The initial phases of the ENCODE project resulted in the development and establishment of several high throughput strategies for the rapid identification of predicted functional elements in the human genome. Adequate systems to experimentally validate these predictions are currently lacking, especially methods that can test for elements that act as transcriptional silencers and boundary elements. We propose to generate an in vitro and in vivo platform to allow the efficient testing of predicted enhancer, silencers and insulators in the human genome. We will target a panel of 4 human cell lines as well as mouse embryonic stem cells with reporter vectors integrated in site-specific manner through homologous recombination. By using various vector designs we will develop cell lines that can be used for testing genomic sequences both for transcriptional activation properties as well as silencing and boundary properties, independent of variegation and multiple-copy integration effects. We will establish a low cost, efficient in vivo zebrafish transgenic system to test these putative functional noncoding sequences. We will also generate a mouse model to test insulators and silencers, currently not afforded by standard mouse transgenesis technologies. Finally, we will implement a modification on ChIP chip strategies, by integrating epitope tags to various classes of DNA binding proteins, allowing their use in ChIP chip experiments independent of the availability of specific antibodies raised against each factor being tested. Combined, these strategies will generate a comprehensive and powerful toolkit that can be used for the generation of genome-wide cis-regulatory maps of virtually all DNA-binding proteins, providing an efficient system for the identification of their binding sites as well as a variety of experimental assays for the validation of the predictions made by ChIP chip and computational experiments. ? ? ? ?
| Kittler, Ralf; Zhou, Jie; Hua, Sujun et al. (2013) A comprehensive nuclear receptor network for breast cancer cells. Cell Rep 3:538-51 |
| Savic, D; Park, S Y; Bailey, K A et al. (2013) In vitro scan for enhancers at the TCF7L2 locus. Diabetologia 56:121-5 |
| Sakabe, Noboru J; Aneas, Ivy; Shen, Tao et al. (2012) Dual transcriptional activator and repressor roles of TBX20 regulate adult cardiac structure and function. Hum Mol Genet 21:2194-204 |
| Savic, Daniel; Bell, Graeme I; Nobrega, Marcelo A (2012) An in vivo cis-regulatory screen at the type 2 diabetes associated TCF7L2 locus identifies multiple tissue-specific enhancers. PLoS One 7:e36501 |
| Sakabe, Noboru Jo; Savic, Daniel; Nobrega, Marcelo A (2012) Transcriptional enhancers in development and disease. Genome Biol 13:238 |
| Smemo, Scott; Campos, Luciene C; Moskowitz, Ivan P et al. (2012) Regulatory variation in a TBX5 enhancer leads to isolated congenital heart disease. Hum Mol Genet 21:3255-63 |
| Distler, Margaret G; Plant, Leigh D; Sokoloff, Greta et al. (2012) Glyoxalase 1 increases anxiety by reducing GABAA receptor agonist methylglyoxal. J Clin Invest 122:2306-15 |
| Auton, Adam; Fledel-Alon, Adi; Pfeifer, Susanne et al. (2012) A fine-scale chimpanzee genetic map from population sequencing. Science 336:193-8 |
| Shen, Tao; Aneas, Ivy; Sakabe, Noboru et al. (2011) Tbx20 regulates a genetic program essential to adult mouse cardiomyocyte function. J Clin Invest 121:4640-54 |
| Taher, Leila; McGaughey, David M; Maragh, Samantha et al. (2011) Genome-wide identification of conserved regulatory function in diverged sequences. Genome Res 21:1139-49 |
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