RESUMO
Sequence comparisons and functional analysis of the 5' upstream regions of tyrosinase genes have revealed the importance of cis-regulatory elements acting to control the spatiotemporal expression of tyrosinase in the melanocytes and retinal pigmented epithelium of developing embryos. To date there are no reports addressing the control of tyrosinase gene transcription in zebrafish, a vertebrate model organism of increasing importance. To exploit the tyrosinase gene as a marker in zebrafish we set out to clone its promoter and analyse its regulation during embryogenesis. Amplification of a zebrafish tyrosinase complementary DNA fragment by reverse transcriptase polymerase chain reaction allowed us to isolate and sequence a 1041 nt genomic DNA fragment that includes a transcription initiation site and 73 nt of the open reading frame. Bioinformatic analysis of this genomic sequence revealed five E-box motifs, including one CATGTG type E-box present in a putative initiation region. These are conserved positive regulatory elements in vertebrate tyrosinase promoters. We show that a region of 814 nt upstream from the translation start site of the zebrafish tyrosinase gene can drive expression in retinal pigmented epithelium in transiently transgenic zebrafish embryos but that its activity is not restricted to melanin-producing cells. This region is unable to drive transcription in human melanoma cell lines. Ectopic expression from this zebrafish tyrosinase promoter fragment is probably due to the absence of positive and negative cis-regulatory elements, such as a tyrosinase distal element, which is known to function as a pigment cell-specific enhancer.
Assuntos
Embrião não Mamífero/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação Enzimológica da Expressão Gênica/genética , Melaninas/biossíntese , Monofenol Mono-Oxigenase/genética , Epitélio Pigmentado Ocular/embriologia , Regiões Promotoras Genéticas/genética , Peixe-Zebra/embriologia , Animais , Sequência de Bases/genética , Elementos E-Box/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/enzimologia , Genes Reguladores/genética , Humanos , Melanócitos/enzimologia , Melanoma/enzimologia , Melanoma/genética , Dados de Sequência Molecular , Epitélio Pigmentado Ocular/citologia , Epitélio Pigmentado Ocular/enzimologia , Sítio de Iniciação de Transcrição/fisiologia , Células Tumorais Cultivadas/enzimologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
The p14(ARF) tumor suppressor is a key regulator of cellular proliferation and is frequently inactivated in human cancer. This tumor suppressor functions in the p53 and pRb cell cycle regulatory pathways and can effectively activate both pathways to induce growth arrest or cell death. We now report that p14(ARF) forms a complex with the E1A-regulated transcriptional repressor, p120(E4F). p120(E4F) contacts p14(ARF) and p53 to form a ternary complex in vivo and enhances p14(ARF)-induced G(2) cell cycle arrest in a p53-dependent manner. We suggest that the interaction of p14(ARF) and p120(E4F) forms an important link between the p14(ARF) and p53 tumor suppressor proteins, both of which exhibit enhanced cell cycle inhibitory activity in the presence of this transcriptional repressor.
