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.

Methylation of histone H3 lysine 4 (H3K4) in Saccharomyces cerevisiae is implemented by Set1/COMPASS, which was originally purified based on the similarity of yeast Set1 to human MLL1 and Drosophila melanogaster Trithorax (Trx). While humans have six COMPASS family members, Drosophila possesses a representative of the three subclasses within COMPASS-like complexes: dSet1 (human SET1A/SET1B), Trx (human MLL1/2), and Trr (human MLL3/4). Here, we report the biochemical purification and molecular characterization of the Drosophila COMPASS family. We observed a one-to-one similarity in subunit composition with their mammalian counterparts, with the exception of LPT (lost plant homeodomains [PHDs] of Trr), which copurifies with the Trr complex. LPT is a previously uncharacterized protein that is homologous to the multiple PHD fingers found in the N-terminal regions of mammalian MLL3/4 but not Drosophila Trr, indicating that Trr and LPT constitute a split gene of an MLL3/4 ancestor. Our study demonstrates that all three complexes in Drosophila are H3K4 methyltransferases; however, dSet1/COMPASS is the major monoubiquitination-dependent H3K4 di-and trimethylase in Drosophila. Taken together, this study provides a springboard for the functional dissection of the COMPASS family members and their role in the regulation of histone H3K4 methylation throughout development in Drosophila.Histone H3 lysine 4 methylation (H3K4me) is associated with the transcriptionally active regions of the genome in yeast, flies, and mammals (3,23,35). Set1 was identified as a component of a macromolecular protein complex named COMPASS (complex of proteins associated with Set 1), as the first H3K4 methylase, and it is responsible for all mono-, di-, and trimethylation of H3K4 in yeast (22,31,40,52). In Drosophila melanogaster, four SET domain-containing proteins, namely, Trithorax (Trx), Trithorax-related (Trr), dSet1, and Ash1, have been reported to implement H3K4 methylation (10). All but Ash1, which has subsequently been demonstrated to be an H3K36 methyltransferase (49,59), are related to subunits of the six COMPASS and COMPASS-like complexes in mammals. trx was originally characterized as a gene that when mutated caused homeotic transformations (6, 18). Detailed genetic and molecular analyses showed that Trx is required to maintain activation states of its target genes throughout development and counteracts the repressive effects of the Polycomb group proteins (PcG) (39, 41). Trr was identified based on sequence similarity to Trx but was shown to function in the regulation of hormone-responsive gene expression (42). dSet1 was identified based on sequence homology to the Saccharomyces cerevisiae and mammalian Set1 proteins (53, 58).In mammals, there are at least six SET1-related proteins that form COMPASS-like complexes, namely, SET1A, SET1B, and MLL1 to MLL4. SET1A and SET1B are orthologous to dSet1; MLL1 and MLL2 are orthologous to Drosophila Trx; MLL3 and MLL4 (also known as ALR) are orthologous to Drosophila Trr (33,43,45). All of the m...

The cohesin protein complex is a conserved structural component of chromosomes. Cohesin binds numerous sites along interphase chromosomes and is essential for sister chromatid cohesion and DNA repair. Here, we test the idea that cohesin also regulates gene expression. This idea arose from the finding that the Drosophila Nipped-B protein, a functional homolog of the yeast Scc2 factor that loads cohesin onto chromosomes, facilitates the transcriptional activation of certain genes by enhancers located many kilobases away from their promoters. We find that cohesin binds between a remote wing margin enhancer and the promoter at the cut locus in cultured cells, and that reducing the dosage of the Smc1 cohesin subunit increases cut expression in the developing wing margin. We also find that cut expression is increased by a unique pds5 gene mutation that reduces the binding of cohesin to chromosomes. On the basis of these results, we posit that cohesin inhibits long-range activation of the Drosophila cut gene, and that Nipped-B facilitates activation by regulating cohesin-chromosome binding. Such effects of cohesin on gene expression could be responsible for many of the developmental deficits that occur in Cornelia de Lange syndrome, which is caused by mutations in the human homolog of Nipped-B.

The importance of the interdomain connector loop and of the carboxy-terminal domain of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) for functional interaction with DNA polymerases ␦ (Pol␦) and (Pol) was investigated by site-directed mutagenesis. Two alleles, pol30-79 (IL126,128AA) in the interdomain connector loop and pol30-90 (PK252,253AA) near the carboxy terminus, caused growth defects and elevated sensitivity to DNA-damaging agents. These two mutants also had elevated rates of spontaneous mutations. The mutator phenotype of pol30-90 was due to partially defective mismatch repair in the mutant. In vitro, the mutant PCNAs showed defects in DNA synthesis. Interestingly, the pol30-79 mutant PCNA (pcna-79) was most defective in replication with Pol␦, whereas pcna-90 was defective in replication with Pol. Protein-protein interaction studies showed that pcna-79 and pcna-90 failed to interact with Pol␦ and Pol, respectively. In addition, pcna-90 was defective in interaction with the FEN-1 endo-exonuclease (RTH1 product). A loss of interaction between pcna-79 and the smallest subunit of Pol␦, the POL32 gene product, implicates this interaction in the observed defect with the polymerase. Neither PCNA mutant showed a defect in the interaction with replication factor C or in loading by this complex. Processivity of DNA synthesis by the mutant holoenzyme containing pcna-79 was unaffected on poly(dA) ⅐ oligo(dT) but was dramatically reduced on a natural template with secondary structure. A stem-loop structure with a 20-bp stem formed a virtually complete block for the holoenzyme containing pcna-79 but posed only a minor pause site for wild-type holoenzyme, indicating a function of the POL32 gene product in allowing replication past structural blocks.