Fish Physiol Biochem (2005) 31:149–152 DOI 10.1007/s10695-006-0017-2
RESEARCH ARTICLE
Isolation of dopamine D2 receptor (D2R) promoters in Mugil cephalus J. N. Nocillado Æ B. Levavi-Sivan Æ A. Avitan Æ F. Carrick Æ A. Elizur
Springer Science+Business Media B.V. 2006 Abstract This paper reports the isolation of two putative D2R promoters from grey mullet, one 5¢ flanking and the other an intronic sequence immediately upstream of the first coding exon. Promoter activity of the intronic sequence was confirmed in vitro through functional analysis using luciferase as reporter gene. The functional characteristics of the region flanking the 5¢-UTR is currently under investigation.
Introduction Dopamine receptors are G-protein-coupled receptors that are classified either as D1- or D2-like depending J. N. Nocillado Æ A. Elizur (&) Department of Primary Industries and Fisheries, Bribie Island Aquaculture Research Centre, QLD 4507, Australia e-mail:
[email protected] F. Carrick Æ J. N. Nocillado School of Integrative Biology, The University of Queensland, Brisbane, QLD 4072, Australia B. Levavi-Sivan Æ A. Avitan Faculty of Agricultural, Food and Environmental Quality Sciences, Department of Animal Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel A. Elizur Faculty of Science, Health and Education, University of the Sunshine Coast, Maroochydore DC, QLD 4558, Australia
on their property to either stimulate or inhibit adenylate cyclase activity, respectively (Missale et al. 1998). In fish species in which reproductive function is inhibited by dopamine (DA), D2R is the relevant receptor as shown by studies that utilized specific D2R antagonists (Vidal et al. 2004; Aizen et al. 2005). D2R is predominantly expressed in the fish brain-pituitary-gonadal axis (Levavi-Sivan et al. 2005). The onset of puberty is under dopaminergic influence in some species of fish (Schreibman et al. 1990). DA turnover in the hypothalamus declined in maturing spadefish, Chaetodipterus faber (Marcano et al. 1995). Physiological studies in immature European eel (Anguilla anguilla) demonstrated that DA impedes the onset of puberty by inhibiting the synthesis and release of luteinizing hormone (Vidal et al. 2004). We aimed to study the promoter regulation of the grey mullet D2R (muD2R) as a means towards elucidating the mechanism of the inhibitory effect of DA on the reproductive function in fish, including the onset of puberty. DA inhibition of the reproductive function is known to be particularly strong in grey mullet (Aizen et al. 2005).
Materials and methods The muD2R cDNA was isolated by Levavi-Sivan et al. (2005; AY673984) and its 5¢-UTR sequence (DQ277008) by our laboratory. Following the BD
123
150
Biosciences (Palo Alto, CA) universal genome walking PCR protocol, the partial intronic sequence 5¢ flanking the first coding exon was isolated from genomic DNA (StuI library) using gene-specific reverse primers (5¢-GACGATGACAAAGATGAGC AGAGTGAGC-3¢ and 5¢-AGTTGTAAGGGTGCTT TTGCTCCCGACC-3¢ for the primary and nested reactions, respectively) and the kit’s universal forward primer. The region 5¢ flanking the 5¢-UTR was isolated in the same manner from genomic DNA (PvuII library) using primers 5¢-CCACAGTTAGC TCCCACTGTATCCTTC-3¢ and 5¢-GAGTCGGTGC AGCGGCTCGGGTGC-3¢ for the primary and nested reactions, respectively. Isolated sequences were analyzed for putative transcription factor binding sites by MatInspector (http://www.genomatix.de), TFSEARCH (http://www.cbrc.jp) and Neural Promoter Predictions Program (http://www.fruitfly.org/ cgi-bin/seq_tools/promoter.pl). Five serial deletion constructs were generated by PCR from the intronic sequence, and these were ligated into the promoterless pGL3Basic vector fused to luciferase reporter gene (Promega). Detailed procedures for generating the reporter constructs and for transient transfection using Fugene 6 (Roche) are described in Nocillado et al. (submitted).
Results The sequence 5¢ flanking the 5¢-UTR of muD2R contained a number of putative regulatory elements, including an Sp1, a progesterone receptor binding site, two CREB (cAMP-response element binding protein) sites, several GATA sites, STAT 6 (signal transducers and activators of transcription), GC boxes and TATA- and CAAT-like sequences (DQ277009). The intronic sequence 5¢ flanking the first coding exon revealed consensus initiator (Inr) and downstream promoter elements (DPE) as well as putative transcription factor binding sites for AP4, glucoccorticoid receptor (GR), NFkappaB, CREB, AP1, thyroid transcription factor 1 (TTF1), CAAT enhancer binding protein (CEBP), pituitary specific pou domain transcription factor (Pit1, GHF-1), aryl hydrocarbon/Arnt heterodimers (Ahr/Arnt), pituitary homeobox 1 (Ptx1), retinoic acid receptor-related receptor a (RARa), GATA, GC boxes and TATA-
123
Fish Physiol Biochem (2005) 31:149–152
like sequences (DQ277010). It also contained elements identical to the enhancers reported in the rat D2R promoter (Wang et al. 1997). Transient transfection assays of serial deletion constructs from this intronic sequence demonstrated promoter activity in COS-7, aT3 and TE671 cell lines except for the shortest construct, which did not include the putative transcription initiation site (Fig. 1).
Discussion In contrast with known mammalian D2R promoters that are GC-rich and TATA-less and CAAT-less (Ben-Jonathan and Hnasko 2001), the muD2R promoters contain less than 50% GC, as reported in fugu (Macrae and Brenner 1995). The muD2R promoters also have scattered TATA- and CAAT-like sequences. The regulatory elements observed from both promoters were similarly identified in the rat D2R promoters (Valdenaire et al. 1994; Ben-Jonathan and Hnasko 2001). Pit1, GHF-1 and Ptx1 binding sites were not previously reported. Pit1, GHF-1 and Ptx1 are pituitary-specific transcription factors that modulate the expression of pituitary hormones (Martinez-Barbera´ et al. 1997; Melamed et al. 2002). These transcription factors may be relevant to the reported pituitary expression of D2R (Vacher et al. 2003; Levavi-Sivan et al. 2005). In vitro luciferase reporter gene assays demonstrated promoter activity of the partial intronic sequence immediately upstream of the second exon. This region contained consensus Inr and DPE, components that can act as a core promoter unit (Smale and Kadonaga 2003). Analysis using the Neural Promoter Predictions Program identified a putative transcription initiation site on one of the Inr sequences, 315 bp upstream of ATG. One of the proposed functions of intronic promoters is the regulation of tissuespecific gene expression (Kolb 2003). This may be the case in muD2R, which is expressed in the brain, pituitary and ovarian tissues in grey mullet (unpublished data). A functional role of the introns of D2-like dopamine receptor genes particularly of the first intron has been suggested because of its exceptionally large size, which is 15–160 kb in fugu and 250 kb in humans (Macrae and Brenner 1995). We have yet to confirm the size of muD2R first intron. The isolation
Fish Physiol Biochem (2005) 31:149–152
151
(a)
*
* *
(b)
a
-848/+195
b b c
-430/+195
d
e
fb
-353/+195 g
gf b
-115/+195 g
+30/+195
g
0
gf
TE671
g g
2
4
6
8
10
12
αΤ3
14
COS-7
16
18
Fold over pGL3-Basic
Fig. 1 (a) Schematic diagram of the regulatory elements identified on the intronic promoter of muD2R (GenBank Accession no. (DQ277010). Asterisk indicates putative transcription initiation site identified by Neural Promoter Predictions Program (http://www.fruitfly.org/cgi-bin/seq_tools/ promoter.pl). (b) In vitro basal promoter activity of 5¢ deletion
constructs was confirmed in COS-7, aT3 and TE671 cells. Data are expressed as means–SEM. b-galactosidase (pCMV-bgalactosidase) was used as internal control during transient transfection. Means marked with different letters are significantly different (ANOVA; P < 0.05)
of muD2R promoters would allow us to fully investigate their regulation.
Macrae AD, Brenner S (1995) Analysis of the dopamine receptor family in the compact genome of the puffer fish Fugu rubripes. Genomics 25:436–446 Marcano D, Guerrero HY, Gago N, Cardillo E, Requena M, Ruiz L (1995) Monoamine metabolism in the hypothalamus of the juvenile teleost fish, Chaetodipterus faber (PISCES: Ephippidae). In: Goetz FW, Thomas P (eds) Proceedings of the fifth international symposium on the reproductive physiology of fish, Fish Symposium 95, Austin, pp 64–66 Martinez-Barbera´ JP, Vila V, Valdivia MV, Castrillo JL (1997) Molecular cloning of gilthead seabream (Sparus aurata) pituitary transcription factor GHF-1/Pit-1. Gene 185:87– 93 Melamed P, Koh M, Preklathan P, Bei L, Hew C (2002) Multiple mechanisms for Pitx-1 transactivation of a luteinizing hormone b subunit gene. J Biol Chem 277:26200–26207 Missale C, Nash SR, Robinson SW, Jaber M, Caron MG (1998) Dopamine receptors: from structure to function. Physiol Rev 78:189–225 Schreibman MP, Holtzman S, Cepriano L (1990) The life cycle of the brain-pituitary-gonad axis in teleosts. Progress in Comparative Endocrinology. Wiley-Liss, Inc., New York, pp. 399–408 Smale ST, Kadonaga JT (2003) The RNA polymerase II core promoter. Annu Rev Biochem 72:449–479
Acknowledgements We acknowledge support to J. Nocillado from the Australia–Israel Scientific Exchange Foundation for the work done at The Hebrew University in Israel, and from The University of Queensland for the post-graduate scholarship. This project was funded by an Aquaculture Industry Development Initiative (AIDI) grant to A. Elizur. We sincerely thank H. Thaggard for maintaining the mullet fish stocks.
References Aizen J, Meiri I, Tzchori I, Levavi-Sivan B, Rosenfeld H (2005) Enhancing spawning in the grey mullet (Mugil cephalus) by removal of dopaminergic inhibition. Gen Comp Endocrinol 142:212–221 Ben-Jonathan N, Hnasko R (2001) Dopamine as a prolactin (PRL) inhibitor. Endocrine Rev 22:724–763 Kolb A (2003) The first intron of the murine b-casein gene contains a functional promoter. Biochem Biophys Res Commun 306:1099–1105 Levavi-Sivan B, Aizen J, Avitan A (2005) Cloning, characterization and expression of the D2 dopamine receptor from the tilapia pituitary. Mol Cell Endocrinol 236:17–30
123
152 Vacher C, Pellegrini E, Anglade I, Ferrie´re F, Saligaut C, Kah O (2003) Distribution of dopamine D2 receptor mRNAs in the brain and the pituitary of female rainbow trout: an in situ hybridization study. J Comp Neurol 458:32–45 Valdenaire O, Vernier P, Maus M, Edwards JDM, Mallet J (1994) Transcription of the rat dopamine-D2receptor gene from two promoters. Eur J Biochem 220:577–584
123
Fish Physiol Biochem (2005) 31:149–152 Vidal B, Pasqualini C, Le Belle N, Holland MCH, Sbaihi M, Vernier P, Zohar Y, Dufour S (2004) Dopamine inhibits luteinizing hormone synthesis and release in the juvenile European eel: a neuroendocrine lock for the onset of puberty. Biol Reprod 71:1491–1500 Wang J, Miller JC, Friedhoff AJ (1997) Differential regulation of D2 receptor gene expression by transcription factor AP1 in cultured cells. J Neurosci Res 50:23–31
