All genetic processes within the nuclei of a eukaryotic cell occur in the context chromatin. Post-translational modifications of histones, the primary component of chromatin, are involved in the regulatory functions of chromatin. Histone methylation at specific lysines residues is associated with both the transcriptional stimulation and repression of genes. Methylation at two lysine sites on histone H3 (K9 and K27) are implicated in the formation of pericentric heterochromatin, X inactivation, Polycomb-mediated gene silencing, and transcriptional repression at euchromatin positions. All these function involve the formation of repressive chromatin. Two classes of proteins heterochromatin protein 1 (HP1) and the polycomb group complex (PcG) interact with histone H3 methylated at lysine 9 (H3-K9) and 27 (H3-K27). Data indicate that HP1 is specific for H3-K9 and PcG is specific for H3-K27. Both HP1 and PcG can establish and propagate repressive heterochromatin. In this proposal we examine the histone methylation at lysine 9 and 27, the HP1 and PcG proteins that interact with these modified histones, and a DMA regulatory region that modulates these interactions. To accomplish this, chromatin immunoprecipitations with antibodies specific to these proteins and histone modifications are performed across the chicken beta-globin neighborhood. Because this neighborhood is well characterized and developmentally regulated, it is an ideal model system to determine the role of these modifications in the regulatory functions of chromatin during cell differentiation. ? ?
