Abstract
Purpose :
Animal models have indicated that eye growth is driven locally by signals emanating from the retina in response to external visual cues. Using experimental models of myopia, several molecules have been postulated to form part of these growth pathways, including the transcription factors early growth response-1 (Egr-1), FBJ osteosarcoma oncogene (cFos), and NGFI-A-binding protein 2 (NAB2). However, it is unknown how environmental cues, such as visual defocus, are translated into changes in the expression of such genes. We examined one mechanism by which this may occur, DNA methylation at CpG dinucleotides.
Methods :
Retinal tissue was collected from chicks (n=3 per group) following 4 and 24 hours of form-deprivation (growth induction) or recovery from form-deprivation (growth suppression). DNA was extracted and bisulfite converted prior to PCR amplification of regions of interest within the Egr-1, cFos, and NAB2 gene promotors. This was followed by next generation sequencing (Illumina MiSeq platform).
Results :
26 individual CpG sites showed significant changes in methylation levels, with an even split seen between form-deprivation and recovery. All but one of these methylation sites were associated with transcription factor binding domains. Twelve of these sites were associated with Egr-1 binding domains, with methylation changes in these domains correlating strongly with the known changes in Egr-1 and NAB2 mRNA expression seen during the development or suppression of experimental myopia. Of specific interest, 6546 base-pairs upstream of the transcriptional start site of Egr-1, a bi-directional change in methylation was seen within an Egr-1 self-binding site in response to opposing growth stimuli (FDM (+3.87% increase in methylation, p<0.05), diffuser-removal (-5.15% decrease in methylation, p<0.01)). Finally, this study demonstrated the presence of multiple sub-patterns of methylation within the retina, which showed significant changes in frequency during form-deprivation in Egr-1 and NAB2 (MANOVA=0.001, F(1,5)=440.371, p<0.05). This is indicative of methylation changes occurring within specific subpopulations of retinal neurons.
Conclusions :
The findings of this study support a potential role for DNA methylation in the translation of external visual cues into the molecular signals that may ultimately modulate the rate of ocular growth.
This is a 2020 ARVO Annual Meeting abstract.