The lawc Gene Is a New Member of the trithorax-Group That Affects the Function of the gypsy Insulator of Drosophila

Corresponding author: Victor G. Corces, Department of Biology, The Johns Hopkins University, 3400 North Charles St., Baltimore, MD 21218., corces{at}jhu.edu (E-mail)

Communicating editor: J. A. BIRCHLER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Mutations in the lawc gene result in a pleiotropic phenotype that includes homeotic transformation of the arista into leg. lawc mutations enhance the phenotype of trx-G mutations and suppress the phenotype of Pc mutations. Mutations in lawc affect homeotic gene transcription, causing ectopic expression of Antennapedia in the eye-antenna imaginal disc. These results suggest that lawc is a new member of the trithorax family. The lawc gene behaves as an enhancer of position-effect variegation and interacts genetically with mod(mdg4), which is a component of the gypsy insulator. In addition, mutations in the lawc gene cause alterations in the punctated distribution of mod(mdg4) protein within the nucleus. These results suggest that the lawc protein is involved in regulating the higher-order organization of chromatin.

INSULATOR elements are DNA sequences that interfere with the ability of an enhancer to act on a promoter when placed between the two (JACK et al. 1991 ; GEYER and CORCES 1992 ; KELLUM and SCHEDL 1992 ). It has been hypothesized that insulators define separate areas of gene activity by establishing higher-order chromatin domains. In such a way, promoters from one gene could be sheltered from action of enhancers from a neighboring gene (GDULA et al. 1996 ; GERASIMOVA and CORCES 1996 ). In addition, transgenes flanked by insulator elements are expressed regardless of the integration site in the chromosomes, i.e., insulators can block repression of transgene expression due to adjacent sequences (KELLUM and SCHEDL 1991 ; ROSEMAN et al. 1993 ). Several insulator elements have been identified, including the scs and scs' sequences of Drosophila (KELLUM and SCHEDL 1991 ), a subset of the Fab-7 DNA sequences of the Drosophila bithorax complex (HAGSTROM et al. 1996 ; ZHOU et al. 1996 ; MIHALY et al. 1997 ), and sequences present in the gypsy retrotransposon (JACK et al. 1991 ; GEYER and CORCES 1992 ). In vertebrates, a DNA sequence at the 5' end of the chicken ß-globin gene (CHUNG et al. 1997 ) and sequences present in the human apolipoprotein B gene (KALOS and FOURNIER 1995 ) have also been shown to affect enhancer-promoter communication.

The gypsy insulator is present in the 5' transcribed untranslated region of the gypsy retrotransposon of Drosophila (GDULA et al. 1996 ). Three components of this insulator have been identified: a 350-bp sequence of the gypsy retrotransposon, the su(Hw) protein that binds to gypsy DNA via its zinc fingers, and the mod(mdg4) protein that interacts with su(Hw). In the case of the yellow² (y²) mutation, the gypsy insulator causes a unidirectional repression of yellow gene enhancers such that those distal to the yellow promoter with respect to the gypsy insulator are repressed, whereas the enhancers proximal to the promoter relative to the insulator are active. In the absence of su(Hw) protein, the enhancers are all active and the y phenotype reverts to wild type, indicating that su(Hw) is an essential component of the gypsy insulator. In the presence of hypomorphic mutations in the mod(mdg4) gene, the gypsy insulator is only partially functional, and all enhancers (both upstream and downstream of the insulator) become partially active; for example, in the posterior abdominal segments, the y body cuticle enhancer is active in some cells and pigment is produced, whereas the enhancer is inactive in other cells, resulting in a loss of pigmentation. This effect manifests itself in a variegated abdominal pigment phenotype in y² mod(mdg4) flies (GERASIMOVA et al. 1995 ). This phenotype is reminiscent of the position-effect variegation (PEV) seen when a gene is juxtaposed to heterochromatin through chromosomal rearrangements (CASPERSON and SCHULTZ 1938 ). In agreement with this effect, mutations in mod(mdg4) act as typical Enhancer of variegation [E(var)] mutants by enhancing the phenotype of the white-mottled4 (w^m4) allele, suggesting that the mod(mdg4) protein acts at the level of chromatin organization (DORN et al. 1993 ; GERASIMOVA et al. 1995 ).

In support of this function at the level of chromatin structure, mod(mdg4) has been shown to display the properties characteristic of members of the trithorax-Group (trx-G; GERASIMOVA and CORCES 1998 ) whose products [as well as those of the Polycomb-Group (Pc-G)] are thought to maintain homeotic gene expression through stable chromatin conformation changes (reviewed by KENNISON 1995 ). Homeotic genes determine segment identity along the anterior-posterior axis (LEWIS 1978 ; KAUFMAN et al. 1980 ). The gap and pair-rule genes initially determine the expression domains of the homeotic genes (INGHAM 1983 ); for example, fushi tarazu is necessary for the activation of both Antennapedia complex and bithorax complex genes (INGHAM and MARTINEZ-ARIAS 1986 ). However, gap and pair-rule genes are not expressed beyond early stages of embryogenesis. The trx-G and Pc-G gene products help maintain homeotic gene expression domains beyond the time when the gap and pair-rule genes themselves are no longer expressed, and indeed, throughout development (KENNISON and TAMKUN 1988 ; KUZIORA and MCGINNIS 1988 ). Null mutations in Pc-G and trx-G genes are lethal, but phenotypes can be observed in heterozygous flies. The trx-G and Pc-G gene products act antagonistically and mutations in one group suppress mutations in the other (INGHAM 1983 ; CAPDEVILA et al. 1986 ). The trx-G gene products maintain homeotic gene expression within their normal domains (BREEN and HARTE 1993 ) and, when mutant, cause a variety of homeotic phenotypes, for example, haltere-to-wing transformation (SHEARN 1989 ). The Pc-G gene products repress homeotic gene expression outside the normal domains (KUZIORA and MCGINNIS 1988 ) and, when mutant, cause an extra sex combs phenotype indicative of transformation toward the first leg (DUNCAN 1982 ). trx-G and Pc-G gene products are thought to form large protein complexes (see PARO and HARTE 1996 for a review) and these complexes compete for the same binding sites in and around homeotic genes called Pc response elements (PREs; ORLANDO and PARO 1993 ; GINDHART and KAUFMAN 1995 ).

Members of the trx-G include trithorax (trx; CAPDEVILA and GARCIA-BELLIDO 1981 ), brahma (brm; KENNISON and TAMKUN 1988 ), absent, small, or homeotic discs1 and 2 (ash-1 and ash-2; SHEARN 1989 ), and mod(mdg4) (GERASIMOVA and CORCES 1998 ). Another member, trithorax-like (Trl), encodes the GAGA factor (FARKAS et al. 1994 ), which has been shown to participate in chromatin remodeling during transcription (TSUKIYAMA et al. 1994 ). The Pc-G includes Polycomb (Pc; LEWIS 1978 ), polyhomeotic (ph; DURA and BROCK 1985 ), Polycomblike (Pcl; DUNCAN 1982 ), and extra sex combs (esc; STRUHL 1981 ). The Pc protein has a domain homologous to the nonhistone heterochromatin-associated protein HP1, which is encoded by the suppressor of PEV gene Su(var)205 (JAMES and ELGIN 1986 ; EISSENBERG et al. 1990 ).

For a gene to be classified as a member of the trx-G, certain genetic criteria (described by SHEARN 1989 ) need to be met: (1) the gene should have a homeotic mutant phenotype; (2) mutations in the gene should enhance the severity and frequency of homeotic transformations due to other trx-G members (e.g., flies doubly heterozygous for trx and ash-1 have a higher number of, and more extreme, transformations than either trx or ash-1 heterozygotes alone); (3) trx-G mutants should suppress the dominant extra sex comb phenotype of Pc mutants. The lawc mutation has been mapped to position 23.0 on the X chromosome (SIMONOVA et al. 1992 ). We noted that the homeotic phenotype of lawc mutations is enhanced by mod(mdg4) and decided to investigate the relationship between these two genes further. Using the criteria listed above, we have determined that lawc is a trx-G member. lawc enhances the mod(mdg4) phenotype and vice versa, and like mod(mdg4) and Trl, lawc acts as an enhancer of position-effect variegation. In addition, mutations in the lawc gene affect the subnuclear distribution of gypsy insulator components. These results suggest that the lawc protein might play a general and fundamental role in chromatin organization.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Drosophila stocks and crosses:
Flies were kept in standard medium and grown at 23°, 75% humidity. The mod(mdg4)^u1 allele is a spontaneous mutation caused by a Stalker transposable element insertion (GERASIMOVA et al. 1995 ), which is viable and behaves genetically as a hypomorph. lawc⁺¹⁰ is a revertant of lawc^P1 that was generated by crossing lawc^P1 to a transposase-supplying line and the subsequent excising of the P element. l(1)EF520/FM7 was obtained from Dr. Norbert Perrimon; ash-1^VF101/TM3, trx^B11/TM3, trx^B11 ash-1^VF101/TM1, and Df(3L)Pc-Mk/TM6 were received from Dr. Allen Shearn; brm² trx^E2/TM3 was obtained from Dr. Jim Kennison. Pc⁴/TM3, brm²/TM6, In(1)w^M4, and Df(1)RA2/TM3 were obtained from the Bloomington Fly Stock Center. The cytological limits of Df(3L)Pc-Mk are 78A2-78C9.

Immunolocalization of proteins:
Antibodies were obtained from the following investigators. Antp antibodies were received from Dr. Matt Scott, Ubx antibodies from Dr. Juan Botas and Dr. Javier Lopez, and labial antibodies from Dr. Bill McGinnis. Immunolocalization of proteins on fly tissues was performed as described by LAJEUNESSE and SHEARN 1995 . Third instar larvae were dissected in PBS and fixed in 4% formaldehyde for 25 min. The tissue was incubated in 0.1% Triton X-100, 0.3% BSA, and 0.5% sheep serum in PBS for 30 min. The primary antibody was then added and the samples were incubated on a shaker overnight at room temperature. The samples were washed six times for 30 min in PBT (PBS, 0.1% Triton X-100, and 0.3% BSA). FITC-conjugated secondary antibodies were then added and the samples were incubated for 2 hr at room temperature. Samples were washed again and the tissue was examined in a Zeiss (Thornwood, NY) microscope.

For the analysis of the nuclear distribution of mod(mdg4) protein, larvae were dissected in Cohen's buffer (25 mM glycerophosphate, 10 mM potassium phosphate, 30 mM potassium chloride, 10 mM magnesium chloride, 3 mM calcium chloride, 160 mM sucrose, and 0.5% NP40) and tissues were fixed for 25 min at room temperature in 0.1 M sodium chloride, 2 mM potassium chloride, 10 mM phosphate, 2% NP40, and 2% paraformaldehyde and transferred to 45% acetic acid. After 10 min, the imaginal discs were dissected out and placed in a drop of 45% acetic acid on a polylysine-treated slide. A siliconized coverslip was placed over the sample and then firmly pressed down to squash the disc tissue. The slides were then frozen at -80° for an hour. The coverslips were then removed and the slides placed into a Coplin jar containing antibody dilution buffer (130 mM sodium chloride, 10 mM sodium phosphate, 0.1% Triton X-100, and 1% BSA) for 5 min. The buffer was changed twice and the slides were incubated with 20 µl of a 1:250 dilution of mod(mdg4) antirat antibody in a humidity chamber overnight at 4°. The slides were then washed three times in antibody dilution buffer and incubated in 20 µl of Texas Red-conjugated secondary antibodies diluted 1:200 in antibody dilution buffer. After incubation at room temperature in the dark for 1 hr, the slides were washed three times in antibody dilution buffer and then stained with 4',6-diamidino-2-phenylindole (DAPI). Antifade mounting medium (Vectashield) was placed on the slides and covered with a coverslip. Slides were viewed under a Zeiss microscope at x100 magnification.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The lawc^P1 mutation results in homeotic transformations:
Mutations in the lawc gene result in an arista-to-leg homeotic transformation. The lawc^P1 mutation was generated by SIMONOVA and coworkers (1992) and is ~1% penetrant for complete transformation of the arista into leg, such that leg segments and tarsal claws can be observed instead of arista tissue (Figure 1B). lawc^P1 is fully penetrant for partial transformation into leg, which is manifested by a thickening of the arista (Figure 2B). In addition, lawc^P1 mutants often have ectopic bristles on the scutellum and thorax (Figure 1D) and the wings are held apart with multiple incisions in the margins, especially along the posterior margin (Figure 1F). Due to this pleiotropic phenotype, the mutation was called leg, arista, wing complex (lawc). The mutant phenotype is stronger in males than in females. The lawc^P1 mutation is caused by the insertion of a P element at approximately position 7E on the X chromosome (SIMONOVA et al. 1992 ). We used overlapping deficiencies to map lawc and found that Df(1)KA14, which lacks 7F1 to 8C6, complements lawc^P1, whereas Df(1)RA2, which deletes 7D10 to 8A4,5, fails to complement the lawc gene. Most lawc^P1/Df(1)RA2 females die as early larvae. The 10% that do eclose have an enhanced lawc phenotype (Figure 2C) that includes strong arista-to-leg transformation, many ectopic bristles, and larger wing margin incisions. l(1)EF520 (LEFEVRE 1976 ) is an EMS-induced allele that causes early larval lethality. The 3% of lawc^P1/l(1)EF520 females that eclose show an enhanced lawc phenotype, including arista-to-leg transformation (Figure 2D), indicating that l(1)EF520 is an allele of lawc. Because the phenotype of lawc^P1/l(1)EF520 females is the same as that of lawc^P1/Df(1)RA2, the l(1)EF520 mutation is an allele of lawc that behaves genetically as a null. We will refer to this mutation as lawc^EF520.

View larger version (124K): In this window In a new window Download PPT slide   Figure 1. Phenotypes of lawc mutants. (A, C, and E) Wild-type Oregon R flies. (A) Arista with characteristic branched appearance. (C) Scutellum with four macrochaete. (E) Wing with smooth, entire wing margins. (B, D, and F) lawcP1 flies. (B) Complete transformation of arista to leg; note the leg segments and tarsal claw (arrows). (D) Scutellum with five instead of four macrochaete (arrow). (F) Wing with incisions in the posterior wing margin (arrows).

View larger version (98K): In this window In a new window Download PPT slide   Figure 2. Homeotic transformations in lawc mutants. (A) Oregon R. (B) lawcP1 (partial transformation). (C) Df(1)RA2/lawcP1. (D) lawcEF520/lawcP1. (E) lawcP1; mod(mdg4)u1.

lawc has the properties of a trx-G gene:
On the basis of homeotic transformation and the pleiotropic phenotype of the lawc^P1 mutation, we hypothesized that lawc may be a new member of the trx-G family. For a gene to be classified as a trx-G member, mutations in that gene should enhance the phenotype of other trx-G members (SHEARN 1989 ). To test this possibility, lawc^P1/lawc^P1 females were crossed to trx^B11/TM3 males and the resulting progeny were analyzed for transformations of haltere-to-wing, posterior abdominal segments into more anterior, or third leg into second leg. No additional transformations were seen in the hemizygous lawc^P1; trx^B11 /+ males of the F₁ progeny compared to the control lawc^P1; +/+ (Table 1). An increase in transformation frequency or severity was also not observed in lawc^P1; brm²/+ or lawc^P1; ash-1^VF101/+ males (Table 1). We speculated from these results that lawc^P1 might be too weak an allele to show an effect in combination with single heterozygous mutant trx-G members such as trx^B11, ash-1^VF101, or brm². To test this possibility, we analyzed whether combinations of two trx-G mutations could enhance the lawc phenotype. trx^B11 ash-1^VF101/++ and brm² trx^E2/++ double transheterozygous mutants result in homeotic transformations such as haltere to wing or third leg to second leg manifested by bristles on the haltere and apical bristles on the third leg, respectively (Figure 3A). In the case of lawc^P1; trx^B11 ash-1^VF101/+ + or lawc^P1; brm² trx^E2/++ males, both the frequency and severity of the homeotic transformations are increased (Figure 3 and Table 1). The occurrence of homeotic transformations increases at least threefold, from 14 to 64% in the case of trx^B11 ash-1^VF101 and from 10 to 29% in the case of brm² trx^E2. In addition, there is a dramatic increase in the severity of the homeotic transformations, with many halteres completely transformed into wings (Figure 3B). This increase in frequency and severity of homeotic transformations is also seen in lawc^P1/+; trx^B11 ash-1^VF101/+ + or lawc^P1/+; brm² trx^E2/++ females (Figure 3C). This enhancement of the trx-G mutant phenotypes was stronger in a lawc^P1 maternal background (Table 1). In addition to the homeotic transformations described, the arista-to-leg transformation characteristic of lawc mutants is enhanced in flies heterozygous for two trx-G mutations, which show transformed aristae and many ectopic bristles (Figure 3D and Table 2), suggesting that lawc^P1 enhances the phenotype of trx-G genes and vice versa.

View larger version (142K): In this window In a new window Download PPT slide   Figure 3. Genetic interactions between lawcP1 and trx-G and Pc-G genes. (A) Partial haltere-to-wing transformation in an ash-1VF101 trxB11/++ male. (B) More complete haltere-to-wing transformation in a lawcP1; ash-1VF101 trxB11/++ male. (C) Third leg to second leg transformation in a lawcP1/+; ash-1VF101 trxB11/+ + female; note the apical bristle on upper third leg (arrow). (D) Ectopic macrochaete on the scutellum of a male of the genotype lawcP1; ash-1VF101 trxB11/++. (E) Df(3L)Pc-Mk/+ male with ectopic sex combs on second and third leg (arrows). (F) lawcP1; Df(3L)Pc-Mk/+ male with no ectopic sex combs on the second and third legs.

When females carrying the null lawc^EF520 allele are crossed to single trx-G mutant males, the resulting lawc^EF520/+; trx-G/+ females show no homeotic transformations (Table 1). The number of transformations increases relative to the control in lawc^EF520/+; trx^B11 ash-1^VF101/++ or lawc^EF520/+; brm² trx^E2/++ females (Table 1). The lawc^EF520 allele is lethal by itself, but lawc^EF520/lawc^P1 females are viable. In combination with single trx-G mutants, no lawc^EF520/lawc^P1; trx-G/+ females eclose, suggesting that heterozygosity in a trx-G gene such as trx, ash1, or brm is sufficient to cause lethality in combination with strong lawc alleles (Table 3). A similar effect can be observed in females carrying Df(1)RA2, which uncovers the lawc gene. Females heterozygous for this deficiency in combination with two trx-G mutations show an increase in the rate of transformations with respect to the control (Table 1). In addition, Df(1)RA2/lawc^P1 females are viable, but they are also lethal in combination with single or double mutants in trx-G genes (Table 3). The ability of lawc mutations to enhance the phenotype of trx-G mutants suggests that lawc might be a new trx-G gene.

To further test this hypothesis, we analyzed the possibility of genetic interactions between lawc and Polycomb (Pc). The ability to suppress the dominant phenotype of Pc mutants is another criterion for trx-G membership (SHEARN 1989 ). About 83% of Df(3L)Pc-Mk/+ heterozygous males have ectopic sex combs on the second and/or third legs, denoting a transformation toward the first leg (Figure 3E). The vast majority of lawc^P1; Df(3L)Pc-Mk/+ males (97.4%) have no ectopic sex combs on the second or third legs (Figure 3F, Table 1), indicating that lawc^P1 is capable of suppressing the phenotype of Df(3L)Pc-Mk. The same result was obtained using the Pc⁴ mutation (Table 1), i.e., lawc^P1 suppresses the Pc⁴ phenotype. Flies carrying the Pc⁴ chromosome had a 57% frequency of ectopic sex combs and this was reduced to 2% in lawc^P1; Pc⁴/ + males. These results further support the hypothesis that lawc might be a new trx-G gene.

To confirm that the lawc^P1 mutation is responsible for the observed interactions with trx-G and Pc-G members, lawc⁺¹⁰/lawc⁺¹⁰ revertant females (see MATERIALS AND METHODS) were crossed to trx^B11 ash-1^VF101/TM1 males and the subsequent lawc⁺¹⁰; trx^B11ash-1^VF101/++ male offspring were examined for homeotic transformations. The rate and severity of transformation were on a par with that seen when Oregon R females were used in the same cross (12% vs. 14% in the control; Table 1). The same result was obtained when lawc⁺¹⁰/lawc⁺¹⁰ females were crossed to brm² trx^E2/TM3 males, i.e., there was no significant increase in the frequency of homeotic transformations in the F₁ males (9% vs. 10% in the control; Table 1). The number of ectopic sex combs was not reduced in the male offspring of lawc⁺¹⁰/lawc⁺¹⁰ mothers crossed to Df(3L)Pc-Mk/TM3 males (80% compared to 83% of male offspring from Oregon R mothers; Table 1). The frequency of 59% ectopic sex combs in lawc⁺¹⁰; Pc⁴/+ males was similar to the 57% frequency seen in controls (Table 1). lawc⁺¹⁰ was therefore unable to suppress the phenotype of Pc mutants or enhance the phenotype of trx-G mutants, confirming that the results shown in Table 1 are due only to the lawc mutation.

Effect of lawc mutations on the expression of homeotic genes:
trx-G members are positive regulators of homeotic genes, and, therefore, if mutant, result in a decrease of homeotic gene expression. If lawc is a member of the trx-G, the level of homeotic gene expression may be influenced in the background of lawc mutations. To test this possibility, we determined the level of tissue-specific expression of various homeotic gene products using immunofluorescence microscopy. Immunostaining of Df(1)RA2/lawc^P1 female larval imaginal discs and brains was performed with various antibodies to homeotic proteins. No effect of the lawc mutation was found on the level of Scr protein in the central nervous system (CNS) or first leg discs (data not shown). The level of Ubx protein was slightly reduced in the CNS and haltere discs (Figure 4A and Figure B, and data not shown) of Df(1)RA2/lawc^P1 larvae. Labial protein was present but reduced in the eye-antenna disc (Figure 4C and Figure D) and deformed protein was likewise reduced (data not shown). Antp protein levels were not reduced in the brain or leg discs, but ectopic expression of Antp protein was observed in the eye-antenna disc (Figure 4E and Figure F). The ectopic accumulation of Antp protein in the eye-antenna disc correlates with the arista-to-leg transformation seen in lawc^P1 adults. The level of some homeotic gene products is therefore influenced by lawc mutations, whereas other homeotic gene product levels are not altered. This variable ability to affect levels of homeotic products is seen with other members of the trx-G (LAJEUNESSE and SHEARN 1995 ) and confirms the suggestion that lawc is a trx-G gene.

View larger version (56K): In this window In a new window Download PPT slide   Figure 4. Expression of homeotic proteins in wild-type and lawc mutant larvae. Expression of Ubx in the CNS of wild-type (A) and lawcP1/Df(1)RA2 (B) larvae. Expression of Labial in the eye-antenna imaginal disc of wild-type (C) and lawcP1/Df(1)RA2 (D) larvae. Expression of Antp in the eye-antenna disc of wild-type (E) and lawcP1/Df(1)RA2 (F) larvae.

lawc^P1 enhances position-effect variegation:
Mutations in the trithorax-like (Trl) and mod(mdg4) genes, both members of the trx-G, enhance position effect variegation (PEV; DORN et al. 1993 ; FARKAS et al. 1994 ; GERASIMOVA et al. 1995 ). To determine whether lawc may also be capable of enhancing PEV we made a recombinant between In(1)w^m4 and lawc^P1. In(1)w^m4 is an inversion on the X chromosome that juxtaposes the white gene next to the centromeric heterochromatin and results in a phenotype characterized by orange/brown dots against a red background (Figure 5A). lawc^P1/lawc^P1 females were crossed to In(1)w^m4 males and the F₁ females were crossed back to lawc^P1 males. The subsequent F₂ progeny were scored for both w^m4 and lawc^P1 phenotypes. A total of 8754 flies were scored before a recombinant was found due to the need to recover a double crossover event in an F₁ female. Flies of the genotype In(1)w^m4, lawc^P1 have eyes with large white/yellow patches against an orange background, i.e., there is less w expression and thus an enhancement of PEV (Figure 5A). The same effect of the lawc^P1 mutation on PEV was observed for other variegating mutations such as brown-Dominant (bw^D) and yellow-v2 (y^v2) (data not shown). This enhancement of PEV by lawc mutations can be rescued by a transgene containing the complete lawc gene (I. ZORIN and V. CORCES, unpublished results), suggesting that the effect is not due to second site mutations present elsewhere in the genome. The observed involvement of lawc in PEV suggests that the lawc protein might function at the level of chromatin organization.

View larger version (90K): In this window In a new window Download PPT slide   Figure 5. Effects of lawcP1 on position-effect variegation and interactions with mod(mdg4). (A) In(1)wm4 (left) and In(1)wm4, lawcP1 (right) males. (B) Abdominal pigmentation in y2 males. (C) Pigmentation of abdominal segments in y2; mod(mdg4)u1 males. (D) Pigmentation of the abdomen in y2 lawcP1; mod(mdg4)u1 males.

Effects of lawc on the gypsy insulator:
The mod(mdg4) product is a component of the gypsy insulator and has been shown to enhance PEV and to be a member of the trx-G of genes (DORN et al. 1993 ; GERASIMOVA et al. 1995 ; GERASIMOVA and CORCES 1998 ). In the case of the gypsy-induced yellow² (y²) mutation, the abdominal pigment of male flies is lighter than usual in color, but uniformly distributed in the last two abdominal segments (Figure 5B). y²; mod(mdg4)^u1 males have variegated abdominal pigmentation with individual spots of dark wild-type pigment against a lighter pigmented background in the last two abdominal segments (Figure 5C). GERASIMOVA and CORCES 1998 showed that some trx-G mutations, in heterozygous combinations with y²; mod(mdg4)^u1, result in an increase in the number of cells with lighter pigmentation relative to the darkly pigmented regions, i.e., an enhancement of the variegated phenotype. To test whether this is also the case with the lawc mutation, we analyzed the phenotype of y² lawc^P1; mod(mdg4)^u1 males. In the same manner as for other trx-G mutants, the variegated phenotype is enhanced in y² lawc^P1; mod(mdg4)^u1 males in that the areas of lighter pigmentation increase at the expense of the darkly pigmented regions (Figure 5D). lawc^P1 therefore suppresses the mod(mdg4)^u1 variegated phenotype, because the phenotype of y² lawc^P1; mod(mdg4)^u1 flies is closer to y² than to y²; mod(mdg4)^u1. This result suggests that in the presence of a lawc^P1 mutation, the functionality of the insulator, which is impaired by mutations in mod(mdg4), is partially restored. In addition, the y² lawc^P1; mod(mdg4)^u1 males also have strong transformation of the aristae to legs and ectopic bristles on the scutellum (Figure 2E), indicating that mod(mdg4) enhances the lawc phenotype. The ability of lawc to partially restore the functionality of the gypsy insulator in the background of a mutation in mod(mdg4) was also tested in the gypsy-induced omb^D11 mutation (TSAI et al. 1997 ). As in the case of y², the lawc^P1 mutation partially suppresses the phenotype of mod(mdg4)^u1 (data not shown). These results suggest that the lawc protein is either a component of the gypsy insulator or it functions at the level of chromatin organization to perturb the effect of the su(Hw) insulator, just as it has been suggested for other trx-G products (GERASIMOVA and CORCES 1998 ).

Mutations in the lawc gene affect the nuclear distribution of mod(mdg4) protein:
The su(Hw) and mod(mdg4) proteins are located in several hundred sites in polytene chromosomes, but both proteins are distributed in a punctated pattern in the nuclei of diploid cells during interphase. Around 20–30 dots are observed in a typical nucleus, suggesting that many chromosomal sites come together in specific regions of the nucleus. We have proposed a model suggesting that the su(Hw)/mod(mdg4) proteins attach the chromatin fiber to a specific nuclear structure, and Pc-G/trx-G proteins are essential in maintaining this organization. Mutations in both Pc-G and trx-G genes cause a disorganization of the punctated pattern, supporting their role in the maintenance of this nuclear architecture (GERASIMOVA and CORCES 1998 ). To test whether the lawc protein is also involved in the maintenance of the nuclear arrangement of the chromatin fiber in a manner that would explain the observed genetic interactions with mod(mdg4), we analyzed the effect of lawc mutations on the nuclear distribution of mod(mdg4) protein. Figure 6 shows the results of this experiment. In mod(mdg4)^u1/+ flies, the nuclear distribution of mod(mdg4) is normal, with several dots visible mostly around the nuclear periphery (Figure 6A). Flies carrying the lawc^P1 mutation show a similar distribution pattern of mod(mdg4) protein, suggesting that a hypomorphic mutation in the lawc gene is not sufficient to disrupt this pattern (Figure 6B). But flies of the genotypes lawc^P1/lawc^P1; mod(mdg4)^u1/+ or lawc^P1/lawc^EF520; mod(mdg4)^u1/+ show a dramatic alteration in the distribution of mod(mdg4) protein. The perinuclear localization of mod(mdg4) protein is lost in these flies, and instead the protein appears to be distributed throughout the nucleus, with a few small dots visible in the central region of some nuclei (Figure 6C and Figure D). These results suggest that lawc, as with other trx-G and Pc-G genes, is involved in the maintenance of the nuclear arrangement of the chromatin fiber imposed by the protein components of the gypsy insulator.

View larger version (138K): In this window In a new window Download PPT slide   Figure 6. Nuclear distribution of mod(mdg4) protein in various genetic backgrounds. Imaginal disc cells from third instar larvae were stained with antibodies against mod(mdg4) protein; red represents mod(mdg4) and blue is DNA stained with DAPI. (A) Nuclei from y2/y w67; mod(mdg4)u1/+ larvae. (B) Nuclei from lawcP1/lawcEF520 larvae. (C) Nuclei from lawcP1/lawcP1; mod(mdg4)u1/+ larvae. (D) Nuclei from lawcP1/lawcEF520; mod(mdg4)u1/+ larvae.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have shown that lawc is a member of the trx-G and regulates homeotic gene expression. The lawc mutant phenotype results in homeotic transformations as well as ectopic bristles and wing margin incisions, indicating that lawc probably has functions in the regulation of genes other than homeotic selector genes. Like lawc, mutations in ash-2, another member of the trx-G, also result in the formation of ectopic bristles on the scutellum and thorax. Whereas lawc^P1 mutants have incisions in the tip and posterior wing margin, ash-2 mutants have campaniform sensilla transformed to bristles on their wings. ash-2 is thought to play an additional role in determining external sensory organs (ADAMSON and SHEARN 1996 ). Mutations in little imaginal discs (lid) cause duplicated thoracic macrochaete; lid is a new trx-G gene and the Drosophila homologue to human RBP2 (J. J. GILDEA and A. SHEARN, personal communication). brahma mutant clones often have duplication of bristles and wing defects (ELFRING et al. 1998 ). Both trx and ash-2 mutations result in antenna-to-leg transformations in a manner similar to lawc (INGHAM 1985 ; ADAMSON and SHEARN 1996 ). lawc therefore has a pleiotropic phenotype, some aspects of which are shared with other members of the trx-G; in addition, lawc might also play a role in sensory cell development.

It has been hypothesized that trx-G proteins form a complex in the cell nucleus (reviewed in PARO and HARTE 1996 ) and that there is no hierarchy between them so that mutations in any one member lead to a homeotic mutant phenotype. As has been pointed out by SHEARN 1989 , the penetrance of various transformation phenotypes depends on the allele used and the extent of the maternal contribution. Most experiments designed to demonstrate the identity of various trx-G members have made use of recessive null alleles; for example, double heterozygous combinations of null alleles of ash-1, trx, and/or ash-2 lead to homeotic transformations (SHEARN 1989 ). When lawc^P1 was combined with either ash-1, ash-2, trx, or brm individually, no enhancement of the homeotic phenotype was observed. A clear effect was seen when lawc^P1 was used in combination with mutations in two trx-G genes, showing that lawc^P1 enhanced the trx-G mutant phenotypes and vice versa. A deficiency for trx as well as null alleles of ash-1 and ash-2 can suppress the dominant Pc phenotype as heterozygotes (CAPDEVILA and GARCIA-BELLIDO 1981 ; SHEARN 1989 ). Similarly, lawc^P1 hemizygotes are able to suppress the phenotype of Pc, even though lawc^P1 is not a null allele.

As expected from its properties as a trx-G gene, mutations in lawc affect the expression of homeotic genes. The observation that the lawc^P1/Df(1)RA2 background leads to the reduction of some homeotic gene products (Ubx, Lab, Dfd) and not others (Scr, Antp) is not exceptional. In the case of ash-2, the level of Antp is not reduced in the first leg disc, and there is no change in the level of Ubx expression in the CNS, although there is patterned loss in the haltere and third leg disc. ash-2 also causes a reduction of Scr in the first leg disc. ash-1 mutations lead to a reduction of Antp in the first leg disc and lower levels of Ubx in the CNS, but only variable loss of Scr in the first leg disc (LAJEUNESSE and SHEARN 1995 ). Because the trx-G gene products appear to form a complex, it is possible that different trx-G gene proteins interact with different homeotic genes. In forming this complex, some trx-G products such as Trl might bind to DNA, whereas others bind to each other. trx-G members are diverse and range from transcription factors such as the Trl GAGA factor to putative nucleosome displacement factors such as brahma. trx-G proteins could exert their effects on gene expression at various levels in the process of regulating transcription. Some trx-G products must have a general role in transcription because they bind to many sites on polytene chromosomes, other than the sites of homeotic genes (TSUKIYAMA et al. 1994 ; GERASIMOVA and CORCES 1998 ).

Because the trx-G products maintain preestablished patterns of gene expression through multiple cell divisions, it has been assumed that they function at the level of chromatin (ORLANDO and PARO 1993 ; GINDHART and KAUFMAN 1995 ). The fact that lawc^P1 enhances PEV supports the idea that the lawc protein might play a role at the level of chromatin in the same manner as other trx-G products. The observed genetic interactions between lawc and mod(mdg4) suggest the possibility that lawc could be a component of the gypsy insulator. It is likely that lawc regulates insulator effects but it is not a structural component per se. Other trx-G proteins have been shown to participate in insulator function indirectly by contributing to the maintenance of a specific arrangement of the chromatin fiber within the nucleus (GERASIMOVA and CORCES 1998 ) and results presented here support a similar function for lawc. Molecular characterization of the lawc gene is in progress to determine its pattern of nuclear localization and its function in the context of other trx-G proteins.

	ACKNOWLEDGMENTS

We thank Drs. Allen Shearn, Norbert Perrimon, and Jim Kennison for providing various strains used in the studies described here. We also thank Drs. Matt Scott, Juan Botas, Javier Lopez, and Bill McGinnis for supplying antibodies. Work reported here was supported by U.S. Public Health Award GM35463 from the National Institutes of Health.

Manuscript received November 16, 1998; Accepted for publication April 7, 1999.

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