Functional Anatomy of Polycomb and Trithorax Chromatin Landscapes in Drosophila Embryos

Abstract

Polycomb group (PcG) and trithorax group (trxG) proteins are conserved chromatin factors that regulate key developmental genes throughout development. In Drosophila, PcG and trxG factors bind to regulatory DNA elements called PcG and trxG response elements (PREs and TREs). Several DNA binding proteins have been suggested to recruit PcG proteins to PREs, but the DNA sequences necessary and sufficient to define PREs are largely unknown. Here, we used chromatin immunoprecipitation (ChIP) on chip assays to map the chromosomal distribution of Drosophila PcG proteins, the N- and C-terminal fragments of the Trithorax (TRX) protein and four candidate DNA-binding factors for PcG recruitment. In addition, we mapped histone modifications associated with PcG-dependent silencing and TRX-mediated activation. PcG proteins colocalize in large regions that may be defined as polycomb domains and colocalize with recruiters to form several hundreds of putative PREs. Strikingly, the majority of PcG recruiter binding sites are associated with H3K4me3 and not with PcG binding, suggesting that recruiter proteins have a dual function in activation as well as silencing. One major discriminant between activation and silencing is the strong binding of Pleiohomeotic (PHO) to silenced regions, whereas its homolog Pleiohomeotic-like (PHOL) binds preferentially to active promoters. In addition, the C-terminal fragment of TRX (TRX-C) showed high affinity to PcG binding sites, whereas the N-terminal fragment (TRX-N) bound mainly to active promoter regions trimethylated on H3K4. Our results indicate that DNA binding proteins serve as platforms to assist PcG and trxG binding. Furthermore, several DNA sequence features discriminate between PcG- and TRX-N–bound regions, indicating that underlying DNA sequence contains critical information to drive PREs and TREs towards silencing or activation.

Transcriptional read-through is not sufficient to induce an epigenetic switch in the silencing activity of Polycomb response elements

“…S5). These findings are consistent with genome-wide studies showing that both proteins preferentially localize to PREs but are not found at high levels elsewhere in silenced domains (19,26,29,30). We next examined the association of PH and dSfmbt under conditions in which constitutive expression of GAL4 from the tubulin promoter disrupts the silencing activity of the bxdPRE.…”

“…The PRC1 complex PC protein specifically interacts with histone 3 trimethylated at K27 (H3K27me3), the nucleosome mark stably associated with PcGrepressed chromatin (30,31). Unlike other PRC core components, the distribution of PC and H3K27me3 in PcG silenced domains is not limited to the PRE but instead extends outward from the PRE (26,30,31). We obtained similar results: ChIP experiments indicate that PC and H3K27me3 are associated with transgene sequences to either side of the PRE including white gene (Fig.…”

“…The PRC1 complex PC protein specifically interacts with histone 3 trimethylated at K27 (H3K27me3), the nucleosome mark stably associated with PcGrepressed chromatin (30,31). Unlike other PRC core components, the distribution of PC and H3K27me3 in PcG silenced domains is not limited to the PRE but instead extends outward from the PRE (26,30,31).…”

Drosophila O -GlcNAc transferase (OGT) is encoded by the Polycomb group (PcG) gene, super sex combs ( sxc )

“…PcG proteins regulate many targets, including genes encoding proteins that function throughout development (17,18). Some of the pleiotropic phenotypes of Ogt mutants may thus be a consequence of improper silencing of PcG targets, although our data do not rule out other roles for OGT and O-GlcNAc modification in the many important cellular processes linked to OGT function.…”

“…To determine whether the addition of O-GlcNAc residues by OGT might act in concert with PcG protein binding to downstream target genes, we used an anti-O-GlcNAc antibody for in situ analysis of polytene chromosomes. This antibody was chosen because the available anti-OGT (17,18), and suggests that 1 or more PcG proteins could be candidate substrates. The resolution of in situ hybridization is not sufficient to show that OGT acts at the known sites of PcG protein binding, the PcG response elements (PREs).…”