RESUMO
Meis1, a conserved transcription factor of the TALE-homeodomain class, is expressed in a wide variety of tissues during development. Its complex expression pattern is likely to be controlled by an equally complex regulatory landscape. Here we have scanned the Meis1 locus for regulatory elements and found 13 non-coding regions, highly conserved between humans and teleost fishes, that have enhancer activity in stable transgenic zebrafish lines. All these regions are syntenic in most vertebrates. The composite expression of all these enhancer elements recapitulate most of Meis1 expression during early embryogenesis, indicating they comprise a basic set of regulatory elements of the Meis1 gene. Using bioinformatic tools, we identify a number of potential binding sites for transcription factors that are compatible with the regulation of these enhancers. Specifically, HHc2:066650, which is expressed in the developing retina and optic tectum, harbors several predicted Pax6 sites. Biochemical, functional and transgenic assays indicate that pax6 genes directly regulate HHc2:066650 activity.
Assuntos
Proteínas do Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Neoplasias/genética , Fatores de Transcrição Box Pareados/metabolismo , Elementos Reguladores de Transcrição , Proteínas Repressoras/metabolismo , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Imunoprecipitação da Cromatina , Biologia Computacional , Olho/embriologia , Olho/metabolismo , Proteínas do Olho/genética , Humanos , Hibridização In Situ , Proteína Meis1 , Especificidade de Órgãos , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Proteínas Repressoras/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismoRESUMO
Non-coding DNA conservation across species has been often used as a predictor for transcriptional enhancer activity. However, only a few systematic analyses of the function of these highly conserved non-coding regions (HCNRs) have been performed. Here we use zebrafish transgenic assays to perform a systematic study of 113 HCNRs from human chromosome 16. By comparing transient and stable transgenesis, we show that the first method is highly inefficient, leading to 40% of false positives and 20% of false negatives. When analyzed in stable transgenic lines, a great majority of HCNRs were active in the central nervous system, although some of them drove expression in other organs such as the eye and the excretory system. Finally, by testing a fraction of the HCNRs lacking enhancer activity for in vivo insulator activity, we find that 20% of them may contain enhancer-blocking function. Altogether our data indicate that HCNRs may contain different types of cis-regulatory activity, including enhancer, insulators as well as other not yet discovered functions.
