Distinct Cytoplasmic and Nuclear Fractions of Drosophila Heterochromatin Protein 1: Their Phosphorylation Levels and Associations with Origin Recognition Complex Proteins

The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions, yet we have only rudimentary molecular details of its structure. A powerful tool in the analyses of its structure in Drosophila has been a group of mutations that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several genes from this group are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin-associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent upon other proteins. To examine HP1 interacting proteins, we isolated three distinct oligomeric species of HP1 from the cytoplasm of early Drosophila embryos and analyzed their compositions. The two larger oligomers share two properties with the fraction of HP1 that is most tightly associated with the chromatin of interphase nuclei: an underphosphorylated HP1 isoform profile and an association with subunits of the origin recognition complex (ORC). We also found that HP1 localization into heterochromatin is disrupted in mutants for the ORC2 subunit. These findings support a role for the ORC-containing oligomers in localizing HP1 into Drosophila heterochromatin that is strikingly similar to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in Saccharomyces cerevisiae.

Molecular cloning of a human homologue of Drosophila heterochromatin protein HP1 using anti-centromere autoantibodies with anti-chromo specificity

We have identified a novel autoantibody specificity in scleroderma that we term anti-chromo. These antibodies recognize several chromosomal antigens with apparent molecular mass of between 23 and 25 kDa, as determined by immunoblots. Anti-chromo autoantibodies occur in 10–15% of sera from patients with anti-centromere antibodies (ACA). We used anti-chromo antibodies to screen a human expression library and obtained cDNA clones encoding a 25 kDa chromosomal autoantigen. DNA sequence analysis reveals this protein to be a human homologue of HP1, a heterochromatin protein of Drosophila melanogaster. We designate our cloned protein HP1Hs alpha. Epitope mapping experiments using both human and Drosophila HP1 reveal that anti-chromo antibodies target a region at the amino terminus of the protein. This region contains a conserved motif, the chromo domain (or HP1/Pc box), first recognized by comparison of Drosophila HP1 with the Polycomb gene product. Both proteins are thought to play a role in creating chromatin structures in which gene expression is suppressed. Anti-chromo thus defines a novel type of autoantibody that recognizes a conserved structural motif found on a number of chromosomal proteins.