The chromatin configurations of Polycomb Response Elements (PREs) define epigenetic states. Kami Ahmad¹, Guillermo Orsi¹, Steven Henikoff², Jorja Henikoff². 1) Dept BCMP, Harvard Medical Sch, Boston, MA; 2) FHCRC, Seattle, WA.

   PREs are regulatory elements that are essential to establish and maintain repression of large chromatin domains. A number of transcription factors bind at PREs and facilitate K27-trimethylation of histone H3, Polycomb recruitment, and gene repression. However, it is thought that both activating trithorax-Group (trxG) and repressing Polycomb-Group (PcG) factors bind simultaneously at PREs, and antagonistic interactions between these factors determine PRE activity. How these factors interact and are developmentally regulated is unknown. We have used micrococcal nuclease digestion of chromatin and paired-end sequencing (MNase-Seq) to define the occupancy of nucleosomes and transcription factors in two Drosophila cell lines, at base-pair resolution. We find that PREs are clusters of protected factor particles in both activating and repressing states. However, the specific configuration of factor binding differs in the two states. Analysis of underlying sequence motifs suggests that the trxG protein Trl binds and destabilizes nucleosomes at both activating and repressing PREs. Strikingly, at repressing PREs a novel occupied motif implicates an additional factor in reorganizing PRE-bound proteins into a more elaborate and stable bound configuration. To determine the composition of these factor complexes, we have developed a method for native immunoprecipitation of transcription factor and non-histone protein chromatin particles (MNase-IP-Seq), which we use to define the sites and modes of chromatin-complex interactions. We propose a model where trxG factors potentiate the chromatin of regulatory elements by increasing nucleosome dynamics, and cooperative interactions between PcG-engaged PREs and target promoters stabilize repressive complexes.