Rhythmic transcription: The molecular basis of circadian melatonin synthesis

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Biology of the Cell (1997) 89, 487-494 o Elsevier, Paris

Review

Rhythmic transcription: The molecular basis of circadian melatonin synthesis Nicholas S Foulkes, David Whitmore and Paolo Sassone-Corsi * lnstitut de Gh%que et de Biologie Molkulaire et Cellulaire, CNRS-INSERM-UP, I, rue Laurent-Fries, CU de Strasbourg, 67404 lllkirch cedex, France

Adaptation to a changing environment is an essential feature of physiological regulation. The day/night rhythm is translated into hormonal oscillations governing the physiology of all living organisms. In mammals the pineal gland is responsible for the synthesis of the hormone melatonin in response to signals originating from the endogenous clock located in the hypothalamic suprachiasmatic nucleus (SCN). The molecular mechanisms involved in rhythmic synthesis of melatonin involve the CREM gene, which encodes transcription factors responsive to activation of the CAMP signalling pathway. The CREM product, ICER, is rhythmically expressed and participates in a transcriptional autoregulatory loop which also controls the amplitude of oscillations of serotonin Nacetyl transferase (AANAT), the rate-limiting enzyme of melatonin synthesis. In contrast, chick pinealocytes possess an endogenous circadian pacemaker which directs AANAT rhythmic expression. CAMP-responsive activator transcription factors CRJZB and ATFl and the repressor ICER are highly conserved in the chick with the notable exception of ATFl that possesses two glutaminerich domains in contrast to the single domain encountered to date in mammalian systems. ICER is CAMP inducible and undergoes a characteristic day-night oscillation in expression reminiscent of AA-NAT, but with a peak towards the end of the night. Interestingly CREB appears to be phosphorylated constitutively with a transient fall occurring at the beginning of the night. Thus, a transcription factor modulates the oscillatory levels of a hormone. (0 Elsevier, Paris) AANAT / CREM / melatonin / pineal

Day-night and seasonal changes in the environment dominate the lives of plants and animals, thus many facets of physiology are adapted to anticipate these changes. In vertebrates, the endocrine system plays a key role in directing temporal changes in physiology; thus circadian and seasonal rhythmicity character&e the action of many hormones. Since long-term physiological adaptations are ultimately mediated by changes in gene expression (Krieger 1979; Felig ef nl, 1987), the dynamic properties of transcription factors and the signalling pathways which regulate them, constitute an essential link in the relay of temporal information.

‘Correspondence

and reprints

Circadianmelatoninsynthesis

Fundamental to plant and animal physiology is the presence of an endogenous circadian pacemaker or clock (Aschoff, 1981). Daily input of light and other stimuli continually reset this clock and synchronise it with the environment. Clock output pathways subsequently modulate various aspects of physiology. A principal output from the clock is the night-time secretion of the hormone melatonin which is synthesised by the pineal gland. In birds, reptiles, amphibians and fish the pineal gland is directly light sensitive earning it the popular name ‘the third eye’ (Collin, 1971; Dodt, 1973; Okshe, 1984). In these lower vertebrates the pineal possessesalso an endogenous clock function (Takahashi et al, 1980; Menaker and Wisner, 1983). In mammals, pinealocytes are neither light-sensitive nor possess a clock. The clock is instead located in a separate hypothalamic structure, the suprachiasFoulkeset al

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CH2CH

(NH&OOH Tryptophan

Tryptophan

hydroxylase

t 544ydroxytryptophan

t

Aromatic amino decarboxpase

acid

Serotonin

t

N-Acetyltransferase (AA/VAT)

N-Acetyleerotonin

Hydroxy-0-methyltrans (HIOMT)

ferase

(1 CH30

CH,CH2NHCOCH3 H

Fig 1. Melatonin synthesis pathway. Serotonin is acetylated by serotonin N-acetyltransferase OAANAT)to produce N-acetylserotonin. N-acetylserotonin is in turn methyiated by the enzyme hydroxyindole-Cl-methyltransferase (HOMTl.

matic nucleus (SCN). Light stimuli are received by the SCN indirectly via the retinohypothalamic pathway. Via a multisynaptic pathway, neurons of the SCN project to the intermediolateral cell column of the spinal cord which contains cell bodies that innervate the superior cervical ganglion. Sympathetic postganglionic neurons then ascend to innervate the pineal gland. The synthesis of melatonin in the pineal gland begins with the N-acetylation of serotonin-by sero-, tonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) followed by addition of a methyl group at the 5-hydroxy position uia the enzyme, hydroxyindole-0-methyltransferase (HIOMT) (Axelrod and Weissbach, 1960) (fig 1). The rate limiting step in melatonin biosynthesis is catalysed by AANAT and thus it is a rhythm in the activity of this enzyme which underlies the rhythmic production of melatonin (Klein and Weller, 1970; Klein, 1985). This enzyme is remarkable in its tight regulation by CAMP. CAMP stimulates enzyme activity as well as its transcription and translation (Takahashi, 1994). In the rat AANAT activity displays a diurnal rhythm with night-time peak levels up to 100 times higher than daytime values (Klein and Weller, 1970). The AANAT and melatonin rhythms derive from activation of the pineal’s sympathetic innervation in mammals during the night. Norepinephrine binds to padrenoceptors and thus stimulates adenylate cyclase activity. The resulting increase in CAMP stimulates AANAT activity (Takahashi, 1994). ul,-Adfenergic Circadianmelatoninsynthesis

receptors also participate in AANA’I‘ stimuiation (Klein el ali 1983), apparently by activating the phosphoinositide (PI) cycle and protein kinasc C (PKC) which potentiates @receptor-induced cAMi’ production Coon pi al (1995) and Borjigin et al (1995) independently cloned AANAT from sheep and rat pineal glands, respectively. Subsequently chick, human, and fish clones have been isolated bv homology and characterised in detail (see Klein r;! u/, 1997 and references cited therein). Originally the sheep enzyme was- cloned by using a cDNA expres sion library (Coon rf al, 1995). Positive clones wcrc~ identified by an enzymatic assay for their ability to acetylate arylalkylamine substrates. ‘The rat cDNA was isolated using a PCR based subtractive hybridisation technique using day and night pineal gbnd RNA (Borjigin rf nl, 1995). Interestingly, while irt the ovine pineal levels of the AANAT transcript change only slightly between day and night, in tht: rat there is a loo-fold day-night oscillation in mRNA levels even though in both systems AANA’I activity oscillates strongly (Borjigin ef crl, 1995; Coon :>bal, 1995; Roseboom et 171, 1996; Klein Pt al, 19973

Night-time intracellular rise in cAMP is a key player in subsequent upregulation of AANAT and melatonin synthesis (Klein, 1985; Sugden ct izl, 1985; Vanecek et rzl,1985). increases in intracellular CAMP levels lead to activation of CAMP-dependent prvtein kinase A (PKA) and the transport of active catnlytic subunits to the nucleus (Krebs and Beavn,~ 1979). Nuclear phosphorylation targets include ,I group of transcription factors which modulate the expression of CAMP responsive genes (Foulkes and Sassone-Corsi, 1996). These factors constitute a family of both activators and repressors which bind as homo- and heterodimers to CAMP responsive elements (CREs). They belong to the basic leucine zipper (bZip) class of transcription factors. These pro teins contain a leucine zipper, an a-helical coiled-coil structure, which is needed for parallel dimerization; and an adjacent basic domain, rich in lysine and arginine residues, needed. for direct contact with DNA. Their function is tightly regulated by phosphorylation (Foulkes and Sassone-Corsi, 1996). ConstitutiveIy expressed factors such t~s CREB (CRE-binding protein) and ATFl (activating transcription factor 1) are phosphorylated by PKA and thereby converted into powerful transcrip tional activators. Their transcriptional activatjon domain contains a phosphorylation box (I’-bax) Foulkeset ai

Biology of the Cell (1997) 89, 487-494

with consensus phosphorylation sites for several protein kinases including PKA (Foulkes and Sassone-Corsi, 1996). In CREB this is flanked by two glutamine-rich zones, termed Ql and Q2 which are believed to make contacts with the basal transcription machinery while ATFl includes only a single Q domain (Q2) (Rehfuss et aI, 1991). The CRE-modulator (CREM) gene is closely related to CREB but appears to play a privileged role in cells of the pituitary-hypothalamic-gonadal axis (Foulkes et al, 1991). The CREM gene generates a family of alternatively spliced isoforms (Laoide et al, 1993; Foulkes and Sassone-Corsi, 1996). A unique feature of CREM is the presence of two alternative DNA binding domains, interchanged by the differential use of splicing acceptor sites (Foulkes et al, 1991). CREM isoforms function as both activators and repressors of CAMP directed transcription and have a characteristic cell- and tissue-specific pattern of expression, with high levels of CREM activators notably in the testis (Foulkes et al, 1991,1992). In addition, the use of an alternative CAMP-inducible promoter (P2) at the 3’ end of the CREM gene generates the factor inducible CAMP early repressor (ICER) (Molina et al, 1993; Stehle et al, 1993). This small factor contains only the CREM DNA binding domain and functions as a dominant repressor of CAMP induced transcription. It acts by binding to CRE elements either as a homodimer or as heterodimeric complexes with other CRE activators. Since it lacks the transcriptional activation domain, ICER’s repression function is primarily regulated by its intracellular concentration (Molina ef al, 1993; Stehle et al, 1993). Importantly, ICER participates in a negative autoregulatory loop since the ICER protein binds with high affinity to the CRE elements in its own promoter (Molina et al, 1993).

ICER AND AANAT RHYTHMS Like AANAT, ICER mRNA displays dramatic diurnal rhythmicity in the rat pineal gland (Stehle et al, 1993). The peak of ICER mRNA occurs during the second part of the night, just preceding the decline of melatonin synthesis. Interestingly, this pattern is developmentally regulated, being absent at birth and maturing only between the first and second week of postnatal development (Stehle ef al, 1995). This coincides with the maturation of a functional sympathetic innervation linking the SCN and pineal as well as with the maturation of CAMP inducibility of gene expression within the pineal and with the appearance of elevated night-time melatonin synthesis (Stehle et al, 1995). Together these observations suggested that ICER might function as a downregulator of melatonin production by repressing CAMP-induced AANAT transcription Circadianmelatoninsynthesis

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at the end of the night (Takahashi 1994; Stehle et aI, 1995). A direct evaluation of the relationship between ICER and AANAT has been made possible by the generation of mice which carry a null mutation at the CREM locus (Nantel et al, 1996). This mutation truncates the C-terminal DNA binding domain and thereby inactivates all CREM isoforms, including ICER. Male mutant mice are completely sterile. Their testes display a complete arrest at the first step of spermiogenesis resulting in a lack of developing spermatids (Blendy et al, 1996; Nantel et nl, 1996). This is consistent with the abundant expression of CREM activator protein in haploid male germ cells and points to CREM functioning as a master controller of postmeiotic gene expression. The majority of inbred mouse strains used for transgenic and homologous recombination experiments have genetic defects in melatonin synthesis (Goto et al, 1989). By biochemical and genetic analyses, defects in AANAT or AANAT regulators and HIOMT have been implicated in this deficiency (Ebihara et al, 1986). Thus the first step was to test whether AANAT mRNA is expressed in the 129/sv strain used for the CREM knockout studies. By RNAse protection assay using a rat AANAT riboprobe to analyse mouse pineal RNA, it was demonstrated that this mouse strain does indeed show a night-time induction in AANAT expression, the timing of which is identical to that in the rat. The same AANAT expression pattern was encountered in C3H/He mice, an outbred mouse strain which does produce melatonin (Foulkes et al, 1996a). This indicates that the genetic defect in melatonin biosynthesis can not be accounted for at the level of AANAT transcription. The two mice strains also display identical profiles of elevated ICER nighttime expression, again with the precise timing being the same as that in the rat (Foulkes et al, 1996a). Expression of the transcription factor Fra-2 (Fos-related antigen) was also tested in the mouse pineal. Fra-2 mRNA and protein have been documented to vary diurnally in the rat pineal gland with an elevation in the early part of the night cycle which appears to be directed by adrenergic signals (Baler and Klein, 1995). Furthermore, like ICER, Fra-2 has been implicated as a negative regulator of AANAT expression (Baler and Klein, 1995). The mouse kinetics of Fra-2 expression are the same as those in the rat. Thus, the patterns of ICER, AANAT and Fra-2 expression indicate that rat and mouse pineal glands can be considered equivalent in terms of adrenergically regulated gene expression (Foulkes et al, 1996a). In the CREM mutant mice, with the exception of time points during the day where mutant and wildtype control animals display an equivalent low basal level of expression, the mutant animals have Foulkeset al

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significantly higher levels of night-time AANA-I mRNA than their wild type counterparts (Foulkes et nl, 1996a). Specifically, in the CREM null mutants a rise in AANAT transcript is detected earlier at the beginning of the night, reaches a higher peak of expression and then persists longer than in wild type siblings. Thus the consequence of removal of ICER protein seems to be the relief of a general dampening effect upon night-time AANAT kxpression. In contrast, the timing and magnitude of Fra-2 expression is equivalent in wild-type and mutant animals. Normal Fra-2 expression in the mutant animals demonstrates that the deregulation of AANAT expression does not extend to all adrenergically regulated genes. Furthermore, the Fra-2 result indicates that clock-derived adrenergic signals are not grossly altered in the knockout animals.

To understand the molecular mechanisms whereby 1CER downregulates AANAT expression, the AANAT promoter was cloned and sequenced. A CRE element (TGACGCCA), different from the consensus (TGACGTCA; Sassone-Corsi, 1995) by only one mismatch, was identified at position -108. A 378 bp promoter fragment including this region is sufficient to direct CAMP inducible transcription of a reporter gene and also the downregulation of CAMP-activated transcription by co-expressed ICER. ICER protein generated in bacteria binds to the AANAT CRE. Moreover, a high mobility tom plex binds to this CRE element in nuclear extracts prepared from mouse and rat pineal glands which is absent in extracts prepared from the CREM knockout mice. Thus, endogenous ICER protein clearly binds to the CRE element that regulates AANAT. The AANAT transcript is upregulated at all stages of its night-time induction in CREM knockouts relative to wild type controls. This indicates that ICER dampens AANAT transcription throughout the night and not as originally predicted simply at the end of the night when melatonin synthesis naturally falls. Consistent with this function, the ICER protein persists throughout the day-night cycle with a shallow increase at the end of the night under normal conditions, in contrast to the striking diurnal variations in its mRNA (Foulkes e’t al, 1996b). These findings support the following scenario for ICER function in the rat pineal gland (fig 2). Adrenergic stimulation at night drives CREB phosphorylation and the termination of adrenergic stimulation towards morning is associated with its dephosphorylation (Foulkes et al, ‘I996a). Abundant Circadianmelatoninsynthesis

Biology of the Cell (1997) 89, 487-494

evidence indicates that CREB phosphorylation involves PKA (Zatz et al, 1978) while the phosphatase that dephosphorylates CREB in the pineal has vet to be identified. Phosphorylated CREB binds to and activates the CREM P2 promoter and thereby drives night-time transcription of ICER. Dephosphorylation of CREB and the instability of the ICER transcript causes ICER mRNA levels to fall drama-tically to low basal levels by the beginning of the day. In contrast, the JCER protein is more stable and therefore persists at elevated levels throughout the day and night. By binding directly to the CRE element in the AANAT promoter, ICER modulates the rate and magnitude of melatonin induction in response to adrenergic signals by exerting a damp’ ening effect (fig 2). Thus the negative regulatorv role of ICER operates throughout the 24-h cycle and not exclusively during the downregulation ot melatonin synthesis occurring at the end of thtq night. Normal Fra-2 expression in the CREM mutant mice strongly indicates that negative regulation by ICER in the pineal gland does not extend equally to all adrenergically regulated genes. Diffe rential binding affinities of activators and repressors to the respective CRE elements may explain this observation. This scenario would predict that the downregulation of the AANAT transcript at the end of the night would in part be determined by .t combination of the dampening effect of ICER, dephosphorylation of the transcriptional activator CREB and possibly also the induced expression o! factors such as Fra-2.

The striking day-night oscillation of the ICER transcript underlying only a shallow fluctuation of the 1CER protein is puzzling. One possible explanation for this is that it may enable ICER protein levels ho be modulated according to long-term changes in pineal gland function. The duration of night-time melatonin synthesis is flexible and dictated by night length. In this way, the melatonin signal conveys not onlvi the day-night transition but .also sttasonal information to the animal (Bartness rf ai 19931. Thus change in the timing of adrenorgic stimulation is an important facet of pineal gland function. Since the pool of ICER protein is maintained by night-time adrenergic stimulation, the size of this pool would be predicted to change according to the duration of the night. This prediction seems to be confirmed. When rats are adapted to longer photoperiods, the basal level of ICER protein falls (Foulkes r:t a/, 1996b). Adaptation to increasing photoperiods is associated with an increased rate of TCER mRNA expression during Foulkeset ai

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CREB

Activated

signals

CREM Feedback Loop Repressed

Fig 2. The role of the CREM feedback loop in transducing a rhythmic clock-directed signal into rhythmic hormone synthesis. Schematic representation of the regulatory pathway responsible for generating rhythmic melatonin synthesis. Nighttime adrenergic signals originating from the clock, activate PKA and thus phosphorylate CREB. During the day, dephosphorylation is achieved by phosphatase action. Thus clock-directed signals determine the equilibrium position. Phosphorylated CREB activates the P2 promoter of the CREM gene and thus induces the expression of ICER. ICER downregulates its own expression constituting the CREM feedback loop. The balance between the proportion of phosphorylated CREB (positive effect) and ICER protein levels (negative effect) determines the transcriptional activity of the AANAT promoter. Thus the promoter cycles between activated and repressed states as a function of time. In this way, AANAT mRNA oscillates between high night-time and low basal daytime levels and determines the characteristic day-night oscillation of AANAT activity. This ensures rhythmic melatonin synthesis (adapted from Foulkes et al, 1996a).

the first part of the night (fig 3). This is entirely consistent with a reduction in the dampening effect of ICER resulting from a decrease in the basal level of ICER protein. Furthermore, there also seems to be an accompanying rise in the speed and magnitude of CREB phosphorylation with increasing photoperiod. This is also consistent with a more rapid night-time CAMP-induced expression (Foulkes et al, 199613). Together these findings support the notion that the nuclear response to CAMP can either be sub- or supersensitive to adrenergic signals depending on the duration of previous nights (fig 3). Injection of rats with the Padrenergic agonist isoproterenol and measurement of the induced levels of the ICER transcript reveals a refractory period to induction following the night, during which isoproterenol fails to elicit a maximal transcriptional response (Foulkes et al, 199613). Similar refractory periods have already been documented in cellular systems (Lamas and Sassone-Corsi, 1997) The duration of this refractory phase increases with decreasing photoperiod. Furthermore, when the refractory period overlaps the beginning of the following night, this is predicted to constrain the timing and magnitude of induced gene expression Circadian melatonin synthesis

(fig 3). Thus in summary, day length is reflected at the transcriptional level by the duration of the transient night-time ICER transcript peak which in turn sets the basal level of the persistent ICER protein and thereby determines the sensitivity of the CAMP transcriptional response (Foulkes et al, 199613).

CAMP RESPONSIVE FACTORS AND THE CHICK PINEAL The ‘intrinsic’ transcriptional mechanisms within mammalian pinealocytes documented here are potentially of great importance. However, the mammalian pineal gland remains an organ driven by the SCN clock rather than an oscillator in its own right. It is thus of great interest to assess the relative contribution of ICER and other transcriptional regulators to melatonin rhythmicity in lower vertebrates such as the chick, where CAMP fluxes which drive melatonin synthesis are generated at least in part by a pinealocyte endogenous circadian clock (Deguchi, 1979; Takahashi et al, 1980). The chick pineal gland has been the subject of extensive studies of photoreception, clock function and melatonin synthesis. The ability to prepare primary Foulkes et al

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Short Delay until maximum inducibility of gene expression

Long photoperiod (Supersensitive)

+

Short photoperiod (Subsensitive)

Long Relay until maximum inducibflity of gene expression

Fig 3. Schematicrepresentationof ICERexpressionand Inducibility in the rat pineal gland following adaptation of animals to short and long photoperiods. Under different photoperiods, peak levels of tCERtranscript are comparable,however, ICERexpressionincreasesvery rapidly at the beginningof the night (indicatedby a black bar) underlong photoperiod,while under short photoperiods the increase is more gradual. The dotted arrows delineate the refractory period: during this phasemaximuminducibilityof ICERmRNA(tested by injection of the adreoergic agonist, isoproterenol)is regainedfollowing a fall which accompaniesthe night-timerise of ICERtranscript. Increasingnight length dictates a slowergain in inducibility. Thereby, ICERdisplays different modes of transcriptional regulation, being supersensitiveto adrenergic stiinulation under long photoperiodand subsensitiveduringthe short photoperiod(adaptedfrom Foulkeset al, 1996b).

cultures of chick pinealocytes which continue to synthesise melatonin with a 24-h rhythmicity and retain photoreceptive properties has been central to their utility. The recent cloning of the chick AANAT gene represents an important advance in understanding the molecular basis of clock regulatory systems (Bernard et ul, 1997a). The first results analysing the expression pattern of chick AANAT have revealed that the daytime basal mRNrl, expression is much higher than in the rat and that at night there is a IO-fold increase in these levels (compared to loo-fold in the rat). This oscillation persists under constant darkness or light in intact birds and furthermore light pulses phase shift the expression profile (Bernard et al, 1997a). Combined these findings suggest that the AANAT promoter is clock regulated (Bernard et a!, 1997a). The observation that forskolin treatment day or night leads to a strong increase in AANAT enzyme activity while the transcript is not induced significantly has lead Circadianmelatoninsynthesis

to the conclusions that cAMI’ acts posttranscriptionally in the activation of AAN AT activity (Bernard et ad,1997b). In order to better clarify the role of CAMP in the chick pineal at the transcriptional level, we chose to study the expression of CAMP responsive transcription factors. We screened a chick pineal cDNA library with a rat ICER cDNA ~probe at low stringency and isolated a series of clones which corresponded to CREB, ATFl as well as ICER (Foulkes ef nl, unpublished observations). The conservation between the predicted chick proteins and their mammalian counterparts is extremely high with the exception of ATFl. In the activation domain of ATFl we document two glutamine-rich domains, while in the rat and human gene, only one domain has been defined (Rehfuss et aI, 1991; Foulkes ct 6~1, unpublished observations). Thus the chick ATFl is more closely related to CREB. Of the three factors, only ICER expression is CAMP-inducible as tested Foulkeset ai

Biology of the Cell (1997) 89, 487-494

in chick embryo fibroblast primary cultures. In the chick pineal, while CREB and ATFl levels are nonfluctuating throughout the 24-h cycle, basal daytime ICER mRNA expression is induced to reach a peak in the second part of the night. The kinetics and magnitude of this increase are reminiscent of those for chick AANAT. Interestingly, levels of the CREB protein are extremely high compared with the rat pineal. Furthermore, analysis of phosphorylation of serine 133 within the CREB P-Box, reveals that levels are constitutively elevated and drop transiently within 3 h from the beginning of the night (Foulkes et al, unpublished observations).

SUMMARY AND PERSPECTIVES There is a high degree of variability in the contribution of transcriptional control to the expression of AANAT even between mammalian species. Thus, there is still much to be learned about how the various transcriptional feedback loops described here regulate signal transduction originating from the clock itself. However, the highly conserved role of CAMP in regulating melatonin synthesis immediately implicates CAMP responsive transcription factors as important regulatory targets. Comparison of the roles of these factors in different species should thus provide important insights into the evolution and plasticity of signal transduction mechanisms at the nuclear level. Importantly, the principles of rhythmic transcription in the pineal gland may apply to other endocrine systems, eg the ability of a transcription factor to modulate the kinetics of hormone synthesis. Long-term changes in gene expression in response to hormonal signals in turn modulate the subsequent pattern of hormone production. In this way, CREM is a key element of a relay system permitting temporal adaptations of endocrine function in response to a changing environment.

ACKNOWLEDGMENTS We wish to thank SH Snyder, J Borjigin, JS Takahashi and K Seidenman for gifts of materials and discussions. This work was supposed by grants from CNRS, INSERM, CHUR, Rhane-Poulenc Rorer (Bioavenir), Fondation pour la Recherche Medicale and Association pour Recherche sur le Cancer (I%-C).

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