Copyright © 2005 by the Genetics Society of AmericaDOI: 10.1534/genetics.103.023341

A Genetic and Molecular Characterization of Two Proximal HeterochromaticGenes on Chromosome 3 of Drosophila melanogaster

Sandra R. Schulze,1 Donald A. R. Sinclair, Kathleen A. Fitzpatrick and Barry M. Honda2

Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada

Manuscript received July 8, 2004Accepted for publication January 12, 2005

ABSTRACTHeterochromatin comprises a transcriptionally repressive chromosome compartment in the eukaryotic

nucleus; this is exemplified by the silencing effect it has on euchromatic genes that have been relocatednearby, a phenomenon known as position-effect variegation (PEV), first demonstrated in Drosophila melano-gaster. However, the expression of essential heterochromatic genes within these apparently repressiveregions of the genome presents a paradox, an understanding of which could provide key insights intothe effects of chromatin structure on gene expression. To date, very few of these resident heterochromaticgenes have been characterized to any extent, and their expression and regulation remain poorly under-stood. Here we report the cloning and characterization of two proximal heterochromatic genes in D.melanogaster, located deep within the centric heterochromatin of the left arm of chromosome 3. One ofthese genes, RpL15, is uncharacteristically small, is highly expressed, and encodes an essential ribosomalprotein. Its expression appears to be compromised in a genetic background deficient for heterochromatinprotein 1 (HP1), a protein associated with gene silencing in these regions. The second gene in this study,Dbp80, is very large and also appears to show a transcriptional dependence upon HP1; however, it doesnot correspond to any known lethal complementation group and is likely to be a nonessential gene.

HETEROCHROMATIN was originally defined cy- chromatin protein 1 (HP1). This protein has beenshown to localize primarily to the heterochromatin oftologically as those regions in eukaryotic chromo-

somes that appear compacted throughout the cell cycle polytene chromosomes (Fanti et al. 2003 and referencestherein), bind repetitive transgene arrays (Dorer and(Heitz 1928) and has since been shown to be virtually

ubiquitous in animal and plant genomes. In the fruit Henikoff 1994), and silence gene expression (reviewedby Eissenberg and Elgin 2000). The phenomenon offly Drosophila melanogaster, it comprises �30% of the

genome, is rich in middle and highly repetitive se- PEV suggests that heterochromatin forms a transcrip-tionally repressive environment within which the pres-quences, and is gene poor. Repetitive DNA poses formi-

dable technical difficulties for both molecular analysis ence of active, resident heterochromatic genes posessomething of a paradox. Moreover, these genes exhibitand sequence assembly; therefore, it is not surprising

that heterochromatin remains incompletely defined in “reciprocal” heterochromatic PEV; i.e., a heterochro-matic gene will variegate when translocated into a eu-most of the model genomes that have been sequenced

to date (Mardis et al. 2002). chromatic environment (Eberl et al. 1993; Howe et al.1995) and this effect is enhanced in a Su(var) mutantHeterochromatin can silence gene expression, as ex-

emplified by the phenomenon of position-effect variega- background. In fact, mutations in Su(var) genes appearto compromise heterochromatic gene expression in thetion (PEV) in Drosophila, in which the expression of a

euchromatic gene is compromised when it is relocated absence of any genetic rearrangements, at least forthe well-characterized heterochromatic genes light andnear or within a block of heterochromatin (reviewed

by Grewal and Elgin 2002). Genetic screens designed rolled (Clegg et al. 1998; Lu et al. 2000; Sinclair et al.2000). This suggests that heterochromatic genes haveto isolate modifiers of this effect have identified genes

that encode proteins involved in maintaining chromatin evolved a transcriptional dependence on factors thatstructure (Sinclair et al. 1983; Wustmann et al. 1989; normally silence gene expression, underscoring the par-Schotta et al. 2003). One well-characterized example, adoxical nature of gene regulation in this region.Suppressor of variegation 2-5 (Su(var)2-5), encodes hetero- A combination of genetic, cytological, and molecular

studies has been undertaken to better understand howheterochromatic genes function. Work on the organiza-

1Present address: Department of Biochemistry, University of Iowa, tion and density of genes in Drosophila autosomal het-Iowa City, IA 52242. erochromatin has identified lethal complementation

2Corresponding author: Department of Molecular Biology and Bio-groups through classical genetic screens, using chromo-chemistry, Simon Fraser University, 8888 University Dr., Burnaby, BC

V5A 1S6, Canada. E-mail: honda@sfu.ca somal deficiencies in pericentromeric heterochromatin

Genetics 169: 2165–2177 (April 2005)

2166 S. R. Schulze et al.

for chromosome 2 (Hilliker and Holm 1975; Hilliker1976; Coulthard et al. 2003) and chromosome 3 (Mar-chant and Holm 1988a,b; Schulze et al. 2001). Com-plementary mutagenesis with P elements has provided“tagged” genes as well as single-copy entry points formolecular characterization of heterochromatic DNA(Zhang and Spradling 1994; Schulze et al. 2001;Konev et al. 2003). In addition, studies using in situhybridization of mitotic chromosomes have allowed pre-liminary correlations between the cytological and ge-netic maps (e.g., Koryakov et al. 2002).

Figure 1.—Schematic genetic map of 3L heterochromatinIn terms of molecular characterization, heterochro-showing a subset of the deficiencies used to position lethalmatic genes tend to be relatively large, embedded with-complementation groups. Euchromatin is indicated by smallin repetitive environments (Devlin et al. 1990a,b; Ris-ovals to the left of region 47. MH superscript indicates defi-

inger et al. 1997; Warren et al. 2000; Tulin et al. 2002), ciencies generated by Marchant and Holm (1988a,b), andand fall into a wide range of biological functions (Parks superscript D indicates deficiencies generated by Vilinsky et

al. (2002). Approximate alignment to cytological heterochro-and Wieschaus 1991; Biggs et al. 1994; Warner et al.matic subdivisions is based on Koryakov et al. (2002). Un-1998; Rollins et al. 1999); however, relatively little isknown gene order is indicated by large round parentheses;known about their expression and regulation. Release 3regions where complete knockouts cannot be recovered are

of the Drosophila genome sequence has provided a large indicated by light brackets. The right-most boundary ofnumber of pieces of heterochromatic DNA sequence and Df(3LR)6B-29 within 3L heterochromatin is unknown (indi-

cated with lighter line); this Df also removes portions of 3Rallowed the prediction of �300 genes within Dro-heterochromatin.sophila heterochromatin (Hoskins et al. 2002). Com-

plementary cytological mapping by these authors andothers (e.g., Dimitri et al. 2003a,b) has also roughly are more consistent with what has been reported fordefined the positions of many of these predicted genes. heterochromatic gene structure: it is very large, appar-An intriguing issue has been the apparent discrepancy ently due to the expansion of repetitive DNA in itsbetween the small number of autosomal “vital loci,” as many introns (Dimitri et al. 2003a,b), is moderatelydefined genetically, and the much larger number of expressed, and appears to possess a conventional pro-predicted gene models from the Release 3 sequence moter structure. However, it does not correspond toannotation (Hoskins et al. 2002). A recent report sug- any known lethal complementation group and thus maygests that there may be several more proximal, essential not encode an essential function. The expression ofloci than previously reported in 2R heterochromatin both genes is compromised in a genetic background in(Myster et al. 2004), but confirmation of this surprising which the HP1 (Su(var)2-5) dose has been reduced,new estimate may require more extensive genetic analy- which is consistent with the results obtained for the well-ses to rule out the complex intragenic complementation studied heterochromatic genes light and rolled (Lu et al.patterns possible in these regions (Coulthard et al. 2000). Thus, heterochromatic genes may have evolved a2003). dependence on factors that maintain heterochromatin

We are working toward a better understanding of the structure, and this dependence appears to be irrespec-organization, expression, and regulation of genes in tive of gene function or promoter type.centric heterochromatin, with particular emphasis on The results reported in this study provide informationchromosome 3. One focus of our efforts has been a on two more heterochromatic genes, located deepmolecular and genetic analysis of the lethal complemen- within proximal centric heterochromatin. This worktation group l(3)80Fi (referred to hereafter as lethal 2), could lead to insights into the mechanisms by whichthe second most proximal genetic locus in 3L hetero- genes maintain activity in an otherwise transcriptionallychromatin (see Figure 1 for a map of the region). On repressive environment and shows the utility of geneticsthe basis of the complex phenotypes displayed by lethal in supporting annotation of molecularly intractable ge-2, we initially focused on two candidate DNA sequences nomic regions. As more genetically mutable loci in het-that might correspond to this essential gene: Dbp80, erochromatin are linked with annotated gene models,which encodes a DEAD box RNA helicase, and RpL15, we will also be in a better position to address questionswhich encodes a large subunit ribosomal protein. concerning the ratio of essential to nonessential genes

We report here that lethal 2 corresponds to RpL15 ; this in this region and the role that chromatin structuregene is uncharacteristically small for a heterochromatic plays in regulating gene expression.gene, is highly expressed, and possesses regulatory fea-tures characteristic of or unique to other ribosomal

MATERIALS AND METHODSprotein genes. RpL15 is embedded in a repetitive envi-ronment and is located �10 kb upstream of the other Culture conditions: Flies were grown on standard cornmeal-

sucrose medium with either tegosept or proprionic acid as acandidate gene Dbp80. The properties of this latter gene

2167Two Drosophila Heterochromatic Genes

mold inhibitor. Stocks were routinely maintained and crosses 5.2 software, using the “volume analysis” option for quantita-tion (http://www.mdyn.com). In all cases, a probe for rp49performed at room temperature unless otherwise indicated.

Drosophila stocks and strains: Descriptions of most muta- was used as a loading control.Transgene constructs: One genomic and two cDNA con-tions, special chromosomes, and deficiencies used in this work

can be found at the FlyBase website (http://flybase.bio.indiana. structs were prepared and established in multiple differenttransgenic lines. A 2.2-kb Bgl II-HindIII RpL15 genomic frag-edu:82/). Stocks, strains, and screens used to identify third

chromosome heterochromatic genes are described in Mar- ment was subcloned from genomic DNA isolated from a phagelibrary (generously provided by Ron Blackman) and ligatedchant and Holm (1988a,b), Schulze et al. (2001), and Vilin-

sky et al. (2002). Thirteen essential 3L heterochromatic genes into appropriately cut pUAST. Two different cDNA transgeneconstructs were also prepared using an RpL15 cDNA (obtainedfor which we have mutant alleles are designated as lethal 1 [afrom Resgen, RE01373), which was cut out of pFlc-1 usingsimplification of l(3L)h1, from Marchant and Holm 1988b,BamHI and EcoRI. The BamHI/EcoRI fragment was then li-or l(3)80Fj used in FlyBase] to lethal 8, from proximal togated into Bgl II/EcoRI-cut pUAST or Klenow end filled anddistal relative to the centromere (see Figure 1). The threeblunt ligated into StuI-cut shrimp alkaline phosphatase-treateddeficiencies used to map lethal 2 molecularly are Df(3L)FX3,pCaSpeR-hs. Each of the three different constructs (andwhich removes lethal 3 to lethal 8 ; Df(3L)9-56, which removeshelper construct p�25.7 wings clipped) were purified for injec-lethal 1 and lethal 2 ; and Df(3L)K2, which removes lethal 2 andtion using an endotoxin-free plasmid maxi prep kit (QIAGEN,lethal 3. The Su(var)2-5 alleles used in this study have all beenChatsworth, CA).described previously (Lu et al. 2000 and references therein).

Germline transformation: Fertilized eggs from establishedPCR mapping: Deficiency stocks were balanced over a TM3stocks of iso-yw or w1118 flies were collected, dechorionated,chromosome bearing a GFP transgene. Genomic DNA fromdehydrated briefly, and injected using a Leitz laborflux micro-individual embryos from the GFP balanced stock was obtainedscope and Eppendorf model 5242 injection controller. Survi-using the method of Hatton and O’Hare (1999) and PCRvors were crossed back to flies from the same white minus stockwas used to identify embryos homozygous for the deficiencythat was injected, and white� progeny were crossed to the(no GFP PCR product). PCR was then used to test for thedouble-balanced T(2:3) apXa/CyO; TM3 stock for segregationpresence or absence of RpL15 or Dbp80 coding sequences. Inanalysis and homozygosis.both cases, the unrelated X-linked gene Grip 84 was used as

a DNA control. All PCR reactions were carried out in a reactionvolume of 25 �l, using 2 �l of genomic template and 1 �l of10 �m stock for each of four primers: Grip 84 (5�-ACGCTTCT RESULTSCGCTGATGGAC-3� and 5�-GTCGCAGTAACTGGATTGAGT-

lethal 2 mutants show complex complementation, and3�), GFP (5�-CAAGAGTGCCATGCCCGAAG-3� and 5�-GACAGGGCCATCGCCAATTG-3�), Dbp80 exon 9 (5�-GAACTGCT heteroallelic combinations are Minute : lethal 2 is definedGCTTGGCTTGC-3� and 5�-ATATTTGTAGTGATAAGCACC by six alleles: two EMS mutants, 72 and 1-166-37, behaveTTC-3�), or RpL15 (5�-CCGTGCTGTAAGTTGGTTGT-3� and genetically as nulls and display an L1 lethal phase, while5�-GTACCGATAAGCCCCCATC-3�), 1 �l 25 mm MgS04, and

four P alleles [natural P elements from the Birmingham1–2 units of Taq polymerase (GIBCO, Gaithersburg, MD).2 strain (Robertson et al. 1988)] are denoted P�2,Sequencing the mutant alleles: P-mutant alleles were iso-

lated by PCR using a P-element specific primer (consisting of P�8, 7-1, and 8-1 and behave as hypomorphs, whichthe entire 31-bp inverted repeat) and a gene-specific primer typically die at later larval stages. P�8 and P�2 appearfor RpL15 (5�-TCTATATCCCTTGCCAATG-3�). In addition, to be the weakest alleles of all six, as indicated by thethe entire region surrounding the P element was amplified

occasional eclosion of sterile homozygotes in the respec-by PCR from P�2/P�2 homozygous DNA, using flankingtive stocks. All pairwise combinations display a complexprimers. All PCR products were cloned into the pCR2.1 TA

vector according to the manufacturer’s specifications (In- complementation pattern (Schulze et al. 2001), andvitrogen, San Diego) and overlapping regions were sequenced several hypomorphic combinations survive to adult-several times to ensure accuracy (University of Calgary Core hood, displaying a classical Minute syndrome (FigureProtein and DNA services: http://www.ucalgary.ca/�dnalab).

2), including delayed development, fine bristles, roughThe EMS mutant stocks were balanced over a GFP balancer,eyes, reduced or gapped sex combs, misrotated genita-DNA representing mutant alleles was amplified from homozy-lia, and sterility. A weak Minute phenotype is also ob-gous mutant embryo DNA, and the products were cloned into

the pCR2.1 TA cloning vector and sequenced, as described served when a deficiency for l2 is placed in trans to a wild-above. type chromosome (data not shown), suggesting that this

Southern analysis: Genomic DNA from adult flies was iso- gene is haplo-insufficient (see the discussion for morelated according to the method of Jowett (Roberts 1998).on the Minute phenotype). Heteroallelic combinationsSouthern blots using Hybond N� nylon membrane (Amer-show variable viability, ranging from complete lethalitysham, Buckinghamshire, UK), were as described in Sambrook

et al. (1989). Probes were labeled using a random priming to 82% survival and, in addition, display a distinct sexlabeling kit (Roche) and [32P]dCTP (Amersham). skew (Table 1) with one genotype (EMS allele/hypo-

Northern analysis and signal quantitation: Total RNA was morph) producing only males.obtained from specific genotypes and wild-type flies usingWe identified two candidate genes on heterochromaticTrizol. Poly(A�) RNA was extracted using oligo(dT) cellulose,

genomic scaffold AABU01002497 from the Release 3 ge-as described in Sambrook et al. (1989). RNA samples (30 �gfor total and 2 �g for poly(A�) were fractionated in formalde- nome sequence assembly, defects in which might showhyde agarose gels and transferred to Hybond N� nylon mem- phenotypes consistent with mutations in lethal 2. The first,branes. Labeling and hybridization were carried out as de- Dead box protein 80 (Dbp80) was identified by Eisen et al.scribed above for Southern analysis. Signal quantitation was

(1998), and mutations in DEAD box helicases can becarried out by exposing labeled Northern blots to a storageassociated with a Minute-like phenotype (Dorn et al.phosphor screen (Amersham), which was scanned by a Ty-

phoon 9410 phosphorimager and analyzed with ImageQuant 1993; Zaffran et al. 1998). The ortholog of Dbp80 in

2168 S. R. Schulze et al.

Figure 2.—Wild type (top) compared to Minute phenotypes of various lethal 2 “escapers.” Genotypes: 8-1/P�8 or P�2/P�8in A–C; P�2/P�2 escapers in D and E. Note the fine bristles (A), misrotated genitalia (B), reduced or gapped sex combs (C),and wing phenotypes (D and E).

yeast (Dbp5) appears to be involved in mRNA export ing transposable elements. The 61-bp intron betweenfollowing heat shock (Rollenhagen et al. 2004). It was exons 5 and 6 may be an ancient one, as it is shared bymapped in Drosophila by polytene chromosome in situ the mammalian homologs and other Drosophila spe-to 3L heterochromatin (Eisen et al. 1998). cies. There are also large portions within the introns

A schematic of Dbp80 gene structure is shown in Fig- that have not yet been sequenced, and the 3�-mosture 3. From this picture it can be seen that Dbp80 shares exon is not present within the annotated assembly. How-many structural characteristics with other heterochro- ever, this last 3� exon can be found in the trace archivematic genes that have been studied. It is very large, (http://www.ncbi.nlm.nih.gov/BLAST/tracemb.shtml);exceeding 140 kb in length, and it is embedded within it is therefore depicted in Figure 3 as separated froma repetitive environment. These repetitive sequences the rest of the gene by an intron of unknown length.are primarily middle repetitive, consisting of degenerat- A second gene, RpL15, is located �10 kb upstream

of Dbp80 and encodes a large subunit ribosomal pro-tein. Since defects in many ribosomal proteins have

TABLE 1 been correlated with a Minute phenotype (Lambertsson1998), this gene is also a suitable candidate for lethal 2.Sex-ratio tests for lethal 2 trans-heterozygotesLike Dbp80, RpL15 is characterized by the presence of

Genotype RV a SRb N c n d middle repetitive sequences upstream, downstream,and within its introns. However, it is unusually small forP�2/P�8 0.82 0.92 2080 567a heterochromatic gene, occupying �2 kb of genomic7-1/P�8 0.24 0.77 1323 105DNA (for a schematic, see Figure 7).7-1/P�2 0.12 2.0 1551 62

8-1/P�8 0.40 1.6 954 148 We used a PCR-based strategy to position Dbp80 and8-1/P�2 0.29 0.97 661 63 RpL15 against the corresponding genetic map, using1-166-37/P�8 0.14 All males 1296 61 genomic DNA from embryos that were either homozy-

gous or heterozygous for various deficiencies (Dfs) thata Relative viability is the observed frequency/expected fre-quency. covered this region. Results suggest that either RpL15

b Sex ratio: number of males divided by the number of or Dbp80 could correspond to lethal 2 (Figure 4). So,females. for example, Dbp80 sequences are absent from embryosc Total number of progeny.

that carry Dfs removing lethal 2 (9-56/9-56, K2/K2, andd Number of trans-heterozygotes. A dramatic decrease inviability correlates with sex-ratio shift. 9-56/K2), but these sequences are present in other Df

2169Two Drosophila Heterochromatic Genes

Figure 3.—Gene organization of Dbp80 : solidboxes indicate exons (sizes in base pairs); linesconnecting them represent introns. This map(not to scale) was derived by aligning the cDNAsequence (Eisen et al. 1998) to genomic scaffoldAABU01002497 in the Release 3 genome se-quence assembly. The exception is the 3�-mostexon of Dbp80, which has not yet been linkedto sequences in Release 3. It is therefore shownseparated from exon 10 by an intron of indetermi-nate length.

combinations. A preliminary characterization shows that any P-element consensus (O’Hare and Rubin 1983),but this consensus is considered weak and is based onboth genes are single copy and expressed (data not

shown), but they appear to have contrasting regulation. euchromatic insertions. The insertions represent, how-ever, at least two different events, since both orientationsRpL15 is highly expressed in all tissues and stages exam-

ined, as is typical of ribosomal protein genes (data not are represented. Insertion 7-1 may have been isolatedas a duplicate of 8-1, given that both insertions wereshown), whereas Dbp80 is moderately expressed and

may be developmentally regulated (Figure 5A). This isolated in the same screen and are identical in sizeand orientation (opposite to the direction of RpL15correlates with the differences in their promoters: Dbp80

possesses initiator and TATA box sequences at �1 and transcription). However, genetic evidence suggests thatthey might behave differently (see, for example, in Ta-�33 (Figure 5B), whereas RpL15 transcription appears

to initiate within the vicinity of a polypyrimidine tract ble 1, relative viability numbers for 8-1 and 7-1 in combi-nation with P�8). Either there are subtle differences(see Figure 7), a feature it shares with ribosomal protein

gene initiators across taxa (Barakat et al. 2001; Yoshi- within the P insertions themselves or years of separationof stocks have resulted in changes in genetic back-hama et al. 2002).

lethal 2 mutations are lesions in RpL15 : PCR and ground (e.g., accumulated modifiers). P�2 and P�8 areinserted in the same orientation as the gene, but P�2Southern analysis of genomic DNA from the P alleles

of lethal 2 established that this gene likely encodes RpL15 possesses a further internal deletion that may have oc-curred before or after mutagenesis, so it may or may(Figure 6). In all cases, complete viability was restored

following reversion/precise excision of the P element. not be a separate event. The EMS alleles were also se-quenced and found to possess mutations in RpL15 se-The P alleles were sequenced, and all the P elements

appear to have inserted into exactly the same place quences: 72 encodes a mRNA with a nonsense mutationin the second exon; 1-166-37 is a G-to-A substitution in(Figure 7). The insertion site does not correspond to

Figure 4.—PCR mapping of Dbp80 and RpL15 under deficiencies that remove lethal 2. FX3 is Df(3L)FX3, which removes lethal3–lethal 8 ; 9-56 is Df(3L)9-56, which removes lethal 1 and lethal 2 ; K2 is Df(3L)K2, which removes lethal 2 and lethal 3. RpL15 wasnot tested under Df(3L)K2 since it was confirmed to be lethal 2. Grip84 is a gene on the X chromosome used as a DNA control.For Dbp80, the PCR primers used derive from exon 9, which is the exon farthest to the left (and most distal to RpL15) inFigure 3.

2170 S. R. Schulze et al.

Figure 5.—Dbp80 expression and promoter analysis. (A) Developmental Northern analysis of Dbp80 expression. L1, L2, andL3 refer to larval instar stages. (B) The Dbp80 promoter region, showing initiator and TATA-box sequences (underlined). Positionof transcription initiation is based on alignment with ESTs that were made using the “cap-trapper” method (Carninci et al.2000).

the 5� splicing consensus of the first intron (Figure 7). al. 2001). In addition, we observed no in vivo effectsBoth these mutants have the same lethal phase (L1) as following either Act-5C or heat-shock GAL4-driven ex-a homozygous deficiency for the region (Df(3L)9-56). pression of UAS-Dbp80-RNAi transgenic lines at 18�, 25�,

Does Dpb80 correspond to another essential gene in or 29� (data not shown).the vicinity? A series of lethal excisions in RpL15 were Expression of RpL15 cDNA transgenes enhances via-obtained by remobilizing the P element and generating bility of mutant lethal 2 trans-heterozygote combinations:a variety of molecular lesions in this locus, and Southern Germline rescue of mutant alleles with transgenic con-blots demonstrate that at least two of these lesions ap- structs would constitute further evidence establishingpear to have removed significant portions of the Dbp80 lethal 2 ’s molecular identity. To this end, three kindsgene (data not shown). All of these excisions, including of germline transformation constructs were generated.those that affect Dbp80, are completely viable over muta- One contained a genomic insert: a 2.2-kb BglII-HindIIItions in the flanking genes lethal 1 and lethal 3 ; this fragment containing 800 bp upstream and 530 bp down-suggests that the Dbp80 gene does not correspond to stream of coding region cloned into pUAST, which pos-either of these essential genes. This is supported by the sesses upstream activating sequences for the yeast tran-PCR mapping data (Figure 4): Dbp80 cannot be lethal 3, scription factor GAL4. A full-length cDNA was alsobecause it is present under a deficiency that removes cloned into both pCaSpeR-hs and pUAST. For the geno-lethal 3 (Df(3L)FX3), and cannot be lethal 1, because Dbp80 mic transgene and the two cDNA constructs, a numbersequences are absent in a deficiency that does not re- of transgenic lines were established for each (eight,move lethal 1 (Df(3L)K2). RNA interference (RNAi) ex- nine, and eight, respectively). Lines carrying the geno-periments also suggest that Dbp80 may not be an essen- mic transgene were tested in the absence of any GAL4tial gene: when expressed in cell culture, Dbp80 RNAi driver, since the few previous successful efforts to rescueconstructs have no effect on cell viability (Gatfield et ribosomal protein genes were carried out using genomic

constructs in uninducible vectors (Voelker et al. 1989;Schmidt et al. 1996; Torok et al. 1999). In addition, anumber of the genomic and cDNA transgenic lines weresubjected to various driver protocols: by direct heatshock in the case of pCaSpeR-hs, by crossing to variousdriver lines expressing GAL4 under heat-shock control,or from an actin-5C (constitutive) promoter. None ofthese attempts was successful in rescuing the lethalityof strong alleles of lethal 2.

Given these problems and the reported difficulty inFigure 6.—Genomic DNA from lethal 2 P mutants and re-

rescuing mutations in many ribosomal protein genesvertants probed with the RpL15 cDNA. Rev indicates a viable,(Lambertsson 1998), we undertook an alternative ap-fully revertant line derived from the mutant line shown imme-

diately to its left. So, for example, from left to right, Rev5 and proach. Heteroallelic combinations of lethal 2 P allelesRev12 are P-revertants of the P�8 mutation. ry[506]/ry[506] do produce a certain number of escapers, so it wasis the wild-type control. DNA from the indicated genotypes possible to attempt to demonstrate partial rescue viawas cut with EcoRI, blotted, and probed with RpL15 cDNA,

enhanced viability—increasing the proportion of es-as indicated in materials and methods. In all cases theP-mutant band is absent in the revertant lines. caper offspring. The crossing scheme is shown in Figure

2171Two Drosophila Heterochromatic Genes

Figure 7.—Composite diagram of RpL15 genestructure. The promoter region is expanded toindicate the insertion site for all four P alleles oflethal 2 and the position of the polypyrimidinetract (transcription initiation). Also shown are thelocations and classifications of the two lethal 2EMS alleles.

8A, and a statistical interpretation of the results in Figure lier, lethal 2 trans-heterozygotes show variability in rela-tive viability (see Table 1), and this is reflected by the8B. For this particular experiment, an X-linked trans-

gene was brought in through the male parent, so in the variability in the sex ratios for the controls.Gene expression of both RpL15 and Dbp80 is compro-test generation, only the females inherit the complete

complement of transgene, driver, and lethal 2 alleles, mised when the HP1 dose is reduced: The expressionof the well-characterized heterochromatic genes lightand the males serve as an internal control. The test

generation was removed from a white minus background and rolled appears to be negatively affected in a geneticbackground in which the HP1 dose is reduced on theto score the eye-color mutation (rosy506) that marks the

P-mutant chromosomes. In addition, three external con- basis of both genetic (Clegg et al. 1998; Sinclair et al.2000) and molecular studies (Lu et al. 2000). It is oftrols were set up at the same time, one possessing neither

transgene nor driver, one with just the transgene, and interest to know if any other heterochromatic genesrespond in a similar manner, particularly RpL15 andone with just the driver. Rescue is reflected by an in-

crease in the female:male ratio of rosy/rosy flies in the Dbp80, which have contrasting promoters, expressionpatterns, and gene organization. In addition to beingtest generation, and this is depicted in Figure 8B. While

crosses involving other genomic and cDNA transgene essential, small, and highly expressed, RpL15 also pre-sents a clear hotspot for transposon insertion, sug-lines showed similar results (consistently enhanced trans-

heterozygote viability relative to controls; data not shown), gesting that its chromatin environment might be moreeuchromatic and might thus respond differently to re-only a cDNA transgene, UAS24-1, gave a statistically signifi-

cant result (95% confidence level). As mentioned ear- ductions in HP1 dose.

Figure 8.—Rescue (enhanced viability) of lethal2 trans-heterozygotes with RpL15 transgenes. (A)Genetic crossing scheme for rescue experimentsusing P�2/P�8 lethal 2 trans-heterozygotes. RpL15cDNA and genomic transgenes were cloned intopUAST and expressed under UAS regulation as aresult of GAL4 expression via an ACT-5C (constitu-tive) driver. (B) Statistical analysis of rescue data.These results are significant (95% confidence) andderive from use of a constitutively driven X-linkedcDNA transgene, UAS24-1.

2172 S. R. Schulze et al.

tion. It is enhanced in a genetic background heterozy-gous for a mutation in HP1 (Su(var)2-501), which is con-sistent with the effects observed for heterochromaticPEV of other translocated heterochromatic genes (Eberlet al. 1993; Howe et al. 1995).

In a similar experiment, lethal 2 mutations can beshown to enhance the wing-margin defect associatedwith the weak Notch allele N 55e11. Mutations in Minutegenes have been shown to affect wing morphogenesisgene expression (Sinclair et al. 1984; Hart et al. 1993),and it has been proposed that this is due to the sensitivityof these genes to reductions in protein synthesis levels.The effect of lethal 2 on Notch is further enhanced in agenetic background in which a single dose of HP1 hasbeen removed (Figure 11). The more severe effects seenin this case are consistent with a further loss of lethal 2function (resulting from the reduced HP1 dosage).

Figure 9.—Quantitation of Dbp80 and RpL15 expressionin a Su(var)2-5 mutant background. RNA was extracted fromthe genotypes listed on the x-axis, as described in materials DISCUSSIONand methods. The y-axis represents expression levels, which

lethal 2 encodes RpL15 and represents an examplewere measured as described in the text: relative to loadingcontrols, averaged from three separate experiments, and nor- of a small heterochromatic gene: lethal 2 is the firstmalized to wild type. Error bars represent standard error (abso- complementation group to be thoroughly characterizedlute value of variance of each reading from the average). at the molecular level in a set of 10 essential 3L hetero-

chromatic genes identified by Marchant and Holm(1988b). It is the second most proximal gene and likely

Our molecular results suggest that the expression of resides in cytological position h51 on the basis of mitoticboth the RpL15 and Dbp80 genes is reduced in an HP1 mapping of deficiencies that remove it (Koryakov etmutant background. Total RNA was blotted in a North- al. 2002). All six lethal 2 alleles are lesions in RpL15,ern analysis from L3 larval genotypes trans-heterozygous which encodes a large subunit ribosomal protein.for functional nulls of HP1: Su(var)2-504/Su(var)2-5149

Heterochromatic genes tend to be very large due to(no functional HP1 dose), their heterozygous sibs (one the expansion of repetitive sequences in their intronsfunctional HP1 dose), and wild-type larvae from the (Devlin et al. 1990a,b; Warren et al. 2000; Tulin et al.same developmental stage (two functional HP1 doses). 2002). Nevertheless, although RpL15 is embedded inWhen these Northerns were probed with the cDNAs for a repetitive environment, this does not seem to haveRpL15 or Dbp80 and the signals quantified by phos- affected its size. On the basis of its essential housekeep-phorimaging, the same trend was observed: gene expres- ing function, there is likely to be a strong selective pres-sion was compromised. Figure 9 shows quantitative re- sure to keep this gene small (Castillo-Davis et al.sults taken from three separate total RNA Northern 2002). The ribosome plays a fundamental role in con-experiments per gene, where the ratio of Dbp80 or trolling cell growth and development, and its constit-RpL15 signal intensity is expressed relative to a loading uent parts must be tightly and coordinately regulated.control (rp49). Expression of both genes is clearly re- In bacteria, these genes are clustered in operons, whileduced in the Su(var) mutant background, as has been in eukaryotes, they are widely dispersed throughout theobserved for light and rolled by Lu et al. (2000). genome. However, the requirement for coordinate ex-

These molecular results are supported genetically for pression has not been lost in eukaryotes, so they haveRpL15 by two assays, which show that lethal 2 gene func- likely evolved mechanisms that allow them to be ex-tion is compromised in a background in which the HP1 pressed in a variety of chromatin environments. In yeast,dose has been reduced by half. The first experiment there is evidence that ribosomal protein genes possessmade use of an inversion that breaks in euchromatin insulating sequences in their promoter regions, whicharound cytological position 62D and, in heterochroma- may render them relatively resistant to position effectstin, in the vicinity of lethal 1, effectively relocating lethal (Bi and Broach 1999). While insulators have not been2 near distal euchromatin. This inversion is semilethal found associated with Drosophila ribosomal proteinin combination with lesions in lethal 1 and exhibits a genes, a common upstream feature is a polypyrimidineposterior wing-margin phenotype in combination with tract, which has been demonstrated to play a criticallesions in lethal 2. The phenotype varies in both pene- role in both transcriptional and translational regulationtrance (Table 2) and expressivity (Figure 10) and also across taxa (Hariharan and Perry 1990; Levy et al.

1991). It remains to be seen whether this sequencein severity, with increasing strength of the lethal 2 muta-

2173Two Drosophila Heterochromatic Genes

TABLE 2 1998); however, it is currently thought that the majorityof Minutes probably encode ribosomal proteins (Lam-Penetrance data for genetic interaction between lethal 2bertsson 1998) and that the number of Minutes couldmutations and the inversion In(3L)C90formally approach the number of ribosomal proteingenes. However, a number might not have been de-Penetrance b Penetrance b

Genotype a males (%) females (%) tected due to their nonadditive properties (i.e., a defi-ciency might remove more than one Minute gene).1-166-37/In(3L)C90 0 13

Some ribosomal proteins appear to have more impor-72/In(3L)C90 13 60tant roles in the ribosome than others, and it has alreadySu(var)2-5 01/�; In(3L)C90/� 0.01 15

Su(var)2-5 01/�; 1-16-0/In(3L)C90 6 23 been demonstrated that a few of them have more thanSu(var)2-5 01/�; 1-166-37/In(3L)C90 0 81 one biological function (Wool 1996; Yacoub et al.Su(var)2-5 01/�; 72/In(3L)C90 100 100 1996a,b). Genetically, different Minute genes have phe-

notypes of varying severity, implying that although ribo-a Only third chromosome genotypes are shown. 1-166-37and 72 are EMS point mutations in lethal 2 (see Figure 7); somal protein genes are constitutively expressed, differ-1-16-0 is an allele of the more distal 3L heterochromatic gene ent genes may express at levels closer or farther fromSNAP25, used as a non-lethal 2 control.

some threshold below which the phenotype will mani-b Calculated as the number of observed trans-heterozygousfest itself (Saebøe-Larssen and Lambertsson 1996;progeny divided by the expected number for a given popula-

tion scored (which, for each cross, consisted of at least 150 Saebøe-Larssen et al. 1998 and references therein).flies). Difficulties in rescuing lethality for RpL15 might be

explained by the lack of an appropriate heterochro-matic environment for the genomic construct in the(or others in the vicinity) can also confer an insulatingvarious transgenic lines tested. However, GAL4-drivenfunction on adjacent genes.expression was also problematic; therefore, failure toRpL15 and the Minute syndrome: A perusal of therescue lethality is more likely to be attributable to theDrosophila genome database indicates that there mayabove-mentioned extreme dose sensitivity. This may bebe at least five to six other ribosomal protein genes ina common feature of mutations in ribosomal proteinthe heterochromatin of this organism: these includegenes, for which relatively few successful transgene res-CG12775 (40F), Yip6 (CG17489, 40D), and RpS3A (CG-cues have been reported (Lambertsson 1998).2168, 102A3), distal genes that may be heterochromatic;

We attempted to address this issue by quantitatingand Qm (CG17521), CG18001, and CG40278 (MinuteRpL15 expression in various genetic contexts; however,41C), which are more proximal and therefore likely todoing this to within statistically significant marginsbe heterochromatic. This has recently been confirmedproved difficult, due to the very high levels of expressionfor two of these genes by a report linking RpL38 withof this gene. For example, Northern analysis of wild-typeCG18001 and RpL5 with CG17489 (Marygold et al.and mutant mRNA levels showed less mRNA present in2005). One of the most notable biological characteris-mutant combinations, but these results were not statisti-tics of lethal 2 mutants is the classical Minute phenotypecally significant, except in the case of P�2/P�2 adultexhibited by certain trans-heterozygote combinations ofescapers (data not shown). A precise molecular analysisalleles. In Drosophila, mutations in some ribosomal pro-of haplo-insufficiency was therefore not possible, al-teins have been shown to cause a dominant Minute phe-though a subtle phenotypic effect can consistently benotype, consisting of thin, weak bristles, rough eyes,observed in the adult (lethal 2 null/Df; data not shown).wing-vein and sex-comb defects, delayed development,Similarly, the high endogenous expression levels forand recessive lethality (Lambertsson 1998). A fewRpL15 made it difficult to accurately quantitate trans-other genes that play various roles in the global regula-gene expression, and we could see a demonstrable in-tion of protein synthesis have also been observed tocrease in RpL15 mRNA levels only in pUAST lines ex-mutate to a Minute-like phenotype (Ritossa et al. 1966;

Dudick et al. 1974; Dorn et al. 1993; Zaffran et al. pressing via heat-shock-driven GAL4 (data not shown).

Figure 10.—Wing-margin phenotypes resulting from interaction between mutations in lethal 2 and In(3L)C90. This inversionbreaks in or near lethal 1 and in distal euchromatin at cytological position 62D, likely placing lethal 2 close to a large block ofeuchromatin. The phenotype is more severe in females than in males, in terms of both penetrance (Table 2) and expressivity(shown here). (A) Wild type (female); (B) In(3L)C90/72 (female); (C) Su(var)2-5 01/�; In(3L)C90/�(female); (D) Su(var)2-5 01/�;In(3L)C90/72 (male); (E) Su(var)2-5 01/�; In(3L)C90/72 (female). The results suggest a strong heterochromatic position effecton lethal 2, which is enhanced in the Su(var) background.

2174 S. R. Schulze et al.

suggests that the evolution of the Drosophila genomemay have been marked by considerable DNA diminution(Petrov 2002).

Is Dbp80 an essential gene? We believe that Dbp80 isunlikely to be an essential gene for a number of reasons.In terms of genetics, PCR deficiency mapping suggeststhat Dbp80 can be neither lethal 1 nor lethal 3, and thisresult is supported by the genetics of lethal excisionsfrom RpL15 affecting Dbp80, which are completely viablein combination with mutations in lethal 1 and lethal 3.This proximal region has been subjected multiple timesto extensive mutagenesis screens (Marchant and Holm1988a,b; Schulze et al. 2001; M. Syrzycka, unpublishedresults) and we have found no other lethal mutationsin the region that might be candidates for Dbp80.

In terms of gene function, although the ortholog ofDbp80 encodes an essential gene in yeast that plays a rolein mRNA export (Rollenhagen et al. 2004), attempts toshow a similar function in insect cell culture (by RNAidepletion) indicate that it is not essential for this path-Figure 11.—Wing genotypes show interactions between le-

thal 2 alleles 166-37 and 72 (see Figure 7) and the weak Notch way in flies (Gatfield et al. 2001). The Caenorhabditisallele N 55e11. Controls: (A) Wild type. (B) N 55e11/�; ri p p/�; elegans ortholog also appears to be nonessential, as therethis Notch allele shows variable expressivity of which this image is no phenotype from an RNAi knockout experimentrepresents a moderate form. (C) N 55e11/�;1-16-0/�; 1-16-0 is

(http://www.wormbase.org/db/seq/rnai?nameJA%3an allele of the more distal heterochromatic gene SNAP25,AT07D4.4;classRNAi). The functional role of Dbp80used as a non-lethal 2 control. (D) N 55e11/�; Su(var)2-5 01/�;

1-16-0/�; a weak interaction is slightly enhanced and SNAP25 protein may be important, but a number of relatedmay interact with Notch. Experimental: (E) FM7/�; Su(var)2- DEAD box genes could provide functional redundancy.5 01/�; 72ri p p/�. (F) N 55e11/�; Su(var)2-5 01/�. (G) N 55e11/�; Our results, showing no effects of RNAi in vivo, are also72ri p p/�. A mutation in lethal 2 causes a slight enhancement

consistent with this view. Finally, the WGS heterochro-of the weak Notch phenotype: (H) N 55e11/�. Su(var)2-5 01/�.matin assembly (Hoskins et al. 2002) places many more72ri p p/�. The effect of lethal 2 loss of function on Notch is

further enhanced in a background in which the HP1 dose gene models in 3L heterochromatin than there are le-has been reduced by half. thal complementation groups. While not all of these

models may be confirmed as genuine after repeatedrounds of annotation, these results would be consistent

We conclude that problems with rescue arise from some with the presence of a significant number of genes thatcombination of dose sensitivity and/or other peculiari- would be nonessential in heterochromatin (Dbp80 andties of ribosomal protein gene expression in Drosophila. others).

Global (organism-wide) phenotypic manifestions due Are there other candidates for essential genes in theto defects in ribosomal protein function appear to be region? The current annotation of Drosophila hetero-unique to Drosophila [although one report identifies chromatin does list two small gene models nested withina Minute -like phenotype caused by an Arabidopsis ribo- Dbp80 (CG40336 encoding 98 amino acids and CG40337somal protein gene mutation (Weijers et al. 2001)]. encoding 103 amino acids) and while we cannot for-This may in part be due to the fact that, with two excep- mally rule out the possibility that these might encodetions (Brown et al. 1988; Yokokura et al. 1993), Droso- essential functions, we think it is unlikely. A BLASTphila appears to have only single-copy ribosomal protein search against a nonredundant protein database pro-genes, while yeast, plants, and humans have multiple cop- duces little homology with genes of known function; ities, including pseudogenes (Zhang et al. 2002). Defects is interesting to note that a third gene model, CG40335,in human ribosomal protein genes have been implicated was initially described, but subsequent annotation hasin a number of inherited disorders [for example, diamond identified it as a repeat sequence. It is possible that allblackfan anemia (RpS19), Turner syndrome (RpS4), and three represent degenerate transposable element se-Noonan syndrome (RpL6) (Zhang et al. 2002 and refer- quences; this provides an example of the difficulties inences therein)], implying that mammalian ribosomes may annotating gene models in a repetitive environment.exist in tissue/stage-specific isoforms. However, no general Both Dbp80 and RpL15 are negatively affected whenMinute syndrome as such appears in organisms with multi- the HP1 dose is reduced: These two genes have veryple-copy ribosomal protein genes. It is not clear why Dro- different promoters and expression patterns, suggestingsophila has retained only single copies, but this may be that there may be no simple, common mechanism to

regulate their expression. However, our molecular evi-explained in part by a growing body of evidence that

2175Two Drosophila Heterochromatic Genes

dence suggests that expression of both is compromised (87A, 87C, and 95D). Formaldehyde crosslinked chroma-tin immunoprecipitation experiments using primers toin a genetic background deficient for HP1. This is con-

sistent with results observed for the well-characterized either the promoter or the coding regions of the Hsp70gene (which maps to 87A and C) show that, after heat-genes light and rolled (Lu et al. 2000) and provides fur-

ther evidence that genes in heterochromatin may have shock induction, HP1 protein is enriched in the codingregion, and not in the promoter of this gene. Moreover,evolved a transcriptional dependence on factors that

are known to silence gene expression. This effect can this enrichment appears to depend on the presence ofalso be shown genetically for lethal 2, using a sensitized RNA and an intact chromodomain in HP1. Thus, HP1background. Ribosomal proteins have been shown to in fact may act as a regulator of transcription by control-enhance wing morphogenesis mutations (Sinclair et ling the stability of the transcript (elongation, pro-al. 1984; Hart et al. 1993), and this appears to be the cessing, etc.) rather than by inducing or repressing genecase for lethal 2 in combination with a weak Notch allele. expression per se, and its function as a repressor or activa-The severity of the interaction with Notch is enhanced tor may well depend on a combination of factors, includ-when only one copy of HP1 is removed. We also report ing but not limited to, chromatin environment.results consistent with this interpretation, using the in- Our work provides two more informative gene modelsversion In(3L)C90, which breaks proximally in the lethal to study the expression and regulation of genes in het-1 gene, and distally in euchromatin at position 62D, erochromatin. In addition, it provides an example ofplacing lethal 2 relatively close to a block of euchromatin. how genetic and molecular analyses can complementIn(3L)C90 complements all lethal 2 alleles for viability and enrich the annotation of this difficult genomic re-and fertility, but presents a posterior wing-margin de- gion. Comparative evolutionary studies have also beenfect. This phenotype increases in severity with the informative in studying the structure and regulation ofstrength of the mutant lethal 2 allele (i.e., the strongest gene expression. In a parallel report (S. R. Schulze,phenotype in terms of penetrance and expressivity is B. F. McAllister, D. A. R. Sinclair, K. A. Fitzpatrick,observed with the EMS allele 72, which appears to be- M. Marchetti, S. Pimpinelli and B. M. Honda, unpub-have as a functional null). It is not clear what the source lished results), we have observed dramatically differentof this particular phenotype is, since it does not resem- chromosomal localizations for RpL15 and Dbp80 inble any of the wing defects resulting from lethal 2 hypo- other Drosophila species, showing that the same genemorphy. However, In(3L)C90 also possesses a deletion can evolve and function in contrasting chromatin envi-for �60 genes near the distal euchromatic breakpoint, ronments. Taken together, these complementary ap-and it is possible that lethal 2 interacts with a reduction proaches lay the foundation for further research intoin dose of a gene from this region in a manner similar the relationship between chromatin structure and geneto its observed interaction with Notch. Once again, the regulation.severity of this phenotype is enhanced in a background

We thank David Deitcher for fly stocks, Ron Blackman and Johnin which the HP1 dose has been reduced by half. This Tamkun for Drosophila genomic libraries, Jerome Dejardin, Gregoryinversion has no effect above a background one on any Chanas, and Giacomo Cavalli for their assistance with transgenic ex-

periments, Rob Hollebakken for RNAi work, and a number of col-mutants or lesions distal to lethal 2, unless combinedleagues for their advice and support. This work was supported by awith a reduction in HP1 dose, in which case the effectDiscovery grant from National Science and Engineering Researchis less severe than that for lethal 2. The variegation phe-Council (NSERC) Canada to B.M.H. and a NSERC Postgraduate schol-notype is thus consistent with an interpretation of the arship B grant to S.R.S.

PEV effects on the translocated lethal 2 gene and issimilar to what has been observed genetically for both.

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