The Large Isoform of Drosophila melanogaster Heterochromatin Protein 2 Plays a Critical Role in Gene Silencing and Chromosome Structure

Abstract

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...

The histone deacetylase Rpd3 regulates the heterochromatin structure ofDrosophilatelomeres

“…In polytene chromosomes HP2 localizes at the chromocenter, at distinct chromosomal bands and at the cap of telomeres (Andreyeva et al, 2005;Shaffer et al, 2006). However, unlike HP1, HP2 is not a capping protein and is not required to prevent chromosome fusions (Shaffer et al, 2006). Surprisingly, immunofluorescence ( Fig.…”

“…Because specific histone modifications are crucial for the localization of heterochromatin proteins, we sought to determine whether alterations in both AcH4 and trimeH3K9 caused by loss of Rpd3 influenced the distributions of heterochromatin proteins that, like HP1, associate with chromosome ends in Drosophila. We therefore analyzed the telomere localization pattern of HP2, an HP1-interacting factor with a role in the structural organization of chromosomes and in heterochromatin-induced gene silencing (Shaffer et al, 2006). In polytene chromosomes HP2 localizes at the chromocenter, at distinct chromosomal bands and at the cap of telomeres (Andreyeva et al, 2005;Shaffer et al, 2006).…”

“…We therefore analyzed the telomere localization pattern of HP2, an HP1-interacting factor with a role in the structural organization of chromosomes and in heterochromatin-induced gene silencing (Shaffer et al, 2006). In polytene chromosomes HP2 localizes at the chromocenter, at distinct chromosomal bands and at the cap of telomeres (Andreyeva et al, 2005;Shaffer et al, 2006). However, unlike HP1, HP2 is not a capping protein and is not required to prevent chromosome fusions (Shaffer et al, 2006).…”

Position-Effect Variegation, Heterochromatin Formation, and Gene Silencing in Drosophila

“…The approach has been used to discover heterochromatin-associated proteins (e.g., HP1a; James and Elgin 1986) and to determine whether other candidates, identified genetically (by Su(var) phenotype) or by interaction with known heterochromatin proteins, show such localization (Fig. 3A) (e.g., HP2, identified by interaction with HP1a; Shaffer et al 2006). Whereas genome-wide ChIP techniques described below have higher resolution, polytene chromosome staining remains a quick and inexpensive way (given specific antibodies in hand) to show distribution patterns.…”

A Distinct Type of Heterochromatin Within Drosophila melanogaster Chromosome 4

“…Stable heterochromatin formation requires products encoded by suppressors of variegation [Su(var)s]. In pericentric regions, antipodal dosage dependence is seen for HP1, HP2, SU(VAR)3-7, and SU(VAR)3-9 (a histone H3K9 methyltransferase, HMT), suggesting that these are core structural proteins of heterochromatin (Locke et al 1988;Eissenberg et al 1990;Reuter et al 1990;Cleard et al 1997;Shaffer et al 2006); HP1 interacts directly with the other three and binds H3 dimethylated at lysine 9 (H3K9me2) as well (reviewed by Schotta et al 2003;Stephens et al 2005). Telomere position effect (TPE) requires a distinct set of Mod(var)s and is generally unaffected by pericentric PEV modifiers (Wallrath and Elgin 1995;Cryderman et al 1999;Boivin et al 2003).…”