Marianne Bernard

Avian melatonin synthesis: Photic and circadian regulation of serotonin N-acetyltransferase mRNA in the chicken pineal gland and retina

Abstract: The circadian rhythms in melatonin production in the chicken pineal gland and retina reflect changes in the activity of serotonin N‐acetyltransferase (arylalkylamine N‐acetyltransferase; AA‐NAT; EC 2.3.1.87). Here we determined that the chicken AA‐NAT mRNA is detectable in follicular pineal cells and retinal photoreceptors and that it exhibits a circadian rhythm, with peak levels at night. AA‐NAT mRNA was not detected in other tissues. The AA‐NAT mRNA rhythm in the pineal gland and retina persists in constant darkness (DD) and constant lighting (LL). The amplitude of the pineal mRNA rhythm is not decreased in LL. Light appears to influence the phase of the clock driving the rhythm in pineal AA‐NAT mRNA in two ways: The peak is delayed by ∼6 h in LL, and it is advanced by >4 h by a 6‐h light pulse late in subjective night in DD. Nocturnal AA‐NAT mRNA levels do not change during a 20‐min exposure to light, whereas this treatment dramatically decreases AA‐NAT activity. These observations suggest that the rhythmic changes in chicken pineal AA‐NAT activity reflect, at least in part, clock‐generated changes in mRNA levels. In contrast, changes in mRNA content are not involved in the rapid light‐induced decrease in AA‐NAT activity.

Characterization of the chicken serotonin N-acetyltransferase gene: activation via clock gene heterodimer/E-box interaction

The abundance of serotoninN-acetyltransferase (arylalkylamineN-acetyltransferase, AANAT) mRNA in the chicken pineal gland exhibits a circadian rhythm, which is translated into a circadian rhythm in melatonin production. Here we have started to elucidate the molecular basis of the circadian rhythm in chicken AANAT (cAANAT). The 5′-flanking region of the cAANAT gene was isolated and found to contain an E box DNA element that confers strong luciferase reporter activity. In transfection experiments using chicken pineal cells, an E box mutation dramatically decreased reporter activity. Northern blot analysis indicated that several putative clock genes (bmal1, Clock, and MOP4) are co-expressed in the chicken pineal gland. bmal1 mRNA is expressed in a rhythmic manner in the chicken pineal gland, with peak levels at early subjective night, coincident with the increase in cAANAT expression. Co-transfection experiments in COS cells demonstrated that chicken BMAL1/CLOCK and human BMAL1/MOP4 heterodimers bound the AANAT E box element and enhanced transcription. These observations suggest that binding of clock gene heterodimers to the cAANAT E box is a critical element in the expression of the cAANAT genein vitro.

Human hydroxyindole-O-methyltransferase in pineal gland, retina and Y79 retinoblastoma cells.

Hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4) was studied in extracts of human pineal gland, retina and Y79 retinoblastoma cells. HIOMT enzyme activity and immunoreactive protein (approximately 42 kDa) were undetectable in the human retina; very low levels of HIOMT mRNA were detected using a highly sensitive RT-PCR/Southern blot method, as has been reported. Analysis of extracts of Y79 cells indicated that HIOMT enzyme activity, immunoreactivity (approximately 42 kDa) and mRNA (approximately 1.3 kb) were detectable at approximately 1/5-1/40 the levels found in the pineal gland. This unambiguously establishes that the HIOMT gene is expressed in Y79 cells. Kinetic analysis of Y79- and pineal-derived HIOMT indicates that the enzyme is generally similar in both tissues; one difference, however, is that substrate inhibition by N-acetylserotonin is greater with the Y79-derived enzyme. These studies show that Y79 cells represent a valid model to study the regulation of human HIOMT protein and mRNA; the differences detected may reflect the existence of tissue-specific regulatory mechanisms or differential patterns of expression of HIOMT isoforms.

Circadian expression of tryptophan hydroxylase mRNA in the chicken retina

Many aspects of retinal physiology are controlled by a circadian clock located within the eye. This clock controls the rhythmic synthesis of melatonin, which results in elevated levels during the night and low levels during the day. The rate-limiting enzyme in melatonin biosynthesis in retina appears to be tryptophan hydroxylase (TPH)[G.M. Cahill and J.C. Besharse, Circadian regulation of melatonin in the retina of Xenopus laevis: Limitation by serotonin availability, J. Neurochem. 54 (1990) 716-719]. In this report, we found that TPH mRNA is strongly expressed in the photoreceptor layer and the vitread portion of the inner nuclear layer; the message is also expressed, but to a lesser extent, in the ganglion cell layer. The abundance of retinal TPH mRNA exhibits a circadian rhythm which persists in constant light or constant darkness. The phase of the rhythm can be reversed by reversing the light:dark cycle. In parallel experiments we found a similar pattern of expression in the chicken pineal gland. However, whereas a pulse of light at midnight suppressed retinal TPH mRNA by 25%, it did not alter pineal TPH mRNA, suggesting that there are tissue-specific differences in photic regulation of TPH mRNA. In retinas treated with kainic acid to destroy serotonin-containing amacrine and bipolar cells, a high amplitude rhythm of TPH mRNA was observed indicating that melatonin-synthesizing photoreceptors are the primary source of the rhythmic message. These observations provide the first evidence that chick retinal TPH mRNA is under control of a circadian clock.

Characterization of melatonin binding sites in chicken and human intestines

The radioligand 2-[125I]iodomelatonin was used to study melatonin binding sites in chicken and human intestines. In the chicken duodenum, 2-[125I]iodomelatonin binding sites were enriched in the musculosa layer (Bmax approximately 1 fmol/mg protein) as compared to the mucosa/submucosa layer (Bmax approximately 0.2 fmol/mg protein). 2-[125I]iodomelatonin bound with a Kd of 68 +/- 18 pM (mean +/- S.E.M., n = 13) and was displaced by melatonin with a Ki of 0.3 nM. The Kd value for 2-[125I]iodomelatonin was increased 2- to 4-fold by a GTP analog, suggesting that the binding sites might be coupled to a G-protein. The affinity order of nine melatonin analogs at the enteric binding sites was in agreement with the pharmacological profile of melatonin receptors described in other tissues. In the human jejunum, 2-[125I]iodomelatonin binding could be observed in the mucosa/submucosa layer (Kd = 150-200 pM, Bmax = 0.7 fmol/mg protein). The radioligand was efficiently displaced by melatonin (Ki = 0.6 nM) but only marginally by N-acetyltryptamine (Ki = 22 microM) and serotonin (Ki = 14 microM).

Cellular and Molecular Regulation of Serotonin N-Acetyltransferase Activity in Chicken Retinal Photoreceptors

Serotonin N-acetyltransferase (AA-NAT; arylalkylamine N-acetyltransferase; EC 2.3.1.87) is the penultimate enzyme in melatonin synthesis and large changes in the activity of this enzyme appear to regulate the rhythm in melatonin synthesis. Recent advances have made it possible to study the mRNA encoding chicken AA-NAT, which has only been detected in the retina and pineal gland. Within the retina, AA-NAT mRNA is expressed primarily in photoreceptors. The levels of chicken retinal AA-NAT mRNA and activity exhibit 24-hour rhythms with peaks at night. These rhythms appear to reflect circadian clock control of AA-NAT mRNA abundance and independent effects of light and darkness on both mRNA levels and enzyme activity. The effects of darkness and light may occur through alterations in cAMP-dependent protein phosphorylation, which increases AA-NAT activity in photoreceptor cell cultures. The cAMP-dependent increase of AA-NAT enzyme activity reflects, at least in part, increased mRNA levels and inhibition of enzyme inactivation by a posttranslational mechanism. This review discusses a hypothetical model for the cellular and molecular regulation of AA-NAT activity by circadian oscillators and light in chicken retinal photoreceptor cells.

Hydroxyindole-O-methyltransferase in the Chicken Retina: Immunocytochemical- Localization and Daily Rhythm of mRNA

In the vertebrate retina and pineal gland, melatonin production displays diurnal variations with high levels at night. Hydroxyindole‐O‐methyltransferase (HIOMT, EC 2.1.1.4) catalyses the last step of melatonin biosynthesis. In the present study, a cDNA encoding chicken HIOMT was used to examine the effects of environmental lighting on HIOMT mRNA expression in the chicken retina. A day/night rhythm of HIOMT mRNA level was observed, with an average 5‐fold increase during the night. Light strongly suppressed the night‐time rise in HIOMT mRNA concentration while darkness prevented its daytime fall. An antibody directed against chicken HIOMT was used for immunocytochemical identification of retinal melatoninergic cells. HIOMT immunoreactivity could be observed in rods as well as in cones. However, the lowest levels of HIOMT immunoreactivity were always observed in the accessory cones of double cones. A few HIOMT‐positive cell bodies could also be observed in the inner nuclear layer. Altogether, these data indicate that HIOMT gene expression in the retina is organized on a daily basis as a direct response to light, and that the different types of photoreceptors may not be equally involved in melatonin production.

Transcriptional regulation of the chicken hydroxyindole-O-methyltransferase gene by the cone-rod homeobox-containing protein.

Functional differentiation of photoreceptor cells involves the expression of two sets of genes: those encoding the proteins of the phototransduction cascade and those encoding the enzymes of melatonin synthesis. The transcription factor Crx (cone−rod homeobox) plays a major role in the differentiation and maintenance of the photoreceptor phenotype. Previous studies have shown that this effect of Crx is correlated with its ability to transactivate several genes of the phototransduction cascade. Here, we show that Crx can also act on the gene encoding the melatonin‐synthesizing enzyme, hydroxyindole‐O‐methyltransferase (HIOMT, EC 2.1.1.4). Three of the six putative Crx‐binding sites found in the chicken HIOMT promoter interact directly with recombinant Crx and bound a pineal/retina‐specific protein showing DNA‐binding characteristics similar to those of Crx. In transient transfection experiments, Crx transactivated transcription of a reporter gene from the chicken HIOMT promoter. Transactivation was observed even with a portion of promoter carrying only one Crx‐binding site and it was abolished by a mutation in this cis‐regulatory element. These data indicate that Crx may participate in photoreceptor cell differentiation, not only by acting on the genes of the phototransduction cascade, but also by controlling the expression of the genes involved in melatonin synthesis.

Molecular and cellular aspects of hydroxyindole-O-methyltransferase expression in the developing chick pineal gland

The pineal gland influences circadian activity and seasonal breeding through the production of an indolic hormone, melatonin. The terminal step of melatonin biosynthesis is catalyzed by hydroxyindole-O-methyltransferase (HIOMT). Using an antibody directed against HIOMT, we examined the differentiation of the melatoninergic phenotype in the developing chick pineal gland. HIOMT first appeared 4 days before hatch and rose linearly until the 7th day posthatch. This was correlated with an increased immunoreactivity of the 38 kDa enzyme on Western blots and with an accelerated rate of HIOMT biosynthesis as demonstrated by [35S]methionine labeling. Immunocytochemistry revealed a growing number of HIOMT-positive cells between day 2 before hatch and day 15 posthatch. Until hatching HIOMT was expressed almost exclusively in modified photoreceptors. Parafollicular pinealocytes became HIOMT-positive mostly after hatching. Their different timings of functional differentiation emphasize the existence of two populations of melatonin-producing cells in the chick pineal gland.

Photoreceptor‐specific expression, light‐dependent localization, and transcriptional targets of the zinc‐finger protein Yin Yang 1 in the chicken retina

The zinc‐finger transcription factor Yin Yang 1 (YY1) is a multifunctional protein that plays a critical role in embryonic development. Although it has been shown to play a role in eye development, its expression in the retina was not previously described. Here, we investigated YY1 expression in chicken tissues and we identified the neural retina as one of the tissues with highest YY1 protein levels. Immunohistochemical detection of YY1 in the retina revealed a clear‐cut photoreceptor specificity and day/night differences in the cytoplasmic localization of the protein. YY1 was also present at high concentration in the nuclei of some photoreceptors. Gel‐shift assays indicated YY1 bound to regulatory regions of several genes specifically expressed in photoreceptors. One of these genes, hydroxyindole‐O‐methyltransferase (EC 2.1.1.4), encodes the last enzyme of the melatonin synthesis pathway. Although over‐expression of chicken YY1 was not sufficient to activate the chicken hydroxyindole‐O‐methyltransferase promoter in HEK293 cells, the YY1‐binding site contained in this promoter was clearly required for full transcriptional activity in chicken embryonic retinal cells. These results suggest a role of YY1 in regulating the melatoninergic function of retinal photoreceptors.