Circadian Rhythms: ICER is nicer at night (sir!)

JOSEPH S. TAKAHASHI JOSEPH S. TAKAHASHI

CIRCADIAN RHYTHMS CIRCADIAN RHYTHMS

ICER is nicer at night (sir!) Autoregulated expression of a gene encoding multiple transcription factors, including the newly discovered repressor protein, ICER, may play a fundamental role in the regulation of circadian rhythms. Circadian rhythms are 24-hour oscillations in an organism's behavior, physiology and biochemistry that constitute a fundamental organizing feature of all living systems. Evidence from various sources suggests that the circadian clock mechanism itself, the input pathways that synchronize the clock and the pathways through which the circadian clock regulates its output rhythms all involve transcriptional and translational processes [1]. At the output level, the transcription of a number of genes has been shown to be under circadian control in microorganisms [21, plants [3] and animals [4-7]. That transcription is subject to circadian control is important because of the opportunities it offers for investigating how the circadian clock regulates gene expression. Specifically, it should be possible to analyze the cis-acting regulatory elements and trans-acting factors through which the circadian clock controls transcription. Two recent papers from Sassone-Corsi and colleagues [8,9] describe a novel clock-controlled product of the mammalian CREM gene, which is named after its known product, the cAMP-responsive element modulator. The new CREM gene product, which is highly expressed in neuroendocrine cells and which encodes a potent repressor of cAMP-induced gene transcription, is named ICER for inducible cAMP early repressor. A remarkable circadian rhythm of ICER mRNA levels exists in the pineal gland of the rat, with high levels at night and low levels during the day. Molecular analysis of ICER shows that it is transcribed from a novel intronic promoter (2) in the CREM gene, which preserves the DNA-binding domain but excludes regulatory and activation regions. The P2 promoter contains four tandem cAMP-responsive elements (CRE), which confer cAMP inducibility upon ICER. Once its levels are elevated, however, ICER can negatively autoregulate its own expression. These new results highlight two new levels of CREM gene regulation and suggest that dynamic expression of ICER plays an important role in the neuroendocrine axis. For almost three decades, the pineal gland has provided an excellent model system for studying environmental and circadian regulation of a neuroendocrine system [10,11]. The mammalian pineal gland expresses a circadian rhythm in the production of the hormone melatonin, which is controlled by a circadian fluctuation in the enzyme arylalkylamine N-acetyltransferase (NAT). A well-described but tortuous neural pathway governs pineal NAT activity (Fig. 1). A hypothalamic circadian clock located in the suprachiasmatic nucleus

(SCN) drives rhythmic noradrenergic input to the pineal gland by way of the sympathetic nervous system. Photic input also reaches the pineal through the same pathway. At night, clock-driven norepinephrine release activates pineal -adrenergic receptors, which stimulate adenylate cyclase and cause an increase in intracellular levels of cAMP (Fig. 2). At the same time, norepinephrine also activates cal-adrenergic receptors, which cause an activation of the phosphoinositide pathway and a consequent elevation of intracellular Ca 2+ levels. Activated protein kinase C (PKC) then dramatically potentiates the stimulatory effects of receptors positively coupled to adenylate cyclase. Ultimately, this combined - and al-adrenergic receptor stimulation causes a synergistic elevation of intracellular cAMP levels [10]. Interestingly, cAMP appears to act at three different levels to regulate NAT in pineal cells (Fig. 2) [11]. First, cAMP seems to induce the transcription of either NAT itself or a regulator of NAT: NAT induction requires transcription during the early night, when NAT levels are rising, but not during the second half of the night, when NAT levels are elevated. Second, cAMP appears to stimulate the translation of NAT (or a NAT regulator). Unlike the transcriptional requirement, the translational requirement for NAT activity is continuous throughout the day. Third, cAMP appears to maintain NAT in an active form. Acute antagonism of 1-adrenergic stimulation, either by exposure to light during the night or by treatment with -adrenergic antagonists, leads to a rapid inhibition of NAT. This acute reduction in NAT activity is fast relative to the rate of turnover of the enzyme, implying that it is due to an active inactivation

Fig. 1. The neural system regulating pineal N-acetyltransferase in the rat. Light is detected by the eye, generating a signal that is transmitted by the retino-hypothalamic tract to the suprachiasmatic nucleus (SCN), which contains a circadian clock. The neural pathway includes the paraventricular nucleus (PVN), the intermediolateral cell column (IML), the superior cervical ganglion (SCG) and the pineal gland.

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Fig. 2. Adrenergic regulation of melatonin biosynthesis and ICER gene expression in a pineal gland cell. AC, adenylate cyclase; G, trimeric G protein; PKC, protein kinase C; PLC, phospholipase C; CRE, cAMP-responsive element; CREBP, CRE-binding protein; NAT, arylalkylamine N-acetyltransferase; HIOMT, hydroxyindole-O-methyltransferase; CREM P2, CREM gene P2 promoter. Yellow arrows indicate tentative interactions.

mechanism. Unfortunately, because NAT has yet to be purified and analyzed at the molecular level, the regulation of NAT by cAMP cannot yet be studied directly. This is one reason for the interest in the paper by Stehle et al. [8] on ICER expression in the pineal gland. CREM is a member of the CREB/ATF (CRE-binding protein/activator transcription factor) family of proteins which bind cAMI'-responsive elements [12]. The best known member of this family, CREB, is constitutively expressed in most cells and binds, either as a homodimer or as a heterodimer with other CREB/ATF members, to CRE sites in the promoters of cAMI'-regulated target genes [131. The CREM gene is interesting because it can generate both transcriptional activators (CREMt) and repressors (CREMca,O,y) by alternative RNA splicing [14,151. Like CREB [16], CREMT is activated by phosphorylation, on residue serine 117 [171 (equivalent to the phosphorylated CREB residue serine 133). Unlike other CREB/ATF members, however, many CREM isoforms are inducible, tissue-specific and developmentally regulated. For example, Foulkes et al. [18] have shown that CREM expression in the testes switches during spermatogenesis from repressor forms (CREMc,3,y) to an activator form (CREM), which has

two additional glutamine-rich regions. The developmental switch in CREM expression during the pachytene spermatocyte stage is regulated by the pituitary hormone, follicle-stimulating hormone (FSH), which causes a high level of CREMt expression by alternative polyadenylation and an increase in transcript stability [191. The levels of CREM regulation thus include alternative splicing, alternative translational initiation and alternative polyadenylation [15]. The two recent papers [8,9] document a surprising new level of CREM regulation. ICER is transcribed from a second intronic promoter, P2, and lacks all exons 5' of the y-exon. A new ICER-specific exon, located between the Q2 domain-encoding exon and the y-exon, contains the translational initiation site. The ICER gene products are therefore 108 and 120 amino-acid proteins that include the CREM DNA-binding domains, but not the amino-terminal phosphorylation domain (P-box) or the glutamine-rich domains (Q1 and Q2). Like other CREM isoforms, multiple ICER mRNAs are generated by differential splicing; this involves the exons encoding the two alternative DNA-binding domains and the y-exon, so that four isoforms of ICER are generated in total. ICER is the first example of a CRE-binding protein that lacks the phosphorylation domain, which suggests that

DISPATCH

in this case the level of expression may be the major determinant of its activity. In functional experiments using transient transfection of tissue culture cells, Stehle et al. [8] show that ICER is a powerful repressor of cAMP-induced transcription. These experiments are elegant because in addition to the typical use of cell lines, they were also performed with primary cell cultures from rat pineal gland. The results suggest that ICER is the most potent repressor of the CREM family to be tested. It also appears that ICER is the predominant form of CREM expressed in most tissues, with the highest levels in neuroendocrine cells such as the pineal, pituitary and adrenal glands. The results reported by Molina et al. [9] show that ICER is rapidly induced by the activation of the cAMP signaling pathway in a number of neuroendocrine cell lines. The kinetics of ICER induction are characteristic of an immediate early gene, peaking two to three hours after stimulation followed by a decline to baseline four to six hours later. The induction of ICER does not require protein synthesis; however, the subsequent decline is blocked by protein synthesis inhibitors, leading to superinduction of ICER in a manner similar to that seen with other immediate early genes. Interestingly, a cluster of four tandem CRE sites within the intronic P2 promoter region of the CREM gene are sufficient for the transcriptional activation of ICER by cAMP. Furthermore, Molina et al. [91 also show that ICER can repress its own production through a negative autoregulatory mechanism. Thus Sassone-Corsi and colleagues propose that activation of the cAMP pathway triggers a cascade of generegulatory events with the following scenario. First, upon activation by cAMP, protein kinase A would phosphorylate pre-existing CREB/ATF factors, causing transcriptional activation of target genes. These activators would also induce the transcription of ICER, eventually leading to a shift in the balance between CRE activators and repressors. The elevated levels of ICER could then repress the expression of CRE-regulated target genes. Finally, ICER would block its own transcription, leading to a return to the basal state. This positive and negative regulatory loop could underlie the temporal regulation of a number of cAMP-induced gene-expression programs in neuroendocrine cells. In the rat, Stehle et al. [81 show that ICER is under circadian-clock control, which is mediated neurally by the adrenergic input to the pineal gland. Like NAT, ICER mRNA is induced at night, peaking about six to nine hours after the light is switched off. The rhythmic variation of the ICER mRNA level persists in constant darkness, showing that it is circadian. Both constant light and acute light exposure at night cause dramatic decreases in ICER mRNA levels. The nocturnal elevation of ICER mRNA is under adrenergic regulation, because it is blocked by superior cervical ganglionectomy and by -adrenergic antagonists. Furthermore, treatment with -adrenergic agonists or with cAMP analogues in

organ culture mimics the nocturnal induction of ICER, showing that elevation of cAMP can induce ICER gene expression in the pineal gland. These experiments immediately suggest a role for ICER in the pineal gland. The time course of ICER expression in the pineal can explain a long-known paradox concerning the pineal gland. Continuous treatment of pineal glands with norepinephrine or dibutyryl cAMP causes an increase in NAT activity which plateaus after six to twelve hours and then decreases to baseline levels [20]. The decrease in NAT activity cannot be explained by desensitization of receptors, or by other steps upstream of cAMP, because it is seen in the presence of cAMP analogues. ICER expression, which peaks during the time NAT activity is declining, could lead to repression of NAT (or a regulator of NAT) gene expression (Fig. 2). Because ICER itself is induced by cAMP treatment in the pineal gland, the negative autoregulatory mechanism described by Molina et al. [91 would be expected to operate. If so, then the inability to induce ICER expression in the pineal gland during the day may reflect ICER autorepression. Although circadian regulation of gene expression appears to be a widespread phenomenon, there are still only a few examples where the control has been shown to be exerted at the level of transcription [1-7]. Along with the Drosophilaperiod gene [5,21], ICER provides an interesting example in which circadian regulation of gene expression is accompanied by a negative autoregulatory feedback loop. It is intriguing that the pineal gland, which contains circadian oscillators in non-mammalian vertebrates [1], uses such a feedback mechanism. Perhaps the neurally driven circadian rhythm of ICER gene expression in the rat pineal gland is a remnant of an oscillator feedback loop that operates in lower vertebrates. In any case, autoregulatory feedback loops in which both positive and negative regulation occur may be a common feature of rhythmic gene expression. References 1.

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THE DECEMBER 1993 ISSUE OF CURRENT OPINION IN NEUROBIOLOGY included the following reviews, edited by Alain Berthoz and Allen Selverston, on Neural Control:

Selected topics on the neural control of saccadic eye movement by D.L. Sparks and EJ. Barton Basal ganglia intrinsic circuits and their role in behaviour by J.W. Minx and W.T. Thach Presynaptic modulation of spinal reflexes by P. Rudomin, J.N. Quevedo and J.R. Eguibar Immediate representations in the formation of arm trajectories by Emilio Bizzi Hormonal control of neural function in the adult brain by Jean-Didier Vincent Cortical mechanisms controlling limb movement by Eberhard E. Fetz Tracing premotor brain stem networks of orienting movements by Alexej Grantyn, Etienne Oliver and Toshihiro Kitama Pattern generation by Ronald M. Harris-Warrick Subcortical motor control by Rodolfo Llinas Neural mechanisms of behaviour by Jeffrey M. Camhi Motor learning by Hans-Joachim Freund and Ulrike Halsband New transmitters and new targets on the autonomic nervous system by Carlos Barajas-L6pez and Jan Huizinga Movement dynamics by Stan C.A.M. Gielen Regulation of motor units by Mark Binder Circadian rhythms by Hugo Arechiga