Methylation of DNA cytosine residues results in epigenetic transcriptional repression, and aberrant methylation plays a role in human cancers. During development, DNA methylation is a mechanism whereby cell-type specific gene expression patterns are set during terminal differentiation. However, the specific role of DNA methylation in terminal differentiation and organogenesis has so far been studied in very few cell types. This is due in part to the early embryonic lethality of knockout mice lacking genes required for DNA methylation. Unlike mouse models, zebrafish with mutations in two key epigenetic regulators, DNA Methyltransferase 1 (dnmt1) and Ubiquitin-like, Containing PHD and RING Finger Domains 1 (uhrf1), survive to late embryonic stages, at which time many complex organs (including the eye) have formed. I have taken advantage of these mutant zebrafish lines to study the role of DNA methylation in development of the vertebrate lens. Consisting of only two cell types: proliferative epithelial cells and terminally differentiated lens fibers, the lens is an ideal tissue in which to study gene regulation. Despite this, little is currently known about the role of DNA methylation in lens development. My preliminary data show that loss of Dnmt1 and Uhrf1 function leads to cataracts and morphologically abnormal lenses that contain disorganized and apoptotic lens fibers. Methylation of genomic DNA in dnmt1 and uhrf1 mutants is reduced to 25% of wild type levels, supporting a model in which DNA methylation is required for normal lens development. The goal of the proposed research is to determine how DNA methylation functions to silence genes during lens fiber terminal differentiation. This will be determined with the following specific aims:
Specific Aim 1 : To determine whether lens epithelial cell genes, which are downregulated during lens fiber terminal differentiation, are silenced by DNA methylation.
Specific Aim 2 : To determine whether the lens defects in dnmt1 and uhrf1 mutant zebrafish are caused by deficient histone H3K9 tri-methylation. The experiments proposed are novel in that they will begin to elucidate the role and mechanism of DNA methylation-mediated gene silencing in the process of lens formation. This topic has relevance as a mechanism for understanding cataract formation, and it will increase our understanding of how deregulated DNA methylation can contribute to cancer.

Public Health Relevance

When the normal process of lens development goes awry in humans due to genetic mutations or other factors, cataracts result. The zebrafish models utilized in this study link cataracts to the epigenetic regulatory mechanism of DNA methylation. Very little is currently known about the role of DNA methylation in lens development, and the results of this study will improve medical understanding of cataract formation, and may additionally lead to improved therapeutic options. Beyond lens development and cataractogenesis, aberrant DNA methylation plays a role in human cancers, and the results of this study will provide an additional in vivo system through which tissue- specific roles for methylation during differentiation can be studied.

National Institute of Health (NIH)
National Eye Institute (NEI)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1-F05-C (20))
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Agarwal, Neeraj
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University of Texas Austin
Schools of Arts and Sciences
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Lee, Jiwoon; Cox, Ben D; Daly, Christina M S et al. (2012) An ENU mutagenesis screen in zebrafish for visual system mutants identifies a novel splice-acceptor site mutation in patched2 that results in Colobomas. Invest Ophthalmol Vis Sci 53:8214-21
Tittle, Rachel K; Sze, Ryan; Ng, Anthony et al. (2011) Uhrf1 and Dnmt1 are required for development and maintenance of the zebrafish lens. Dev Biol 350:50-63