RESUMEN
DNA microarrays allow to simultaneously determine the expression level of thousands of genes. A nycthemeral study must enable to conclude which ones show a circadian rhythm. Two aspects prove this to be quite difficult: firstly, what does "circadian" exactly mean and how to quantify this qualification, and secondly which genes pertain to this definition. Our method, derived from linear optimisation procedures, consists in determining a cost function, depending from magnitudes characterising the notion of circadian rhythm. Given number of genes present on the microarray are known to be expressed rhythmically; their time series are considered as reference series. We have further constructed random series having the same temporal structure as the circadian gene series. We then carried out an optimisation procedure to determine the weighting coefficients in order to obtain a cost function value which orders the time series as follows: the reference series are in the first rows and the random series have low scores. We have tested this method on over 6000 genes expressed in mouse liver. We obtained a circadian gene detection probability of 100% with a false positive rate inferior to 1%.
Asunto(s)
Ritmo Circadiano/genética , Perfilación de la Expresión Génica , Algoritmos , Animales , Hígado/fisiología , Ratones , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Hydroxyindole-O-methyltransferase (HIOMT) plays an important role as the final enzyme in the synthesis of melatonin. In the chicken pineal gland, HIOMT mRNA concentration exhibits a circadian rhythm with a threefold peak at midday. The present study sought to evaluate the possible role of cyclic AMP in this transcriptional rhythm. In cultured pineal glands from 4-day-old chicks, cyclic AMP analogs and the adenylate cyclase activator, forskolin, increased HIOMT mRNA levels twofold to threefold in a dose-dependent manner. Vasoactive intestinal polypeptide increased HIOMT mRNA levels by 50%. Actinomycin-D chase experiments indicated that cyclic AMP did not affect the stability of HIOMT mRNA, thus providing indirect evidence that the effect of cyclic AMP was exerted at transcriptional level. In cultured pineal glands from 11 days embryos, HIOMT mRNA levels failed to respond to cyclic AMP. However, a daily rhythm of HIOMT mRNA, with an endogenous component in constant darkness was clearly observed at this developmental stage. This observation indicates that cyclic AMP is not required for circadian rhythmicity of HIOMT gene transcription.
Asunto(s)
Acetilserotonina O-Metiltransferasa/genética , Ritmo Circadiano/fisiología , AMP Cíclico/farmacología , Glándula Pineal/efectos de los fármacos , ARN Mensajero/metabolismo , Animales , Northern Blotting , Embrión de Pollo , Pollos , Colforsina/farmacología , AMP Cíclico/análogos & derivados , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Modelos Logísticos , Técnicas de Cultivo de Órganos , Glándula Pineal/metabolismoRESUMEN
The mRNAs encoding three enzymes of the melatonin synthesis pathway (tryptophan hydroxylase (TPH), arylalkylamine-N-acetyltransferase (AANAT) and hydroxyindole-O-methyl-transferase (HIOMT)) are expressed with a day/night rhythm in the chicken pineal gland and retina. TPH and AANAT mRNA levels reach their peak at night. HIOMT mRNA levels peak at night in the retina, but during the day in the pineal gland. In this tissue, the rhythm of TPH, AANAT and HIOMT mRNA levels persisted in constant darkness (DD), both in vivo and in vitro, indicating that the three genes are controlled by the circadian oscillator of the chicken pineal. In the retina, the rhythms of TPH and AANAT mRNA levels also persisted in DD in vivo, suggesting that they are driven by a circadian oscillator. In contrast, the rhythm of HIOMT mRNA in the retina appeared to be controlled only by light. The clones of chicken AANAT and HIOMT genes that we have isolated should help us to understand the molecular mechanisms of: 1) their transcriptional regulation by circadian oscillators and by light; 2) their tissue-specific expression in the pineal gland and the retina.
Asunto(s)
Acetilserotonina O-Metiltransferasa/biosíntesis , Arilamina N-Acetiltransferasa/biosíntesis , Ritmo Circadiano/genética , Proteínas de Drosophila , Proteínas del Ojo/biosíntesis , Regulación Enzimológica de la Expresión Génica , Melatonina/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Glándula Pineal/enzimología , Retina/enzimología , Triptófano Hidroxilasa/biosíntesis , Factores de Transcripción ARNTL , Acetilserotonina O-Metiltransferasa/genética , Animales , Arilamina N-Acetiltransferasa/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteínas CLOCK , Pollos , Inducción Enzimática/efectos de la radiación , Proteínas del Ojo/genética , Regulación Enzimológica de la Expresión Génica/efectos de la radiación , Proteínas de Insectos/biosíntesis , Proteínas de Insectos/genética , Luz , Modelos Biológicos , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Especificidad de Órganos , Proteínas Circadianas Period , Fotoperiodo , Glándula Pineal/inervación , ARN Mensajero/biosíntesis , Retina/efectos de la radiación , Núcleo Supraquiasmático/fisiología , Sistema Nervioso Simpático/fisiología , Transactivadores/fisiología , Factores de Transcripción/fisiología , Transcripción Genética , Triptófano Hidroxilasa/genéticaRESUMEN
Hydroxyindole-O-methyltransferase (HIOMT) catalyzes the final step of melatonin synthesis, a neurohormone involved in photoperiodism and produced specifically in the pineal gland and in the retina. In the chicken, HIOMT gene transcription appears to be controlled by a circadian oscillator located in the pineal gland. We have characterized the chicken HIOMT gene over 17 kb, including 2.9 kb of 5'-flanking sequence. The major transcript (1.6 kb) is composed of eight exons distributed over 7.5 kb of genomic DNA. A ninth alternative exon was identified 6 kb downstream of exon 8. It was found in minor transcripts in the pineal gland and in the retina. Sequence similarity between exons 8 and 9 suggests their origin by exon duplication. Due to early stop codons, inclusion of exon 9 truncates the open reading frame by up to 33%. A restriction fragment length polymorphism was detected for a BglII site in intron 8. Fluorescence hybridization localized the HIOMT gene on chicken chromosome 1q22. The 5'-flanking region contains GATTAA and TAATCC sequences that may be related to tissue-specific expression. An ATTTAAAT sequence at position -29 would play the role of a TATA box, as evidenced by electrophoretic mobility shift assay. Information obtained in this study open the way to further studies aimed at analyzing the circadian rhythm of transcription at promoter level.
Asunto(s)
Acetilserotonina O-Metiltransferasa/genética , Animales , Secuencia de Bases , Línea Celular , Embrión de Pollo , Mapeo Cromosómico , ADN Complementario , Exones , Hibridación Fluorescente in Situ , Intrones , Datos de Secuencia Molecular , Polimorfismo Genético , Empalme del ARN , Mapeo RestrictivoRESUMEN
The production of the pineal hormone melatonin displays circadian variations with high levels at night. The last enzyme involved in melatonin biosynthesis is hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4). The expression of the mRNA encoding chicken HIOMT was investigated in vivo and in vitro throughout the light/dark cycle, in constant darkness and with light interruption of the dark phase. The stability of HIOMT mRNA was also examined. A day/night rhythm of HIOMT mRNA levels, with a peak at the midlight phase, was observed in vivo as well as in vitro. Constant darkness did not abolish this rhythm in vivo. One cycle of the HIOMT mRNA rhythm could be observed in constant darkness in vitro. In addition, a stimulatory effect of light on HIOMT mRNA levels during the dark phase could be observed in vivo as well as in vitro. HIOMT mRNA stability was not affected by light or dark conditions, as demonstrated by chase experiments with actinomycin D. The results indicate that the daily changes in HIOMT mRNA concentration reflect transcriptional regulation by circadian oscillators and photosensory mechanisms that are endogenous to the pineal gland.
Asunto(s)
Acetilserotonina O-Metiltransferasa/biosíntesis , Ritmo Circadiano , Regulación Enzimológica de la Expresión Génica , Glándula Pineal/fisiología , Animales , Northern Blotting , Pollos , Dactinomicina/farmacología , Oscuridad , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Luz , Técnicas de Cultivo de Órganos , Glándula Pineal/enzimología , ARN Mensajero/biosíntesis , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Acetilserotonina O-Metiltransferasa/genética , Acetilserotonina O-Metiltransferasa/metabolismo , Pollos/metabolismo , Ritmo Circadiano , ARN Mensajero/metabolismo , Retina/enzimología , Animales , Oscuridad , Inmunohistoquímica , Luz , Retina/citología , Retina/efectos de la radiación , Distribución TisularRESUMEN
Hydroxyindole-O-methyltransferase (HIOMT), the enzyme which catalyzes the final step of melatonin biosynthesis, constitutes a marker of the functional differentiation of pineal cells. In addition, a day/night rhythm of HIOMT mRNA concentration, previously described in the chicken pineal gland [6], would suggest that HIOMT gene transcription is one output of the circadian system that controls pineal function. The study sought to monitor the developmental expression of HIOMT mRNA in the chick pineal gland and to investigate a possible role of instructive signals in this differentiation process. RT-PCR analysis indicated that HIOMT mRNA is expressed at embryonic day 8 (E8). At E12, HIOMT mRNA became detectable on northern blots and traces of HIOMT activity could be measured. HIOMT mRNA concentration increased 100-fold between E14 and day 10 post-hatch, then levelled off. A day/night rhythm of HIOMT mRNA concentration was readily observed in the pineal gland of 2-day-old chicks. Pineal glands isolated on minimum culture medium at E11 stopped developing HIOMT gene expression. However, the addition of serum to the culture medium restored HIOMT mRNA concentration to the levels observed in vivo. The data suggest that the functional differentiation of melatoninergic cells observed during the second week of embryonic life may be controlled [correction of controled] by serum factors.
Asunto(s)
Acetilserotonina O-Metiltransferasa/genética , Fenómenos Fisiológicos Sanguíneos , Regulación del Desarrollo de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Glándula Pineal/embriología , ARN Mensajero/metabolismo , Animales , Diferenciación Celular/fisiología , Embrión de Pollo , Ritmo Circadiano/genética , Glándula Pineal/crecimiento & desarrollo , Transducción de Señal/fisiologíaRESUMEN
Hydroxyindole-O-methyltransferase (HIOMT) catalyzes the final step of melatonin biosynthesis and appears to be specifically expressed in the pineal gland and in the retina. This review deals with the regulation of HIOMT by environmental light and with the developmental aspects of HIOMT expression in chicken and rat. Early studies based on HIOMT activity measurements and more recent studies involving cDNA hybridization to HIOMT mRNA are taken into consideration. Together, the data reveal that long term regulation of HIOMT by light would rely on a day/night rhythm of HIOMT gene transcription, coupled to a slow turnover of the protein. Rapid changes in HIOMT mRNA levels and early expression during embryonic development suggest that further studies on this gene may shed light on the molecular mechanisms involved in the differentiation of the melatoninergic function and in its regulation by light, both in the pineal gland and in the retina.