The EMBO Journal Vol.18 No.13 pp.3724–3735, 1999 Silencing at Drosophila telomeres: nuclear organization and chromatin structure play critical roles Diane E.Cryderman, Eric J.Morris, rich tracks, but instead possess tandem repeats of the Harald Biessmann1, Sarah C.R.Elgin2 and retrotransposons HeT-A and TART (Biessmann et al., 1992; Lori L.Wallrath3 Levis et al., 1993; Walter et al., 1995; Danilevskaya et al., 1997). Many parallels have been noted between Department of Biochemistry, 4-772 Bowen Science Building, telomerase-based telomere elongation and this retrotran- 1 University of Iowa, Iowa City, IA 52242, Developmental Biology sposon-based system (Pardue et al., 1997). Center, University of California, Irvine, CA 92697 and 2Department of Biology, Washington University, St Louis, MO 63130, USA Telomeric and pericentric regions of most eukaryotic 3 genomes are packaged into heterochromatin, the relatively Corresponding author gene-poor, late replicating material that remains condensed e-mail: [email protected] throughout the cell cycle (Weiler and Wakimoto, 1995). Transgenes inserted into the telomeric regions of Droso- In contrast, most regions between centromeres and telom- phila melanogaster chromosomes exhibit position effect eres are packaged into euchromatin, the gene-rich, early variegation (PEV), a mosaic silencing characteristic of replicating material that decondenses during interphase. euchromatic genes brought into juxtaposition with When euchromatic genes are brought into juxtaposition heterochromatin. Telomeric transgenes on the second with heterochromatin by chromosomal rearrangement or and third chromosomes are flanked by telomeric associ- transposition, they can exhibit position effect variegation ated sequences (TAS), while fourth chromosome telom- (PEV), a silencing of the gene in a subset of the cells in eric transgenes are most often associated with which it is normally expressed (Weiler and Wakimoto, repetitious transposable elements. Telomeric PEV on 1995). Such silencing is not gene specific, and can affect the second and third chromosomes is suppressed by flanking DNA up to 100 kb from the breakpoint (Weiler mutations in Su(z)2, but not by mutations in Su(var)2-5 and Wakimoto, 1995). Silencing is observed when genes (encoding HP1), while the converse is true for telomeric are near telomeres in Trypanosoma brucei (Horn and PEV on the fourth chromosome. This genetic distinc- Cross, 1995; Rudenko et al., 1995), Saccharomyces tion allowed for a spatial and molecular analysis of cerevisiae (Gottschling et al., 1990; Palladino and Gasser, telomeric PEV. Reciprocal translocations between the 1994), Schizosaccharomyces pombe (Nimmo et al., 1994) fourth chromosome telomeric region containing a and Drosophila melanogaster (Gehring et al., 1984; transgene and a second chromosome telomeric region Hazelrigg et al., 1984; Karpen and Spradling, 1992; Levis result in a change in nuclear location of the transgene. et al., 1993; Wallrath and Elgin, 1995); at the silent mating While the variegating phenotype of the white transgene type loci in S.cerevisiae (Stone and Pillus, 1998); and at is suppressed, sensitivity to a mutation in HP1 is pericentric regions of the chromosomes in S.pombe retained. Corresponding changes in the chromatin (Allshire et al., 1994), D.melanogaster (Wallrath and structure and inducible activity of an associated hsp26 Elgin, 1995; Zhang and Spradling, 1995) and mammals transgene are observed. The data indicate that both (Dobie et al., 1997; Kioussis and Festenstein, 1997). nuclear organization and local chromatin structure Although telomeric and pericentric PEV share common play a role in this telomeric PEV. properties, different responses to genetic modifiers have Keywords: chromatin structure/Drosophila/nuclear been noted (Nimmo et al., 1994; Wallrath and Elgin, 1995). organization/PEV/telomeres To understand the molecular mechanisms underlying PEV, a P-element mobilization screen was performed to recover D.melanogaster stocks in which well-studied euchromatic genes were placed within regions inducing Introduction PEV (Wallrath and Elgin, 1995). The P-element included Telomeres were first identified as distinctive structures at a white1 reporter gene expressed from an hsp70 promoter, the natural ends of chromosomes by cytological and allowing detection of a variegating eye phenotype, and a genetic studies in Drosophila (Muller, 1938). Telomeres tagged version of the hsp26 gene. The hsp26 gene, play important roles in maintaining chromosome stability, encoding one of the small heat shock proteins, has been complete DNA replication, correct chromosome segre- extensively characterized; the heat shock genes can be gation and correct positioning of chromosomes within induced in most cell types, facilitating molecular analysis the nucleus (Kipling, 1995). Organisms must possess of gene expression and chromatin structure. In situ hybrid- mechanisms to maintain telomere length. Without such ization of polytene chromosomes from stocks showing mechanisms, telomeric sequences are lost during each PEV of the hsp70–white1 transgene revealed that the P- round of replication (Lingner and Cech, 1998). In most element had inserted within pericentric regions, telomeric metazoans, short G-rich tracks are laid down at the ends sites, or the fourth chromosome (Wallrath and Elgin, of chromosomes by telomerase, using an RNA primer as 1995). [The small fourth chromosome contains blocks of a template (Zakian, 1995). Drosophila telomeres lack G- repetitious DNA with characteristics of heterochromatin 3724 © European Molecular Biology Organization Drosophila telomeric PEV (Carmena and Gonzalez, 1995; Pimpinelli et al., 1995).] The telomeric inserts from this collection have been used Table I. Effects of Su(z)2 and Psc alleles on telomeric PEV here to identify cis- and trans-acting components involved Gene Mutant allele Suppression of in this gene silencing. telomeric PEV A large number of dominant mutations that modify the expression of variegating genes juxtaposed to pericentric Supressor 2 of zeste Su(z)1 – heterochromatin have been characterized (Weiler and Su(z)4 – 1.a1 Wakimoto, 1995). Such mutations are frequently found to Su(z)2 1 Su(z)21.a5 – be in genes that encode chromosomal proteins or modifiers Su(z)21.b7 1 of chromosomal proteins (Wallrath, 1998). Mutations that Su(z)11.b8 – decrease or increase the extent of gene silencing are Su(z)2Deos – designated suppressors or enhancers of PEV, Su(var)sor aSu(z)25 11 Posterior sex combs Psc1 – E(var)s, respectively. In general, those mutations that Psc1.d19 1 suppress the PEV associated with chromosomal rearrange- Psc1.d20 1 ments also suppress variegation of hsp70–white1 trans- Psc14P4 – genes in pericentric regions or along the fourth Pscepb – e22 chromosome, including those in fourth chromosome telom- Psc – Psc1433 – eric locations. In contrast, these Su(var)s have no effect Psch27 – 1 on variegating hsp70–white transgenes associated with Psch28 – second and third chromosome telomeres (Wallrath and Psch30 – Elgin, 1995). Two such examples are Su(var)2-502,a Psce23 – Psce24 – missense mutation in heterochromatin protein 1 (HP1) Psce25 – and Su(var)2-101, which causes an increase in the amount of acetylated histone H4 (Dorn et al., 1986; Eissenberg –, no effect; 1, weak suppression; 11, strong suppression. et al., 1992; Wallrath and Elgin, 1995). These data suggest Su(z)2 and Psc alleles are described in Wu and Howe (1995). a that fourth chromosome telomeric PEV is mechanistically Also deletes Psc. similar to that operating in pericentric regions, but that PEV at the second and third chromosome telomeres differs, Mutations tested that showed no effect on telomeric PEV perhaps involving a different set of chromosomal proteins. were Pc1, Pcl1, Asx1, Scm01, z1, ze, brm2, ISWI1, ISW2, Here we use transgenes as molecular entry points k431, k43γ4, E(z)1 and E(z)53 (mutations are defined in to examine the characteristics of silencing at different Flybase and Lindsley and Zimm, 1992). In contrast, telomeres. We find that a particular class of DNA sequences particular mutations of the Polycomb group genes Psc surrounds the variegating transgenes in the second and and Su(z)2 suppressed second and third chromosome third chromosome telomeres; a different class of DNA telomeric PEV (Table I). Effective Psc alleles include sequences is associated with the variegating transgenes Psc1.d19 and Psc1.d20; effective Su(z)2 alleles include at the fourth chromosome telomere. Genetic analysis Su(z)21.b7 and Su(z)21.a1. The most pronounced effect was implicates different sets of chromosomal proteins as the observed with a deletion of both Psc and Su(z)2, designated limiting factor(s) in silencing at these two types of Su(z)25 (Wu and Howe, 1995). This suppression of PEV telomere. Using X-rays to generate translocations between was observed for all telomeric variegating hsp70–white1 distal chromosome regions (telomere swapping), we transgenes tested on the left arm of chromosome two, the assayed for effects on several chromosomal properties. right arm of chromosome two and the right arm of Changes in nuclear organization, gene expression and chromosome three (Table II). It is unlikely that the effects chromatin structure are observed upon translocation. How- observed with these mutations are due to background ever, sensitivity to specific modifiers of PEV is unaltered. differences, since several of these mutant stocks were These data suggest that both nuclear organization and derived