The overarching goal of this study is to understand how BEST1 expression is modulated by cis-regulatory elements in the retinal pigment epithelium (RPE). The variability of retinal phenotypes and age of onset of visual loss, even in individuals who carry the same causative mutation, is one of the biggest mysteries for BEST1 related diseases. Genetic variations in non-coding regulatory elements can serve as an attractive model to provide the molecular basis for the observed various onsets of BEST1 vitelliform macular dystrophy (BVMD) in patients. Therefore understanding the transcriptional control mediated by cis-regulatory elements in the RPE will give us insight into novel target regions for therapeutic editing of the enhancer elements identified by this study. We are utilizing human induced pluripotent stem cells (iPSCs)-derived RPEs as a model to understand cell type-specific transcriptional controls of BEST1. We are also utilizing integrative, unbiased, and high throughput epigenomic and genetic tools to achieve the following aims: (1) We will use a high resolution 4C-seq analysis to identify potential regulatory elements that interact with the BEST1 promoter in human primary RPE. (2) In Aim 2, we will generate BEST1 dual allele reporter lines and use these lines to interrogate cis-regulatory elements of BEST1 in their native chromatin contexts utilizing high throughput CRIPSR/Cas9 mediated genome editing approach. (3) Finally, we will test the utilities of allelic specific enhancer deletion to modulate allelic expression of BEST1. We expect these analyses will significantly advance our knowledge of the precise control of BEST1 transcription in RPEs.
Elucidating the molecular mechanism of BEST1 gene regulation will greatly advance our understanding of various onset of incurable BEST1-related blindness. The outcome of this study will provide the molecular basis for new drug targets and places for therapeutic editing via modulating allelic expression of BEST1. The proposed system will have a wide implication to other dominant negative eye disease with various clinical phenotypes and various ages of onset.
| Li, Yun; Hu, Ming; Shen, Yin (2018) Gene regulation in the 3D genome. Hum Mol Genet 27:R228-R233 |
