HP1 was first described in Drosophila as a heterochromatin-associated protein with dosage-dependent effects on heterochromatin-induced gene silencing. Recently, membership of the HP1 protein family has expanded tremendously. A number of intriguing interactions between HP1 and other proteins have been described, implicating HP1 in gene regulation, DNA replication, and nuclear architecture.

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...

Recent studies show that heterochromatin‐associated protein‐1 (HP1) recognizes a ‘histone code’ involving methylated Lys9 (methyl‐K9) in histone H3. Using in situ immunofluorescence, we demonstrate that methyl‐K9 H3 and HP1 co‐localize to the heterochromatic regions of Drosophila polytene chromosomes. NMR spectra show that methyl‐K9 binding of HP1 occurs via its chromo (chromosome organization modifier) domain. This interaction requires methyl‐K9 to reside within the proper context of H3 sequence. NMR studies indicate that the methylated H3 tail binds in a groove of HP1 consisting of conserved residues. Using fluorescence anisotropy and isothermal titration calorimetry, we determined that this interaction occurs with a KD of ∼100 μM, with the binding enthalpically driven. A V26M mutation in HP1, which disrupts its gene silencing function, severely destabilizes the H3‐binding interface, and abolishes methyl‐K9 H3 tail binding. Finally, we note that sequence diversity in chromo domains may lead to diverse functions in eukaryotic gene regulation. For example, the chromo domain of the yeast histone acetyltransferase Esa1 does not interact with methyl‐ K9 H3, but instead shows preference for unmodified H3 tail.

Covalent modification of histones on chromatin is a dynamic mechanism by which various nuclear processes are regulated. Methylation of histone H3 on lysine 4 (H3K4) implemented by the macromolecular complex COMPASS and its related complexes is associated with transcriptionally active regions of chromatin. Enzymes that catalyze H3K4 methylation were initially characterized genetically as regulators of Hox loci, long before their catalytic functions were recognized. Since their discovery, genetic and biochemical studies of H3K4 methylases and demethylases have provided important mechanistic insight into the role of H3K4 methylation in HOX gene regulation during development.

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.