Developmental regulation of lens gene expression

ABSTRACT The long-term goal of this research program is to uncover the transcriptional and posttranscriptional mechanisms coordinating the regulation of tissue-specific gene expression during mammalian embryonic lens development and differentiation. Despite the simple composition of the lens that contains only two mature cell types, the full range and spectrum of mechanisms and pathways governing lens gene expression remain to be identified. A hallmark feature of lens development and cellular differentiation is the temporally and spatially regulated expression of a- and b-/g-crystallins required for lens structure, homeostasis and transparency. This application seeks to identify novel cis-acting DNA regulatory sites and novel RNA-binding proteins (RBPs) that regulate the expression of mammalian crystallins in vivo during lens differentiation. We have now linked the expression of the bB3-crystallin gene in the lens with in vivo binding of transcription factor Pax6 to its “open” promoter chromatin domain in lens enabling prediction of novel cis-acting sites and transcription factors and their posttranslational modifications (PTMs) that spatially and temporally control its expression. We have also discovered transient nuclear accumulation of spliced Crybb3 mRNAs in early lens fiber cell nuclei along with multiple spatially and temporally regulated RBPs that likely regulate bB3- and other crystallin mRNAs transport, stability, translation and decay. These general mechanisms control expression of other lens-specific genes making their discovery an essential first step towards understanding both transcriptional and posttranscriptional regulatory mechanisms underlying lens differentiation. To test our predictions, Aim 1 is designed to uncover the lens-specific cis-regulatory grammar regulating the mouse Crybb3 as a model crystallin. Aim 2 is designed to examine posttranscriptional regulation of lens gene expression by identifying novel RBPs and their sites in crystallin mRNAs particularly within their 3’-UTRs, determining function of identified differentiation-regulated RBPs, and employing a state-of-art MS2-MCP system to in vivo track individual crystallin mRNAs from their birth in the nucleus throughout their nuclear export and localization in the vast space of lens fiber cells at single molecule resolution. These results will reveal those mechanisms linking a-, b/g-crystallin mRNA regulation, identify novel and diverse RBPs that interact with crystallin mRNAs, establish molecular mechanisms of Crybb3 transcription, and provide new insights into the 3D-distribution of individual crystallin mRNAs in lens fiber cells at specific stages of lens cell differentiation. Together, these studies will generate transformative insights into the molecular and cellular mechanisms required for mammalian lens morphogenesis, transparency and refraction and provide a basis for comprehensive understanding of gene expression in more complex tissues in and outside of the eye.