CrebB-17A

EVOLUTIONARY HOMOLOGS

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CREB and photoperiod response

Synchronization between the environmental lighting cycle and the biological clock in the suprachiasmatic nucleus (SCN) is correlated with phosphorylation of the Ca2+/cAMP response element binding protein (CREB) at the transcriptional activating site Ser133. Mechanisms mediating the formation of phospho-CREB (P-CREB) and their relation to clock resetting are unknown. To address these issues, the signaling pathway between light and P-CREB was probed. Nocturnal light rapidly and transiently induces P-CREB-like immunoreactivity (P-CREB-lir) in the rat SCN. Glutamate (Glu) or nitric oxide (NO) donor administration in vitro also induce P-CREB-lir in SCN neurons only during subjective night. Clock-controlled sensitivity to phase resetting by light, Glu, and NO is similarly restricted to subjective night. The effects of NMDA and nitric oxide synthase (NOS) antagonists on Glu-mediated induction of P-CREB-lir parallel their inhibition of phase shifting. Significantly, among neurons in which P-CREB-lir is induced by light are NADPH-diaphorase-positive neurons of the SCN's retinorecipient area. Glu treatment increases the intensity of a 43 kDa band recognized by anti-P-CREB antibodies in subjective night but not day, whereas anti-alpha CREB-lir of this band remain constant between night and day. Inhibition of NOS during Glu stimulation diminishes the anti-P-CREB-lir of this 43 kDa band. Together, these data couple nocturnal light, Glu, NMDA receptor activation and NO signaling to CREB phosphorylation in the transduction of brief environmental light stimulation of the retina into molecular changes in the SCN resulting in phase resetting of the biological clock (Ding, 1997).

Photic resetting of the adult mammalian circadian clock in vivo is associated with phosphorylation of the Ser133 residue of the calcium/cyclic AMP response-element binding-protein (CREB) in the retinorecipient region of the suprachiasmatic nucleus (SCN). Western blotting and immunocytochemistry were used to investigate whether agonists known to reset the clock of neonatal hamsters in vivo are also able to influence the phosphorylation of CREB in the suprachiasmatic hypothalamus in vitro. Antisera raised against synthetic CREB peptide sequences were used to differentiate between total CREB and the Ser133 phosphorylated form of CREB (pCREB). Western blot analysis of proteins isolated from suprachiasmatic tissue of 1-day-old Syrian hamsters reveal bands at approximately 45 kDa corresponding to total CREB and pCREB. Treatment of the tissue with a mixture of glutamatergic agonists [N-methyl-D-aspartate (NMDA), amino-methyl proprionic acid (AMPA) and kainate, all at 1 microM], or native glutamate (1 microM) have no effect on the total CREB signal, but increase the pCREB signal, indicative of agonist-stimulated phosphorylation of CREB on Ser133. A similar effect is seen following treatment of the suprachiasmatic blocks with either dopamine (1 microM) or forskolin (1 microM). Simultaneous treatment with melatonin (1 microM) significantly attenuates stimulation by forskolin. The effect of the agonists on nuclear pCREB-immunoreactivity (-ir) was investigated in primary cultures containing a mixture of cell types characteristic of the suprachiasmatic nuclei in vivo. Basal expression of nuclear total CREB-ir is high, whereas expression of pCREB-ir is low. Treatment with glutamate (1 microM) or dopamine (1 microM) has no effect on total CREB-ir, but increases pCREB-ir in approximately 50% and 30% of cells, respectively, whereas forskolin (1 microM) increases pCREB-ir in almost all cells (> 90%). The effects of all three agonists are rapid (< 15 min), and dose and time dependent. Melatonin reverses the effects of forskolin in mixed cultures, but not in pure astrocyte cultures. Dual-immunocytochemistry (ICC) reveals that glutamate (1 microM) increases nuclear pCREB-ir in cells immunoreactive for microtubule-associated protein II (MAP II-ir), but not other cells, indicating an effect predominantly on neurons. This occurs equally in gamma-amino butyric acid (GABA)-ir and non-GABA-ir neurons. Dopamine (1 microM) is more selective, increasing pCREB-ir only in GABA-ir neurons, whereas forskolin increases pCREB-ir in all cells. The specific stimulation of pCREB-ir in GABA-ir neurons by dopamine is reversed by melatonin, but melatonin has no effect on the increase in pCREB-ir induced in GABA-ir neurons by glutamate. These results demonstrate that agonists known to entrain the circadian clock in vivo modulate phosphorylation of CREB in GABA-ir neurons derived from the neonatal suprachiasmatic nuclei (McNulty, 1998).

Mammalian circadian rhythms are regulated by a pacemaker within the suprachiasmatic nuclei (SCN) of the hypothalamus. The molecular mechanisms controlling the synchronization of the circadian pacemaker are unknown; however, immediate early gene (IEG) expression in the SCN is tightly correlated with entrainment of SCN-regulated rhythms. Antibodies were isolated that recognize the activated, phosphorylated form of the transcription factor cyclic adenosine monophosphate response element binding protein (CREB). Within minutes after exposure of hamsters to light, CREB in the SCN becomes phosphorylated on the transcriptional regulatory site, Ser133. CREB phosphorylation is dependent on circadian time: CREB becomes phosphorylated only at times during the circadian cycle when light induces IEG expression and causes phase shifts of circadian rhythms. These results implicate CREB in neuronal signaling in the hypothalamus and suggest that circadian clock gating of light-regulated molecular responses in the SCN occurs upstream of phosphorylation of CREB (Ginty, 1993).

The suprachiasmatic nucleus (SCN) is a central pacemaker in mammals, driving many endogenous circadian rhythms. An important pacemaker target is the regulation of a hormonal message for darkness, consisting of the circadian rhythm in synthesis of the pineal hormone melatonin. The endogenous clock within the SCN is synchronized to environmental light/dark cycles by photic information conveyed via the retinohypothalamic tract (RHT) and by the nocturnal melatonin signal that acts within a feedback loop. The sensitivity of the SCN to the resetting actions of neurochemical factors implicated in entrainment is restricted to discrete temporal windows. These windows, overlapping with equally discrete molecular gates for distinct signaling pathways, are notably out of phase with one another. During daytime, phase shifts can be induced by activation of the cAMP-signaling pathway, whereas the prevailing stimulus for phase shifts during nighttime results from activation of the Ca2+- and/or the cGMP-signaling pathway(s). In the rodent RHT, glutamate signals 'light' to the pacemaker during nighttime. Conversely, the pituitary adenylate cyclase-activating polypeptide (PACAP) makes the SCN sense 'darkness' during daytime, by elevating the intracellular cAMP concentration. At dusk and possibly also at dawn, a sensitivity window exists for the pineal hormone melatonin to affect clock activity. Thus, melatonin, the hormonal message for darkness, may fine-tune circadian timing via interference with other pathways, thereby defining SCN sensitivity to resetting cues. Because phase shifts in SCN activity require protein synthesis and induce DNA-binding proteins, transcription is part of pacemaker adjustment. Therefore, a molecular interface for neuronal (RHT neurotransmission) and/or endocrine (melatonin) cues must be able to serve different signaling pathways, to react fast, and to convey external stimuli rapidly to the transcriptional machinery necessary for consolidation or even initiation of phase shifts (von Gall, 1998 and references). There is increasing evidence that the transcription factor Ca2+/cAMP response element-binding protein (CREB) may act as an integrator involved in resetting circadian rhythms (Ginty, 1993; McNulty, 1998).

How do melatonin levels intersect the temporally gated resetting actions of two RHT transmitters: pituitary adenylate cyclase-activating polypeptide (PACAP) and glutamate? The inducible phosphorylation of the transcription factor Ca2+/cAMP response element-binding protein (CREB) was investigated in the SCN of a melatonin-proficient (C3H) and a melatonin-deficient (C57BL) mouse strain. In vivo, light-induced phase shifts in locomotor activity are consistently accompanied by CREB phosphorylation in the SCN of both strains. However, in the middle of subjective nighttime, light induces larger phase delays in C57BL than in C3H mice. In vitro, PACAP and glutamate induce CREB phosphorylation in the SCN of both mouse strains, with PACAP being more effective during late subjective daytime and glutamate being more effective during subjective nighttime. Melatonin suppresses PACAP (but not glutamate-induced) phosphorylation of CREB. The distinct temporal domains during which glutamate and PACAP induce CREB phosphorylation imply that during the light/dark transition the SCN switches sensitivity between these two RHT transmitters. Because these temporal domains are not different between C3H and C57BL mice, the sensitivity windows are set independent of the rhythmic melatonin signal (von Gall, 1998).

Adaptation of the circadian pacemaker in the mammalian SCN to changing environmental light/dark cycles is fundamental for survival. These analyses of signaling events in the mouse SCN highlight a major role for the transcription factor CREB as a molecular interface between various resetting cues accessing the circadian clock. In particular, light and two important transmitters of the RHT, PACAP and glutamate, activate CREB in the SCN by phosphorylation during discrete time windows that match with the corresponding temporal domains for these signals to induce phase shifts in vivo. The comparative analyses of melatonin-deficient and -proficient mice show that sensitivity windows for resetting cues are determined predominantly by cell-autonomous mechanisms within the SCN rather than by a phasic melatonin signal (von Gall, 1998 and references).

The principal natural stimulus for the phase adjustment of the circadian oscillator is light. The light-induced phase delays in locomotor activity of both mouse strains are consistent with previous reports. Exogenous melatonin attenuates photically induced phase delays in C3H mice at CT14 (two hours after the start of darkness) and CT18. Because C3H mice show an elevated melatonin synthesis only during the second half of the night, the endogenous hormone effect on light-induced phase shifts should not be detectable at CT14. Indeed, phase delays are attenuated at CT18 as compared with CT14. Although this interpretation awaits further experimentation, it is supported by consistently large phase delays in melatonin-deficient C57BL mice at CT14 and at CT18. In both mouse strains, resetting light pulses are always associated with CREB phosphorylation in the SCN, a molecular link originally observed in the Syrian hamster. Notably, the dynamic profile of phosphorylated CREB is not different between the two mouse strains and, thus, independent of the size of the phase delays. These results imply that endogenous melatonin may modulate the magnitude of light-induced phase shifts, but not the acute cellular responses of the SCN to light, including the induction of pCREB. These observations profile a feedback function for the pineal hormone, with melatonin potentially setting the gain of SCN sensitivity to resetting stimuli on a diurnal and a seasonal basis (von Gall, 1998 and references).

Glutamate, a principal transmitter of the RHT, induces CREB phosphorylation in the SCN probably via activation of the Ca2+/calmodulin-signaling pathway. In both mouse strains glutamate-induced CREB phosphorylation is observed during subjective night but not during subjective daytime. This observation extends the results of a previous study in rat, which compared the effects of glutamate at CT07 (seven hours into the light cycle) and CT20 (Ding, 1997). The temporal window of sensitivity to glutamate coincides with the period of glutamate-induced phase shifts in rat SCN explants. Notably, CREB phosphorylation is a consistent cellular response to resetting glutamatergic cues, regardless of the direction of the phase response, because it can be induced at early subjective night (this study) when glutamate induces maximal phase delays and at CT20 when glutamate induces phase advances (von Gall, 1998 and references).

PACAP is an RHT transmitter that stimulates cAMP production in the SCN via the PACAP-R1 receptor. Because PACAP levels in the rat SCN are reduced by light, any release of PACAP by RHT fibers would make the SCN "sense darkness" and allow the pacemaker to readjust rapidly to ambient lighting conditions. PACAP induces a maximal CREB phosphorylation at ZT10, and the effect is not different between the two mouse strains. Therefore, as for glutamate and light, the gate of sensitivity is set independently of any rhythmic melatonin signal from the pineal gland. The sensitivity window for PACAP overlaps temporally with phase advances induced either by dark pulses in vivo or by application of cAMP analogs in vitro. The phasic induction of phosphorylated CREB by PACAP described here for mice conforms to studies in rat showing that the resetting action of this peptide is restricted to subjective daytime. All data support the concept that phosphorylation of CREB mediates PACAP-mediated retinal signaling to the clock (von Gall, 1998 and references).

These investigations with the two RHT transmitters imply a switch in SCN sensitivity between PACAP (daytime), activating the cAMP/adenylate cyclase-signaling pathway, and glutamate (nighttime), stimulating the Ca2+/calmodulin-signaling pathway. This switch occurs endogenously around the light/dark transition and is set independent of a phasic melatonin signal, because it is present in C3H and also in C57BL mice. All these findings support the notion that transcription is part of the mechanism for adjustment of oscillator timing (von Gall, 1998 and references).

Exogenous melatonin applied around dusk is also a potent resetting cue in rats, in Siberian hamsters and in some strains of mouse. In the current study, melatonin reverses PACAP-induced phosphorylation of CREB when applied at ZT10 in both C3H and C57BL mice. This conforms to an autoradiographic demonstration of melatonin-binding sites in the SCN of both strains. Characterization of the two melatonin receptor subtypes expressed in the mouse SCN, the Mel1a and the Mel1b receptor, has shown that both are coupled to inhibition of adenylate cyclase activity. It is therefore likely that in the SCN of both mouse strains, molecular cross talk exists between melatonin- and PACAP-regulated signaling at the level of cAMP accumulation but not between melatonin and the glutamate-activated Ca2+-dependent pathway. The insensitivity of this signal transduction pathway toward melatonin may secure the responsiveness of the SCN to light-induced phase shifts at night. This is consistent with a recent report that melatonin cannot affect glutamatergic induction of phosphorylated CREB in the SCN of neonatal Syrian hamsters (von Gall, 1998 and references).

The data in this paper suggest that CREB serves in the mouse SCN as a molecular interface to translate a gated transmitter preference for adjustment of the phase of the pacemaker. This convergence of multiple and separately inducible signaling pathways onto CREB phosphorylation is well known. In the mouse SCN such convergence seems to be achieved via a rapid and efficient but restricted intracellular molecular cross talk and may affect and adjust clockwork transcription. The evidence supporting this idea can be derived from observations that in the SCN of both mouse strains, CREB phosphorylation is induced within a few minutes by (1) light stimuli that reset the SCN pacemaker; (2) glutamatergic receptor activation, and (3) PACAPergic receptor activation. Importantly, melatonin interferes selectively with the PACAP-induced CREB phosphorylation in both mouse strains. The case for granting CREB a central role for the integration of photic information into the clockwork can also be inferred from known molecular details of this transcription factor. It should be noted, however, that data do not allow the elimination of a possible parallel processing of clock resetting and CREB phosphorylation, both affected by PACAP and glutamate signaling (von Gall, 1998 and references).

The very rapid stimulus-induced CREB phosphorylation in the rodent SCN allows this event to intersect with clock mechanisms at the earliest time point possible. CREB phosphorylation induced by photic stimulation at nighttime occurs before the transcriptional induction of immediate early genes that is associated with light-induced phase shifts in clock function and before the rapid elevation of mPerI and mPerII mRNA levels, the putative mouse ortholog of the Drosophila clock gene period. Since CREB phosphorylation is known to be a key step in coupling short-term neuronal stimuli to long-term intracellular responses, the light-induced CREB phosphorylation in rodent SCN may be the molecular initiator to reset the phase of circadian behavioral cycles. It may be envisioned that pCREB induction affects clock genes like mPer to cause phase delays during the early night by retarding the spontaneous decline in SCN activity and to cause phase advances during late night by activating a precocious increase in the molecular oscillation, based on mPer (von Gall, 1998 and references).

Neurotransmitter-driven activation of transcription factors is important for control of neuronal and neuroendocrine functions. Norepinephrine cAMP-dependent rhythmic hormone production in rat pineal gland is accompanied by a temporally regulated switch in the ratio of a transcriptional activator, phosphorylated cAMP-responsive element-binding protein (pCREB), and a transcriptional inhibitor, inducible cAMP early repressor (ICER). pCREB accumulates endogenously at the beginning of the dark period and declines during the second half of the night. Concomitant with this decline, the amount of ICER rises. The changing ratio between pCREB and ICER shapes the in vivo dynamics in mRNA and, thus, protein levels of arylalkylamine-N-acetyltransferase, the rate-limiting enzyme of melatonin synthesis. Consequently, a silenced ICER expression in pinealocytes leads to a disinhibited arylalkylamine-N-acetyltransferase transcription and a primarily enhanced melatonin synthesis (Maronde, 1999).

Circadian clock genes are regulated through a transcriptional-translational feedback loop. Alterations of the chromatin structure by histone acetyltransferases and histone deacetylases (HDACs) are commonly implicated in the regulation of gene transcription. However, little is known about the transcriptional regulation of mammalian clock genes by chromatin modification. This study shows that the state of acetylated histones fluctuate in parallel with the rhythm of mouse Per1 (mPer1) or mPer2 expression in fibroblast cells and liver. Mouse CRY1 (mCRY1) represses transcription with HDACs and mSin3B, which is relieved by the HDAC inhibitor trichostatin A (TSA). In turn, TSA induces endogenous mPer1 expression as well as the acetylation of histones H3 and H4, which both interact with the mPer1 promoter region in fibroblast cells. Moreover, a light pulse stimulates rapid histone acetylation associated with the promoters of mPer1 or mPer2 in the suprachiasmatic nucleus (SCN) and the binding of phospho-CREB in the CRE of mPer1. TSA administration into the lateral ventricle induces mPer1 and mPer2 expression in the SCN. Taken together, these data indicate that the rhythmic transcription and light induction of clock genes are regulated by histone acetylation and deacetylation (Naruse, 2004).

microRNAs (miRNAs) are a class of small, noncoding RNAs that regulate the stability or translation of mRNA transcripts. Although recent work has implicated miRNAs in development and in disease, the expression and function of miRNAs in the adult mammalian nervous system have not been extensively characterized. This study examined the role of two brain-specific miRNAs, miR-219 and miR-132, in modulating the circadian clock located in the suprachiasmatic nucleus. miR-219 is a target of the CLOCK and BMAL1 complex, exhibits robust circadian rhythms of expression, and the in vivo knockdown of miR-219 lengthens the circadian period. miR-132 is induced by photic entrainment cues via a MAPK/CREB-dependent mechanism, modulates clock-gene expression, and attenuates the entraining effects of light. Collectively, these data reveal miRNAs as clock- and light-regulated genes and provide a mechanistic examination of their roles as effectors of pacemaker activity and entrainment (Cheng, 2007).

As a starting point to examine miRNA expression in the SCN, data from a genome-wide screening technique was used to identify CREB-regulated miRNAs. CREB was the initial focus because photic stimulation has been shown to elicit robust CRE-dependent gene expression in the SCN. For these studies, miR-132, a CREB-regulated miRNA that is induced by neurotrophins, was studied. First, the CREB Serial Analysis of Chromatin Occupancy (SACO) results were verified by using a combination of chromatin immunoprecipitation (ChIP) and real-time PCR. The miR-132 enhancer region was selectively immunoprecipitated by a CREB antibody but not IgG. The abundance of a bona fide CREB target, c-Fos, was analyzed as a positive control. The CREB ChIP did not enrich for a locus near the 18S ribosomal-RNA repeat or other negative control regions. Given the role of CREB as a light-inducible transcriptional factor within the SCN, these data raised the possibility that miR-132 expression might be activated by light (Cheng, 2007).

To discover miRNAs that are regulated by the E box-dependent core timing mechanism, miRNAs were screened by using a CLOCK ChIP. Interestingly, the enhancer region of miR-219-1 was significantly enriched in the CLOCK IP fraction, whereas that of miR-132 was not. The mperiod1 gene, a bona fide target of CLOCK- and BMAL1-mediated transcription, was used as the positive control, and 18S rRNA was used as the negative control. Notably, the miR-219-1 enhancer region was also enriched in the CREB antiserum IP fraction (Cheng, 2007).

Sequence alignment of pre-miR-132 across multiple species, including mouse, human, dog, zebrafish, and puffer fish indicates that the mature miR-132 sequence is highly conserved throughout evolution. With respect to the miR-219-1 gene, vertebrates possess multiple copies, and sequence alignment of pre-miR-219 from varied species shows a high degree of conservation. Zebrafish in situ hybridization has shown that miR-219 is enriched in the brain. As predicted by the ChIP assays, an E box motif (noncanonical) is found in the promoter region of the miR-219-1 gene and two consensus CRE motifs were also identified. These response elements are found in mice, rats, humans, and dogs (Cheng, 2007).

To determine whether the CLOCK and BMAL1 heterodimer regulates miR-219-1 expression, CLOCK and BMAL1 were expressed in PC12 cells and miR-219-1 expression was examined by semiquantitative reverse transcription (RT)-PCR and real-time (RT) PCR approaches. Overexpression of CLOCK and BMAL1 together resulted in a significant increase in pre-miR-219-1 transcript levels as determined by semiquantitative RT-PCR and real-time PCR, whereas expression of CLOCK or BMAL1 singly had limited effect. Both pre-miR-132 and gapdh levels were largely unaffected by CLOCK and BMAL1 coexpression (Cheng, 2007).

The role of CREB as a regulator of miR-132 transcript was examined. In primary cortical neurons, both forskolin (an adenylate cyclase agonist) and KCl depolarization induced expression of pre-miR-132 as assessed by RT-PCR. Expression of A-CREB, which blocks CREB-mediated transcription, suppressed forskolin- and KCl-induced pre-miR-132 expression (Cheng, 2007).

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