We report here that a point mutation in the gene which encodes the heterochromatin-specific nonhistone chromosomal protein HP-I in Drosophila melanogaster is associated with dominant suppression of position-effect variegation. The mutation, a G-to-A transition at the first nucleotide of the last intron, causes missplicing of the HP-1 mRNA. This suggests that heterochromatin-specific proteins play a central role in the gene suppression associated with heterochromatic position effects.The partitioning of eukaryotic chromosomes into regions which differ in their degrees of compaction has long been appreciated. Most of the transcriptionally active chromatin appears to decondense after mitotic telophase into euchromatin, but a substantial fraction of chromosomal material remains condensed as heterochromatin. Heterochromatin replicates relatively late in the cell cycle and, in tissues which undergo polytenization, the heterochromatin may be underreplicated.The potential of heterochromatin formation to result in transcriptional inactivation is inferred from two genetic phenomena: Barr-body formation (Lyonization) in mammalian females and position-effect variegation in a variety of organisms (reviewed in ref. 1). In both cases chromosomal regions which are euchromatic under some circumstances assume the morphology of heterochromatin. The condensed structure observed in these cases is strongly correlated with transcriptional inactivity.In Drosophila, the genetic dissection of heterochromatin is aided by the availability of numerous rearrangements which lead to variegated expression of euchromatic genes that have come to be relocated near the heterochromatic breakpoint. A number of loci have been identified which, when mutated, act as dominant modifiers of such variegating position effects (2-7). Many of these loci are believed to encode chromatin proteins or factors that modify chromatin structure (see refs. 8 and 9 for recent reviews).A heterochromatin-specific chromosomal protein called HP-1 has been identified and characterized in D. melanogaster (10, 11). A cDNA encoding this protein has been cloned (10), and the gene has been localized to cytological position 29A on the polytene chromosome map. In this report, we provide the sequence of the gene$, identifying exon and intron boundaries, and present molecular evidence that a point mutation at one boundary, causing missplicing of the HP-1 pre-mRNA, is associated with dominant suppression of heterochromatic position effect. This indicates a requirement for HP-1 protein in generating normal heterochromatin structure. MATERIALS AND METHODSDrosophila Stocks. Su(var)205/In(2LR)CyO and the iso-2nd line (marked with b It rl) were obtained from T. Grigliatti (University of British Columbia, Vancouver). Flies were cultured in half-pint plastic bottles at room temperature, using a cornmeal-based medium supplemented with dried bakers' yeast.Northern Blot Analysis. Total nucleic acids were purified from several flies essentially according to the method of Meyerowitz and H...

Heterochromatin protein 1 (HP1) is a non‐histone chromosomal protein in Drosophila with dosage‐dependent effects on heterochromatin‐mediated gene silencing. An evolutionarily conserved amino acid sequence in the N‐terminal half of HP1 (the ‘chromo domain’) shares > 60% sequence identity with a motif found in the Polycomb protein, a silencer of homeotic genes. We report here that point mutations in the HP1 chromo domain abolish the ability of HP1 to promote gene silencing. We show that the HP1 chromo domain, like the Polycomb chromo domain, has chromosome binding activity, but to distinct chromosomal sites. We constructed a chimeric HP1‐Polycomb protein, consisting of the chromo domain of Polycomb in the context of HP1, and show that it binds to both heterochromatin and Polycomb binding sites in polytene chromosomes. In flies expressing chimeric HP1‐Polycomb protein, endogenous HP1 is mislocalized to Polycomb binding sites, and endogenous polycomb is misdirected to the heterochromatic chromocenter, suggesting that both proteins are recruited to their distinct chromosomal binding sites through protein‐protein contacts. Chimeric HP1‐Polycomb protein expression in transgenic flies promotes heterochromatin‐mediated gene silencing, supporting the view that the chromo domain homology reflects a common mechanistic basis for homeotic and heterochromatic silencing.

Chromosome rearrangements which place euchromatic genes adjacent to a heterochromatic breakpoint frequently result in gene repression (position-effect variegation). This repression is thought to reflect the spreading of a heterochromatic structure into neighboring euchromatin. Two allelic dominant suppressors of position-effect variegation were found to contain mutations within the gene encoding the heterochromatin-specific chromosomal protein HP-1. The site of mutation for each allele is given: one converts Lys169 into a nonsense (ochre) codon, while the other is a frameshift after Ser10. In flies heterozygous for one of the mutant alleles (Su(var)2-504), a truncated HP-1 protein was detectable by Western blot analysis. An HP-1 minigene, consisting of HP-1 cDNA under the control of an Hsp70 heat-inducible promoter, was transduced into flies by P element-mediated germ line transformation. Heat-shock driven expression of this minigene results in elevated HP-1 protein level and enhancement of position-effect variegation. Levels of variegating gene expression thus appear to depend upon the level of expression of a heterochromatin-specific protein. The implications of these observations for mechanism of heterochromatic position effects and heterochromatin function are discussed.

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