Transcriptional control of circadian hormone synthesis via the CREM feedback loop

Abstract

Transcription factor cAMP-responsive element modulator (CREM) plays a key physiological and developmental role within the hypothalamic-pituitary-gonadal axis. The use of an alternative, intronic promoter within the CREM gene is responsible for the production of a cAMP-inducible repressor, inducible cAMP early repressor (ICER). ICER negatively autoregulates the ICER promoter, thus generating a feedback loop. We have previously documented a striking, clock-driven circadian fluctuation of CREM expression in the pineal gland. Oscillating ICER levels tightly correlate with fluctuations in the synthesis of the pineal hormone melatonin, whose production is also driven by the endogenous clock. Melatonin in turn regulates the hypothalamic-pituitary axis. The enzyme serotonin N-acetyltransferase (NAT) catalyzes the rate limiting step in melatonin synthesis. Thus, oscillations in NAT levels determine the circadian synthesis of melatonin. Here we demonstrate that NAT expression is dramatically increased in CREM-deficient mice that we have generated by homologous recombination. Characterization of the NAT promoter shows the presence of a ICER binding site. In addition, transfection studies show that ICER powerfully represses NAT transcription. Our results implicate CREM as a central regulator of output functions of the clock. Indeed, CREM acts as a key regulator of oscillatory hormonal synthesis.

Figure 1

CREM and NAT rhythmic expression is conserved in melatonin-deficient and normal mouse strains. RNase protection assays of NAT and CREM mRNA expression in a melatonin deficient (129/Sv) and normal (C3H/He) mice (37). Mice were sacrificed at 12:00 (D) or 24:00 (N) as indicated (lanes 2–5 and 7–10). Also, 129/Sv mice were injected with isoproterenol (+Iso) and sacrificed 2, 4, or 6 hr later. Together with a noninjected control (0), these mice were assayed for CREM expression (lanes 11–14). t represents tRNA controls for both probes (lanes 1 and 6). For the CREM probe (p6N1 in ref. 16) the 316- and 218-nucleotide bands correspond to CREM transcripts incorporating DBDI and DBDII, respectively (16). For NAT, the mouse protected fragment is 157 nucleotides as in the rat. In both 129/Sv and C3H/He mice, rhythmic day–night expression of both NAT and CREM is evident. Furthermore CREM expression is induced by isoproterenol injection as reported for the rat (22).

Figure 2

Day–night expression profiles of CREM, NAT and Fra-2 in the mouse and rat are identical. (A–C) Pineal RNA was extracted from mice sacrificed at the times indicated above the lanes and assayed for CREM, Fra-2, and NAT expression. (D) Rat RNA was extracted at the same time points and assayed for NAT mRNA. Both mice and rats were housed for 2 weeks prior to analysis in light/dark 12:12 lighting conditions (night from 19:00 to 07:00) as indicated by the black and white bar above the panels.

Figure 3

Rhythmic NAT expression in CREM mutant mice is deregulated. Pineal RNA from wt (+/+) and CREM mutant (−/−) littermates that were sacrificed in parallel at the times indicated throughout the night, were analyzed for NAT (A) and Fra-2 (B) expression by RNase protection assay. The NAT transcript begins to increase at 23:00 in the mutant mice while a similar increase is detected later in the wt animals between 24:00 and 01:00. The peak of expression at 03:00 is considerably stronger in the mutant than in the wt animals. Furthermore, the NAT transcript remains higher in the mutant than the wt mice at 09:00. In contrast, Fra-2 expression has the same profile in both sets of mice as shown in Fig. 2B. (C). The times of sacrifice relative to the 12-hr night are shown schematically. Above is represented the kinetics of nighttime NAT mRNA expression in the wt (+/+; black shaded curve) and CREM mutant mice (−/−; grey shaded curve). Lack of CREM significantly perturbs the normal profile of NAT expression.

Figure 4

The NAT promoter is directly regulated by ICER. (A) Schematic representation of the exon-intron organization of the rat NAT gene. Black bars denote the coding region while white bars show transcribed, noncoding sequences. An arrowhead indicates the position of the most 5′ transcription start site. Three introns interrupt the cDNA at positions +141, +359, and +514 relative to the transcription start site. A hatched box represents the CRE-like element. Above is shown the sequence of the first exon and the 5′ flanking region. Lower case and upper case letters denote nontranscribed and transcribed sequences, respectively. Italic, lowercase letters show the sequence at the 5′ boundary of the first intron. Numbers in the left-hand margin indicate the position of the adjacent base relative to the transcription start site. A CRE site in the promoter region is boxed and shown in boldface. Two AP-1-like sites are underlined (between positions −70 and −90). The sequence of the 5′ AP-1-like site (TGGGTCA), however, has been previously reported not to constitute a functional AP-1 site (38). (B) Results of transient transfection experiments using plasmids with an NAT promoter fragment cloned upstream of the CAT reporter gene (pPromNAT-CAT) and the NAT CRE element (TGACGCCA) inserted 5′ of a thymidine kinase promoter-CAT heterologous gene (pNAT-CRE-CAT). Transfected cells were either treated with forskolin (Fsk) or cotransfected with an expression vector for the catalytic subunit of PKA, in the presence or absence of a cotransfected expression vector for ICERIIγ (20, 21). CAT assay results were quantified by counting radioactivity of chromatographic plates and expressed as a fold induction. Both the NAT promoter and CRE element direct cAMP-inducible transcription that is strongly repressed by ICER. (C) Gel mobility shift assay of binding to the NAT CRE element. Bacterially generated CREMτ and ICERII protein (CREMτ and ICER, respectively) and nuclear extracts prepared from the pineal glands of rat and wt (+/+) and CREM-mutant (−/−) mice were assayed for binding. Both rat and wt mice show a high mobility binding complex that comigrates with that generated by bacterial ICER protein. This complex is absent in the CREM-mutant extracts.

Figure 5

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 down regulates 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 NAT promoter. Thus the promoter cycles between activated and repressed states as a function of time. In this way, NAT mRNA oscillates between high nighttime and low basal daytime levels and determines the characteristic day–night oscillation of NAT activity (alternating black and white bars below the activity curve denote night and day, respectively). The conversion of serotonin to N-acetylserotonin catalyzed by NAT importantly constitutes the rate limiting step of melatonin synthesis. Therefore this oscillating transcriptional control mechanism ensures rhythmic melatonin synthesis.