of polyhomeotic with polycomb group genes of drosophila ... · copright 0 1994 by the genetics...

Copright 0 1994 by the Genetics Society of America

Interactions of polyhomeotic With Polycomb Group Genes of Drosophila melanogaster

Niansheng Nick Cheng,' Donald A. R. Sinclair,' Rod B. Campbell and Hugh W. Brock

Department of Zoology, University of British Columbia, Vancouver, British Columbia, V6T 124 Canada Manuscript received March 6, 1994

Accepted for publication August 12, 1994

ABSTRACT The Polycomb ( P C ) group genes of Drosophila are negative regulators of homeotic genes, but individual

loci have pleiotropic phenotypes. It has been suggested that PC group genes might form a regulatory hierarchy, or might be members of a multimeric complex that obeys the law of mass action. Recently, it was shown that polyhomeotic ( p h ) immunoprecipitates in a multimeric complex that includes PC. Here, we show that duplications o f p h suppress homeotic transformations of Pc and Pcl, supporting a massaction model for PC group function. We crossed ph alleles to all members of the Polycomb group, and to E(Pc) and Su(z)2 to look for synergistic effects. We observed extragenic noncomplementation between ph503 and PC, Psc' and Su(z) 2l in females, and between ph409 and Sce', ScmD' and E(%)' mutations in males, suggesting that these gene products might interact directly with ph. Males hemizygous for a temperature-sensitive allele, ph2, are lethal when heterozygous with mutants in Asx, PC, Pcl, Psc, Sce and Scm, and with E(Pc) and Su(z)2. Mutations in trithorax group genes were not able to suppress the lethality of ph2/Y; Psc'/+ males. ph2 was not lethal with extra sex combs, E(%), super sex combs (sxc) or l(4)IOZEFc heterozygotes, but did cause earlier lethality in embryos homozygous for E(z), sxc and l (4) 102EFc. However, ph503 did not enhance homeotic phenotypes of esc heterozygotes derived from homozygous e s t mothers. We examined the embryonic phenotypes of ph2 embryos that were lethal when heterozygous or homozygous for other mutations. Based on this phenotypic analysis, we suggest that p h may perform different functions in conjunction with differing subsets of PC group genes.

M UTATIONS in the Polycomb ( P C ) group of genes cause homeotic transformations in embryos or

adults that resemble those exhibited by gain-of-function mutations in homeotic genes (LEWIS 1978; STRUHL 1981; DUNCAN 1982; INGHAM 1984; DURA et al. 1985; JURGENS

1985). They take their name from the presence of sex combs on the second and third legs of mutant males, presumably because of ectopic expression of Sex combs reduced (STRUHL 1982). Subsequent molecular analyses have shown that homeotic genes are ectopically expressed in PC group mutations, confirming that PC group genes are negative regulators of homeotic genes (STRUHL and AKAM 1985; WEDEEN et al. 1986; CARROLL et al. 1986; RILEY et al. 1987; DURA and INGHAM 1988; GLICKSMAN and BROWER 1988; MCKEON and BROCK 1991; SIMON et al . 1992). At least eleven PC group genes have been described: Additional sex combs ( A s x ) UURGENS 1985); Enhancer of zeste (E(%)) (JONES and GELBART 1990), also known as polycombeotic (PHILLIPS and SHEARN 1990); extra sex combs (STRUHL 1981); 1(4)102EFc (GEHRING 1970), recently renamed pleiohomeotic (GIRTON andJEoN 1994); PC (LEWIS 1978); Polycomblike (Pc l ) (DUNCAN 1982); polyhomeotic ( p h ) (DURA et al. 1985); Posterior sex combs (Psc) UURGENS 1985); Sex combs extra (Sce) (BREEN and DUNCAN 1986) ; Sex combs on midleg (Scm) UURGENS 1985); and super sex combs

' These two authors contributed equally to this work.

Genetics 138: 1151-1162 (December, 1994)

( sxc) (INGHAM 1984). It has been estimated that there may be up to 30-40 PC group genes in total (JURGENS

1985; LANDECKER et al . 1994). JURGENS (1985) showed that double homozygous com-

binations of Pcl, Asx, Psc and Scm had more severe ho- moeotic phenotypes than any single mutant, and proposed that the PC group genes might form a regulatory hierarchy. Similar results have been observed with Psc and Pcl, Scm, Sce and l(4) I02EFc (ADLER et al . 1989). Despite their similarities, phenotypes of PC group mutants are rather variable, and include segmentation defects (INGHAM 1984; BREEN and DUNCAN 1986; SINCWR et al . 1992), nervous system defects (SMOUSE et al. 1988; GIRTON andJEoN 1994), small disc phenotypes (PHILLIPS and SHEARN 1990), and modification of the interaction between zeste and white (WU et al. 1989). We are not aware of a systematic examination of phenotypes of PC group double mutants for the purpose of classifylng members of the group. STRUHL (1983) made the important observation that esc appears to function indepen- dently of PC, a suggestion supported recently by MOAZED and O'FARRELL (1992).

PARO and HOGNESS (1991) showed that PC shares se- quence similarity with Su(var)205, leading to sugges- tions that PC group genes might function analogously to modifiers of position-effect variegation in establishment or maintenance of chromatin structure (PARO 1990; GAUNT and SINGH 1990; REUTER et al. 1990), but there has

1152 N. N. Cheng et al.

been no direct evidence for this idea. Recently FAWARQUE and DURA (1993) showed that variegation of transposons carrying the white mini-gene and p h regulatory sequences was dependent on p h and PC, which supports the suggestion that PC group genes regulate chromatin structure, but KASSIS (1994) interprets these data differently. As an alternative model, BIENZ (1992) has argued that PC group genes might form silencing complexes, and that chromatin changes are a secondary consequence of silencing.

LOCKE et al. (1988) proposed that the Polycomb group genes might form a multimeric complex that obeyed the law of mass action. This was based on observations by KENNISON and RUSSELL (1987) who showed that duplications that include the Pcl locus suppress the extra sex comb phenotype of PC, as well as on observations that many of the genes exhibit haplo-abnormal homeotic phenotypes. Support for the idea that PC group genes might form a complex has come from observations that PC and p h bind to identical sites on polytene chromosomes and coimmunoprecipitate (FRANKE et al. 1992), and that Psc binding sites on polytene chromosomes also substantially overlap those of PC and p h (MARTIN and ADLER 1993; RASTELLI et al . 1993). If p h is a member of a complex that obeys the laws of mass action as proposed by LOCKE et al. (1988), increased dosage of p h should enhance complex formation, and thus suppress homeotic transformations exhibited by other PC group genes.

The observation that p h protein forms part of a multimeric complex that includes PC protein suggests that PC group genes that are also members of the complex might be especially sensitive to changes in p h dosage. Therefore, we crossed various p h alleles to PC group mutants, and looked for synergistic effects in double heterozygous flies. One problem arises from the fact that all PC group genes tested have maternal effects (STRUHL 1981; HAYNIE 1983; LAWRENCE et al. 1983; INGHAM 1984; BREEN and DUNCAN 1986; DURA et al . 1988; JONES and GELBART 1990; MARTIN and ADLER 1993). Therefore the absence of a detectable interaction between p h and any other PC group mutation does not necessarily mean that these genes do not interact, as it is always possible that if the amount of one or other protein was further reduced, an interaction would be detected. The maternal contribution also makes it difficult to look for epistatic interactions.

Duplications that include the p h locus suppress homeotic phenotypes of PC and Pcl, suggesting that p h is a member of a complex at equilibrium. We observe extragenic noncomplementation in females with Ph5", and in males with ph409 with some but not all members of the PC group, and with Su(z)2. We show that a temperature-sensitive allele o f p h is lethal in males when doubly heterozygous for some, but not all PC group mutations. p h is also lethal with Su(z )2 (ADLER et al. 1989;

TABLE 1

List of mutants

Allele Type Reference

Asx' Df(2R)trix

E(z) E(z) E(Pc)

E ( 4 '

Df(22R)en-A esc es c l(4) 1 02EFc2

5

PC4

P C I h P C P

Ph DfLZR)Pcl-W5

pscArP.'

Df(2R)vg-B

See ' Scm"' S U ( Z ) 3 0 2

S U ( Z ) 2 ' S U ( Z ) 2 1 . b X

~ ~ ( ~ ) 2 ~ sxc sxc

3 4

Gain of function Deletion of Asx Gain of function Null Antimorph Unknown Deletion of E(Pc) Null Null Unknown Probable null

Null Probable null Null Temperature-

sensitive Hypomorph Hypomorph Null Complex Gain of function

Null Deletion of Psc

and Su(2)P Unknown Null Gain of function

allele of Scm Gain of function Deletion of Psc,

Gain of function Null Null

su (2) 2, su (2) 3

SINCWR et al. (1992) LINDSLEY and ZlMM (1992) Wu et al. (1989) PHILLIPS and SHFARN (1990) Wu et al. (1989) LINDSLEY and ZIMM (1992) LINDSIXY and ZIMM (1992) STRUHL (1981) STRUHI. (1981) LINDSLEY and Z l M M (1992) KENNISON and RUSSELI.

LINDSLEY and ZIMM (1992) SATO et al. (1984) SATO et al. (1983) DURA et al. (1985)

DURA et al. (1987) DURA et al. (1987) DURA et al. (1987) ADLER et al. (1989) C.-T. Wu (personal

communication) BRUNK et al. (1991a) LASKO and PARDUE

BREEN and DUNCAN (1986) BREEN and DUNCAN (1986) WU et al. (1989)

BRUNK et nl. (1991a) BRUNK et al. (1991a)

Wu et al. (1989) INCHAM (1984) INCHAM (1984)

(1987)

(1988)

BRUNK et al . 1991a,b) and Enhancer ofPolycomb (E(Pc)) (SATO et al. 1984), genes that are not themselves homeotic. The phenotypes of the lethal embryos suggest that p h has at least three other functions besides regulation of homeotic genes.

MATERIALS AND METHODS

Fly strains and culture: Flies were grown on standard cornmeal-sucrose medium, with Tegosept added as a mold inhibitor. Except where indicated, stocks were maintained at 22". Crosses were performed at 25" and 29" where indicated in the text. Descriptions of mutations can be found in LINDSEY and ZIMM (1992) or as indicated below. The strains used in this study are listed in Table 1. Where possible we tried to use a null allele or a deficiency, and gain-of-function alleles if they were available and this information is also listed in Table 1. In addition, we used a strain containing a recombinant chromosome synthesized by JURGENS (1985) that is mutant for Psc (Df(2R)ug-D), Asx (Asx ' ) , and Pcl (Pc17), termed Psc- Asx- Pcl- hereafter, and a strain containing both the w+Y chromosome that is duplicated for the ph+ locus, and an attached X chromosome ( C ( 1 ) R M ) .

Crosses and scoring: To assess the effects of the w+ Yon the extra sex combs phenotype of PC group mutations, C ( l ) R M / w+Y females were crossed to PC group mutant males in un- crowded bottles, and the number of legs with extra sex combs

ph-PC Group Interactions 1153

was determined for a sample of male flies of the appropriate genotype. All the flies in a bottle were allowed to eclose, and the sample was picked at random. We did not score the number of uneclosed flies, but by not scoring these, we should bias the result toward making it more difficult to see repression, since the more mutant flies are the most likely to fail to eclose. The experimental values were compared to control values obtained from crossing PC group mutant males to five different strains, including awild-type strain, and four strains containing a compound Xand miscellaneous duplications, and determining the average number of legs with sex combs. We chose the value obtained from the cross giving the fewest extra sex combs for comparison to the experimental value, which biases the result against detection of an effect of the duplication to re- duce the number of sex combs. The control values for Pel, PC and Psc-, Asx- Pcl-, respectively, were 2.23,2.26,2.36,2.4,2.5; 2.22, 2.28, 2.39, 2.44, 2.5; and 5.0, 5.06, 5.34, 5.5 and 5.77. To confirm that the effects of suppression by the w+Y chromosome resulted from duplications of the p h locus and not some other gene containedwithin the du li~ation,ph~~j/FM7~; L Bc Pe l fo /+ females were crossed toph /w+Ymales. The average number of legs with sex combs ofph503/w+ Y; L BcPcl"/ + flies were compared to those of + / Y; L Bc Pclfo/ + flies. For all the above crosses, a log-likelihood test was used to compare the distributions of flies containing 2 ,3 ,4 ,5 or 6 sex combs to determine if the distributions were significantly different at P = 0.05.

In other crosses, phSo3/FM7 females were crossed to PC group males, and the number of viable females of each genotype were compared. The viability of the experimental genotype was calculated relative to the viability of the average of the control genotypes. If the viability was less than 3% of the control values, the cross was scored as lethal. Finally, homozygous phz or ph409 females were crossed to PC group mutant males, and the number of ph/Y; PcC/+ flies were compared to the number of ph/Y; Balancer/ + flies. For comparison, the number of females of each genotype were also counted, as PC group mutant males are often less viable than females. Other crosses are described in the text.

Cuticle preparations: Cuticles were prepared as described previously (SINCWR et al . 1992). All crosses with ph2 were performed at 29" to maximize the likelihood of recovering double-mutant embryos. Generally, ph2 females were crossed to mutant males. The exceptions are described in the text.

P,

RESULTS

Effects of p h dosage changes on PC group homeotic phenotypes: LOCKE et al. (1988) proposed that if proteins associate in a complex that is at equilibrium, then dosage changes in any member of the complex should obey the laws of mass action. A decrease in dosage of a constituent should decrease the amount of complex formed, and enhance the phenotype caused by loss of the complex, whereas an increase in the dosage should favour complex formation, and suppress the phenotype caused by loss of the complex. Because we have suggested that p h is part of a multimeric complex that con- tains PC, and perhaps other members of the PC group ( F M E et al. 1992), we examined the effects of dosage changes of the p h f locus on the extra sex combs phenotype of mutations in other PC group genes. We used the w+Y chromosome which is duplicated for the 2D1-2 to 3D34 region of the X chromosome that includes the

TABLE 2

Suppression of extra sex comb phenotypes by w+Y

Average no. of legs Genotype n with sex combs

PC'6/+ 100 2.22 + / w+ u; P C P / i 100 2.0 PCt2 / + 100 2.23 4- / W+ r; PCt2 / 4- 100 2.0

Df(2R)vg-D Am' Pc17/+ 100 5.0 i / w + Y ; Df(2R)vg-D A m 1 P e l 7 / + 100 2.6 ph503/w'r; L Bc P c l ' o / i 65 2.52 + / w + Y ; L Bc Pcl'O/+ 97 2.0

p h locus. It has been shown previously that the p h gene carried on the w+ Y chromosome is dosagecompensated (DURA et al. 1988), so +/ w+ Y males have twice as much product as wild-type males. Initially, C(l)RM/w'Y females were crossed to P c ' ~ , Pcl", and Psc- Asx- Pel- triple mutant males (Table l), and the average number of legs with extra sex combs in the w+ Y; PC group/ + class was determined. Because all males carry the p h duplication, there are no internal controls. Therefore, control values were obtained by crossing the PC group mutant strains to five different strains, including a wild-type strains and four strains carrying a compound X , and determining the number of legs with sex combs for the heterozygous PC group mutants in each of these five strains. The control cross with lowest number of legs with extra sex combs was used as the control value for each PC group mutation listed in Table 2. It can be seen that the w+ Y chromosome strongly suppresses the extra sex comb phenotype of the Psc- Asx- Pcl- triple mutant, and completely suppresses that of Pcl and PC mutants. To control for the possibility that some other locus in the duplication was responsible for the suppression of the extra sex combs phenotype, we examined the effect of the wf Y duplication in a background with and without a p h null mutation, ph503. Accordingly, ph5O3/+; L Bc Pcl''/ + females were crossed to ph503/w+ Y males. As seen in Table 1, males that are euploid for p h have extra sex combs, whereas males with a ph+ duplication do not, demonstrating that the suppression of the extra sex combs phenotype of Pcl by the w+ Y chromosome is specific to the p h locus. These data support the conclusion that ph is at equilibrium with a multimeric complex containing PC group genes. Other PC group mutants express the extra sex combs phenotype at such a low frequency that they could not be reliably tested for suppression by w+ Y.

Tests for extragenic noncomplementation with p h alleles: In light of biochemical evidence that p h protein is found in a multimeric complex containing PC, and the genetic evidence above that p h duplications suppress the extra sex comb phenotypes of some PC group genes, it seems reasonable to predict that PC group proteins that are members of such a complex might be especially sensitive to mutations in ph. As reported by DURA et al.

1154 N. N. Cheng et nl.

TABLE 3

Lethal interactions of ph503

Progeny

Cross ph503/+; PcG/+ FM 7/ + ; PcG/ + phSo3/+; Bal/+ a FM7c/+; Bal/+

ph503/FM7c X Pc4/TM6 Sb 0 284 301 ph5"/FM7c X Pc'6jTM3 0 216 207

253 204

ph503/FM7c X Psc /Cy0 4 164 231 209 ph503/FM7c X Su(z)2'/CyO 0 81 87 97

a Bal = Balancer chromosome.

TABLE 4

Lethal interactions of ph409

Male progeny' Female progeny" Total Relative

Mutant ph409/Y; Peg/+ ph409/+; Bal/+ ph4OY/+; PcG/+ ph409/+; Bal/+ progeny viability

Asx' 25 163 200 197 585 0.15 Df(2R)trix 44 172 290 298 804 0.26

Df(2F)en-A 80 291 398 454 1223 0.27 E(Pc) 181 404 468 504 1581 0.45

E(4 4 163 512 547 1226 0.02

E ( 6 ;* 268 100 412 318 1098 2.68

W z ) 112 103 217 224 656 1.09 esc 48 33 7 2 79 232 1.45 l (4 ) I02Efc2 154 109 565 369 1197 1.41 PC 2 210 365 326 903 0.01

50 188 548 301 1087 0.27 Df(fR)Pcl-W5 54 188 532 276 1050 0.29 Psc 0 284 289 378 951 0 Pscp22 52 288 505 475 1320 0.18

pseA*P. 1 233 205 321 31 1 1070 1.14

Scrn"' 9 127 485 504 1125 0.07

su ( 2 ) 2 78 279 429 370 1156 0.28 215 8 358 274 855 26.8

sxc 212 17 29 1 21 1 731 12.5

PC16 3 173 539 509 1224 0.02

p s e l . d 2 0 89 316 332 421 1158 0.28

Df(;2R)vg-B 167 203 321 330 1021 0.82

su (z) 372 2 208 561 499 1270 0.01

See 1 229 373 315 918 0.004

sxc 3

' Progeny from crosses of ph409/ph409; +/+ X + / X PcG/Balancer. Calculated as: number of ph409/Y; PcG/+ divided by number of ph409/Y; Bal/+.

(1985), ph2/Y; PC?/+ males die as pharate adults. Therefore we crossed various p h alleles to strains carrying mutations in all known PC group genes, and to E(Pc) and Su(z)2. E(Pc) mutants do not have a homeotic phenotype in embryos or adults, but are strong enhancers of PC, Pcl and 1(4)102EFc (SATO et al. 1984). The phenotype of embryos derived from mothers with germlines homozygous for E(Pc) has not been determined, so it is not yet clear if E(Pc) is a member of the PC group. Su(z) 2 is a homolog of Psc (BRUNK et al. 1991b), but mutations at this locus do not have homeotic phenotypes or enhance the homeotic phenotypes of PC group mutants (ADLER et al. 1989).

ph null mutations are lethal (DURA et al. 1987). There- fore we initially crossed ph503/+ females to males heterozygous for the eleven known PC group mutations and to Su(z) 2 and scored for lethality in double heterozygous females, compared to their sibling controls. As

shown in Table 3, no ph503/+; Pc4/+, ph503/+; Pc16/+ or ph503/+; Su(z)2'/ + females were recovered, and fewer than 3% of the expected number of ph503/+; Psc'/ + females were recovered, showing extragenic noncomplementation for these loci. However, Df(2R) trix, E(Pc), es2, l(4) 1 02EFc2, P C ~ ' ~ , Psd'P.', Df(2R)vg-B, Sce', ScmD' and sxc? were all viable when heterozygous with ph503 (data not shown). It is noteworthy that Df(BR)vg-B, which deletes both Psc and Su(z)2, is viable when heterozygous with ph503, suggesting that the lethal interactions observed with Psc' and Su(z)2' are gain of function interactions. Few progeny were recovered when ph503/w+Y males were crossed to E ( z ) ~ females, so viability could not be reliably assayed.

Strong hypomorphic mutations of p h are homo- and hemizygous viable, and are thought to arise when only one of the two homologous transcription units that com- prise the p h locus are mutant, thus reducing the dosage

ph-Pc Group Interactions 1155

TABLE 5

Lethal interactions of ph* at 25" ~ ~

~ ~ ~ ~~ ~

~ ~

Male progeny' Female progeny" ~~

Total Relative Mutant p h '/ Y; PcG/ + p h '/ Y; Bal/ + p h 2 / + ; P c G / + p h ' / + ; Bal /+ progeny viability

Asx' 86 206 221 238 75 1 0.42 Df(2R)trix 74 170 197 121 562 0.44

Df(2F)en-A 74 134 159 191 568 0.55

E(Pc) 95 169 182 180 626 0.57

E(z) 57 117 130 143 447 0.49

E(Z);O 312 164 340 99 915 1.90

- W j 103 102 120 103 428 127

1 .o esc 144 180 163 614 l (4) 1 OZEfc' 93 51 112 105 361

0.91

PC 0 444 567 517 1528 0 1 .80

PC'6 0 165 204 148 517 Pc1"

0 43 144 294 192 673

Df(fR)Pcl-WS 25 176 268 255 724 0.15 0.30

Psc 0 186 193 208 587 0.01 PSce'2 p s ~ l . " o

2 80 154 123 1

359 0.03

pscA7P. ' 228 1 49 275 206 858 144

1.58 Df(fR)vg-B 128 212 160 644 1.12 Sce 1 168 258 230 65 7 Scm"' 135 268 35 1 225 979

0.01

Su(z)3?2 62 207 194 21 1 674 0.50 0.30

su ( z ) 2 0 423 258 475 1156 Su(z)Z5 99 176 200 723 0.56

0

su(r)21.bR

s x c 4 73 223 94 559 2.20

250 239 264 754 0

248

sxc 96 52 119 84

' Progeny from crosses: ph2 /ph2 . +/+ X +/E PcG/Balancer.

163

160 3

244 164 286 857 0.67 351 1.90

Relative viability: viability of ph'/Y, PcG/+ divided by viability of p h 2 / Y; Balancer/+.

of p h protein (DURA et al. 1987). ph409 is an inversion that breaks in the proximal transcription unit (DURA et al. 1987). We have shown using northern analysis that only transcripts from the distal transcription unit are produced in ph409 individuals (NEEL RANDSHOLT, SALLY FREEMAN and H. W. BROCK, unpublished data). Thus ph409 individuals have wild-type protein from the distal transcription unit only, compared to ph503/+ females which have a mixture of proteins synthesized from the proximal and distal transcription units. Without know- ing the relative transcription and translation rates of the proximal and distal transcription units, the absolute amount of wild-type ph protein cannot be estimated in ph409 homozygotes relative to ph503 heterozygotes. We crossed ph409 females to males mutant for different alleles of the known PC group genes, and to E(Pc) and Su(z)2. The results are shown in Table 4. Loss of function mutations of PC are inviable when heterozygous in Ph4O9 males, as are gain-of-function mutations of E(z) ' , Psc (Psc') and Scm (Su(z)302) . Sce' is also inviable in ph409 males, but the nature of Sce' is unknown as there are no deficiencies for the locus (BREEN and DUNCAN 1986).

Finally, we crossed ph2 females to PC group mutant males, and scored for viability ofph'/Y; Pcgroup/+ flies compared to their sibling controls. Because ph2 is a temperature sensitive mutation (DURA et al. 1985) the crosses were performed at 25" and 29". Molecular analy-

sis shows that the ph' mutation is a deletion that removes most of the distal and part of the proximal transcription unit. Consequently, all of the ph protein in ph2 individuals is mutant, and the amount of protein is reduced in ph' flies relative to wild-type flies. The results are shown in Tables 5 and 6. Combining the results from both temperatures, mutations in Asx, PC, Pcl, Psc, Sce and Scm are lethal when heterozygous with ph'. Strik- ingly, E(%)', which is lethal when heterozygous with ph409 is viable in combination with ph', suggesting that the interaction between ph409 and E(z)' is allele-specific. It is also noteworthy that only gain of function mutations of Psc (Psc', PsceZ2) are lethal when heterozygous with ph'. The E(Pc) and Su(z)2' mutations are also lethal in combination with ph'. In contrast, the Pcgroup mutations, esc, E(z), 1(4)102EFc and sxc were all viable with ph2.

p h is a duplicated locus, and both transcription units are required for wild-type phenotypes. We have proposed a dosage model to account for hypomorphic and lethal phenotypes observed with various combinations of mutations that affect either the proximal or distal transcription unit (DURA et al. 1987). We tested the ability of a transformed line containing a copy of the distal transcription unit, generously supplied by NEEL RANDSHOLT, to rescue the lethal interaction between ph' and Psc'. Ph'/Y; +/+; P[phdisSL'']/ + males were crossed to C( l )RM/Y; Psc'/Cyo; +/+ females at 25" and 29" and the viability of Ph'/Y males was scored. Because the re-


TABLE 6

Lethal interactions of ph2 at 29"

Male progeny' Female progeny'

p h 2 / Y ; PcG/+ Total Relative

Mutant ph'/Y; B a l / + p h 2 / + ; PcG/+ p h 2 / + ; B a l / + progeny viability

Ass' 0 53 152 128 Df(2R) trix 0 65

Df(2P)en-A

333 0 162 103 330

0 82 212 211 0

506 1 61 154 162 377

0 0.02

14 17 131 23 185 0.82 21 5 29 10 65 15 26 134 51 226 0.58

4.2

es c 76 81 117 99 373 l (4) 1 02EFc2 3

0.94 11

0 102 218

3 0.27

Df(2R)Pcl-W5 101

0 362 0

3 117 59 179 0 0 0

1.34

74 167 121 363 0.01 0.1

0

E(Pc)

E ( 4 E ( 4 E (z]

PC112

P S P PSCA'P.'

ScmD' Su(z)302 0 22 125 130 277 su(z)21.b8 sxc3 56 0 185 78 318 Viable s x c 4 90 4 142 54 290 22.5

102 258

75 136 146 357 117 87 167 148 519

299 Df(2R)vg-B 6 60 119 114 1

28 129 137 194 488 0.2

' Progeny from crosses: p h 2 / p h 2 . +/+ X + / E PcG/Balancer. Relative viability: viability of p h 1 / Y; PcG/+ divided by viability of ph2 /Y; Balancer/+.

sults were equivalent at both temperatures, the data were pooled. We recovered 151 ph2/Y; Psc ' l /+; P[phd'stal]/+, no ph2/Y; Psc'/+; +/+; 154 ph2/Y; CyO/+; P[phdista'] /+; and 93 ph2/Y; CyO/+; +/+ males. These results show that the products of the distal transcription unit are sufficient to rescue the lethal interaction between ph' and Psc', and support the idea that the products of the proximal and distal transcription units are similar. Moreover, combined with the observation that ph2/+; Psc ' /+ females are viable, the observations above show that the interaction observed above is unlikely to be due to antimorphy of ph .

KENNISON and TAMKUN (1988) identified mutations in many trithorax ( t r x ) group genes that suppressed the extra sex comb phenotype of PC. We asked if alleles of brahma (brm'), moira (mor'), osa2 or tr8' could suppress the lethal interaction between ph' and Psc'. ph2/Y; trx group/+ males were crossed to C ( l ) R M / Y ; Psc'/ C y 0 females, and the number of ph2 /Y males of each genotype was scored. None of the four mutations res- cued the lethality of the ph2-Psc' interaction (data not shown), demonstrating that trx group mutations cannot suppress all PC group phenotypes.

Analysis of embryonic cuticle phenotypes: The interactions between p h alleles and a subset of PC group mutants could be explained if non-interacting genes have more product, either through maternal contribution or greater synthesis or stability of the mRNA or protein, or if these genes are less dosage sensitive. This possibility is suggested by observations that some of the ph2/Pc group mutants had reduced viability compared to control genotypes. We predicted that if this dosage argument was true, further reductions in the dosage of PC group

genes would allow us to detect synergistic interactions between p h and the remaining PC group genes. Sec- ondly, we predicted that if synergistic interactions between p h and other mutants reflected the role of the mutants in a common process or complex, then the phenotypes of the lethal embryos should be similar. On the other hand, if different PC group genes, or E(Pc) and Su(z)2 have different functions, then different embryonic phenotypes might be observed in double mutants. Accordingly, we examined the effects of further reductions in dosage of PC group genes that were viable with ph alleles, and then compared the embryonic phenotypes of all lethal interactions detected.

Four of the genes tested, esc, E(%), l(4) 102EFc and sxc were viable or semi-viable in combination with ph2. ph2 males heterozygous for E(%), l(4) 102EFc and sxc mutations show modest to strong enhancement of the extra sex combs phenotype (data not shown), consistent with the idea that these genes interact with ph', but that the amount of PC group protein needs to be further reduced to detect lethal interactions. Therefore, we examined E(%), 1(4)102EFc and sxc homozygotes that were also ph2/Y. E(%) UONES and GELBART 1990) and 1(4)102EFc (HOCHMAN et al. 1964) homozygotes die as late larva or early pupae, and sxc homozygotes die as pharate adults (INGHAM 1984), so we reasoned that if ph' enhances the phenotypes of any of these genes, they might exhibit embryonic lethality.

ph2/+; PC group/+ females were crossed to PC group/+ males at 29", and their progeny were examined for embryos that failed to hatch. In the S X ~ crosses, about 25% of the embryos failed to hatch, and two classes of dead embryos were observed. Embryos of the


FIGURE 1 .--Embryonic phenotypes of homozygous PC group mutant.. hemizygous for ph’. All embryos have anterior up, and are viewed under phase contrast illumination. The embryo shown in has not been removed from the vitelline membrane. (A) Presumed ph’ /+; sxc3 embryo. It is essentially wild type, but shortened. (R) Presumed p h 2 / Y ; sxc3 embryo. Note the complete absence of head. The abdominal denticle belts are compressed, but do not show evidence of posterior transformation. (C) Putative ph’/Y; 1(4) I02LFc embryo. Loss of anterior structures including most of the thorax; some abdominal segments are also missing. Note that the sixth abdominal denticle belt appears to be absent. (D) A putative Ph’/Y; E ( z ) ~ embryo showing partial loss of head. The U-shaped embryo has not undergone gehhand retraction.

first class (Figure 1A) looked wild type, but were shorter than usual, and might represent P h 2 / + ; sxc3/sxc3 embryos. The second class, that were less abundant and that are probably ph2/Y; sxc3/sxc3 embryos, were shortened, did not exhibit homeotic transformations in thoracic and abdominal segments, and entirely lacked the head (see Table 7). The anterior cuticle and mouth parts a p pear to be absent (Figure 1B). Similar results were o h tained with l (4 ) 1021:’I;c, except that most embryos hatched, suggesting that the dead embryos represent the extreme phenotypes. Most of the dead embryos were essentially normal, but some lacked all anterior structures and cuticle (Table 7). These embryos were more severely affected than the sxc embryos, as they usually lacked all thoracic and some anterior abdominal segments as well. It was difficult to score for homeotic transformations because the setal belts were usually distorted (Figure 1C). Over half of ph2/Y; I < ( z ) ~ / E ( z ) ~ embryos had head defects, and 45% of the abnormal embryos failed to complete germband retraction, and were thus U-shaped (Table 7). About 18% of embryos failed to hatch. Homeotic transformations of thoracic and a h domina1 segments were not observed, but this was com- plicated by the frequency of abnormal denticle patterns in the denticle belts.

esc product is required only early in development (STRUHL and BROM’ER 1982). To look for interactions between ph and e s ~ , P S ? / P S C ~ females were crossed to ph5”’/ w+ Y males, and embryos were scored for severity of homeotic transformations compared to those found in embryos derived from a control cross of esc2/~sc5 females

to wild-type males. No differences in severity of transformation were found (data not shown). esc was the only PC group gene for which we could not demonstrate an interaction with ph, but it is not possible to examine the double homozygote because homozygous p h null embryos lack ventral cuticle.

Next, we compared the phenotypes of embryos resulting from crosses between ph2 and all loci that failed to complement it as heterozgyotes. ph2 does not have an embryonic phenotype, nor do heterozygotes of any of the PC group or other genes tested. The lethal embryos were recovered and these are presumed to be phz/Y; mutan,t/+ embryos. In all crosses except with Pc1I2 and Psc’, less than 5-10% of the embryos failed to hatch, suggesting that not all ph2/Y; mutant/+ embryos die. Therefore the results shown represent the embryos with the most extreme phenotypes. The results are shown in Figure 2, and are described in Table 7. For PC, Pcl, Sce, Scm, Psc and Asx, the embryos were very similar, but not identical. All showed modest posterior transformations of abdominal segments six and seven, but no obvious posterior transformations were observed in thoracic segments (Figures 2, B-G). Sce showed the strongest homeotic transformations of abdominal segments. All crosses exhibited variable head defects ranging from essentially wild type, to failure of head involution, through to complete loss of the head. Generally, these phenotypes are similar to the transformations exhibited by homozygous Asx or Psc. In addition, at least some lethal embryos from most crosses exhibited segmentation defects similar to those observed in gap, pair rule or


TABLE 7

Phenotypes of php/y; PC group/+ embryos

Cross f l Headless Homeotic

ph2 X Asx' p h t X E(Pe)/+ p h /+; E$z)/+ X +/Y; E(z ) /+ ph2 X PC [+ p h z X Pel 2 / + ph2/+; 1(4)102EFe2/+ X

+ / E 1(4)102EFc2/+ ph2/X P s c ' / + ph2 X See%? ph2 X Scm /+ ph2 X Su(z )Z ' /+ ph2/+; s x c / + X +/E s x c / + I

18 44 74 31 83

14 91 16 25 10

.66

17 4

61 13 0

14 0 6

16 0

13

100 100

3 100 87

28 100 100 100

0 0

U-shaped

6 35 45 0 0

Gap defects

6 0 0 6 0

0 0 0 2

100 0

Segment defects

6 16 55

0 5

7 8

12 4 0 1

All data are expressed as percent, except the second column which gives the number of embryos scored for defects. In most crosses, most embryos hatched, so embryos that died represent the most extreme class of progeny of the given genotype. An embryo was scored as headless if the head was completely absent, and the interior of the embryo was completely exposed. Other head defects were scored as homeotic transformations. Gap phenotypes were scored if embryos were shortened and had fewer than the expected number of segments. Missing segments, fused segments, and segment polarity defects were lumped and scored as segmentation defects.

segment polarity mutants. Details are given in Table 7. The results with E(Pc) and Su(z)2 are quite different.

Embyros that are ph2/Y; E ( P c ) / + exhibit two phenotypes. About a third of the embryos failed to complete germ band retraction, and remain U-shaped, as shown in Figure 2H, reminiscent of the results seen with embryos that are homozygous for E(z) and hemizygous for ph2. All embryos show homeotic transformations similar to those seen with PC group mutations, and occasional embryos have missing or fused segments (Figure 21). On the other hand, the rare ph2/Y; Su(z)2' embryos that fail to hatch have a dramatic phenotype that resembles the phenotype of Kruppel mutants. The head, thorax and telson are normal, but most abdominal segments are missing, and the two that remain have mirror image symmetry. However, most of the embryos resulting from a cross of ph2 females with Su(z) 2' females resulted in eggs that appeared to be unfertilized or did not develop, and less than 5% of unhatched embryos exhibit the gap phenotype. Interestingly, about 5% of ph2/Y; Asx/ +; ph2/Y; Pc4 /+ and ph2/Y; Scm/ + embryos show similar Kruppel-like phenotypes (Table 7). It should be noted that null mutations of Su(z) 2, including Df(2R) ug-B and Su(~)2 ' . '~ are not lethal in ph2 males, although viability is greatly reduced. We examined dead embryos resulting from crossingph2/ph2; +/+ females to +/Y; Df(2R)vg- B/ + males, which are deleted for Psc and Su(z) 2. Some rare lethal embryos were recovered, and they typically had extreme head defects, and pair-rule defects (data not shown), suggesting that the gap gene phenotype seen in ph2/Y; Su(z)2'/+ embryos is a gain of function phenotype.

DISCUSSION

JURGENS (1985) showed that some PC group genes act synergistically as double homozygotes, and proposed that the PC group genes form an integrated system. He

suggested that loss of a single component destabilizes the system, and that loss of two components causes the system to break down. Under this hypothesis, it is not necessary that PC group genes interact directly, as they could have different roles affecting the same process or processes. A more specific hypothesis is that PC group genes might form a multimeric complex. The observation that ph and PC proteins co-immunoprecipitate as part of a multimeric complex containing about 10-15 proteins (FRANKE et al. 1992) provides biochemical evidence for the complex model. Locm et aL (1988) proposed that PC group genes were members of a multimeric complex at equilibrium that obeys the laws of mass action.

A prediction of the mass-action model (LOCKE et al. 1988) is that duplications of a PC group gene should favour complex formation, and thus suppress PC group mutations. KENNISON and RUSSELL (1987) showed that duplications from 55B to 55E that include the Pel locus suppress the extra sex comb phenotype of PC, consistent with the idea the PC group proteins are members of a complex. However, the effects of duplications of the 55B to 55E region were not examined in a Pel- background to confirm that the suppression was owing to dosage changes of Pel and not some other gene included in the duplications tested. Our observations show that duplications containing the ph+ locus do suppress the extra sex combs phenotypes of Psc, Pel and a Psc- Asx- Pel- chromosomes. For the latter chromosome, it is not known if all PC group loci on it are partially suppressed by the ph duplication, or if only a subset of the loci are suppressed. At least for suppression of Pel, we show that suppression of the extra sex combs phenotype is specific to the p h locus, and by extension, this is likely to be true for suppression of PC and Pse- Asx- Pet . According to the mass-action model, only members of a complex should be suppressed by duplications for a gene encoding one of its constituents. Therefore these data suggest that ph, PC and

ph-Pc Group Interactions 1159

FKUKE 2.-Phenotype of embryos hemizygous for ph’ and heterozygous for other mutations. All embryos are mounted with anterior up, and are viewed under dark- field illumination. (A) Wild-type (Canton S) embryo. (B) p h 2 / Y ; PC“/+ embryo. Abdominal segments six and seven have more rec- tangular denticle belts indicating a posterior transformation, and mouth hooks are splayed laterally. (C) ph’/Y; Pcl” /+ embryo. Mild posterior transformation of the seventh abdominal denticle belts is seen, together with a reduction and fusing in the cephalopharyn- geal apparatus. (D) ph2/Y; Sce’/+ embryo. Clear homeotic transformation of abdominal segments five through seven are seen, together with severe loss of ce halopharyn- geal skeleton. (E) p h P . / Y , Scrn”’/+ embryo with phenotype very similar to that observed in B. (F) ph’/Y; Psc’/+ embryo resembling that seen in B. ( G ) p h 2 / Y ; A d / + embryo with very modest transformation of the seventh abdominal segment, but exhibiting nearly complete loss of cephalopharyn- geal skeleton. ( H ) p h - / Y ; I ; (Pc) /+ embryo showing failure to complete germband retraction and loss of mouthparts. ( I ) Same as H, to show homeotic transformation of sixth and seventh abdominal segments. u) ph’/Y; Su(z)P’/+ embryo. This phenotype was fully pen- etrant. The head, telson and thorax appear essentially normal, but there has been loss of abdominal segments coupled with mirror image symmetry of fused abdominal segments.

Prlare members of a complex at equilibrium, and are consistent with the idea that Am and A c might also be members of this complex. We favor the view that the molecular observations (FKIZNKE at d. 1992) and the dosage sensitivity of ph provide strong support for the idea that at least some Pr group gene products form a multimeric complex at equilibrium.

Nevertheless, not all genes whose products form complexes show the dosage sensitivity exhibited by ph and

Prl, or by modifiers of positioneffect variegation (LOCKE P t 0.1. 1988). For example, the S W I I , SW12, SW13, SNFS and SNF6 genes are believed to be members of a complex, but null mutations at any one of the S W I I , SW12 or SW3 loci have the same phenotype as a triple SWI mutant (PETERSON and HERSKOWITZ 1992). Similarly, with one exception flies doubly heterozygous for mutations in the Minula loci, which probably encode ribosomal proteins ( KONGSUWAN et nl. 1985), have phenotypes that

1160 N. N. Cheng et nl.

are no more severe than either single heterozygote alone (see discussion under Minute in LINDSLEY and ZIMM 1992). Furthermore, there is little evidence for general suppression of PC group phenotypes by duplications of PC group genes. KENNISON and RUSSELL (1987) showed that duplications of PC did not suppress the extra sex combs phenotype of Pel. Moreover, no other duplication that included other PC group genes suppressed the extra sex combs phenotypes of Pcl or PC, although the authors point out that weak suppressors would not be detected in their screen.

As an alternative to the complex hypothesis, suppres sion of extra sex comb phenotypes by three doses of ph' could be explained if the additional ph protein can sim- ply substitute for the absence of another PC group protein if it is assumed that ph and the other PC group protein have similar functions. Given the differences in the molecular structure between p h (DECAMILLIS et al. 1992) and PC (PARO and HOGNESS 1991), this explanation is less convincing. Because regions containing PC group loci are recognized by antibodies to ph (DECAMILLIS et al. 1992) it is also possible that PC group expression levels might be altered by changing the dose of ph. This possibility has not been tested.

Extragenic non-complementation can occur if gene products interact directly, as has been shown for tubu- lins (STEARNS and BOTSTEIN 1988; HAYS et al. 1989) and for proteins that interact with actin (WELCH et al. 1993). The failure to complement might arise from poison complexes or result from dosage effects (STEARNS and BOTSTEIN 1988; FULLER et al. 1989; and see discussion in VINH et al. 1993). Scoring for lethality in ph503/+; mutant/ + females clearly detects extragenic noncomplementation of PC, Psc and Su(z)Z. Null alleles of PC fail to complement ph503, which is itself is a null mutant, and this is consistent with a dosage interaction. However, only the gain of function mutations Psc' and Su(z)2' fail to complement ph503 and ph2, which is more consistent with poison complex model.

The results with ph4"9 provide further evidence for extragenic non-complementation. The results shown above show that ph409/Y males are not equivalent to phsn3/+ females because E(%)', Sce' and Su(z)302 fail to complement ph4OY/Y males, but do complement ph5"'/+ females. This could be because the proximal and distal transcription units produce functionally different proteins, or that they are transcribed in different amounts, or both. It is interesting that ph4"', which is a deletion removing the distal transcription unit of ph (DURA et al. 1987) is viable with PC, Psc' and Su(z)Z' (D. A. R. SINCWR, unpublished results), confirming that there is a difference in amount or quality of proximal and distal products of the ph locus. In view of these results, it is interesting that a transformant containing the distal ph transcription unit rescues the lethal interaction between ph2 and Psc', supporting the idea that there is

more likely to be a quantitative than a qualitative difference in the products of the proximal and distal transcription units.

In yeast, extragenic noncomplementation has been shown to arise from interaction of proteins encoded by tubulin genes (STEARNS and BOTSTEIN 1988) and between proteins that interact with actin (WELCH et al. 1993). It is tempting to interpret all examples of extragenic noncomplementation as evidence for protein-protein interactions. Apart from interactions of tubulin proteins (HAYS et al. 1989), extragenic noncomplementation has been observed among genes encoding myofibrillar proteins (HOME and EMERSON 1988), at a locus in the 37DF region (GAYand CONTAMINE 1993), and between haywire and tubulin mutants (REGAN and FULLER 1988). It is probable that the myofibrillar proteins that show lethal interactions are constituents of a multimeric protein complex. The haplo-abnormality of the loci, their sensitivity to dosage changes in the gene encoding the myo- sin heavy chain, and the complexity of the interactions are very reminiscent of the interactions reported above for the PC group genes. The molecular basis for interaction with the locus at the 37DF region is not known. Finally, the interaction of haywire, which encodes the fly homolog of a human excision repair gene, and a testis specific tubulin gene is unlikely to be explained on the basis of direct protein interactions (see discussion in MOUNKES et al. 1992). The latter example is instructive because mutations in haywire affect very general processes, and interactions may arise from indirect effects, a possibility that may also be true for genes that have pleiotropic effects like the PC group. Therefore, direct evidence is required before extragenic noncomplementation can be used as a criterion for showing that gene products interact.

There is suggestive evidence that two of the genes that show extragenic noncomplementation with p h are members of a complex. The failure of PC null alleles to complement ph503 is consistent with the evidence that the antibody-binding sites of ph and PC on polytene chromosomes overlap completely, and p h and PC products co-immuneprecipitate as part of a multimeric complex (FRANKE et al. 1992). The two other genes that fail to complement ph5"', Psc and Su(z)Z also have antibody- binding sites on polytene chromosomes that largely overlap with those of p h (MARTIN and ADLER 1993; RASTELLI et al. 1993), and are strong candidates to interact with p h protein.

ph2 is a temperature-sensitive mutation associated with a deletion that includes part of the proximal and distal p h transcription units (DURA et al. 1987), and thus all protein is mutant in ph2 males. But it is not clear if the interactions between ph2 and other PC group genes at restrictive temperatures arises from the decrease in the amount of functional product, or because the product is abnormal, or both. Therefore, we do not know if


the synergistic interactions we observe result from extragenic noncomplementation, or from poisoning effects of abnormal ph2 protein. Nevertheless, it is striking that, with the exception of esc, we were able to demonstrate enhancement of lethal phenotypes in all ph2; PcG double mutants. Our data suggest that a screen for extragenic noncomplementing mutations of ph2 might be a powerful way to identify other dominant members of the PC group. We predicted that if all p h interactions with other mutants upset the same process, then the lethal phenotypes should be similar. This was true for many of the interactions as the lethal phenotypes of ph2 and Asx, PC, Pel, Psc, Sce and Scm embryos were very similar.

Different results were obtained with the other mutants tested. E @ ) , l(4) 102Efc and sxc homozygous embryos that were also hemizygous for ph2 showed missing anterior structures, including the head and sometimes the thorax, but did not show clear homeotic phenotypes. This result suggests that the process(es) required for head formation in embryos is more sensitive to loss o f p h and these three genes than is regulation of homeotic genes. These data point to a striking difference between the relative sensitivities of the process(es) that require these three genes and the other PC group genes. There- fore we argue that ph, E(z), l (4) 102EFc and sxc partici- pate in a second process that is upset by mutations in only a subset of PC group genes. This view does not rule out participation of these three genes in regulation of homeotic loci, because as noted above, heterozygotes for each of these three genes show enhanced expression of extra sex combs in ph2 hemizygotes. In addition, ph2/Y; E(z) and ph2/Y; E(Pc) embryos often failed to complete germband retraction, a phenotype peculiar to these double mutant combinations, showing that there is a third process that requires only a subset of genes that interact with ph.

The phenotype of ph2; Su(z )2 /+ embryos strongly resembles those of Kmppel mutant embryos and r e p resents a fourth phenotype seen in p h double mutant embryos. This result strongly suggests a specific role for Su(z)2 in the regulation of gap genes. As noted above, occasional ph; PC group mutant/+ embryos also show a very similar phenotype, suggesting that other PC group genes may also be required for the regulation of gap genes. The recent results of PELECRI and LEHMAN (1994) showing that some alleles of E(z), the Su(z)2 complex, and some alleles of 1(2)102EFc are required for regulation of other gap genes by the maternal hunchback protein are consistent with the observations reported above. They suggest that definitive evidence about the role of other PC group genes in the regulation of gap genes awaits testing of embryos lacking maternal and zygotic PC group products. We suggest that we detected rare embryos exhibiting phenotypes resembling those caused by gap genes because we examined double mutant embryos.

We thank PAUL ADLER, BRUCE BAKER, IAN DUNCAN, PHIL INGHAM, RICK

JONES, JIM KENNISON, GARY STRUHL and TING Wu for stocks. In particular we thank NEEI. RANDSHOLT for her generous gift of the p h distal transformant. This work was supported by grants from the Medical Research Council of Canada, the Natural Sciences and Engineering Research Council of Canada, and the National Cancer Institute of Canada to H.W.B.

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Communicating editor: R. E. DENELL

of polyhomeotic with polycomb group genes of drosophila ... · copright 0 1994 by the genetics...

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