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A Cis-Acting Element, Directs Pineal-Specific Gene Expression in Zebrafish

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A Cis-Acting Element, Directs Pineal-Specific Gene Expression in Zebrafish Pineal expression-promoting element (PIPE), a cis-acting element, directs pineal-specific gene expression in zebrafish Yoichi Asaoka*, Hiroaki Mano*, Daisuke Kojima†, and Yoshitaka Fukada‡ Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan Edited by Jeremy Nathans, Johns Hopkins University School of Medicine, Baltimore, MD, and approved September 26, 2002 (received for review July 25, 2002) The pineal gland, sharing morphological and biochemical similar- photoreceptor cells (9–13). These observations illustrate re- ities with the retina, plays a unique and central role in the markable parallels in the pineal and retinal phototransduction photoneuroendocrine system. The unique development of the pathways, but very little is known about the molecular basis that pineal gland is directed by a specific combination of the expressed accounts for the characteristic development of the pineal gland. genes, but little is known about the regulatory mechanism under- The regulatory mechanism responsible for tissue-specific gene lying the pineal-specific gene expression. We isolated a 1.1-kbp expression and its evolutionary background are, therefore, im- fragment upstream of the zebrafish exo-rhodopsin (exorh) gene, portant issues providing clues to the developmental control which is expressed specifically in the pineal gland. Transgenic specifying the pineal or retinal identity. analysis using an enhanced green fluorescent protein reporter In recent years, a number of studies have been reported about gene demonstrated that the proximal 147-bp region of the exorh transcriptional regulation of the retinal genes (reviewed in ref. promoter is sufficient to direct pineal-specific expression. This 14), among which the rhodopsin (rh) promoter has been studied region contains three copies of a putative cone rod homeobox extensively. Biochemical approaches and in vitro transcription (Crx)͞Otx-binding site, which is known to be required for expres- assays have identified several cis-acting DNA elements, such as sion of both retina- and pineal-specific genes. Deletion and muta- Ret-1 (15), BAT-1 (16), and Ret-4 (17). These studies suggest a tional analyses of the exorh promoter revealed that a previously cooperative interplay of multiple cis-acting elements and tran- uncharacterized sequence TGACCCCAATCT termed pineal expres- scription factors for the retinal photoreceptor cell-specific gene sion-promoting element (PIPE) is required for pineal-specific pro- expression. Consistent with this idea, several transcription fac- moter activity in addition to the Crx͞Otx-binding sites. By using the tors have been identified and shown to regulate the rh gene zebrafish rhodopsin (rh) promoter that drives retina-specific ex- expression. Subtraction cDNA cloning resulted in identification pression, we created a reporter construct having ectopic PIPE in the of neural retina leucine zipper (Nrl), a basic leucine zipper rh promoter at a position equivalent to that in the exorh promoter (bZIP) transcription factor that is expressed in all cell layers of by introducing five nucleotide changes. Such a slight modification adult mammalian retina (18, 19). Nrl binds to a well conserved in the rh promoter induced ectopic enhanced green fluorescent cis-acting element Nrl response element (NRE) and transacti- protein expression in the pineal gland without affecting its retinal vates the rh promoter (20, 21). On the other hand, cone rod expression. These results identify PIPE as a critical cis-element homeobox (Crx) is an Otx-related homeodomain protein ex- contributing to the pineal-specific gene expression, in combination pressed exclusively in the retinal photoreceptor cells and pine- with the Crx͞Otx-binding site(s). alocytes (22–24). Crx transactivates the rh promoter by binding to BAT-1 and Ret-4 (22) and is implicated in the regulation of iving organisms use environmental light signals for multiple photoreceptor cell-specific gene expression (25). In addition to Lphysiological functions such as vision, photoentrainment of these transcription factors, Rx and Erx seem to participate in the circadian rhythms, regulation of body color, and detection of regulation of the rh gene expression (26–28). seasonal changes in photoperiod. These diverse functions are In contrast to these advances in the studies on the retina- mediated not only by the retina but also by extra-ocular photo- specific gene expression, transcriptional regulation of the pineal receptive organs, such as the pineal gland. The retina and pineal gene remains poorly understood. The only cis-acting element gland probably arose via divergence from a common ancestral identified so far is a pineal regulatory element (PIRE), which is recognized by Crx (29, 30). PIRE with a consensus sequence of photoreceptive organ, and consistently, the pineal gland acts as ͞ Ј a photosensory organ in the lowest vertebrate (1–3). In the TAATC T is present in 5 -flanking regions of several pineal course of vertebrate evolution, the physiological role of the genes such as rat arylalkylamine-N-acetyltransferase and human pineal gland has been changed from a photosensory organ to a hydroxyindole-O-methyltransferase genes (29). Recently, it has photoendocrinal organ in the lower vertebrates and eventually to been reported that circadian gene expression in the zebrafish pineal complex requires Otx5, which is closely related to Crx a neuroendocrinal organ in mammals (4, 5). Generally, the retina ͞ receives visual images and transmits them to the brain, whereas (31). These studies suggest that a member of the Crx Otx family the primary role of the pineal gland is the rhythmic production and its binding site(s) play a common role in the transcription of of circulating melatonin, which regulates numerous physiological activities (6). Despite such a dynamic change in the physiological This paper was submitted directly (Track II) to the PNAS office. function, the pineal gland displays many similarities to the retina ͞ Abbreviations: rh, rhodopsin; Nrl, neural retina leucine zipper; NRE, Nrl response element; in tissue cellular morphology and biochemical properties (7, 8). Crx, cone rod homeobox; exorh, exo-rhodopsin; PIPE, pineal expression-promoting ele- In fish, amphibians, and lacertilian reptiles, their pineal photo- ment; EGFP, enhanced GFP; dpf, days postfertilization; hpf, hours postfertilization. receptor cells possess well developed and lamellar outer seg- Data deposition: The sequences reported in this paper have been deposited in the GenBank ments, which are homologous to the outer segments of retinal database (accession nos. AB079551 and AB079552). photoreceptor cells. These photoreceptor cells form an ordered *Y.A. and H.M. contributed equally to this work. layer structure in both tissues. Biochemically, the retinal pho- †Present address: Department of Molecular and Cellular Biology, Harvard University, totransduction proteins such as opsin, ␣-subunit of transducin, Cambridge, MA 02138. arrestin, and recoverin also have been localized in the pineal ‡To whom correspondence should be addressed. E-mail: [email protected]. 15456–15461 ͉ PNAS ͉ November 26, 2002 ͉ vol. 99 ͉ no. 24 www.pnas.org͞cgi͞doi͞10.1073͞pnas.232444199 Downloaded by guest on September 24, 2021 Fig. 1. The proximal promoter sequences of exorh and rh genes of vertebrates. The upstream sequence of the zebrafish exorh (zEx) was aligned with those of rh genes of the zebrafish (zRh), Xenopus (xRh), chicken (cRh), mouse (mRh), rat (rRh), bovine (bRh), and human (hRh). The upstream sequence of the European eel exorh (eEx) was determined in the present study and included in the alignment. Nucleotides conserved among at least six sequences are shown with white characters on black backgrounds. Horizontal lines indicate TATA box, ATG initiation codon, and conserved cis-elements identified previously in the rh promoters. Potential Crx͞Otx-binding sites found in the zebrafish exorh promoter are double-lined. The PIPE sequence in the zebrafish exorh gene is boxed. The nucleotide numbers are relative to the translation initiation site of the zebrafish exorh gene. Accession numbers of the sequences obtained from GenBank are U23808 (xRh), M98497 (cRh), M55171 (mRh), U22180 (rRh), and U49742 (hRh). The bRh sequence was obtained from the original paper (17). both pineal and retinal genes. It should be stressed, however, that to confirm the sequence with no PCR error. In a similar manner, the transcriptional regulation operated by Crx͞Otx is inadequate a 238-bp region upstream from the ATG initiation codon of the to explain the mechanism segregating the pineal- and retina- European eel exorh gene (GenBank accession no. AB079552) specific gene expression. As yet unidentified transcription fac- was obtained from the eel genomic DNA by using the LA PCR tor(s) and cis-acting element(s) should strictly determine pineal- in vitro Cloning Kit and sequenced. On the other hand, screening specific gene expression, most probably in combination with the of ␭-Fix II zebrafish genomic library resulted in isolation of a Crx͞Otx-dependent regulation. 10.5-kbp fragment containing a 4,558-bp sequence upstream of We previously demonstrated that the zebrafish has two dis- the rh coding region. tinct rhodopsin genes that are highly similar in coding sequence to each other (74% identical) but show unique tissue distribu- Microinjection and Generation of Germ-Line Transgenic Zebrafish. tions (32). One is the canonical rh
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