1. Cis-acting control of epigenetic states

There is a lot of information known about where DNA methylation and histone modifications lie in the mammalian genome, but almost nothing is known about how they get there. Critical enzymes needed to place these marks have been identified, but the enzymes don't act randomly in the genome; there are some essential instructions that tell them where to act. What provides those instructions? The process of transcription provides a useful analogy to appreciate this question: 

We know a lot about where genes are located, the mRNAs transcribed from them, the promoters and other regulatory sequences that direct transcription and soluble transcription factors that act at those regulatory sequences. But in the epigenetics field, though there are rapidly accumulating data describing the locations of epigenetic modifications and the soluble factors that are able to place them, there are at most three known regulatory sequences that can program or direct local placement of those marks.

Our lab has identified one of them at the Rasgrf1 locus in mouse that is able to program the local DNA methylation state through the piRNA pathway. Now we seek to understand how that methylation programmer directs the piRNA pathway to control local DNA methylation and the consequences of perturbing the mechanism. Some of our papers in this area are:

Park, Y. J., Herman, H., Gao, Y., Lindroth, A. M., Hu, B. Y., Murphy, P. J., Putnam, J. R., and Soloway, P. D. (2012) Sequences sufficient for programming imprinted germline DNA methylation defined. PLoS ONE 7, e33024.

Watanabe, T., Tomizawa, S., Mitsuya, K., Totoki, Y., Yamamoto, Y., Kuramochi-Miyagawa, S., Iida, N., Hoki, Y., Murphy, P. J., Toyoda, A., Gotoh, K., Hiura, H., Arima, T., Fujiyama, A., Sado, T., Shibata, T., Nakano, T., Lin, H., Ichiyanagi, K., Soloway, P. D., and Sasaki, H. (2011) Role for piRNAs and noncoding RNA in de novo DNA methylation of the imprinted mouse Rasgrf1 locus. Science 332, 848-852.

Drake, N. M., Devito, L. M., Cleland, T. A., and Soloway, P. D. (2011) Imprinted Rasgrf1 expression in neonatal mice affects olfactory learning and memory. Genes Brain Behav 10, 392-403.

Brideau, C. M., Kauppinen, K. P., Holmes, R., and Soloway, P. D. (2010) A Non-Coding RNA Within the Rasgrf1 Locus in Mouse Is Imprinted and Regulated by Its Homologous Chromosome in Trans. PLoS ONE 5, e13784.

Drake, N. M., Park, Y. J., Shirali, A. S., Cleland, T. A., and Soloway, P. D. (2009) Imprint switch mutations at Rasgrf1 support conflict hypothesis of imprinting and define a growth control mechanism upstream of IGF1. Mamm Genome 20, 654-663.

Lindroth, A. M., Park, Y. J., McLean, C. M., Dokshin, G. A., Persson, J. M., Herman, H., Pasini, D., Miro, X., Donohoe, M. E., Lee, J. T., Helin, K., and Soloway, P. D. (2008) Antagonism between DNA and H3K27 Methylation at the Imprinted Rasgrf1 Locus. PLoS Genetics 4, e1000145.

Holmes, R., Chang, Y., and Soloway, P. D. (2006) Timing and Sequence Requirements Defined for Embryonic Maintenance of Imprinted DNA Methylation at Rasgrf1. Mol Cell Biol 26, 9564-9570.

Yoon, B.-J., Herman, H., Hu, B., Park, Y. J., Lindroth, A. M., Bell, A., West, A. G., Chang, Y., Stablewski, A., Piel, J. C., Loukinov, D. I., Lobanenkov, V., and Soloway, P. D. (2005) Rasgrf1 Imprinting is Regulated by a CTCF-dependent Methylation-Sensitive Enhancer Blocker. Mol Cell Biol 25, 11184-11190.

Herman, H., Lu, M., Anggraini, M., Sikora, A., Chang, Y., Yoon, B. J., and Soloway, P. D. (2003) Trans allele methylation and paramutation-like effects in mice. Nat Genet 34, 199-202.

Yoon, B. J., Herman, H., Sikora, A., Smith, L. T., Plass, C., and Soloway, P. D. (2002) Regulation of DNA methylation of Rasgrf1. Nat Genet 30, 92-96.

2. Epigenomic analyses on a nanoscale device

Existing methods for performing genome wide epigenomic analyses rely on chromatin immunoprecipitations (ChIP) analyzed on a microarray (chip) or by deep sequencing (seq). ChIP-chip and ChIP-seq approaches have been extraordinarily useful, but they suffer from inherent limitations: (1) they require abundant materials, making analysis of single embryos, rare populations of cells or microdissected material impossible; (2) they query only one epigenetic mark at a time, making simultaneous analysis of multiple epigenetic marks impossible. In collaboration with Harold Craighead's lab in Engineering Physics, we are developing methods to overcome these limitations.

Cipriany, B. R., Murphy, P. J., Hagarman, J. A., Cerf, A., Latulippe, D., Levy, S. L., Benitez, J. J., Tan, C. P., Topolancik, J., Soloway, P. D., and Craighead, H. G. (2012) Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel. Proc Natl Acad Sci U S A 109, 8477-8482.

Cipriany, B. R., Zhao, R., Murphy, P. J., Levy, S. L., Tan, C. P., Craighead, H. G., and Soloway, P. D. (2010) "Single Molecule Epigenetic Analysis in a Nanofluidic Channel" Anal Chem 82: 2480-2487

Funding for Our Work

Epigenetic Control of a Ras Activator
National Institutes of Health CA098597 / GM105243

Epigenomic Analysis on a Nanoscale Device
National Institutes of Health DA025722

Tools for Single Molecule and Single Cell Epigenomic Analysis
National Institutes of Health HG006850