Gene name - Cyclic-AMP response element binding protein B at 17A
Synonyms - CREB, dCREB2
Cytological map position - 17A
Function - transcription factor
Symbol - CrebB-17A
Genetic map position -
Classification - basic leucine zipper
Cellular location - nuclear
Memory involves synthesis of new proteins. In organisms as diverse as Aplasia, Drosophila and humans, the enzyme cyclic-AMP response element binding protein (CREB) is directly involved in the establishment of the capacity for long term memory (Bailey, 1994). CREB is so named because of the part it plays in binding the cyclic-AMP response element in the promoter of the mammalian proto-oncogene Jun. CREB was originally described as a factor stimulating the transcription of genes in response to growth factors and phorbol esters.
Suppose a Superfly were to exist, one that could learn from a single experience without need for repetition. In fact, such an animal has been created. Precocious long term memory has been observed after a single cycle of training when a transgene for a PKA-responsive activator form of CREB is expressed before training takes place (Yin, 1995b). This activator is one isoform found in multiple splicing of CREB.
It seems that flies need to rest during training periods in order to establish long term memory. Mass training, that is, repeated experience without rest, fails to produce long term memory. Spaced training, training with rest periods between repeated tests, produces two functionally independent forms of consolidated memory: long term memory (LTM) and anesthesia resistant memory (ARM). LTM is disrupted by cyclohexamide, a drug that blocks protein synthesis, in contrast to ARM, which remains unaffected. LTM, and not ARM is disrupted by induced expression of a dominant negative transgene of CREB (Yin, 1995b).
Understanding of gene activation in the memory pathway comes from an analysis of transcriptional regulation of the Jun gene. A classic model for the formation of heterodimers is the Jun/Fos heterodimer, the predominant form of the gene regulator AP-1 (activator protein-1). Analysis of the Drosophila Jun promoter reveals binding sites for AP-1, CREB and DTF1, a protein that acts as an enhancer binding factor of the D2 promoter of Antennapedia (Perkins, 1988a and b). Thus Jun is regulated by JUN itself combined with FOS and by two other proteins: DTF1 and CREB. Three of these proteins (FOS, JUN and CREB) are responsive to the phosphorylation cascade that results from cAMP signaling. Additional information on the role of cyclic-AMP in fly memory (Davis, R. L., 1996) will be found in the dunce and rutabaga sites.
The role of dCREB2 (CrebB-17A) in circadian rhythms has been examined. dCREB2 activity cycles with a 24 hr rhythm in flies, both in a light:dark cycle and in constant darkness. A mutation in dCREB2 shortens circadian locomotor rhythm in flies and dampens the oscillation of period, a known clock gene. Cycling dCREB2 activity is abolished in a period mutant, indicating that dCREB2 and Period affect each other and suggesting that the two genes participate in the same regulatory feedback loop. It is proposed that dCREB2 supports cycling of the Period/Timeless oscillator. These findings support CREB's role in mediating adaptive behavioral responses to a variey of environmental stimuli (stress, growth factors, drug addiction, circadian rhythms, and memory formation) in mammals and long-term memory formation and circadian rhythms in Drosophila (Belvin, 1999).
To measure dCREB2 activity in vivo, transgenic Drosophila lines were constructed carrying the luciferase reporter gene driven by an enhancer element comprised of consensus CREB binding sites. Three cAMP response elements (CREs), 5'-TGACGTCA-3', were placed upstream of the TATA box region of the hsp70 gene promoter, followed by the luciferase reporter gene. This sequence was flanked by the scs and scs' insulator elements to reduce potential positional effects caused by the random insertion site of the transgene. The transfected lines are referred to as CRE-luc lines. A mutant CRE-luc reporter construct (mCRE-luc) was also generated in which the consensus CRE sites were mutated to TGAAATCA. dCREB2 protein binds this mutant CRE site with at least 20-fold lower affinity in gel shift experiments. This construct is otherwise identical to wild-type CRE-luc (Belvin, 1999).
The expression of luciferase in the wild-type CRE-luc flies oscillates in a 24 hr rhythm, both in a light:dark cycle and in constant darkness. The main peak of activity occurs just after lights out, with the nadir just before the main peak. Since this rhythmic transcription pattern is sustained in constant darkness, it is regulated by the circadian system, rather than simply being a response to light. In light:dark conditions, a second peak is observed in the middle of the day; however, these two peaks gradually blend together under conditions of constant darkness. This pattern is very similar to that seen for per activity. The per-luc reporter also exhibits a similar secondary peak under light:dark conditions, even though per RNA peaks only once per cycle. It is likely that the secondary peaks of both reporters, which occur during the day, are due to a light response of luciferase rather than a circadian response. The expression level of the mCRE-luc reporter is drastically reduced relative to the wild-type reporter, indicating that the CRE sites mediate the high-level expression of the wild-type reporter (Belvin, 1999).
These experiments show that dCREB2 activity is under circadian control, but they do not show whether dCREB2 plays a role in maintaining the rhythms or is just responsive to them. The A dCREB2 mutation, S162, one that allows for survival of a few flies to adulthood, was used to address this question. To test for behavioral effects of the mutation, S162 escaper males were assayed for circadian locomotor activity. The flies were tested for 10 days in constant darkness to determine whether they display normal circadian fluctuations in activity. Of the 34 S162 mutants tested, 13 (38%) were arrhythmic while the 21 that were rhythmic had a short period averaging 22.8 hr. None of these flies had a wild-type 24 hr rhythm. The high percentage of arrhythmicity is typical of mutations that affect period length. To verify that this behavioral phenotype is specific for the S162 mutation, the phenotype was rescued by induction of the hs-dCREB2-10 transgene. To induce the transgene, larvae and pupae were subjected to a daily 60 min heat pulse of 37°C during development. All of the rescued flies (12/12) were rhythmic, and they all displayed normal circadian locomotor rhythms of 23.5-24 hr. This demonstrates that the short period phenotype is caused by the S162 mutation rather than a second site mutation elsewhere on the chromosome. It also shows the involvement of dCREB2 in the timing of the clock (Belvin, 1999).
If S162 is indeed acting in the clock, then it should affect the per clock gene. The effects of S162 on two different per-dependent reporters were examined. The first is a transcriptional fusion with a 4.2 kb fragment of the per promoter upstream of the luciferase reporter gene, referred to as per-luc. The second is a translational fusion containing the same promoter fragment, plus the 5' untranslated region and the first 2.4 kb of the per coding region fused in frame to the luciferase gene, referred to as BG-luc. When the expression of these reporters was compared, it was found that the BG-luc reporter cycles much more robustly than the per-luc reporter, consistent with the interpretation that there are at least two mechanisms contributing to the cycling of Per: one mediated by the promoter, and the other(s) mediated by sequences in either the per transcript or Per protein itself. S162 affects the two reporters differently. The S162 mutation reduces both the expression level and cycling pattern of the per-luc reporter. However, its effect on the BG-luc reporter is weaker. In S162 flies, the BG-luc reporter maintains a robust cycling pattern, although its expression level and amplitude are reduced. The peak in the mutant background also occurs in advance of the peak in wild-type flies, consistent with the short period phenotype of these flies (Belvin, 1999).
In order to demonstrate a direct effect of the S162 mutation on the clock, its effects on the Per protein itself were examined. Wild-type and S162 flies were entrained on a 12 hr light:12 hr dark cycle and aliquots were frozen every 2 hr throughout the cycle. Head extracts were prepared and analyzed by Western blot using an antibody directed against Per. Per is present at very low levels at ZT 6 and ZT 8 (Zeitgeber time; 6 and 8 hours after lights on), increasing through the lights of period to peak levels before lights on, which occurs at ZT 0. A corresponding change in phosphorylation, and protein mobility, accompanies the change in absolute levels, with Per becoming more highly phosphorylated as it accumulates. This temporal pattern of Per is altered in the S162 mutant background, where Per is present at more equal levels throughout the circadian cycle. At the peak time, ZT 20, the amount of Per is at least comparable to that in wild-type flies; however, it decreases less at ZT 6 and ZT 8, when Per is virtually absent in wild-type flies. At these trough periods of Per expression, a discrete doublet protein band persists in S162, perhaps representing preservation of certain phosphorylated forms. There also seems to be a general increase in the amount of Per protein throughout the cycle in the mutant flies. The change in both per-luc expression and Per protein levels in the S162 mutant background demonstrates that Per activity is under the influence of the dCREB2 gene. The effects of S162 on Tim protein were assayed in the same experiment. The effect of S162 on Tim is much more subtle than the effect on Per. The Tim protein appears to accumulate slightly sooner in the mutant than in the wild type (ZT 12 versus ZT 14); however, its overall oscillation remains fairly normal (Belvin, 1999).
What is the biological significance of dCREB2's participation in the clock? Since a mutation in the dCREB2 gene affects the clock, the possibility exists that stimuli that activate CREB could affect circadian rhythmicity. This is clearly true for light pulses that reset the clock when delivered at the appropriate times during the nighttime period and have been shown to induce CREB phosphorylation in mammals. What about 'noncircadian' stimuli? One example of this phenomenon is work showing that rats can learn an association between air puffs delivered to the eye (conditioned stimulus) and light (unconditioned stimulus). After acquisition, the conditioned stimulus alone, when delivered during the nighttime period, phase shifts the clock. This suggests that there may be much more overlap in the conditioning circuitry and the circadian circuitry than is generally appreciated. Since the molecules within all of these neurons are similar, the molecular pathways may also overlap. CREB-mediated transcription occurs in response to stimuli that induce stress, long-term memory formation, and growth factor responses. What is the significance of the fact that this transcription factor also responds to circadian signals, which happen regularly over a 24 hr period? One speculation is that the cyclical pattern in CREB activity means that there are optimal periods during the 24 hr cycle for CREB-responsive physiological processes. One physiological process for which there is evidence that the nighttime period is important is the consolidation of long-term memory formation. Over the years, a large number of experiments, primarily on rodents and humans, suggest a possible involvement of some aspect of nighttime sleep in the consolidation of memory. A unifying interpretation of the physiological and behavioral data is that the brain utilizes the sleep period to 'replay' plasticity-related events, thereby ensuring their total consolidation and maintenance. This replay occurs during sleep, when there is minimal external input into the brain. It is speculated that the circadian system controls some of the neuronal activity that occurs during the sleep period. This activity in turn leads to activation of CREB-responsive transcription, which may be important in consolidating and maintaining preexisting circuits and memories. Experiments to test these types of ideas are underway (Belvin, 1999 and references).
CrebB-17A consists of seven isoforms (alternatively spliced forms). The isoforms fall into two categories. In one category, four of the seven isoforms have the basic leucine zipper domain of exon 7. In the second category the b zip domain is absent. The basic zip domain consists of a basic activation domain followed by a leucine zipper domain. A short amino acid motif is a target for phosphorylation by kinases (P-box). The P-box is located carboxyl terminal to a glutamate-rich region (Yin 1994).
date revised: 10 July 99
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