Identification and characterization of DNA binding proteins necessary for epigenetic silencing by Polycomb group proteins. Payal Ray, Judith A Kassis. Eunice Kennedy Shriver National Institutes of Child Health and Human Development, NIH, Bethesda, MD.

   Polycomb group proteins (PcG) are a class of transcriptional regulators thought to mediate epigenetic inheritance of a repressed transcriptional state. PcG proteins play an important role in Drosophila and have been shown to have similar functions in vertebrates. PcG proteins act through specific DNA sequences known as PcG response elements (PREs]. PREs range from several hundred to a few thousand base pairs and often can be subdivided into smaller fragments with similar activities. PREs are made up of binding sites for multiple proteins. Our goal is to determine the identity of all the DNA binding proteins that bind to the engrailed PREs. The engrailed gene contains a PRE that has been studied extensively by our group. Previous studies have shown that a minimal 139 bp region contains binding sites for Pho (Pleiohomeotic), Spps (Sp1-like factor for Pairing Sensitive-silencing) GAF (GAGA Factor) and two unknown factors. We aim to identify these unknown proteins that bind to this region and characterize them. To this end, we performed a pull-down using a biotin-tagged oligonucleotide containing the binding site in parallel with an oligonucleotide containing a mutated site. The pull-down samples were analyzed by mass spectrometry and we identified a few candidates that potentially bind to the 139bp fragment. Currently, we are validating these candidate genes by biochemical and genetic approaches and will be presenting the results.