Sorted cell ChIP-seq shows the molecular organization of Polycomb-repressed chromatin in the bithorax complex. Sarah K. Bowman¹, Aimee M. Deaton¹, Heber Domingues², Welcome Bender², Robert E. Kingston¹. 1) Dept. of Molecular Biology, Massachusetts General Hospital, and Dept. of Genetics, Harvard Medical School, Boston, MA; 2) Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA.

   The 300 kb of the Drosophila bithorax complex (BX-C) is the original model for studying gene repression by Polycomb group proteins. Decades of genetic experiments have led to the hypothesis that Polycomb organizes BX-C chromatin differently in each of the abdominal parasegments. However, studying the molecular organization of chromatin in individual parasegments has been technically difficult. We solved this problem by developing a sorted nuclei ChIP-seq pipeline. In this system, transgenic embryos produce tagged nuclei in single parasegments, in either PS4, 5, 6, or 7. Using FACS, we sort these tagged nuclei and perform small-scale ChIP-seq. Initial results show that the mark of Polycomb repression, H3K27me3, is lost from the active regions of the BX-C. The proximal H3K27me3-free region becomes progressively larger as we move from PS5 to PS7. The boundaries of the H3K27 methylation correlate precisely with previously identified CTCF binding sites. Correspondingly, the H3K4me3 active mark appears over the transcription start sites of BX-C genes as they emerge from the repressive environment. These results suggest that BX-C chromatin "opens up" at a molecular level in a proximal to distal direction, as previously hypothesized. By extending this study to other histone modifications and regulators of chromatin structure, we hope to gain insight into the molecular organization of repressed and activated chromatin in this uniquely well-annotated gene region.