The recent discovery of an evolutionary highly conserved epigenetic pathway that regulates segmental homeotic gene expression has opened many new experimental avenues to dissect the molecular basis of patterning of the anterior-posterior (A-P) body axis in the mammalian embryo. Constituents of this pathway include genes of the trithorax (trxG) and Polycomb group (PcG). trxG and PcG proteins engage in multimeric protein complexes that regulate transcription of Hox and other unknown downstream target genes by modifying higher-order chromatin structures. Some of the trxG proteins seem to render regulatory regions accessible to transcription factors by displacing nucleosomes from promoter regions. In contrast, PcG proteins appear to package nucleosomes into a heterochromatin-like state, thereby silencing downstream target genes. Evidence is increasing that an intricate interplay between trxG and PcG genes governs long-term maintenance of segment-specific combinatorial codes of Hox gene expression in the developing paraxial mesoderm and neuroectoderm of the mouse embryo. Disruption of the PcG/trxG pathways alters the Hox gene expression domains and causes homeotic transformations. For example, a hypomorphic mutation in eed (embryonic ectoderm development) results in posterior homeotic transformations along the entire axial skeleton. Based on its direct interaction with histone deacetylases, eed represents a pivotal component of the PcG pathway. A combination of in vitro and in vivo approaches is proposed to dissect the function of the complex PcG network in mouse embryonic development using eed as a molecular entry point. Systematic molecular phenotyping of eed mutant alleles will provide novel insight into the upstream mechanisms that control the temporal and spatial regulation of Hox gene expression in the mouse embryo. Double mutant crosses between eed and other murine PcG and trxG genes are designed to dissect their interplay in the specification of Hox gene expression domains. The molecular phenotyping is complemented by chromatin immunoprecipitation experiments to localize the binding sites of the trxG and PcG protein complexes in the Hox clusters. Novel constituents of this pathway will be identified in a chemical mutagenesis screen for dominant modifiers of eed function in vivo. The long-term goal of this work is to dissect the mechanisms of the murine PcG/trxG network as a paradigm of epigenetic regulation of developmental gene expression in mammals.
