Regulation of the tyrosinase promoter in transgenic mice: expression of a tyrosinase-lacZ fusion gene in embryonic and adult brain

Friedrich Beermann
1997
Article

The enzyme tyrosinase is indispensable for pigmentation and the gene is expressed mainly in pigment cells. Regulatory elements, at -12 to -15 kb (enhancer) and within the 270 bp directly upstream of the transcription start site, have been described recently and their importance demonstrated in transgenic experiments. We were interested in tyrosinase promoter activity during development and used beta-galactosidase as reporter gene. Transgenic mice were generated carrying a tyrosinase-lacZ fusion gene, containing 6.1 kb of tyrosinase 5' sequences. In transgenic embryos, beta-galactosidase activity was detected along the entire neural tube, with the most prominent expression in the developing telencephalon, and also in the adult brain. Equivalent expression was observed in the developing retina. Tyrosinase protein was identified in embryonic and adult brain, but no DOPAoxidase or tyrosine hydroxylase activity was detected. From our results we conclude that 1) tyrosinase protein is present in embryonic and adult mouse brain and 2) the tyrosinase promoter can direct expression of a reporter gene to pigment cells and neural tissues.

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Friedrich Beermann
1997
Article

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https://infoscience.epfl.ch/record/115456?ln=fr

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The mouse tyrosinase promoter is sufficient for expression in melanocytes and in the pigmented epithelium of the retina

Friedrich Beermann

The mouse c locus encodes tyrosinase (monophenol monooxygenase; monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1), the key enzyme in melanin synthesis, which is expressed in the pigment epithelium of the retina and in melanocytes derived from the neural crest. To define regulatory regions of the gene that are important for cell type-specific expression, a deletion series of the tyrosinase 5' region was fused to a chloramphenicol acetyltransferase (CAT) reporter gene and electroporated into tyrosinase-expressing and -nonexpressing cell lines. We show that 270 base pairs 5' of the transcriptional start site is sufficient for CAT expression in a human and a mouse melanoma cell line. This 5' flanking fragment, when cloned in the context of a tyrosinase minigene construct and injected into fertilized eggs of an albino mouse strain, is sufficient for cell type-specific expression in mice. The transgenic mice were pigmented in both skin and eyes. In situ hybridization analysis shows that the 270-base-pair regulatory region contains elements sufficient for specific expression of the transgene both in the pigmented epithelial cells of the retina, which are derived from the optic cup, and in neural crest-derived melanocytes.

1991

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Distinct distal regulatory elements control tyrosinase expression in melanocytes and the retinal pigment epithelium

Friedrich Beermann

Pigment cells of mammals are characterized by two different developmental origins: cells of the retinal pigment epithelium (RPE) originate from the optic cup of the developing forebrain, whereas melanocytes arise from the neural crest. The pigmentation gene tyrosinase is expressed in all pigment cells but differentially regulated in melanocytes and RPE. The tyrosinase promoter does not confer strong expression in pigment cells in vivo, while inclusion of a distal regulatory element at position -15 kb is necessary and sufficient to provide strong expression in melanocytes. Nevertheless, the regulatory elements responsible for correct spatial and temporal tyrosinase expression in the RPE remained unidentified so far. In this report, we show that a 186 kb BAC containing the tyrosinase gene provides transgene expression in both RPE and melanocytes indicating the presence of regulatory sequences required for expression in the RPE. A deletion analysis of the BAC was performed demonstrating that a RPE-regulatory element resides between -17 and -75 kb. Using multi-species comparative genomic analysis we identified three conserved sequences within this region. When tested in transgenic mice one of these sequences located at -47 kb targeted expression to the RPE. In addition, deletion of this regulatory element within a tyrosinase::lacZ BAC provided evidence that this sequence is not only sufficient but also required for correct spatial and temporal expression in the RPE. The identification of this novel element demonstrates that tyrosinase gene expression is controlled by separate distal regulatory sequences in melanocytes and RPE.

2007

Chargement

Pigment cells of mammals originate from two different lineages: melanocytes arise from the neural crest, whereas cells of the retinal pigment epithelium (RPE) originate from the optic cup of the developing forebrain. Previous studies have suggested that pigmentation genes are controlled by different regulatory networks in melanocytes and RPE. The promoter of the tyrosinase-related family gene Tyrp1 has been shown to drive detectable transgene expression only to the RPE, even though the gene is also expressed in melanocytes as evident from Tyrp1-mutant mice. This indicates that the regulatory elements responsible for Tyrp1 gene expression in the RPE are not sufficient for expression in melanocytes. We thus searched for a putative melanocyte-specific regulatory sequence and demonstrate that a bacterial artificial chromosome (BAC) containing the Tyrp1 gene and surrounding sequences is able to target transgenic expression to melanocytes and to rescue the Tyrp1b (brown) phenotype. This BAC contains several highly conserved non-coding sequences that might represent novel regulatory elements. We further focused on a sequence located at -15 kb, which we identified as a melanocyte-specific enhancer as shown by cell culture and transgenic mice experiments. In addition, we show that the transcription factor Sox10 can activate this conserved enhancer. The presence of a distal Tyrp1 regulatory element, which specifies melanocyte-specific expression, supports the idea that separate regulatory sequences can mediate differential gene expression in melanocytes and RPE.

2006

Chargement

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Gène

Un gène, du grec ancien (« génération, naissance, origine »), est, en biologie, une séquence discrète et héritable de nucléotides dont l'expression affecte les caractères d'un organisme. L'ensemble

Expression génétique

L'expression des gènes, encore appelée expression génique ou expression génétique, désigne l'ensemble des processus biochimiques par lesquels l'information héréditaire stockée dans un gène est lue po