Rodent Aanat: Intronic E-box sequences control tissue specificity but not rhythmic expression in the pineal gland

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Abstract

Arylalkylamine N-acetyltransferase (Aanat) is the penultimate enzyme in the serotonin–N-acetylserotonin–melatonin pathway. It is nearly exclusively expressed in the pineal gland and the retina. A marked rhythm of Aanat gene expression in the rat pineal is mediated by cyclic AMP response elements located in the promoter and first intron. Intron 1 also contains E-box elements, which mediate circadian gene expression in other cells. Here we examined whether these elements contribute to rhythmic Aanat expression in the pineal gland. This was done using transgenic rats carrying Aanat transgenes with mutant E-box elements. Circadian expression of Aanat transgenes was not altered by these mutations. However, these mutations enhanced ectopic expression establishing that the intronic Aanat E-box elements contribute to the gene's pineal specific expression. A similar role of the Aanat E-box has been reported in zebrafish, indicating that Aanat E-box mediated silencing is a conserved feature of vertebrate biology.

Introduction

Daily rhythms of melatonin production in the pineal gland are governed by a rhythm in the synthesis and activity by the penultimate enzyme in the melatonin pathway, arylalkylamine N-acetyltransferase, Aanat; (Coon et al., 1995, Klein et al., 1997, Klein, 2006). This enzyme is encoded by Aanat which is rhythmically expressed in the rodent pineal gland, resulting in a ∼150-fold increase in the abundance of Aanat mRNA at night (Coon et al., 1995, Roseboom et al., 1996, Klein et al., 1997).

The increase in expression of the Aanat gene in the rat pineal gland appears to result from cis-acting mechanisms that involve a cAMP-responsive element (CRE)-CCAAT complex within the proximal promoter and a CRE located in the first intron (Baler et al., 1997, Baler et al., 1999). Transcription appears to be activated in response to cAMP-dependent phosphorylation of CRE binding protein (CREB) (Roseboom and Klein, 1995, Maronde et al., 1999). The first intron of the rat Aanat gene also contains an E-box element (Baler et al., 1999; Fig. 1A), which is of interest because a sub-set of E-boxes, described as circadian E-boxes (Munoz et al., 2002) mediate rhythmic gene expression through interaction with the trans-acting clock proteins CLOCK and BMAl1 (Jin et al., 1999, Reppert and Weaver, 2002); clock and Bmal1 are both expressed in the rat pineal gland (Namahira et al., 1999). Furthermore, functional Aanat E-box elements have been characterized in other species (Appelbaum et al., 2004, Chong et al., 2000); in the zebrafish these elements appear to form part of an enhancer (PRDM, pineal-restrictive downstream module) that includes photoreceptor conserved elements (PCEs). PCEs are found in genes expressed in the pineal gland and retina and are thought to be essential for this pattern of expression (Appelbaum and Gothilf, 2006). This enhancer contributes not only to the control of rhythmic expression of Aanat but also to the determination of tissue-specific zebrafish (zf) Aanat expression; the mechanism appears to reflect the interaction of OTX proteins and PCE in the enhancer (Appelbaum et al., 2005).

The ∼2 kb region between −218 and +1786 (relative to transcription start site) in the rat Aanat gene is sufficient to mediate both rhythmic and tissue-specific expression of rat Aanat (Burke et al., 1999, Smith et al., 2001). Of special interest is the importance of intron 1 to the rhythmic expression of Aanat. Little is known about this region, other than gross deletion results in decreased pineal expression and increased ectopic expression (Burke et al., 1999) and that it appears to influence the basal level of expression of the gene in non-pineal cells; in addition the CRE in intron 1 amplifies in vitro cAMP responsiveness of reporter constructs containing the CRE/CCAAT complex (Baler et al., 1999). The role of other putative regulatory elements in intron 1 has not been determined.

Here, we examined the role of two related E-box elements in intron 1, a consensus E-box element (CACGTG) and an E-box-like element (CACATG) (Fig. 1A). This was done using a reporter transgene composed of the ∼2 kb sequence that contains the promoter region and intron 1 of Aanat described above. The role of the intronic E-box elements was examined in vivo in animals carrying a reporter construct in which these elements were distrupted by the insertion of one or two bases. The results of these studies are presented here.

Section snippets

Design of constructs and transgenic rat production

The Aanat-luciferase transgene construct used in the production of the novel lines of transgenic rats reported here has been described (Burke et al., 1999). This transgene (−218/Luc, which served as the wild-type control for the current study, contains Aanat genomic sequence from position −218 to +1786 relative to the transcription start site, and includes the entire 1612 nucleotide intron 1 sequence (Fig. 1A and B). A mutant E-box transgene (−218mEB/Luc was derived from the wild-type transgene

Circadian expression

A marked circadian rhythm was detected in the expression of the wild-type transgene with transcripts being minimal during subjective day (08.00 h–18.00 h) and intensely expressed during the middle period of subjective night (22.00 h–03.00 h). For the purposes of the present study we therefore selected a sampling period of 18.00 h–08.00 h. In confirmation of previous findings (Burke et al., 1999) the pattern of expression of the wild-type transgene (−218/Luc) is characterized by a rhythm with peak

Discussion

The present study has provided a number of insights into the regulation of rat Aanat gene expression. First, we have discovered that E-box sequences within intron 1 are not required to drive or modulate the physiological circadian expression of the gene in the pineal gland (Baler et al., 1999, Chen and Baler, 2000, Burke et al., 1999). Second, we have determined that E-box sequences within intron 1 are required for the maintenance of tissue-specific expression because mutation of the +623 E-box

Acknowledgements

Support from the Wellcome Trust (AH, DAC) is gratefully acknowledged. This research was also supported in part by the Intramural Research Program of the National Institute of Child Health and Human Development, National Institutes of Health, USA.

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