Year: 2012
Type: dissertation
Abstract: The expression of retina-specific genes is essential for retinal development during embryogenesis and for maintaining the differentiation of photoreceptor and retinal pigment epithelial (RPE) cells.The regulation of these genes is controlled by retinal transcription factors that bind to specific DNA sequences in the regulatory regions of target genes.Amino acid mutations in some of these factors such as orthodenticle homeobox 2 (OTX2) and microphthalmia-associated transcription factor (MITF) are connected with severe ocular malformations e.g.microphthalmia or anophthalmia.Mutations in DNA-binding sites may also cause aberrant target gene expression and associate with retinal disorders but their significance is currently unclear.In the present studies, several human retina-specific gene promoters (rhodopsin, rod arrestin, rod transducin-D, rod PDEE, IRBP, cone blue opsin and tyrosinase) were cloned and their functionality was studied in cultured photoreceptor and RPE cell models with reporter gene assays.Moreover, the role of transcription factors MITF and OTX2 in regulation of the human tyrosinase gene was studied in detail.Importantly, a new type of gene delivery method, reverse transfection, was developed for post-mitotic retinal cells to facilitate detailed analysis of weak gene promoters.In summary, only the gene promoter of interphotoreceptor retinoid binding protein (IRBP) displayed clear activity and was specifically active in the photoreceptor cell model.The presented data indicates that the cell model had only partially differentiated to photoreceptors.Regulation of the human tyrosinase, a rate-limiting enzyme in melanogenesis, was studied in cell models resembling human RPE and in melanocytes.In RPE cells, OTX2 factor was an essential regulator of the human tyrosinase gene.Transactivation of tyrosinase by OTX2 was mediated by three novel binding elements as well as a novel OTX2-MITF protein-protein interaction that did not require any OTX2 binding sites.A genetic polymorphism in one of the OTX2 binding sites dramatically decreased the activity of tyrosinase expression in the RPE cells.This suggests a functional role of OTX2 in RPE depigmentation.Due to the association of depigmentation with age-related macular degeneration in epidemiological studies, the present findings provide a basis for further investigation of genetic variants associated with abnormal RPE pigmentation.
Author: Mika Reinisalo
Language: English
Subfield: Ophthalmology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W3152546264