Sloan-Kettering Institute // Developmental Biology Program

Mary Goll, PhD

Almost every cell in an organism contains the identical DNA sequence; yet different tissues transcribe unique sets of genes. Differences in transcription are mediated in part through epigenetic marks including DNA methylation and histone modifications. Epigenetic regulation is also important for heritable silencing of transcription at transposons, repeated sequences and the inactive X chromosome. Altered epigenetic profiles lead to abnormal development and are almost always detected in cancer genomes. We are interested in understanding the mechanisms of epigenetic regulation and their roles in vertebrate development with a primary focus on the DNA modification, 5-methylcytosine.

The zebrafish model for study of epigentic regulation

Zebrafish (A) 1-cell embryo, (B) transgenic larva expressing red and green fluorophores in different subsets of cells, C) Adult female

Zebrafish are particularly suited to studies of epigenetic regulation. Each female can lay hundreds of eggs every week, providing large numbers for monitoring inheritance. Embryos develop externally, and are optically clear, which facilitates live monitoring of transcription from fluorescent reporters at cellular resolution. Finally, their small size and rapid generation time make zebrafish amenable to forward genetic screens.

An in vivo system to study epigenetic regulation

Epigenetic regulation of transcription is essential for normal development. Despite its importance, in vivo systems to monitor epigenetic regulation of transcription during the course of development have been lacking in vertebrates. We have developed a transgenic approach to monitor epigenetic regulation of fluorescent reporters in different cells and tissues in live zebrafish. A variety of transgenic lines have established that initially direct robust tissue-specific expression of GFP. However, in subsequent generations, GFP labeling becomes mosaic or lost. DNA cytosine methylation is an important component of the observed silencing, because increased DNA methylation is seen at silenced transgenes and reactivation is observed upon introduction of mutations that decrease DNA methylation. Our aim is to elucidate additional factors required for silencing and to understand the sequence cues that lead to the silencing of these transgenes.

Dorsal views of 3dp zebrafish larvae (A) Larvae carrying a methylated brain-specific GFP transgene do not express GFP due to silencing of the transgene. (B) Reactivation of the silenced transgene in a zebrafish larva with decreased DNA methylation.

The transgenic strategy we have developed will provide an excellent platform for us to assay conditions that cause silencing and reactivation of transgene expression using genetic, molecular and chemical approaches. Moreover, it provides us with a unique opportunity to probe the specific requirements for silencing in distinct cell types and at different stages of development using live imaging techniques. Ultimately, we expect that a greater understanding of how these fluorescent reporters are recognized and silenced will have broader implications for silencing at many endogenous sequences across vertebrate genomes.

Publications

Akitake CM, Macurak M, Halpern ME, Goll MG. Transgenerational analysis of transcriptional silencing in zebrafish. Dev Biol. 2011 Apr 15;352(2):191-201. doi: 10.1016/j.ydbio.2011.01.002. Epub 2011 Jan 9.

Hu G, Goll MG, Fisher S. ΦC31 integrase mediates efficient cassette exchange in the zebrafish germline. Dev Dyn. 2011 Sep;240(9):2101-7. doi: 10.1002/dvdy.22699. Epub 2011 Jul 29.

Goll MG, Anderson R, Stainier DY, Spradling AC, Halpern ME. Transcriptional silencing and reactivation in transgenic zebrafish. Genetics. 2009 Jul;182(3):747-55. doi: 10.1534/genetics.109.102079. Epub 2009 May 11.

Anderson RM, Bosch JA, Goll MG, Hesselson D, Dong PD, Shin D, Chi NC, Shin CH, Schlegel A, Halpern M, Stainier DY. Loss of Dnmt1 catalytic activity reveals multiple roles for DNA methylation during pancreas development and regeneration. Dev Biol. 2009 Oct 1;334(1):213-23. doi: 10.1016/j.ydbio.2009.07.017. Epub 2009 Jul 22.

Goll MG, Kirpekar F, Maggert KA, Yoder JA, Hsieh CL, Zhang X, Golic KG, Jacobsen SE, Bestor TH. Methylation of tRNAAsp by the DNA methyltransferase homolog Dnmt2. Science. 2006 Jan 20;311(5759):395-8.