociation constant of ∼3 μM as assessed by changes in DREAM/KChIP3 intrinsic protein fluorescence in the presence of CaM. Two-dimensional (1)H,(15)N heteronuclear single quantum coherence spectra reveal changes in chemical shifts and line broadening upon the addition of CaM to (15)N DREAM/KChIP3. The amino-terminal portion of DREAM/KChIP3 is required for its binding to CaM because a construct of DREAM/KChIP3 lacking the first 94 amino-terminal residues fails to bind CaM as assessed by fluorescence spectroscopy. The addition of Ca(2+)-bound DREAM/KChIP3 increases the activation of calcineurin (CN) by calcium CaM. A DREAM/KChIP3 mutant incapable of binding Ca(2+) also stimulates calmodulin-dependent CN activity. The shortened form of DREAM/KChIP3 lacking the NH(2)-terminal amino acids fails to activate CN in the presence of calcium CaM. Our data demonstrate the interaction of DREAM/KChIP3 with the important EF-hand protein, CaM, and show that the interaction alters CN activity.

Nitric Oxide Modulated the Expression of DREAM/Calsenilin/KChIP3 in Inflammatory Pain of Rats

Downstream regulatory element antagonistic modulator (DREAM) is a critical transcriptional repressor for pain modulation. The role of nitric oxide (NO) plays in modulating DREAM pain pathway in the periphery is unclear. Therefore, we investigated the role of the NO in modulation of the expression of DREAM in formalin-induced rat inflammatory pain models. Male Sprague-Dawley rats were randomly distributed into four groups: the normal group, formalin test group, Nω-nitro-L-arginine (l-NNA) group, and morphine group. One hundred microliters of 2.5% formalin was injected into the plantar surface of the right hindpaw of rats. l-NNA (40nmol/L) and morphine (40nmol/L) were injected intrathecally in the hindpaw before formalin injection. The nociceptive behavioral reaction was recorded. After the formalin test, the expression of DREAM mRNA and protein in the spinal cord of the four groups were measured. The nociceptive reaction induced by injection of formalin exhibited two phases. Morphine and l-NNA significantly decreased pain scores of the second phase. The expression of DREAM was significantly increased in the rat spinal cord after formalin-induced pain. Morphine significantly upregulated the expression of DREAM, and the formalin-induced upregulation was significantly attenuated by l-NNA. NO may play an important role in the DREAM pathway modulation of inflammatory pain.

Regulation of DREAM Expression by Group I mGluR

DREAM (downstream regulatory element antagonistic modulator) is a calcium-binding protein that regulates dynorphin expression, promotes potassium channel surface expression, and enhances presenilin processing in an expression level-dependent manner. However, no molecular mechanism has yet explained how protein levels of DREAM are regulated. Here we identified group I mGluR (mGluR1/5) as a positive regulator of DREAM protein expression. Overexpression of mGluR1/5 increased the cellular level of DREAM. Up-regulation of DREAM resulted in increased DREAM protein in both the nucleus and cytoplasm, where the protein acts as a transcriptional repressor and a modulator of its interacting proteins, respectively. DHPG (3,5-dihydroxyphenylglycine), a group I mGluR agonist, also up-regulated DREAM expression in cortical neurons. These results suggest that group I mGluR is the first identified receptor that may regulate DREAM activity in neurons.

Downstream regulatory element antagonistic modulator (DREAM); NMDAR (GRIN)

Downstream regulatory element antagonistic modulator (DREAM); NMDAR (GRIN)

Lack of DREAM Protein Enhances Learning and Memory and Slows Brain Aging

Memory deficits in aging affect millions of people and are often disturbing to those concerned. Dissection of the molecular control of learning and memory is paramount to understand and possibly enhance cognitive functions. Old-age memory loss also has been recently linked to altered Ca(2+) homeostasis. We have previously identified DREAM (downstream regulatory element antagonistic modulator), a member of the neuronal Ca(2+) sensor superfamily of EF-hand proteins, with specific roles in different cell compartments. In the nucleus, DREAM is a Ca(2+)-dependent transcriptional repressor, binding to specific DNA signatures, or interacting with nucleoproteins regulating their transcriptional properties. Also, we and others have shown that dream mutant (dream(-/-)) mice exhibit marked analgesia. Here we report that dream(-/-) mice exhibit markedly enhanced learning and synaptic plasticity related to improved cognition. Mechanistically, DREAM functions as a negative regulator of the key memory factor CREB in a Ca(2+)-dependent manner, and loss of DREAM facilitates CREB-dependent transcription during learning. Intriguingly, 18-month-old dream(-/-) mice display learning and memory capacities similar to young mice. Moreover, loss of DREAM protects from brain degeneration in aging. These data identify the Ca(2+)-regulated "pain gene" DREAM as a novel key regulator of memory and brain aging.

Downstream regulatory element antagonistic modulator regulates islet prodynorphin expression

Calcium-binding proteins regulate transcription and secretion of pancreatic islet hormones. Here, we demonstrate neuroendocrine expression of the calcium-binding downstream regulatory element antagonistic modulator (DREAM) and its role in glucose-dependent regulation of prodynorphin (PDN) expression. DREAM is distributed throughout beta- and alpha-cells in both the nucleus and cytoplasm. As DREAM regulates neuronal dynorphin expression, we determined whether this pathway is affected in DREAM(-/-) islets. Under low glucose conditions, with intracellular calcium concentrations of <100 nM, DREAM(-/-) islets had an 80% increase in PDN message compared with controls. Accordingly, DREAM interacts with the PDN promoter downstream regulatory element (DRE) under low calcium (<100 nM) conditions, inhibiting PDN transcription in beta-cells. Furthermore, beta-cells treated with high glucose (20 mM) show increased cytoplasmic calcium (approximately 200 nM), which eliminates DREAM's interaction with the DRE, causing increased PDN promoter activity. As PDN is cleaved into dynorphin peptides, which stimulate kappa-opioid receptors expressed predominantly in alpha-cells of the islet, we determined the role of dynorphin A-(1-17) in glucagon secretion from the alpha-cell. Stimulation with dynorphin A-(1-17) caused alpha-cell calcium fluctuations and a significant increase in glucagon release. DREAM(-/-) islets also show elevated glucagon secretion in low glucose compared with controls. These results demonstrate that PDN transcription is regulated by DREAM in a calcium-dependent manner and suggest a role for dynorphin regulation of alpha-cell glucagon secretion. The data provide a molecular basis for opiate stimulation of glucagon secretion first observed over 25 years ago.

Identification of transcriptional regulators of neuropeptide FF gene expression

Neuropeptide FF (NPFF) is an RF-amide peptide with pleiotropic functions in the mammalian central nervous system, including pain modulation, opiate interactions, cardiovascular regulation and neuroendocrine effects. To gain insights into the transcriptional mechanisms that regulate NPFF gene expression, we cloned and sequenced 9.8 and 1.5 kb of the mouse and rat NPFF 5′-flanking region, respectively. Regions with high sequence homology between mouse, rat and human were expected to have high probability to interact with regulatory proteins and were studied further. Electromobility shift assays revealed one region that may interact with the homeobox proteins Oct-1, PDX1, Pit-1 and MEIS and two consensus DRE sites that bind a nuclear protein, which was identified as the downstream regulatory element antagonistic modulator DREAM by supershift assays. The distribution of NPFF gene expression was examined in the mouse using in situ hybridization and RT-PCR. NPFF expression was also evident during mouse embryogenesis. A fixed transcription initiation site for the mouse NPFF gene was found. A novel splice variant with a retained intron of the NPFF gene was characterized. Chimeric luciferase reporter gene constructs for the mouse NPFF gene revealed a minimal promoter region and a region with transcriptional suppressor features. An NGF responsive area was found using mouse NPFF reporter gene constructs. We postulate that Oct-1, PDX1, Pit-1, MEIS and DREAM are likely transcriptional regulators of NPFF gene expression.

The transcriptional repressor DREAM is involved in thyroid gene expression

Downstream regulatory element antagonistic modulator (DREAM) was originally identified in neuroendocrine cells as a calcium-binding protein that specifically binds to downstream regulatory elements (DRE) on DNA, and represses transcription of its target genes. To explore the possibility that DREAM may regulate the endocrine activity of the thyroid gland, we analyzed its mRNA expression in undifferentiated and differentiated thyroid cells. We demonstrated that DREAM is expressed in the normal thyroid tissue as well as in differentiated thyroid cells in culture while it is absent in FRT poorly differentiated cells. In the present work, we also show that DREAM specifically binds to DRE sites identified in the 5′ untranslated region (UTR) of the thyroid-specific transcription factors Pax8 and TTF-2/FoxE1 in a calcium-dependent manner. By gel retardation assays we demonstrated that thapsigargin treatment increases the binding of DREAM to the DRE sequences present in Pax8 and TTF-2/Foxe1 5′ UTRs, and this correlates with a significant reduction of the expression of these genes. Interestingly, in poorly differentiated thyroid cells overexpression of exogenous DREAM strongly inhibits Pax8 expression. Moreover, we provide evidence that a mutated form of DREAM unable to bind Ca 2+ interferes with thyroid cell proliferation. Therefore, we propose that in thyroid cells DREAM is a mediator of the calcium-signaling pathway and it is involved in the regulation of thyroid cell function.

DREAMing about arthritic pain

The experience of acute pain serves a crucial biological purpose: it alerts a living organism to environmental dangers, inducing behavioural responses which protect the organism from further damage. In contrast,...