Drosophila melanogaster Heterochromatin Protein 1a (HP1a) is essential for compacted heterochromatin structure and associated gene silencing. Its chromo shadow domain (CSD) is well-known for binding to peptides that contain a PXVXL motif. Heterochromatin protein 2 (HP2) is a nonhistone chromosomal protein that associates with HP1a in the pericentric heterochromatin, telomeres and the fourth chromosome. Using NMR spectroscopy, fluorescence polarization and site-directed mutagenesis, we identified an LCVKI motif in HP2 that binds to the HP1a CSD. The binding affinity of the HP2 fragment is approximately two orders of magnitude higher than that of peptides from PIWI (with a PRVKV motif), AF10 (with a PLVVL motif), or CG15356 (with LYPLL and LSIVA motifs). To delineate differential interactions of the HP1a CSD, we characterized its structure, backbone dynamics and dimerization constant. We find that the dimerization constant is bracketed by the affinities of HP2 and PIWI, which dock to the same HP1a homodimer surface. This suggests that HP2, but not PIWI, interaction can drive homodimerization of HP1a. Interestingly, the integrity of the disordered C-terminal extension (CTE) of HP1a is essential for discriminatory binding, whereas swapping the PXVXL motifs does not confer specificity. Serine phosphorylation at the peptide binding surface of the CSD is thought to regulate heterochromatin assembly. Glutamic acid substitution at these sites destabilizes HP1a dimers, but improves the interaction with both binding partners. Our studies underscore the importance of CSD dimerization and cooperation with the CTE in forming distinct complexes of HP1a.

Heterochromatin protein 1 (HP1), first discovered in Drosophila melanogaster, is a highly conserved chromosomal protein implicated in both heterochromatin formation and gene silencing. We report here characterization of an HP1-interacting protein, heterochromatin protein 2 (HP2), which codistributes with HP1 in the pericentric heterochromatin. HP2 is a large protein with two major isoforms of approximately 356 and 176 kDa. The smaller isoform is produced from an alternative splicing pattern in which two exons are skipped. Both isoforms contain the domain that interacts with HP1; the larger isoform contains two AT-hook motifs. Mutations recovered in HP2 act as dominant suppressors of position effect variegation, confirming a role in heterochromatin spreading and gene silencing.

Heterochromatin Protein 2 (HP2) is a nonhistone chromosomal protein from Drosophila melanogaster localized principally in the pericentric heterochromatin, telomeres, and fourth chromosome, all regions associated with HP1. Mutations in HP2 can suppress position effect variegation, indicating a role in gene silencing and heterochromatin formation [Shaffer, C.D. et al. (2002) Proc. Natl. Acad. Sci. 99, 14332-14337]. In vitro coimmunoprecipitation experiments with various peptides from HP2 have identified a single HP1 binding domain. Conserved domains in HP2, including those within the HP1 binding region, have been identified by recovering and sequencing Su(var)2-HP2 from D. willistoni and D. virilis, as well as examining available sequence data from D. pseudoobscura. A PxVxL motif, shown to be an HP1 binding domain in many HP1-interacting proteins, is observed but is not well conserved in location and sequence, and does not mediate HP2 binding to HP1. The sole HP1 binding domain is composed of two conserved regions of 12 and 16 amino acids separated by 19 amino acids. Site-directed mutagenesis within the two conserved regions has shown that the 16 amino acid domain is critical for HP1 binding. This constitutes a novel domain for HP1 interaction, providing a critical link for heterochromatin formation in Drosophila.Regions of every euchromatic genome, prominently the centromeres and the telomeres, are packaged as constitutive heterochromatin. These regions contain relatively few genes, and are made up primarily of repetitious sequences, such as satellite DNA and transposable elements. Heterochromatic domains are characterized by a condensed appearance of the interphase chromatin, a low level of meiotic recombination, and late replication in S phase. In Drosophila, a normally euchromatic gene that is mislocalized to heterochromatin by tranposition or rearrangment will be silenced in some cells but not others, resulting in a variegating phenotype. This phenomenon is known as PEV 1 , or position effect variegation (1). Mutations that suppress PEV (resulting in a loss of silencing) have identified many genes whose products are essential for heterochromatin formation (2,3).Heterochromatin Protein 1 (HP1), one of the best-characterized nonhistone chromosomal proteins, has been implicated in both heterochromatin formation and gene silencing. HP1 is conserved from yeast (Schizosaccharomyces pombe) to humans and is consistently associated with pericentric heterochromatin (4). HP1 has been found to be part of multiprotein complexes that are necessary for the induction of heterochromatin formation. Proteins in the HP1 family contain an amino-terminal chromodomain and a carboxy-terminal chromoshadow domain separated by a linker region of variable length. The chromodomain has been found to bind to histone H3 methylated at lysine 9 (H3-mK9) (5,6); the chromoshadow domain is a proteinCorrespondence to be sent to: Gena Eve Stephens, protein interaction domain that forms a homodimer (7) and binds to many other proteins, in...

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Drosophila melanogaster heterochromatin protein 2 (HP2) interacts with heterochromatin protein 1 (HP1). In polytene chromosomes, HP2 and HP1 colocalize at the chromocenter, telomeres, and the small fourth chromosome. We show here that HP2 is present in the arms as well as the centromeric regions of mitotic chromosomes. We also demonstrate that Su(var)2-HP2 exhibits a dosage-dependent modification of variegation of a yellow reporter transgene, indicating a structural role in heterochromatin formation. We have isolated and characterized 14 new mutations in the Su(var)2-HP2 gene. Using w m4h , many (but not all) mutant alleles show dominant Su(var) activity. Su(var)2-HP2 mutant larvae show a wide variety of mitotic abnormalities, but not the telomere fusion seen in larvae deficient for HP1. The Su(var)2-HP2 gene codes for two isoforms: HP2-L (365 kDa) and HP2-S (175 kDa), lacking exons 5 and 6. In general, mutations that affect only the larger isoform result in more pronounced defects than do mutations common to both isoforms. This suggests that an imbalance between large and small isoforms is particularly deleterious. These results indicate a role for HP2 in the structural organization of chromosomes and in heterochromatin-induced gene silencing and show that the larger isoform plays a critical role in these processes. T HE DNA found inside a eukaryotic nucleus does not exist as such, but is packaged with proteins to form chromatin. By weight, chromatin is approximately one-third DNA, one-third histones, and one-third nonhistone chromosomal proteins, plus a small RNA component. The histones play a major role in packaging and organizing the very long DNA molecules of each chromosome. Analysis of post-transcriptional modifications of histones shows that these basic proteins also play a significant role in determining specific modes of packaging and concomitant gene regulation, interacting with both enzymes and structural proteins that define alternative chromatin states (for reviews, see Richards and Elgin 2002;Khorasanizadeh 2004). One level of packaging, evident by cytological examination of interphase nuclei, is the partitioning of chromatin into euchromatin and heterochromatin. While euchromatin decondenses during interphase, heterochromatin remains relatively more condensed, showing intense staining.In Drosophila melanogaster, the entire Y chromosome, most of the fourth chromosome, the proximal 40% of the X chromosome, and the pericentric 20% of the major autosomes are heterochromatic. Euchromatic DNA has a high proportion of genes and unique sequences and tends to replicate throughout S phase. Heterochromatin has relatively few genes, is rich in repetitive sequences, and tends to replicate late in S phase; it is often found to be underreplicated (compared to euchromatin) in the polytene chromosomes of insects such as D. melanogaster. Heterochromatin is also characterized by a very low rate of meiotic recombination (Weiler and Wakimoto 1995;Zhimulev et al. 2004).Euchromatin and heterochromatin appear t...