Effect of the Suppressor of Underreplication (SuUR) Gene on Position-Effect Variegation Silencing in Drosophila melanogaster

Corresponding author: I. F. Zhimulev, Koptyuga Ave. 2, Novosibirsk 630090, Russia., zhimulev{at}bionet.nsc.ru (E-mail)

Communicating editor: K. GOLIC

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

It has been previously shown that the SuUR gene encodes a protein located in intercalary and pericentromeric heterochromatin in Drosophila melanogaster polytene chromosomes. The SuUR mutation suppresses the formation of ectopic contacts and DNA underreplication in polytene chromosomes; SuUR⁺ in extra doses enhances the expression of these characters. This study demonstrates that heterochromatin-dependent PEV silencing is also influenced by SuUR. The SuUR protein localizes to chromosome regions compacted as a result of PEV; the SuUR mutation suppresses DNA underreplication arising in regions of polytene chromosomes undergoing PEV. The SuUR mutation also suppresses variegation of both adult morphological characters and chromatin compaction observed in rearranged chromosomes. In contrast, SuUR⁺ in extra doses and its overexpression enhance variegation. Thus, SuUR affects PEV silencing in a dose-dependent manner. However, its effect is expressed weaker than that of the strong modifier Su(var)2-5.

HETEROCHROMATIN position-effect variegation (PEV) is a form of epigenetic silence that results from placement of euchromatic genes to pericentric heterochromatin (CH). Relocated genes are silenced in a part of the cell population through successive cell divisions, causing the mosaic phenotype. On the basis of evidence provided over recent years, mosaic silencing of euchromatic genes undergoing PEV is hypothesized to be caused by variable spreading of silence from CH into the relocated euchromatin. Spreading presumably becomes feasible when the rearrangement removes a barrier at the heterochromatin/euchromatin boundary that blocks the spread of the heterochromatin state (NOMA et al. 2001 ; DONZE and KAMAKAKA 2002 ). PEV silencing can be distinguished cytologically by heterochromatization of the euchromatic region. Heterochromatized euchromatin becomes late replicating, even under-replicating when undergoing strong PEV (for a review see ZHIMULEV 1998 ). Thus, silencing in pericentric heterochromatin and PEV heterochromatization have common molecular mechanisms. Advances in this field have been made in recent years. On the basis of studies on modifications of histones and DNA, including histone hypoacetylation and methylation, it was inferred that such covalent modifications mark heterochromatin and generate the basis for silenced domain formation and spreading of silence (for reviews see STRAHL and ALLIS 2000 ; TURNER 2000 ; COWELL et al. 2002 ; DILLON and FESTENSTEIN 2002 ; RICHARDS and ELGIN 2002 ).

Studies on the genetic modifiers of PEV, which were revealed in screens for dominant suppressor and enhancer of PEV, played an important role in the development of this concept (GRIGLIATTI 1991 ; REUTER and SPIERER 1992 ; WALLRATH 1998 ). Certain modifiers have a dose-dependent effect on PEV, suppressing it when present in one dose and enhancing it in three or more doses, thereby suggesting the involvement of their products in protein complexes modifying heterochromatin (LOCKE et al. 1988 ).

The function of a number of modifier genes has been established (WALLRATH 1998 , review). Thus, Su(var)3-6 encodes protein phosphatase PP1 (BAKSA et al. 1993 ); Su(var)2-1, histone acetylase (DORN et al. 1986 ); and Su(var)3-9, the enzyme for methylation of histone H3 on lysine 9 (REA et al. 2000 ; CZERMIN et al. 2001 ; SCHOTTA et al. 2002 ). The missense mutations at the histone deacetylase locus (HDAC1) are suppressors of PEV (MOTTUS et al. 2000 ). Su(var)2-5 encodes the HP1 protein (EISSENBERG et al. 1992 ) required for the assembly of the protein-modifying complex (RICHARDS and ELGIN 2002 , review).

The list of chromatin modifications that control gene expression probably will increase, by including mainly the loci for the heterochromatin-associated proteins. A recently discovered gene, the Suppressor of Underreplication (SuUR; BELYAEVA et al. 1998 ), whose protein is located in CH and in intercalary heterochromatin (IH) in Drosophila melanogaster, is the focus of this study. IH is represented in the polytene nucleus by numerous sites scattered throughout the euchromatin arms of chromosomes and has many distinctive features in common with CH: compaction, late DNA replication, and underreplication, among others. The SuUR mutation causes complete suppression of DNA underreplication in IH regions of polytene chromosomes and loss of ectopic contacts between them, and it partially restores polytenization in CH as a result of which new euchromatic regions appear at the bases of chromosome arms. There are grounds for assuming that synthesis in late-replicating heterochromatin ceases earlier in SuUR mutant DNA than in wild type (BELYAEVA et al. 1998 ; MOSHKIN et al. 2001 ; ZHIMULEV et al. 2003 ). The SuUR cDNA encodes a deduced protein of 962 amino acids. A BLAST search of the protein database does not reveal proteins with significant similarity for the full-length SuUR protein. However, the N-terminal 250 amino acids show a moderate similarity to the N-terminal part of the ATPase/helicase domain found in the SNF2/SWI2 family of proteins (see details in MAKUNIN et al. 2002 ).

Here, our aim was to determine whether SuUR functions in PEV silencing. We studied the influence of SuUR dosage on PEV in classic chromosome rearrangements and also SuUR effect on DNA underreplication in euchromatic regions inactivated by PEV. We demonstrated that the SuUR protein appears in regions heterochromatized by PEV, its absence in the SuUR mutant suppresses underreplication in these regions, and also that SuUR⁺ affects PEV silencing in a dose-dependent fashion: an increase in its dose enhances PEV at the levels of both variegation of the adult morphological characters and euchromatin heterochromatization in the rearranged salivary gland chromosomes.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Fly stocks and constructs:
All stocks and mutations used are described in LINDSLEY and ZIMM 1992 . The SuUR mutation was detected in In(1)sc^V2 stock due to its unusual effect on the phenotype of polytene chromosomes. Its sole effect is observed as suppression of DNA underreplication in IH and CH. SuUR homozygotes are viable and fertile. SuUR mutant stock contains a 6-kb insertion in the last (4) exon of the gene (BELYAEVA et al. 1998 ; MAKUNIN et al. 2002 ; ZHIMULEV et al. 2003 for details).

The PEV-modifying effect of SuUR was studied in rearrangements In(1)w^m4h, In(1)sc⁸, Dp(1;f)1187, Dp(1;f)R, Dp(1;1)pn2b, T(1;2)dor^var7, and T(2;3)Sb^V. The SuUR mutation was introduced in each stock carrying w^m4h, pn2b, dor^var7, and sc⁸. In doing so, considerable care was taken to keep the genetic backgrounds of the mutant and control lines as similar as possible. In this way, we selected the ru h SuUR⁺ and ru h SuUR^- recombinants from the progeny of a single female ru h SuUR⁺ th st sr e ca/SuUR^- (SuUR lies between h and th at 34.8 cM) and synthesized the w^m4h; ru h SuUR⁺ and w^m4h; ru h SuUR^- lines. The second chromosome was replaced using the w; Cy/If; MKRS/TM6, Hu, Tb e ca line. The lines were isogenic for the X, second chromosome, and most of the third, with only the regions between h and th (13 cM) nonidentical. The C(1)RM, y v/0; SuUR⁺ and C(1)RM, y v/0; SuUR^- lines with the attached X chromosomes were sources of males without the Y chromosome.

Flies were maintained at 18° or 25° on standard cornmeal yeast-agar medium. The H7 construct contains the yellow⁺ gene, the entire open reading frame, and a part of the 3' untranslated region (UTR) of the SuUR⁺ gene, which is under the control of the hsp 70 promoter (Fig 1A). The y w⁶⁷ H7-X; H7-3 stock is homozygous for two insertional sites: on the X (in 4F) and the third (in 91F) chromosomes, it contains the transposon in four doses. Overexpression of the hs-SuUR transposon rescues the phenotype of the SuUR mutant (for a detailed description of the construct and the rescue experiments, see MAKUNIN et al. 2002 ). The HsHP1.83C stock is homozygous for the P(neoHSHP1.83C) construct that contains HP1 cDNA under control of the hsp70 promoter (EISSENBERG et al. 1992 ). The X6S1 construct (Fig 1B) is composed of a 4.9-kb XbaI-SalI genomic fragment with the functional SuUR⁺ gene and miniwhite⁺ gene (MAKUNIN et al. 2002 ). The y w⁶⁷ stocks carrying two, four, or six X6S1 transposons in addition to two doses of the endogenous SuUR⁺ gene are referred to as stocks with four, six, and eight doses of the SuUR gene, respectively. The X6S1 insertions were mapped in situ in 8D and 59D; another insertion was not mapped in situ, but it is genetically on the third chromosome. The y w⁶⁷ line used for transformation served as a control.

View larger version (17K): In this window In a new window Download PPT slide   Figure 1. Diagram of the transposons used. (A) H7 transposon contains the entire open reading frame, part of the 3' UTR of the SuUR gene under control of the hsp70 promoter, and yellow marker gene. (B) X6S1 transposon contains a genomic fragment harboring the SuUR gene with its own promoter and mini-white marker gene (see MAKUNIN et al. 2002 for details).

Heat-shock treatment:
Vials were immersed in a 37° water bath for 40 min. Eggs were collected for 2 hr at 25°; then, after 3 hr of development at 25°, the cultures were heat shocked. Thereafter heat shock was repeated every day until third instar larvae developed or imago eclosed. In the control variant, all development proceeded at 25°. The control and experimental samples were composed of progeny of the same parents.

Quantitative Southern hybridization:
DNA was isolated from 50 salivary glands and from 25 sets of larvae brains and imaginal discs (see KARPEN and SPRADLING 1990 ) and digested with HindIII (MBI Fermentas). Restricted DNA was size separated in 0.8% agarose TAE gel and transferred to Hybond-NX (Amersham Pharmacia Biotech) according to manufacturer's protocols.

The following DNA clones were used: T2, 1.45-kb EcoRI-HindIII genomic fragment located between 0 and +1.5 kb in the map of scute region; l'sc, 1.10-kb EcoRI-EcoRI genomic fragment located between +19.0 and +20.0 kb in the map of scute region (GONZALEZ et al. 1989 ); a 2.15-kb BamHI-HindIII genomic fragment from the region between +40.5 and +42.65 kb in the map of scute was amplified from genomic DNA and named sc(RAM); w(1-4), a 2.26-kb genomic fragment including two to six exons of white gene (obtained by S. Demakov in our laboratory); rosy, a 2.8-kb BamHI-BamHI genomic fragment of rosy gene. Gel-purified PCR fragments were labeled with [³²P]dATP by random priming, and the control and the test probes were labeled at the same time (see BELYAEVA et al. 1998 ). Hybridization was performed according to the protocol recommended by manufacturer for Hybond-NX. Blots were exposed for various times at -70° using Agfa CP-BU X-ray film. Signal density was measured using an HP Scan Jet 4C/T scanner, following counting of signal intensity with the Band Leader 3.0 program. Relative DNA abundance was calculated as the ratio of hybridization signal in salivary glands to that in diploid tissues after normalization with rosy signal.

Immunofluorescent staining of polytene chromosomes:
Immunostaining of polytene chromosomes was performed according to ZINK and PARO 1989 with some modifications. Prepared squashes were stored in PBS for not >72 hr at 4°. Slides were incubated in blocking solution [0.5% BSA (albumin from bovine serum; Fluka, Buchs, Switzerland), 0.1% Tween-20, PBS] and then incubated with the SUUR antibody (dilution 1:30 in 0.5% BSA, 0.1% Tween-20, PBS) for 2 hr at room temperature in a humid chamber. The slides were washed in PBS with 0.1% Tween-20 (three times for 5 min) and incubated 1 hr with FITC-conjugated goat anti-rabbit secondary antibody (Sigma, St. Louis) diluted 1:150. After that they were washed in PBS with 0.1% Tween-20 (three times for 5 min) and were covered with a coverslip in antifade reagent (Molecular Probes, Eugene, OR). Chromosomes were examined using epifluorescence optics (Olympus BX50 microscope) and photographed with the CCD Olympus DP50.

Measurements of position-effect variegation:
The extent of white⁺ variegation was analyzed in terms of red eye pigmentation (EPHRUSSI and HEROLD 1944 ). Flies were selected on the day of eclosion and aged for 5 days at 25° and then frozen at -70°. Extraction was performed using 10 fly heads/ml of 30% ethanol at pH 2.0. Ten measurements were done on each variant at 480 nm.

Analysis of the deep orange⁺ variegation in T(1;2)dor^var7 was done visually (see RESULTS for details). Variegation of the yellow⁺ gene in the Dp(1;f)1187 minichromosome was expressed as the percentage of yellow bristles in the middle of the anterior triple row at the wing margin. The number of bristles was determined by counting among the first 30 bristles of the proximal part of the wing in 30 flies, with the wings submerged in glycerol under coverslips. The extent of Stubble variegation was expressed as the number of mutant bristles (posterior supra-alars, anterior post-alars, posterior dorsocentrals, and anterior and posterior scutellars) on each side of the adult (i.e., 10 per fly) in 100 flies of each genotype. Since the full expression of Sb produces mutant bristles, suppression of Sb variegation causes an increase in the number of short bristles. Variegation of scute⁺ was determined as the number of normal bristles among four anterior and posterior scutellars in 100–150 flies of each experimental sample.

Cytological analysis of variegation:
Cytological analysis was performed on acetic orcein-stained salivary gland polytene chromosomes of third instar larvae as described in BELYAEVA and ZHIMULEV 1991 . To estimate the frequency of heterochromatization and the extent of heterochromatin formation, we compared the morphology of all the polytene chromosome bands in the region of interest in the rearranged and normal homologs. Frequency of heterochromatization in different chromosome regions under PEV was expressed as the ratio (%) of number of heterochromatized regions to total number of the regions analyzed (150 and >150 in each case). Ten to 20 nuclei in each of 8–20 larvae were examined. Normally highly variable in different strains, the morphology of the subtelomeric 1A1-8 region was not analyzed.

Statistical treatment:
The significance of the differences was calculated using Fisher's test. Histograms were compared using the chi-square test.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The SuUR protein localizes to regions of chromosomes undergoing PEV:
PEV is manifested cytologically as heterochromatization of euchromatic regions. The normal euchromatic banded appearance is lost, giving rise to bands that become denser and merge into blocks of heterochromatin-like material. To determine the localization of SUUR in the heterochromatized regions of euchromatin, Dp(1;1)pn2b (hereafter pn2b) was used. This stock is convenient because discontinuous compaction often appears in the duplication, when a compacted region is separated from CH by an active region (BELYAEVA and ZHIMULEV 1991 ); thus, the fluorescent signal does not merge with that in the adjacent CH. The range of compaction varied from single heterochromatized bands to complete heterochromatization of all the bands. pn2b contains the 1A-2E region transferred to the right-arm heterochromatin of the X chromosome. Just one site in this region, 1AB, binds the SUUR antibodies in the wild-type chromosome (ZHIMULEV et al. 2003 ). Fig 2A and Fig B, demonstrates the normal pattern of all bands in pn2b without signs of heterochromatization. Two sites of antibody binding are seen: the 1AB region and the site of the eu-/heterochomatin junction in the duplication, i.e., the 2E region. Among 200 squashes investigated we never observed other fluorescent signals in duplications showing normal morphology of the bands. However, when compacted blocks are formed, the SUUR antibody recognizes these blocks: blocks in 2CD (Fig 2C and Fig D) and in 2B (Fig 2E and Fig F) show fluorescent signals. Fig 2G and Fig H, shows pn2b composed entirely of a series of unidentified bands/blocks, which fluoresce brightly. Thus, the SUUR is present in the euchromatic regions compacted by PEV.

View larger version (69K): In this window In a new window Download PPT slide   Figure 2. Immunolocalization of anti-SUUR on Dp(1;1)pn2b with different levels of heterochromatization. (A and B) Normal structure of pn2b. Only 1AB and 2E sites bind anti-SUUR. (C and D) Heterochromatization and fluorescent signal in 2CD. (E and F) Heterochromatization and fluorescent signal in 2B. (G and H) Heterochromatization and fluorescence of the whole 1D-2C region. (A, C, E, and G) Phase contrast. (B, D, F, and H) Staining with anti-SUUR. CH, chromocenter; 4, fourth chromosome. Brackets designate heterochromatized regions of duplication.

The SuUR mutation suppresses underreplication of DNA sequences undergoing PEV:
We investigated whether underrepresentation of DNA sequences undergoing PEV was due to SuUR. We chose In(1)sc⁸ (hereafter sc⁸), which has breakpoints in 1B3-4 within the Achaete-Scute Complex and the h32 segment of pericentric heterochromatin of the X chromosome, to address this issue. In the wild strain Oregon-R underreplication is absent in the region 1AB (ZHIMULEV et al. 2003 ), but appears in sc⁸ in this area adjacent to the CH block containing the centromere. Underreplication is maximal in the immediate proximity to pericentric heterochromatin and decreases with increasing distance from the breakpoint. The underreplicated region under study was of 50 kb (Fig 3A). DNA underreplication was detected in both sc⁸ females and males and is completely suppressed in this region on the background of the SuUR mutation (Fig 3, B–D). It is known that PEV is enhanced in males without a Y chromosome and the enhancement is observed at the levels of both compaction (BELYAEVA and ZHIMULEV 1991 ; ZHIMULEV 1998 for references) and underreplication, for example, in the yellow region in Dp(1;f)1187 (KARPEN and SPRADLING 1990 ). It appeared to be of interest to ascertain whether the SuUR mutation can restore replication in the studied region in XO males. It was found that in XO, sc⁸; SuUR^- males, even the sequence of clone sc(RAM) closest to heterochromatin is fully presented in salivary gland chromosomes (Fig 3E).

View larger version (33K): In this window In a new window Download PPT slide   Figure 3. Action of the SuUR mutation on underreplication in In(1)sc8 (A–E) and In(1)wm4h (F). (A) Scheme of In(1)sc8 and position of DNA probes on physical map; 0, break point. (B–F) Genomic Southern blot hybridizations of salivary gland (SG) and diploid tissue (D) DNA with probes T2, l'sc, sc(RAM), and w(1-4). DNA of the rosy gene was used as standard. *, percentages showing the representation of DNA from salivary glands in comparison to diploid tissue.

A similar effect of the SuUR mutation was observed for the sequence w1-4, which lies 25 kb from the heterochromatin junction point in In(1)w^w4 (TARTOF et al. 1984 ). The sequence w1-4 is highly replicated in the salivary gland polytene chromosomes of the wild Oregon-R strain, but severely underreplicated in those of w^m4h; ru h SuUR⁺ larvae with two normal SuUR alleles. In the larval polytene chromosomes of the isogenic strain w^m4h; ru h SuUR^-, which are SuUR^- homozygotes, the abundance of the w1-4 clone sequence is recovered to 60–77% of the diploid level (Fig 3F).

Thus, we demonstrated that SuUR affects properties of the heterochromatin formed by PEV: (i) the SuUR protein is presented in regions inactivated by PEV and (ii) the SuUR mutation suppresses underreplication caused by PEV.

Variegation depends on SuUR dosage:
In(1)w^m4h (hereafter w^m4h) is a classic model used for isolating and characterizing the PEV modifiers. To minimize the effects of genetic background, we generated w^m4h; ru h SuUR⁺ and w^m4h; ru h SuUR^- isogenic lines (MATERIALS AND METHODS). Data on red eye pigment levels in these lines with and without SuUR⁺ (Table 1) suggest that suppression of the white⁺ variegation in males and females homozygous for SuUR^- is significant (P < 0.01 for females and P < 0.05 for males).

T(1;2)dor^var7 (hereafter dor^var7), the rearrangement with the 1A-2B8-9 region moved to CH of the 2R chromosome, was selected as a strong variegator for the dor⁺ gene and exhibits long-distance PEV (BELYAEVA and ZHIMULEV 1991 ). To study the effect of SuUR on dor⁺ variegation in dor^var7, the y dor; ru h SuUR⁺ and y dor; SuUR^- lines were generated. The females of these lines were crossed to dor^var7; ru h SuUR⁺ or dor^var7; SuUR^- males, and their female progeny, y dor/dor^var7; ru h SuUR⁺ (control) and y dor/dor^var7; SuUR^-, respectively, was analyzed. Their eyes were assigned to types according to dor⁺ variegated expression (Fig 4, A–D). The distribution of frequencies of eye phenotypes suggests significant suppression (P < 0.01) of dor⁺ variegation by SuUR^-. Almost all the eyes of the control females have mosaic phenotype while 16% eyes of flies homozygous for SuUR^- are normal, i.e., dor⁺. The frequency of eyes with the most severe phenotype (type A) is two times higher among all progenies bearing SuUR⁺ than in those with SuUR^-.

View larger version (48K): In this window In a new window Download PPT slide   Figure 4. Effect of SuUR on deep orange+ variegation in T(1;2)dorvar7. Frequencies of different classes of mosaic eyes (A–D) among progeny with SuUR+ and SuUR- are shown. (A) Mosaic eyes with >50% dor facets; (B) 50% and less dor facets; (C) Single dark facets in reddish background; (D) wild type, i.e., without dor+ inactivation.

Dp(1:f)1187 (hereafter Dp1187), a minichromosome with most of the X-euchromatin deleted, displays yellow⁺ PEV (KARPEN and SPRADLING 1990 ) observed as yellow bristles in the y, Dp1187 flies (see MATERIALS AND METHODS). The y, Dp1187 females were crossed to males of the w; ru h SuUR⁺ and w; ru h SuUR^- isogenic lines, and the y, Dp1187; ru h SuUR⁺ (control) and y, Dp1187; ru h SuUR^-/SuUR⁺ males were analyzed (Table 2). The number of yellow bristles is significantly smaller in SuUR^- heterozygotes than in the control. Thus, even when present in one dose, SuUR^- suppresses yellow⁺ variegation in Dp1187.

Dp1187 is advantageous for studying additional doses of SuUR⁺ on yellow⁺ PEV. Extra doses of SuUR⁺ introduced with transposon X6S1 (see Fig 1) enhance synthesis of the SuUR protein. In chromosomes of larvae with two Tn[SuUR⁺] bearing four doses of the SuUR gene (two endogenous and two extra doses), the number of chromosome sites associated with SUUR is greater than two times that observed in the wild-type control containing two doses of SuUR⁺ (see ZHIMULEV et al. 2003 ). This fact is also demonstrated in Fig 6A and B. Females of the transgenic line 4[Tn SuUR⁺] carrying y w⁶⁷ and six SuUR⁺ doses (two genomic and four transgenic) were crossed to the y, Dp1187 males. SuUR⁺ was in four doses in progeny. Females used in the control cross were y w⁶⁷, with SuUR⁺ in two genomic doses. Analysis of their progeny (Table 2) suggests that the extra doses of SuUR⁺ enhance the inactivation of yellow⁺ in duplication: the percentage of yellow bristles in the carriers of SuUR⁺ in four doses is significantly higher than that in the control flies (P < 0.001).

View larger version (54K): In this window In a new window Download PPT slide   Figure 5. Effects of different doses of SuUR+ on the compaction of rearranged chromosomal regions: (A) dorvar7/dor females with two and zero doses of SuUR+ from crosses y dor/FM6; ru h SuUR+ x dorvar7/Y; ru h SuUR+ and y dor/FM6; SuUR- x dorvar7/Y; SuUR-, respectively, at 18°. (B) w/dorvar7 females with two and zero doses of SuUR+ from crosses w; ru h SuUR+ x dorvar7/Y; ru h SuUR+ and w; ru h SuUR- x dorvar7/Y; SuUR- at 25°. (C) y w67/dorvar7 females with two and five doses (two endogenic and three transgenic) of SuUR+ from crosses y w67; SuUR+ x dorvar7/Y; SuUR+ and y w67; SuUR+ + 6[Tn SuUR+] x dorvar7/Y; SuUR+ at 25°. (D) XO, Dp(1;f)R males with two and one doses of SuUR+ from crosses C(1)RM/0, y v; SuUR+ x XY, Dp(1;f)R; SuUR+ and C(1)RM/0, y v; SuUR- x XY, Dp(1;f)R; SuUR+ at 18°. (E) y w pn2b /FM6 females with two and zero doses of SuUR+ from crosses y w pn2b /FM6; ru h SuUR+ x y w pn2b/Y; ru h SuUR+ and y w pn2b /FM6; ru h SuUR- x y w pn2b/Y; ru h SuUR- at 18°. (F) y w67/y w pn2b females with two and four (two endogenic and two transgenic) doses of SuUR+ from crosses y w67; SuUR+ x y w pn2b/Y; SuUR+ and y w67; SuUR+ + 4[Tn SuUR+] x y w pn2b/Y; SuUR+ at 25°.

View larger version (19K): In this window In a new window Download PPT slide   Figure 6. Extra doses and overexpression of SuUR+ induce additional binding sites of anti-SUUR. (A) wild-type Oregon-R with two doses of SuUR+ (control). (B) y w67, SuUR+ + 2[Tn SuUR+] with four doses of SuUR+; the number of binding sites is increased in comparison to control. (C) dorvar7/H7; +/H7 with overexpression of SuUR+ (2 hr after heat shock); most chromosome bands bind anti-SUUR.

In T(2;3)Sb, the mutant allele of Sb exhibits PEV; therefore, reduced PEV would result in an increase in mutant bristle number. We compared the number of Sb bristles in flies carrying different doses of SuUR⁺ (one, two, or four) and observed no significant correlation between the occurrence of the Sb phenotype and SuUR⁺ dosage (data not shown).

In(1)sc⁸ variegates for different classes of bristles, due to PEV associated with genes of the AS-complex. The four scutellar bristles, which can be thinner, shorter than normal, or entirely missing, are most convenient for estimating the extent of PEV. Variegation is enhanced by the removal of the Y chromosome, with males becoming sharply inviable presumably because of PEV spreads into the essential genes (LINDSLEY and ZIMM 1992 ).

We studied the effect of the SuUR⁺ dosage on the viability of sc⁸/0 males. A total of 3.6% of the expected number of males survive in the progeny from a cross of female C(1)RM/0; SuUR⁺ to sc⁸; SuUR⁺ males (8 males: 319 females at the expected 1:1 ratio). In a similar cross, on the background of SuUR^- [C(1)RM/0; SuUR^- x sc⁸; SuUR^-], the portion of surviving males is 24% (70 males:285 females). The portion of male survivors is 8% (16 males:200 females) in the presence of a single maternally derived dose of SuUR⁺ [C(1)RM/0; SuUR⁺ x sc⁸; SuUR^-]. Therefore, a decrease in SuUR⁺ dosage results in an increase in the viability of sc⁸/0 males. This may be interpreted as a weakening of PEV.

The analysis of variegation of four scutellar bristles gave similar results. Variegation was estimated by determining the number of normal bristles and the proportion of mosaic individuals (Table 3). The number of normal bristles is considerably reduced (two times lower) in line sc⁸ in which SuUR⁺ is present in two doses. In contrast, the number approaches the normal number (four) in line sc⁸; SuUR^-. In males in which SuUR⁺ is present in four doses, variegation is enhanced; i.e., the number of normal bristles is reduced. However, the effect of two extra doses of SuUR⁺ is weaker than that of an additional dose of the classic modifier Su(var)2-5 introduced via Dp(2;2)P90 (Table 3, lines 3 and 4). The proportion of mosaic progeny is also dependent on SuUR⁺ dosage (Table 3). Bristle variegation in the XO male is also partly suppressed by the SuUR mutation (Table 3, lines 5 and 6); however, the modifying effect of the absence of the Y chromosome is not completely overcome by SuUR^-. Taken together, the results allowed us to conclude that SuUR affects PEV of classic variegators in a dose-dependent manner, although much weaker than Su(var)2-5 does.

The extent of heterochromatization in a region subject to PEV is dependent on SuUR dosage:
Cytologically, PEV is measured by the frequency of heterochromatization and the amount of compaction in the euchromatic regions juxtaposed to CH by chromosome rearrangement. All the regions that had unusually dark bands, or appeared as blocks of CH-like material, were assumed to be heterochromatic. The structure of the rearranged region and of the normal homolog was compared in the same nucleus.

The compaction criterion is applicable to the rearrangements in which (i) PEV spreads over to several cytologically discernible regions and (ii) PEV is not strong enough to completely heterochromatize the region of interest and to make it merge with the CH material. Regrettably, w^m4, y^V, Sb^V, and sc⁸ (the classic systems for the determination of the effects of modifiers on variegation) are not suitable for analysis of this kind, because of short-distance PEV (w^m4, sc⁸) and the small size of the region (Dp1187). In the T(2;3)Sb^V case, we found no evidence of heterochromatization at the junction of the 3R chromosome (88E1-2 and 89B) and CH of the 2R chromosome (data not shown). Spreading of heterochromatin is obviously undetectable at the cytological level in T(2;3)Sb^V.

Thus, we further pursued the well-studied rearrangements in the distal parts of the X chromosome with long-distance PEV: T(1;2) dor^var7, Dp(1;1)pn2b, and Dp(1;f)R. Removal of the Y chromosome and variation of temperatures (18° or 25°) were used as modifiers of PEV to achieve the degree of compaction optimal for analysis. The frequency of heterochromatization and its extent were shown to correlate with SuUR⁺ dosage in all the rearrangements (Fig 5). There are significant differences among the distributions of the frequencies of compaction for all the rearrangements in all the variants of the experiment (P < 0.01).

SuUR overexpression enhances variegation of morphological characters but does not affect heterochromatization in rearranged polytene chromosomes:
We used two transposons, H7 and HSHP1.83C, which contain the entire open reading frame of the SuUR⁺ and cDNA Su(var)2-5 genes under the control of the hsp70 promoters (see MATERIALS AND METHODS). The transformed line HSHP1.83C is homozygous for the transposon insert. Upon heat-shock treatment HSHP1.83C flies have increased levels of the heterochromatin-associated protein HP1 and the lethality of Su(var)2-5 homozygotes is rescued (EISSENBERG and HARTNETT 1993 ). The H7-X; H7-3 line was homozygous for the transposon inserts H7 containing SuUR⁺ in the X and the third chromosomes; i.e., the transposon was present in four doses. Induction of SuUR⁺ overexpression in the transposon by heat shock rescues the SuUR⁺ phenotype (MAKUNIN et al. 2002 ), which meant that the transposon is functionally active. The level of the SuUR protein substantially rises after the heat shock. In wild strains the SuUR protein is detected in the chromocenter and additionally in 110 late-replicating sites in polytene chromosomes (MAKUNIN et al. 2002 ). Under conditions of our experiments with daily exposure to 37°, anti-SUUR binds to most polytene chromosome bands of third instar larvae, thereby demonstrating that SuUR is, indeed, overexpressed (Fig 6A and Fig C).

SuUR overexpression induced by shock every day beginning with a 5-hr embryo causes a significant enhancement of variegation in the two analyzed rearrangements, In(1)sc⁸ and T(2;3)Sb^V (Table 4). The enhancing effect is comparable to that of HP1 overexpression, although it is weaker. Similar results were obtained for w^m4h (data not shown). Daily heat shock, alone, does not affect the expression of the mutant phenotype sc⁸ and Sb^V (data not shown).

The effect of SuUR⁺ overexpression on heterochromatization was studied in the Dp(1;1)pn2b and T(1;2)dor^var7 rearrangements. The results obtained for dor^var7 are given in Table 5; the data for pn2b are not shown. Flies were allowed to lay eggs for 2 hr, and the embryos were kept for 3 hr at 25°; then, they were heat shocked for 40 min. Developing embryos and larvae were exposed to heat shock daily to midthird larval instar; control individuals developed at 25°.

Analysis of the heterochromatization frequencies of regions subject to PEV in rearrangements demonstrated no differences between the control and the variant with overexpression induction (Table 5). Overexpression of neither HP1 nor SuUR affected heterochromatization. These results were obtained in two independent experiments. It is interesting that heterochromatization in dor^var7 is identical in sibs obtained from mothers heterozygous for PEV modifier mutation and balancer chromosome: Su(var)2-5⁰⁵/+ and Cy0/+; Dp(2;2)P90/+ and Cy0/+; and Su(var)3-9/+ and TM3/+ (Table 5). Similar data were obtained for Dp(1;1)pn2b (data not shown). So, heterochromatization under PEV in salivary gland chromosomes seems to be independent of zygotic dosage of modifiers and is determined by maternal products of these genes.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Overall, the results show that SuUR affects PEV silencing in a dose-dependent fashion. The SuUR⁺ gene in one or zero doses suppresses variegation; SuUR⁺ in extra doses enhances it. The modifying effect of SuUR is weaker than that of the classic modifiers, such as Su(var)2-5. Different variegating markers respond differently to SuUR⁺ dosage. To illustrate, SuUR⁺ in one or four doses did not affect bristle variegation in Sb^V; however, overexpression of SuUR⁺ enhanced PEV in Sb^V. That Sb^V sometimes gives a weak or even no response to PEV modifiers has been previously reported (SINCLAIR et al. 1983 , SINCLAIR et al. 1998 ; BISHOP 1992 ; VERNI et al. 2000 ). One explanation why variegators respond differently may be the structural and functional heterogeneity of CH (LLOYD et al. 1997 ). It is noteworthy that SuUR^- restores polytenization of only a small part of pericentric heterochromatin, which also means that the response of different CH domains to SuUR is not the same. Nevertheless, we established a clear influence of SuUR dosage for most variegators studied, at the level of both morphological characters of the adult and cytological observations of heterochromatization.

As for SuUR⁺ overexpression, enhancement of PEV was observed only for adult morphological traits, and there was no effect on heterochromatization in the salivary gland polytene chromosomes (Table 5). However, this is not surprising, when making allowance for the early differentiation of salivary gland cells during development. According to LU et al. 1998 , PEV silencing starts by gastrulation and is most extensive in the later embryo. The second important step in the establishment of the variegation pattern corresponds to the beginning of cell differentiation after the cessation of mitosis. As demonstrated for the eye imaginal disc, at this time relaxation of PEV occurs, and thereafter the state is maintained unaltered to complete formation of the eye. These two periods correspond to the two temperature-sensitive periods of variegation of eye pigment (LU et al. 1998 ). It is known that the last mitotic division in the salivary gland precursor cells takes place in the 5-hr embryo and the first polytenization round proceeds between 8 and 9 hr (ORR-WEAVER 1994 ). In the current experiments, we were able to induce overexpression of SuUR⁺ only at 5–6 hr of embryonic development because 3-hr embryos cannot tolerate heat shock and die. We propose that by the onset of overexpression, PEV not only is established in salivary gland cells, but also is already relaxed. This interpretation is supported by the observation that HP1 overexpression does not affect heterochromatization (Table 5). It was also shown that PEV heterochromatization does not depend on zygotic dosage of PEV modifiers. So, we suggest that in salivary gland chromosomes compaction of the euchromatic regions under PEV acquires a stable pattern before the beginning of transcription in embryogenesis. Furthermore, PEV heterochromatization in salivary gland chromosomes is temperature dependent mainly during the first 6 hr of embryonic development (for details see ZHIMULEV 1998 ). Thus, the pattern of compaction is maintained stably in salivary gland cells, supporting the assumption that heterochromatin is sensitive to the concentration of products of gene modifiers of PEV during the period when silencing is relaxing (LU et al. 1998 ). This explanation is consistent with the fact that the heat-shock-induced overexpression of SuUR⁺ and HP1 was strong enough to enhance variegation in cells with developmentally late differentiation—the bristle-forming cells.

Another reason why heterochromatization did not appear to be susceptible to the overexpression of modifiers may be limited resolution of visual examination. We can discriminate differences in the extent of heterochromatization only when the process involves at least a single band of the polytene chromosome, i.e., a stretch of 30 kb and longer.

Homozygotes for SuUR and transgenic lines with extra doses of normal gene are not lethal (BELYAEVA et al. 1998 ). MICHAILIDIS et al. 1988 reported a correlation between developmental timing and variegation, which could explain the dose-dependent effects of SuUR on PEV. However, we failed to detect any change in developmental timing with the various SuUR dosages applied (VOLKOVA and ZHIMULEV 2001 ).

The SuUR protein appears in euchromatic regions heterochromatized under PEV. HP1 appears in these regions as well, suggesting the two proteins might function together (BELYAEVA et al. 1993 ). Therefore, SuUR affects not only intercalary and pericentric heterochromatin but also regions that undergo PEV. Does the SuUR protein spread from CH into PEV heterochromatin or does it recognize an already silenced domain? This is an open issue. In any event, the SuUR protein affects the structure of the heterochromatized region, making it less accessible to replication and transcription. We obtained here cytological evidence that the degree of compaction of a region undergoing PEV is dependent on SuUR⁺ dosage. The SuUR protein shows homology to the N-terminal part of the SWI2/SNF2 remodeling protein (MAKUNIN et al. 2002 ). Data indicate that chromatin-remodeling proteins can interact with chromatin modification proteins through the formation of large complexes that regulate high-order chromatin structure and the accessibility of chromatin to various factors (LI 2002 ).

In IH and CH, SuUR locally affects DNA underreplication in salivary gland polytene chromosomes (BELYAEVA et al. 1998 ; MOSHKIN et al. 2001 ). There is reason now for extending this conclusion to regions undergoing PEV. Our results demonstrated that, in the background of the SuUR mutation, DNA underreplication under PEV is completely suppressed in In(1)sc⁸ even in males XO, although variegation of bristles is in part retained in the absence of underreplication. Substantial suppression of DNA underreplication was observed in In(1)w^m4h. Increasing the copy number of the variegating genes in polytene chromosomes could be one possible reason for the modifying effect of SuUR on PEV. Detailed discussion of this problem can be found in KARPEN and SPRADLING 1990 .

SuUR may act in another way—through gene-dosage effects on ectopic pairing. The frequency of ectopic contacts is dependent on SuUR dosage: virtually all the contacts vanish in the SuUR^- homozygotes, and the contacts become more prominent relative to the wild type as the dose of the SuUR⁺ allele increases (ZHIMULEV et al. 2003 ). BELYAEVA and ZHIMULEV 1991 demonstrated that the formation of regions undergoing PEV is associated with the appearance of ectopic fibers that stretch between compacted regions and CH. According to the coalescence model of spreading (SABL and HENIKOFF 1996 ), PEV silencing would be mediated by moderately repetitive elements scattered in euchromatin, which can coalesce with heterochromatin blocks. Heterochromatin formation is nucleated by pairing structures arising between repeated sequences (TALBERT and HENIKOFF 2000 ). It is also possible that the effect of SuUR on PEV might be due to changes in the expression of genes outside the region undergoing PEV. To summarize, we can conclude that SuUR influences PEV silencing through various mechanisms, which are not mutually exclusive, but rather complementary.

	ACKNOWLEDGMENTS

We are very grateful to J. Modolell, S. Campuzano, and S. A. Demakov for DNA clones and advice; J. C. Eissenberg for HP1.83C stock; I. V. Makunin for remarks; and O. V. Demakova for the gift of the unpublished photograph. We express our gratitude to L. Wallrath for reading the manuscript. This work was supported by grants from the program "Frontiers in Genetics of the Russian Federation 2-02PNG-2002," grants from the Russian Foundation for Basic Research program 00-15-97984 and 02-04-48222, Laurentiev's grant for Young Scientists (L.V.B.), and grant PD02-1.4-74 of the Ministry of Education of the Russian Federation.

Manuscript received January 24, 2003; Accepted for publication July 10, 2003.

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