And Abdominal-B in Drosqbhilu Melanogaster

Home , B chromosome, Transheterozygote, Transvection (genetics)

and Trans Interactions Between the iab Regulatory Regions and abdominal-A and Abdominal-B in Drosqbhilu melanogaster

Jd Eileen Hendrickson and Shigeru Sakonju

Department of Human Genetics, Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah 841 12 Manuscript received August 15, 1994 Accepted for publication November 8, 1994

ABSTRACT The infra-abdominal ( iab) elements in the bithorax complex of Drosophilamelanogaster regulate the transcription of the homeotic genesabdominal-A ( abd-A) and Abdominal-B ( Abd-B) in cis. Here we describe two unusual aspects of regulation by the iab elements, revealed by an analysis of an unexpected complementation between mutations in the Abd-B transcription unit and these regulatory regions. First,we find that iab-6 and iab7 can regulate Abd-B in trans. This iab trans regulation is insensitive to chromosomal rearrangements that disrupt transvection effects at the nearby Ubx locus. In addition, we show that a transposed Abd-B transcription unitand promoter on the Ychromosomecan be activatedby iabelements located on the third chromosome. These results suggest that the iab regions can regulate their target promoter located ata distant sitein the genomein a manner that is much less dependent on homologue pairing than other transvection effects.The iab regulatory regionsmay have a very strong affinity for the target promoter, allowing them to interactwith each other despite the inhibitory effectsof chromosomal rearrangements. Second, by generating abd-A mutations on rearrangement chromosomes that break in the iab-7 region, we show that these breaks induce theiab elements to switch their target promoter from Abd-B to abd-A. These two unusual aspects of iab regulation are related by the iab7 breakpoint chromosomes that prevent iab elements from acting onAbd-B and allow them to act on abd-A. We propose that the iab-7 breaks prevent both iab trans regulation and target specificity by disrupting a mechanism that targets the iab regions to the Abd-B promoter.

ROPER development of an organism requires precise other chromosome ( ZACHARet al. 1985; CASTELLI-GAIR P regulation of gene expression. Enhancer and si- et al. 1990; GEYERet al. 1990) . lencer elements provide one level of this regulation, The homeotic genes of the bithorax complex, Ubx, activating and suppressing, respectively, the transcrip- abd-A and Abd-B, specify segmental identitiesof overlap- tion of genes in precise temporal and spatial patterns. ping body segments of the fly from parasegments (PS) Enhancer/ silencer elements can regulate the activity 5 through PS 14, corresponding to the third thoracic of specific target promoters from a relatively long dis- (T3) throughthe ninth abdominal segment (A9) tance in cis on the same chromosome (for review see (LEWIS1978; DUNCAN 1987).Genetic analyses have re- ATCHISON1988) . In Drosophila melanogaster a genetic vealed two typesof complementation groups, lethal and phenomenon calledtransvection suggests thaten- nonlethal, within thebithorax complex (Figure 1; hancers and silencers can also regulate promoters in LEWIS1978; SANCHEZ-HERREROet al. 1985) . Lethal com- trans on a homologous chromosome (see JUDD 1988; plementation groups identify the Ubx, abd-A and Abd-B WU and GOLDBERC1989; PIRROTTA1990; TARTOFand transcription units that encode essential DNA-binding HENIKOFF1991; WU 1993 for reviews). This effect was proteins. Abd-Bcomprises two subfunctions, Abd-Bm and originally described for regulatory mutations for the Abd-Br, provided by four overlappingtranscripts encod- Ultrabithmax ( Ubx) gene (LEWIS1954). In a transvec- ing Abd-BI and Abd-BII proteins in PS10-13 and PS14, tion effect chromosomal rearrangements that disrupt respectively (CASANOVA et al. 1986; CASANOVA and pairing between homologous chromosomesenhance or WHITE 1987; DELORENZIet al. 1988; KUZIORA and suppress the phenotype caused by certain regulatory MCGINNIS1988; SANCHEZ-HERREROand CROSBY1988; mutations. Thus, it has been proposed that homologue CELNIKERet al. 1989; ZAVORTINK and SAKONJU1989; pairing allows interaction between a regulatory element DELORENZIand BIENZ1990; BOULETet al. 1991 ) . Non- on one chromosome and its target promoter on an- lethal complementation groups,which affect subsets of Ubx, abd-A and Abd-B expression domains, identify regu- We dedicate this paper to E. B. Lewis, who began it d, on the latory elements for Ubx, abd-A and Abd-B transcription of his 77th birthday. occasion units. The abx, bx, pbx and bxd elements regulate Ubx Corresponding author: Shigeru Sakonju, Howard Hughes Medical Institute, 5200 Eccles Institute, Bldg. 533, University of Utah, Salt transcriptionin PS 5 (T2/T3)and PS 6 (T3/A1) Lake City, UT 841 12. E-mail: [email protected] ( BEACHYet al. 1985; HOGNESSet al. 1985; WHITEand

Genetics 139: 835-848 (February, 1995) 836 J. E. Hendrickson and S. Sakonju

WILCOX1985) . iab-2, -3 and -4 elements regulate abd- 137 Cs source and crossing them to females carryingthe double A in PS7 (A2), PS8 (A3) and PS9 (A4), respectively mutation Cbx'Ubx'. Cbx'Ubx' heterozygotes show a dominant phenotype of warped and held out wings because Cbx' acts ( KARCH et al. 1990; MACIAS et al. 1990) iab-5-iab-7 ele- . to misexpress Ubx+ on the homologous chromosome via a ments regulate theAbd-B class A transcription unit that transvection effect (LEWIS1955; CABRERAet al. 1985; WHITE codes for the Abd-BI protein in PSlO (A5) through and AKAM 1985; WHITEand WILCOX1985; WTELLI-GAIRet al. PS13 (A8) (CELNIKERet al. 1990; BOULETet al. 1991; 1990). This transvection effect is disrupted by chromosomal SANCHEZ-HERRERO1991 ) . Although specific regulatory rearrangements that break once between Ubxand the centromere and once outsidethis region. We recoveredre- mutations affecting the expression of the class A Abd-B arrangement mutations on the Abd-BDi4chromosome that dis- transcriptin PS13 andthe three longer transcripts rupt transvection between Cbx' and Ubx+ by screening for (class B, C and y) that code for the Abd-BII protein Abd-Bfli4/ Cbx'Ubx' F1 progeny with wild-type wings. The re- in PS14 and PS15 have not been found, they are ex- arrangements were mapped by following segregationwith au- pected to identify iab8 and iab9, respectively. tosomal markers. Some mutations were more finely mapped by cytogenetic analysis of larval polytene salivary gland chro- Genetic and molecular evidence indicate thatreg- the mosomes. ulatory elements in these iab regions act in cis. When To isolate abd-A mutations on an iab7 breakpoint chromo- mutations mapping to the transcription units aremade some, i~b-7~~'/TM3 Sbmale flies, aged1-7 days, were irradi- heterozygous with mutationsin regulatory elements, ated with 4000 rad using a I3'Cs source. They were immedi- they partially fail to complement each other. Further- ately mated en masse to TM? Sb/ TM6 Tb 1-7-day-old virgin females. After 7 days the parents were removed. Individual more, expression patterns of Ubx, abd-A and Abd-B tran- i~b7~~'*/TM6 Tb F1 males were allowed to mate with abd- scripts are altered in embryos mutant for these regula- A['"/ TM6 Tb females. i~b7~~'*/TM6 TbF2 progeny from tory elements ( BEACHYet al. 1985; HOGNESSet al. 1985; vials containin no Tb' upae were crossed separately to abd- A MXI , abd-AMx', abd-AMyand abd-AflZ4to confirm that they WHITEand WILCOX 1985;CELNIKER et al. 1990; KARCH carried an abd-A mutation. From 9735 individual matings we et al. 1990; MACIAS et al. 1990; BOULETet al. 1991; Ski- recovered two abd-A mutations, abd-AP' and abdAF2. CHEZ-HERRERO1991 ) , and aspects of correct expression abd-AP' and abd-A.F2 are null for abd-A function based on patterns can be recapitulated by a lacZ reporter under the following criteria. Cuticle preparations of abd-AF'/ abd- the controlof these regulatory elements in transformed ~D24and abd-A./Xz/ ~bd-A')'~embryos show transformation of flies (SIMONet al. 1990; MULLERand BIENZ1991; QIAN ventral denticle belts towardAl, typical of abd-A mutants (Sh- et ul. 1991; BUSTURIAand BIENZ1993). CHEZ-HERREROet al. 1985). abd-AP' homozygotes also show transformations typical of abd-A mutants, but abd-A,'xz homozy- We describe in this paper an analysis of an unex- gotes show a transformation of denticle belts toward T3. abd- pected complementation between mutations in Abd-the AP2/ Ubx embryos show a transformation of A1 toward T3 B transcription unit andits regulatory regions.We show and die before hatching, but abd-AJX2/bx34' animals live to that the iab regulatory elements can regulate the target adulthood and show no obvious transformation. We suspect promoter intrans. Unlike previously described transvec- that the abd-APz deletion includes pbx and bxd but not bx. The deletion may include part of the Ubx promoter, causing tion effects, this trans regulation by the iab elements, an enhanced transvection from bxt that suppresses the b~'~' which we call ITR, is unusually resistant to disruption phenotype ( MARTINEZ-LABORDA et al. 1992). Analysis of abd- by chromosomal rearrangements. We alsoshow that A-"' or abd-AP2 chromosomes reveals no chromosomal abnor- chromosomal rearrangements that separate theiab ele- malities other than the parental i~b7~"inversions. Southern ments from their normal Abd-B target promoter cause blot analysis using fragmentsof genomic clonesL2235, L2255, L2265, L2279, R8004 and R8019 (KARCH et al. 1985) reveals abd-A to substitute for someAbd-B functions, mostlikely that abd-AJX' contains a deletion of 4-6 kb including the abd- by allowing the iab elements that normally act on Abd- A homebox and that abd-AP' is associated witha deletion with B to act on abd-A instead. the right endpoint between abd-A exons 2 and 4. Neither mutation contains lesions in the iab regions to the right of the abd-A transcriptional unit. Neither mutant expresses abd- MATERIALS AND METHODS A transcripts in embryos as assayed by whole mount in situ Fly stocks. Fly stocks were maintained at 25" in a humidi- hybridization. fied incubator on standard cornmeal medium supplemented In the screen for abd-A alleles, we fortuitously isolated one with fresh yeast. All crosses were performed under these con- insertional transposition mutation, Tp(?; Y)p.Tp(3; Y)p ditions. Descriptions of the mutations used can be found in fully complements abd-A mutations and is genetically identical the following references: abd-AM3 ( SANCHEZ-HERREROet al. to the parental iat~7~''allele, except that it segregates with 1985) ; abd-AMX',abd-A"", abd-ADZ4,i~b7~~', i~b7~'~,iab the Y chromosome. The two components of Tp(?; Y)p?, 7'"A;A62, iab4,5"', Abd-Brli4, Abd-BD16,Df(?R)P9 (KARCH et al. Dp(?; Y)JX? and Df(?)JX?, segregate independently, al- 1985) ; Abd-BM5 (PELAZ1994) ; Abd-Bs4 (TIONGet al. 1985) ; though the Df(?)p is not viable without the Dp(?;Y)p?. Abd-BS7 (TIONGet al. 1988) ; iab7"" ( TIONGet al. 1987) ; Fab Analysis of Tp(?; Y)p? of larval polytene chromosomes re- 7, Fab7R7, Fab7R9, Fab7R41, Fab7R59, Fab7R73( GKJRKCN- veals a transposition of 65A-64A/ 89E-92A,spanning the right ICS et al. 1990) ; i~b7~",iab7" ( GALLONIet al. 1993) ; TM? inversion breakpoint in iab-7MX2,to the Y chromosome and Sb, TM6 Tb, z', z""', zap, P (LINDSLEYand ZIMM 1992); zeste the corresponding deletion on the third chromosome. alleles, 826.2s, 9'77.2s, 1008.lbs (WU 1984). Cuticle preparations: Adult cuticles wereprepared using a Mutagenesis and mapping: We induced transvection-dis- modification of the technique described in DUNCAN( 1982). rupting chromosomal rearrangements on the Abd-BD" chro- The abdomen was split middorsally with a razor blade, teased mosome by irradiating Abd-BDI4males with 4000 rad from a away from the rest of the fly and incubated in 10% KOH for Trans Regulation in the BX-C 837

UbX abd-A A %?I Chx It classB,Cand Y DB Fab7 RI3 Di4 ("Bf)

7sz Fab7R59

FIGURE1.-Map of the bithorax complex. The Ubx, abd-A and Abd-B transcripts are shown. Arrows indicate the positions of breakpoint mutations; - , the regions covered by deletion mutations; A, the positions of insertion mutations; - - -, uncertainty in the locations of the endpoints.

30 min at room temperature. The abdominal cuticles were (from S. SMOLIK-UTLAUT) . We stained the embryos using then splayed out on a slide, covered with 10% KOH and X-gal following the protocol in RUSSELI.et al. ( 1992). If we flattened with a coverslip overnight. The cuticles were then could not follow the chromosomes using this marker, we washed off the slides with water and mounted in CMCPlO determined the ratios of the different staining patterns to (Masters Chemical Company, Inc.,Elk Grove, IL) mountant. confirm that the mutantexpression pattern appeared in the The cuticle pigmentation remained anatural color for expected ratios. months in this mountant. For larval cuticle preparations we followed the protocol described in LAMKAet al. ( 1992). In situ hybridizations and antibody stainings: We synthe- RESULTS sized DNA probes forin situ hybridizations using digoxigenin conjugated dUTP (Boehringer Mannheim) and asymmetric Unexpectedcomplementation at the Abd-B lo- PCR to predominantlyamplify the antisense strand. The class cus: The mutant phenotype caused by rearrangement A and commonprobes were constructed as described in BOU- mutations with a break in the iab7 region (2~"'~'''~) LET et al. ( 1991 ) . The Dl4specific probe includednucleotides +95 through +191 of the Abd-B sequence ( ZAVORTINK and depends on thetype of Abd-B mutations on thehomolo- SAKONJU1989). The abd-A probe included sequences from gous chromosome. When an i~b-7'""~allele is heterozy- +3494 to +4043 (&PEL 1993). We detected the probeswith gous with a deletion of the bithorax complex DfP9, alkaline phosphatase-conjugated sheep anti-digoxigenin anti- abdominal segments A5-A7 transform toward A4. This body fromBoehringer Mannheim ( 1:ZOOO). Monoclonal transformation is evident in adult male flies by the loss mouse anti-invected was provided by J. KASSIS and D.-H. HU- of male-specific pigment in A5 and A6 tergites (dorsal ANG and used at a 1:4 dilution. We detected the anti-invected antibody with biotinylated anti-mouse (used at 1:2000) (Vec- plates) and the presence of A7 segment, which is nor- torLabs) followed by streptavidin-horseradish peroxidase mally suppressed in males (Figure 2B) . It is also indi- (HRP) (used at5 pg/ml) (Chemicon) . We used the ratanti- cated by an increase in trichome density in A6 and A7 Abd-B antibody described in BOULETet al. ( 1991 ) at 1:200 to tergites andthe presence of sternite(ventral plate) 1:400. All antibodies were preadsorbed to Cans embryos at 1:20 for 1 hr at room temperature and stored at 4" (except bristles in A6 and A7. In contrast,i~b-7~'"~ heterozygous for the invected antibody that is preadsorbed at 1:4). with Abd-Bpointor pseudopoint mutations(henceforth We performed in situ hybridizations as described ( TAUTZ referred to as Abd-B- ) that do notaffect the iab regula- and PFEIFLE1989). To double label the embryos with anti- tory regions cause a less severe mutant phenotype. A5 invected antibody anda DNA probe, we modified this proto- segment appears normal, and A6 and A7 tergites show col after the hybridization washes by blocking the embryos with phosphate-buffered saline (PBS) + 0.1% Triton X-100 male-specific pigmentation and reduced trichome den- + 0.1% bovine serum albumin (BSA) (PBSBT) for 1 hr at sity, whereas their sternitesresemble that of A5 (Figure room temperature and incubating the embryos with anti- 2C). We shall call this mixed identity assumed by A6 invected antibody overnight at 4", washing 12 times for 10 and A7 the "A5/A6 identity". Thus, contrary to expec- min with PBSBT, incubating with biotinylatedanti-mouse tations, i~b-7~""~/Abd-B-flies show a less severe trans- and sheep anti-digoxigenin in PBSBT for 1 hr at roomtem- perature, washing again and incubatingwith fresh streptavi- formation of the posterior abdominal segments than din-HRP in PBSBT for 30 min at room temperature. The do iab7be"k/DfP9flies. final wash was without BSA. HRP was detected by reacting We eliminated two trivial explanations for the un- the embryos with 0.03%hydrogen peroxide and0.5 mg/ml expected A5/A6 identityin i~b-7~~"~/Abd-B-flies. diaminobenzidine. TheHRP reaction was stopped with PBS First, this phenotype is not due to uncharacterized plus 0.1 % Tween-20, and the alkaline phosphatase reaction was performed as described(TAUTZ and PFEIFLE1989). mutations outside the bithorax complex,because sev- When possible, we identified specific genotypes by using a eral i~b-7~"~mutations heterozygous with several Abd- TM3 balancer chromosome containing a ftz-lacZ construct B- mutations(listed in Table 1) show the nearly J. E. Hendrickson and S. Sakonju

I \I iab-5 iab-6 iab-7 iab-8.9iab-7iab-6 iab-5 iab-5iab-6iab-7 ia b-8.9 iab-5iab-6iab-7 iab 8 9 nnnt nnn+ Abd-B II I (Deficiency)

\ JLII Frcum 2.-Unexpected complementation at the AldB locus. Adult male phenotypes of wild rype (A), i~b7~"/D/P9(B) and i~b7"".~~/Abd-B"'~(C). Each abdominal cuticle shows the ventral sternites on the right and half of the dorsal tergites on the left. Abdominal segments are labeled. Wild-type male flies show a characteristic dark pigmentation pattern (male-specific pigmentation) on the dorsal part of the sclerotized cuticle (tergite) in abdominal segments A5 and A6 and suppress the formation of A7 cuticle. Small hairs called trichomes cover the A5 tergite hut are very sparse on the A6 tergite, and there are bristles on the A5 sternite (ventral cuticle plate) hut not on the A6 sternite. i~b7"'.~~:2/D/P9males show derepression ofA7 tergite and sternite formation, the loss of dark male-specific pi ment and an increase in trichome density on A5, A6 and A7 tergites, and presence of bristles on A6 and A7 sternites. iab7"~yF/AM-l?"6males show male-specific pigment on A5, A6 and A7. In addition, the A6 tergite has patches devoid of trichomes, the A7 tergite nearly completely lacks trichomes, and the A6 and A7 sternites are larger, indicating a greater amount of posterior identity in A6 and A7 than in A5. identical pigmentation phenotype. Second, the A5/ Abd-B- heterozygotes. We examined i~b7~*~mutations A6 identity is not due to the possible hypomorphic heterozygous with a chromosome carrying the triple nature of the Abd-B- alleles used. All of the Abd-B- mutant chromosome Ubx'"x12abd-A" Abd-BMX,which alleles we have tested behave genetically null for the behavesas Ubx- abd-A-Abd-Bm-r- (CASANOVA et al. Abd-Bm function(see Table 1 and MATERIALS AND 1987). Theseheterozygous flies show the same pheno- METHODS for references). Furthermore, in two cases type as iab7''m"k/Abd-B- flies (data not shown). There- where associated DNA lesions have been determined, fore, neither Ubx, abd-A nor Abd-Bron the Abd-B- chro- one mutant allele ( Abd-B"") has a 411-bp deletion mosome contribute to theless severe phenotype in iab of the Abd-B class A promoter and transcription start 7"r'f'k/Abd-B- flies. Because the only remaining signifi- site, obliterating detectable levels of Abd-B class A cant difference between the Abd-B- chromosome and transcription, and another ( Abd-B"'5) carries a point the DfP9 chromosome is the presence or absence, re- mutation that causes premature termination of the spectively, of the iab regions, we hypothesized that the Abd-B open reading frame (ORF) in the first exon A5 /A6 identity iniab7'""k/ Abd-B- heterozygotes is due (see Table 1 for references). to the activity of the iab regions located on the Abd-B- We also testedthe possibility that otheressential func- chromosome. We further hypothesized that the intact tions of the bithorax complexstill present on the Abd- inb5-iab7regions on theAbd-B- chromosome activate, B- chromosome,but missing on the DfP9 chromo- in trans, the Abd-B promoter on the i~b-7'"~ chromosome, contribute to the A5/A6 identity in iab7'"r''k/ some, normally quiescent in A5 through A7. We shall Trans Regulation in the BX-C 839

TABLE 1 Characterization of iab7 and Abd-B alleles

M utation Phenotype ProteinPhenotype Mutation RNAlesion Molecular Cytology iabPkmutants i~b-7~" iab5,-6,-7 126-129 T(Y;2; 3) i~b7~~'In(89E,89A,64A) 15"' 14,139.5-142 13,iab5,-6,-7 15" 14, 13, iab7SGA62 iab5,-6,-7 133-139.5 In(89C-89E) iab7"" 154-158iab5,-6,-7 (89c-89E;Y) Tp Fab7R 7 iab5,-6,-7 141.5-143 89E) In(87D; Abd-B mutants Abd-pl4* Abd-Bm- Normal15"3'sd+345of15' 14,Deletes 14, to -66 class A transcript' Abd-p5 codonAbd-Bm-Stop Normal 151 14, in first class A exon" Fab7R9" Abd-Bm- 0.5-kb 157-158Normal at insert Fab7R41 Abd-Bm- insert 0.5-kb Normal at 154-156 Abd-Pl6 NormalAbd-Bm-r- None' Abd-Bs4 Abd-Bm-r- Abd-P7 AM-Bm-r- Characterization of iabPkand Abd-B alleles used in this study. The names of alleles according to the nomenclature established by DUNCAN(1987) are shown with their hemizygous phenotype. * denotes two alleles that exhibit a slightly weaker mutant phenotype (a lower density of trichomes in A6 and A7 tergites) than other Abd-B alleles when transheterozygous with iabPk alleles. However, because Abd-pI4 and Fab7R9 behave similarly to other Abd-Bm- alleles when hemizygous, we suspect that this weaker mutant phenotype is due not to these alleles' hypomorphic nature, but rather to an ectopic activation of abd-A (see DISCUSSIONfor a possible explanation). The Protein column indicates in which embryonic parasegments Abd-B protein is detected. TheRNA column indicates in which embryonic parasegmentsAbd-B transcript is detected. We have indicated references for molecular data. References: * CELNIKERet al. (1990), SANCHEZ-HERREROand AKAM (1989), BOULETet al. (1991), SANCHEZ-HERRERO(1991), ZAVORTINKand SAKONJU(1989), 'DELORENZI and BIENZ(1990), PELAZ (1994). refer to this postulated trans activity of the iab regions sequencesdeleted in the Abd-B"14 mutation.In iab as iab trans regulation or ITR. 7Mx2/Abd-BD14embryos we detect this sequence in PS Molecular evidencefor ITR: To test if the Abd-B pro- 11, 12 and 13 (Figure 3E) , whereas we detect this se- moter is activated in trans in ITR, we examined Abd-B quence only in PS 13 in i~b-7~~'homozygotes, and we expression patterns using in situ hybridization in em- do not detect this sequence in Abd-BDI4homozygotes bryos. Although embryonic cells that express Abd-B do (data not shown).Therefore, when i~b7~~~istranshet- not necessarily become imaginal precursor cells ofadult erozygous withAbd-BDI4, the Abd-B class A promoter on tergites and sternites, Abd-B expression patterns in the the iab7hfX2breakpoint chromosome is activated in PS embryo have been previouslyshown to correlate re- 11 and 12. markably well with adult mutant phenotypes( CELNIKER A notabledifference between the expression patterns et al. 1990; BOULET et al. 1991; SANCHEZ-HERRERO of embryosthe and wild-type em- 1991) . In wild-type embryos Abd-B class A transcripts bryos is that Abd-B transcripts are not expressed in PS are expressed in PS10-13, and class B, C and y tran- 10 in the mutantembryos. Our failure to detect hybrid- scripts are expressed in PS14 and PS15 (Figure 3A) ization signal in PS 10 could be due either to absence ( KUZIORAand MCGINNIS1988; SANCHEZ-HERREROand of Abd-B expression or to insensitivity ofthe technique. CROSBY1988; CELNIKERet al. 1989; ZAVORTINKand SA- We suspect, however, that the absence of hybridization KONJU 1989). In i~b-7~~~ homozygous(i~b-7~~~~) em- signal is real, because, as will be shown below, genetic bryos Abd-B class A transcript is not detected in PS10- experiments indicate that Abd-B contributes to A5/A6 12 (Figure 3B), and in Abd-BD14( Abd-B- ) homozygous identity only in A6 and A7 (corresponding to PS11 and embryos, it is no longer expressed in PS10-13 (Figure PS12) and not in A5 of i~b7~~~/Abd-B"'~heterozy- 3C). In contrast,we observe that i~zb7~~~/Abd-B"'~em- gotes. bryos express Abd-B transcripts in PS11-PS15 in a Genetic evidence that Abd-B is activated in trans: To graded mannersimilar to the wild-type embryos (Figure test whether trans activation of the Abd-B promoter can 3D) . These transcripts are translated, because immuno- confer A5/A6 identity in i~b7'""~/Abd-B-adult flies, staining detects Abd-B protein in a similarly graded we used an X-ray-induced insertional transposition de- manner (A. BOULET,unpublished results) . rived from the i~b-7~~'inversion chromosome. This To determinewhether Abd-B on theiab7MX2 chromo- transposition, Tp (3; Y)JU, moves the AM-B transcrip- some is activated, we hybridized i~b7~~~/Abd-B~'~em- tion unit andpromoter to the Y chromosome bryos with a class A-specific probe containing only the [ Dp(3; Y)JU], leaving the iab regions on the third 840 J. E. andHendrickson S. Sakonju

chromosome [ Df(3)JX3] (Figure 44) (MATERIALS AND METHODS) . To ascertain that the transposition eventdid not alter the state of Abd-B, we constructed Tp(3;Y)JX3/ DfP9 flies. These control flies showthe same mutant phenotype (data not shown) as the parental iab7-/ DfP9 flies (Figure 2B), indicating that the original iab7 break mutation still existson this chromosomeand that the transposition did not cause ectopic activation of Abd-B. We then constructed another control genotype, T'(3; Y)JIU/Abd-B- , that is equivalent to iab7-/ Abd-B- heterozygotes, except that the Abd-B transcription unit is transposed to the Y chromosome (Figure 4B). These flies show a phenotype identical tothat of i~b7~/Abd-B-heterozygotes (Figures 2C and 43%). This result indicates that all the genetic factors that contribute to the unexpected A5/A6 identity in iab 7MX2/Abd-B-flies are still present in Tp(3; Y)m/Abd- B- flies. To test whetherthe transposed copyof Abd-B confers A5/A6 identity, we constructed the genotype Dp(3; Y)jX3; Abd-B-/DfP9. In this genotypethe Abd-B transcription unit and promoter from the original i~b7~~chromosome is on the Y chromosome, and the rest of the bithorax complex from the original i~b-7~~'chromosome is missing. Another copy of the bithorax complex, including the iab regions, is supplied on the Abd-B- chromosome. These fliesare fullyviable and show male- specific pigment in A6 and A7 (Figure 4C) . In compari- son, the genotype Abd-B-/DfP9 in female siblings is lethal. In addition, it has beendemonstrated previously that escapers of Abd-B-/DfP9 flies or Abd-B- clones show no male-specific pigment in the abdominal segments ( SANCHEZ-HERREROet al. 1985; YIONGet al. 1985, 1988). Together, these results indicate that Abd-B on the Y chromosome, transposed fromthe iab7- chromosome, provides essential functions for viability and confers male-specific pigmentation in A6 and A7. In support, we have confirmed, usingin situ hybridization, that Abd-B is expressed in Tp(3;Y)JX3/AM-B- mutant embryos in a pattern identical to that of iab7-/Abd- B- embryos (data not shown). Because Abd-B activity has not been altered by the transposition event (see above), the result obtained with Dp(3;Y)p should also reflect the activity ofAbd-Bon the parental chromosome i~b-7~~.We conclude that transactivation of Abd- FIGURE 3.-Expression of Abd-B in embryos with homozygous and transheterozygous combinationsof iab7Mx2 and Abd- B promoter on the i~b.7~~'chromosome explainsITR P14mutations. (A) Wild-type embryos, (B) iab7Mx2homozy- observed in A6 and A7 of i~b7~'/Abd-B-flies. At the gotes, (C)Abd-PI4 homozygotesand (D and E) iab7MX2/ end of RESULTS, we will present our examination of the Abd-P14transheterozygotes. Anterior isto the left, and dorsal factors that confer male specific pigmentation in A5. is up. Embryos in A-D were hybridized to a probe antisense Genetic evidence that iabd and iab-7 activate AbM in to AM-Bm and r sequences (blue) ; the embryo in E was hy- tmm: The phenotype of Dp(3; Y)JX3; Abd-B-/DP9 flies bridized to a probe antisense to thesequences missing in the suggests that the iab regions, located on the AM-B- chro- Abd-PI4 deletion (blue). All embryos were also stained for invected protein(brown). Invected protein isdetected at the mosome, can activate anAbd-B promoter in tram. To test anterior boundary of each parasegment. Parasegments are more directly whether iab elements are responsible for labeled. the tram activation of the Abd-B promoter that confers 7i.cr71.s Regulation in the BX-C 84 1

inb3-iab7 FIGURE4.-Phenotypes of adult males carrying the transposition of a segment from the in/~7"'s'chromosome, including Abd-B, to the Y chromosome, Dp(3;Y)JX3. (A) Map showing gross structure of Tp(3;Y)p mutation induced on the id~-7"'~~chromosome. Adult henotype of Tp(3;Y)JX3/ y,$l& ( B) and Dp(3; Y)JX3; Abd.B'"6/ DfP9 (C) shown above maps depicting each genotype. Note that Tp(3;Y)P/ AM-B- male looks like parental i&71's2/A&" male (Figure 2C). 4(3;Y)jX?; AM-L!""/ m DfP9 male, in contrast, shows pigmentation in A6 and A7 but not in 4 M, indicating that the transposed AM-B on the Y chromosome makes pigment in A6 and A7, whereas factors on the Df(3)jX? chromosome make pigment in M.

iab-8 9 Dp(3;Y)JX3 +

iab-5iab-6iab-7 nn- '4'

M/A6 identity, we constructed the genotype iab7/""k/ To test if iab6 and iab7 elements can each activate Fab7R59 (Figure 5) and compared the phenotype of an Abd-B promoter in trans, we used various iab mutants these flies tothe phenotype of iab7/m"k/AM-B- flies (Fig- in which onlyone of these elements is inactivated, leav- ure 2C).The Fab7R59mutation deletes all sequences left ing the otherstill functional. We compared thedeletion from the 3' end of the AM-B transcript, including all of mutants iab4,5"", iab7" and Fab7R73 and the inser- the iab regions, through bxd but retains a functional AM- tion mutants iab7" and iab6"' (Figure 1 ) as hemizy- B transcription unit (Figure 1) ( GWRKOWCSet 01. 1990) . gotes us. as heterozygotes with Abd-B- mutations. In all iab7/m'k/Fab7R59fliesdo not show male-specificpigmen- cases the phenotype of segments A6 and A7 showed tation in A5-A7 (Figure 5) (see DISCUSSION for an expla- more posterior segment identitywhen the mutants were nation of residual pigmentation) , observed withiab7".yz/ heterozygous with Abd-B- mutations (data not shown). AM-B"'" flies (Figure 2C). We conclude that the iab ele- For instance, iab4,5""/ DfP9 flies show a loss of male- ments on the AM-B- chromosome, which are missing specific pigment in A5 and A6 and a small, pigmented ti-om the Fab7R59 chromosome, contribute to M/A6 seventh tergite. In contrast, iab4,5""/Abd-B- flies show identity in iab7"P"k/Abd-B- flies. a loss of male specific pigment in A5 but not in A6, and 842 J. E. Hendrickso~9 and S. Sakonju

rearrangementsthat apparently disrupt pairing he- tween homologous chromosomes. For example, LEWS ( 1954) has found thattransvection effects are disrupted by chromosomal rearrangements that break once between Ubx and the centromere and once outside this region. To compare ITR directly with the transvection effect at Ubx, we induced chromosomal rearrangements on theAbd-B"'4 chromosome that disrupt the dominant cbx' transvection effect at the ubx locus (see MATERIALS AND METHODS). Because these rearrangements will break between Ulm and thecentromere, theywould be predicted to disrupt a transvection effect at Abd- B because Abd-B is distalto Ubx with respect to the centromere (see Figure 1 ) . We recovered 23 rearrangements, called R(D14), that disrupt transvection at the Ubx locus. To ascertain that our newly isolated transvectiondisrupting mutations conform to the expected type of rearrangements, we mapped these mutations. All of the mutations map to the third chromosome, and 13 of them map to another chromosome as well. We examined larval salivary f \ gland polytene chromosomes from six of the mutations and found that each mutation has one break between the centromere and thebithorax complex at map posi- iab-5 iab-6 iab-7 iab-8.9 nnmt tion 89E and at least one break outside this region I I (Table 2) and conforms to the type of rearrangements 't W' found by LEWIS( 1954) for Cbx' transvectiondisrupting iab-7b'eak mutations. To test if these rearrangements disrupt ITR at Abd- Fab7R59 B, we crossed flies carrying R(D14) chromosomes to flies carrying iab7"'"2. The phenotypes of the F1 iab \ b1 7''''/R(Dl4) flies (data not shown) are identical to the phenotypes of iab7"'X2/Abd-B1)'4flies (Figure 2C). FIGURE5.-Fab7R59mutation, lacking the inbregions, fails to com lement iab7'.'' mutation. Adult male phenotype of To eliminate the possibility that the identical pheno- iab-7"xY / Fub7R59 is shown abovea map depicting the genotypes result from fortouitously pairing ofnew re- type. Note that the loss of male-specific pigmentation on A5- arrangements on the Ahd-B"'4 chromosome with the A7 is similar to that in inb7"sz/DfP9 (Figure 2B), implicat- rearranged iab7MX2chromosome, we tested R(D14) re- ing the iub elements in the transregulation of A6d-B to produce male-specific pigment. arrangements heterozygous with thenonrearranged Fab7R59 chromosome. R(D14)/Fab7R59 flies also show an identical phenotype to that of the parental the seventh tergite is much smaller. iab7'"/ DfP9 flies Al)d-B"'4/Fab7R59 flies, which show pigmentation in have a seventh tergite that looks like A6, whereas iab A6 and A7, like the phenotype of Dp(jX.3); Abd-B-/ 7'"/Abd-B- flies have a much smaller seventh tergite, DfP9 flies (Figure 4C). These results indicate that ITR indicative of an incompletetransformation to A6. These is not sensitive to the same chromosomalrearrangements results indicate that the functionof each of the regions that disrupt the Cbx' transvection effect at the nearby deleted or disrupted on the iab mutant chromosomes Ubx locus. By this criterion ITR at the Abd-B locus is can be partially complemented by the trans regulation distinct from transvection effect at the Ubx locus. of Abd-B by the wild-type iab element located on the As a further test of similarity of ITR to transvection, Abd-B- chromosome. We conclude that both iab6 and we examined iab7"~y2/Abd-B')'4 heterozygotes in zeste iab7 elements can contribute to ITR. backgrounds. reste is a gene that has been shown to Directcomparison of ITR with transvectionat disrupt transvection effects at Ubx and other loci (GEL Ubx: Trans activation of Abd-B by its regulatory regions BART and WU 1982) . We tested several classes of zste suggests that ITR may be similar to previously described alleles (listed in the MATERIALS AND METHODS). None transvection effects. One of the hallmarks of transvec- of these reste alleles altered the phenotype of i~b7~"/ tion effects is that they are sensitive to chromosomal Abd-B"'4 flies. These results also indicate that ITR is not Trans Regulation in the BX-C 843 a transvection effect of the type that has been described in conferring A5 identity in iab7 breakpoint heterozy- for the Ubx locus. gotes aswell. We tested our double mutants abd-AJX' Contribution of abdA on the iab-7'& chromosome iab7MX2and abd-Ap2 i~b-7~~'as heterozygotes with iab 7brmk to Pigmentation in A5: We have shown that ITR is re- chromosomes and find that pigment is reduced sponsible for A5/A6 identity in A6 and A7 of i~b7~'"~/ compared to iab7hp"kheterozygotes (data not shown). Abd-B- flies. Still unexplained is the male-specific pig- This result indicates that abd-A contributes to pigment mentation in A5 in i~b7~~"~/Abd-B-flies. The compari- in these heterozygotes. i~b7~~"~/DfP9 flies also show son ofDp(?;Y)JX3;Abd-B-/DfP9flies(Figure 4C) with patchy pigmentation in the posterior abdominal seg- Dp(?;Y)jX?; Abd-B-/Df(?)jX? flies (Figure 4B) sug- ments (Figure 2B) ; this patchy pigmentation can now gests that the Df(?)JX3chromosome contains some fac- be explained as the result of abd-A activity from the iab tor, not present on theDfP9 chromosome, that contri- 7h"'kchromosome. We suggest that iab5 regulates abd- butes to male-specific pigmentation in A5. A candidate A on i~b7~'"~chromosomes in these genotypes as well for this factor is the abd-A gene. Some geneticdata as in iab7/"""k/Abd-B- heterozygotes. indicate that abd-A may be able to perform some of the To explain the remaining pigmentationin A5 of abd- same functions as Abd-B; for instance, both Abd-B-/ iab A."' iab7MX2/Abd-B- flies (Figure 6), we compared 5 and abd-A-/ iab5 heterozygotes show a partial trans- these flies to Dp(?;Y)JW; Abd-BP/DfP9 flies (Figure formation of A5 toward A4 ( KARCH et al. 1985; DUNCAN 4C) that show a complete loss of male-specificpigment 1987) . We have already discussed that because iab in A5. A major difference between the two genotypes 7hp"k/Ubx- abd-A- Abd-B- flies show a phenotype iden- is the presence of a copy of the iab regions on the abd- tical to iab77meak/Abd-B-flies, abd-A on the Abd-B- chro- AJX~ iab7MX2 chromosome. Although we have presented mosome does not contribute to the A5 male-specific evidence that iab regions on the i~b-7~~~~chromosome pigmentation in these heterozygotes. We hypothesized are not capable of regulating Abd-B, they could still therefore that the abd-A gene on the i~b-7~~"~chromo- regulate abd-A. Therefore, we propose that iab5 on the some contributes to male-specific pigmentation in A5 abd-A.IX' i~b-7~"or abd-AJX2i~b-7~~' chromosome regu- of i~b7'""~/Abd-B-flies. To test this hypothesis, we in- lates abd-A on the homologous chromosome in trans. duced abd-A mutations on the i~b-7~~'chromosome. The trancactivated abd-A would then contribute to the We irradiated iab7h'X2 males and screened for lethality male-specific pigmentation in A5 seen in abd-AJX' iab over abd-ADZ4.We recovered two mutations, abd-AJX' 7MX2/Abd-B-and abd-AJX2 i~b7~~'/Abd-B-flies. We and abd-A.IX2.Both of these mutations are null for abd- tested whether this proposed trans iab5 function is due A based on genetic and molecular evidence (see MATE- to a transvection effect by crossing abd-AJX' and abd-AJX2 RIALS AND METHODS) , and neither contain mutationsin to our rearrangement R(D14) alleles. We observe the the iab regions ( see Figure 1 ) . We crossed flies carrying same pigmentation patterns in abd-AJX' "Jx2/ R(D14) as the double mutant chromosome abd-AJX' iab7MX2or in the parental abd-AJX'w.IX2/Abd-B"'4. Thus, the pro- abd-AJX2 i~b-7~~'to flies carrying Abd-B- chromosome posed trans interaction between iab5 and abd-A is simi- to assess the effect of the new abd-A mutations. We find lar to ITR involving Abd-B and unlike transvection ef- that abd-AJX' iab7"X2/ Abd-B- or abd-Ap2 i~b7~~'/Abd- fects at Ubx. B- heterozygotes show loss of dark pigment from anterior and lateral parts of the A5 tergite (Figure 6) , com- DISCUSSION pared with the parental i~b7~~'/Abd-B-flies (Figure 2C). This pigmentation pattern is similar for all the Similarities between ITR and transvection: We have heterozygous flies with either the abd-Ap' iab7MX2or demonstrated that iab regions, known to regulate Abd- the abd-A.v' chromosome and shows 100% pen- B in cis, can also regulate Abd-B in trans. This trans etrance. We conclude that abd-A on the iab7MX2chro- regulation by the iab regions is reminiscent of previously mosome contributes, at least partially, to the specifica- described transvection effects and related pairing- or tion of male-specific pigmentation in A5 of proximity-dependent phenomena in Drosophila. Many Abd-B- flies. We suggest that the iab5 element on the of these phenomena have been correlated with the al- i~b-7~~'chromosome regulates abd-A to produce this tered transcription of the affected gene ( BINCHAMand pigmentation. ZACHAR1985; KORNHERand BRUTLAG1986; HENIKOFF The contribution of abd-A to A5 identity has been and DREESEN1989; GEYERet al. 1990), leading to the postulated previously ( CELNIKERet al. 1990) , because hypothesis that transvection effects result when tran- posterior abdominal segments in i~b-7~~"~transhetero- scriptional regulatory elements on one chromosome zygotes show an A5 identity rather than the expected influence the expression of their target gene on the A4 identity despite the fact that no Abd-B transcript or (paired) homologouschromosome ( ZACHAR et al. protein can be detected in i~b-7~~~transheterozygote 1985). Transvection at Ubx has been correlated with embryos ( CELNIKERet al. 1990; ~ROSBYet al. 1993). We altered expression of Ubx ( CASTELLI-GAIRet al. 1990). used our new mutants to test whether abd-A is involved Similarly, we haveshown thatthe phenotype of iab 844 J.E. Hendrickscm and S. Sakonju TABLE 2 Induced chromosomal rearrangements

Mutant Linkage Cytolow. .,, R(D14)b 3 In (64,88A) R(D14)c Y, 2, 3 Tp(88D-94;Y), T(59A,94), T(88Di59A) R(D14)i 2, 3 T(24;82) R(D14)j 2, 3 T(58;89A) R(Dl4)k 3 In(61F;82) R(D14)o Y, 3 T(89D;Y) Locations of breakpoints for some R(D14) chromosomes that disrupt transvection at the Ubx locus, as described in the text.

7'""'/Abd-B- flies correlates with transcriptional activation of Abd-B in embryos. The Abd-B class A promoter is activated by iab elements in trans in qualitatively the same way as it is in cis in wild-type animals. However, 7 ~ iab-3iab-4 iab-5 bb-6 iab-7 the expression of Abd-Bin iab7'rp"k/AOd-B-flies appears to be quantitatively less than in wild-type flies, because abd-A + Abd-B * it fails to restore the wild-type phenotype completely. For transvection effects, including those at Uhx, the lab-3 "7 trans interaction appearsto be weaker than the cis inter- /" action as well, resulting in incomplete complementa- abd-A - iab-7 break Abd-B+ tion ( GEYERet al. 1990; MARTINEZ-LABORDA et al. 1992) . L J Thus, ITR is similar to other transvection effects and related phenomena in thattranscription of the affected FIGURE6."abd-A on the iab-7"" chromosome contributes to the male-specific pi mentation in A5. Adult male pheno- gene appears to be regulated in a manner similar to type of abd-A'.'' iab-7''g.z/Abd-I?)'". The arrows point to the but weaker than its wild-type cis regulation. loss of pigment in A5 compared with iab-7"xz/Abd-B'"h or Differences between ITR and transvection: Trans- Tp(3:Y)JX3/Abd-lf)'6 flies shown in Figures 2C and 4R. vection and related phenomena show some degree of pairing or proximity dependence, suggested by their sensitivity to chromosomal rearrangements (LEWIS region that could potentially pair. In Dp(3;Y)p;Abd- 1954). In contrast, ITR is not affected by chromosomal B-/DfP9 flies, for example, the transposed chromo- rearrangements, including those that clearly disrupt a somal segment from position 89E to 92A could pair transvection effect at Ubxor a translocation of the Abd-B with the corresponding chromosomal segment on the promoter and transcription unit to the Y chromosome. third chromosome (Figure 4A). If pairing does occur Furthermore, i~b-7'~"~mutations do not disrupt ITR in this case, a functional copy of iab elements, which between iab elements on an intact homologue and the themselves do not have a homologous copy to pair, Abd-B promoter on the iab7/"p"kchromosome (as in iab might be brought in close enough proximity to activate 7'm"'/Abd-B- flies), even though these rearrangement the AM-B promoter on the homologous chromosome. mutations might be expected to disrupt any pairing to The differential sensitivity of transvectionand ITR to the right of the iab7 breakpoint because they break chromosomal rearrangements may also indicate that once between Abd-B and the centromere and onceout- pairing along homologous chromosome arms need not side this region. These differences between transvection be uniform. Chromatin in the vicinity of Abd-B could and ITR can be interpreted in one of two ways: ITR pair much more strongly than chromatin in the vicinity may not be pairing dependent, at least to the extent of Ubx. Such differential pairing could occur if proteins required for transvection effects at Ubx; alternatively, that mediate pairing have more binding sites in the the Abd-B locus, but not the Ubx locus, has a sufficiently Abd-B region than in the Ubx region. For instance, Poly- strong affinity to pair with its homologous copy despite comb, a chromosomal protein intimately involved with the negative effects of chromosomal rearrangements. regulation of the bithorax complex genes, has been Our data do not rule out the possibility that ITR found to be present at a higher density on chromatin requirespairing between homologous chromosomal in the abd-A/Abd-B regions than on chromatin in the segments. In all the genotypes in which we observed Ubx region (ORLANDOand PARO 1993). Similarly, a ITR, there were two copies of the Ab& chromosomal chromosomal protein involved in ITRcould be present Trans Regulation in the BX-C 845 at a higher density in the Abd-B region, allowing this spect to abdominal phenotypes even though they are region on two chromosomes to pair more avidly. fused to different chromosomal regions. We have shown that ITR is insensitive to chromo- Because neither of these explanations appears fully somal rearrangements except to those with a break- satisfactory, we propose a third model: iab7 chromo- point within the iab7 region. This relative insensitivity somal breaks may disrupt a mechanism that targets iab to chromosomal rearrangements is different from trans- elements to theAbd-B promoter. In this model we spec- vection at the Ubx locus but similar to a few other re- ulate that the iab7breaks interrupt requisitethe mecha- ported cases of tralzsregulatoryphenomena. For instance, nism that initiates at a site on the right-hand side of the zeste-white interaction, a proximitydependent regu- the iab7 breaks and propagates proximally toward the latory phenomenon, is evidently resistant to chromo- iab6 element. In molecular terms this could be initia- some rearrangements except to those with breakpoints tion of transcription across the iab regions. Transcripts in the immediate vicinity of the white locus ( SMOLIK- from the iab regions have beendetected (SANCHEZ- UTLAUTand GELBART1987). Another example has HERREROand AKAM1989) ; at least one of these tran- been suggested byCASTELLI-GAIR et al. ( 1992) , who scripts initiates to the right of iab7 breaks and extends found that a putative negative regulatory element in the entire span of the iab regions (S. SAKONJU,unpub- the bxd region of the bithorax complex can apparently lished results). Alternatively, this mechanism could in- exert its negative effect in trans independent of homolo- volve a nucleation site for formation of chromosomal gous chromosome pairing. Thus ITR may represent an protein aggregates or an element similar to the locus emerging class of tramregulatory interaction phenom- control region of thehuman P-globin gene cluster ena that are not readily disrupted by chromosomal re- ( FEUENFELD1992) that might be required for a tran- arrangements. scriptionally active conformation of chromatin ( PARO A unique propertyof a-7- alleles suggests a mech- 1990). Theexistence of such a mechanism would also anism for ITR: The resistance of ITR to chromosomal explain the behavior of the iab5 element on the iab rearrangements can also be explainedif the iab regions 7 breakpoint chromosomes. We have shown that, on have a very strong affinity for the Abd-B promoter. This unbroken chromosomes, iab5 elements preferentially strong interaction could allow the iab elements to regu- interact with an AM-B promoter in ci5 or in trans, rather late Abd-B either in the absence of pairing between ho- than with an abd-A promoter. On the chromosomes mologous chromosomal regions or during transient or with iab7 breaks, however, iab5 element is released weak pairing. The clue to themechanism of this interac- from this mechanism that directs it to the Abd-B pro- tion may be revealed by the only type of mutations we moter, becoming free to regulate the abd-A promoter have found that disrupt ITR: chromosomal rearrange- in cis or trans. In sum,we suggest that structural discon- ments with breakpoints in the iab7 regions. These re- tinuity between the Abd-B transcription unit and down- arrangementmutations not only inactivate the iab7 stream iab regions may disrupt themechanism required function but also prevent ITR between the adjacent iab for ITR. elements and Abd-B promoter in cis or trans, as indicated The role of abd-A in conferring posterior abdominal by the phenotypes of iab7-/DfP9 or i~b7~'~/Fabsegment identity: CELNIKERet al. ( 1990) have proposed 7R59 flies (Figures 2B and 5). Because iab elements in previously that in flies heterozygous for different iab other genotypes can regulate Abd-B in trans, we might 7- alleles, iab5, unable to regulate Abd-B, regulates expect that structurally intact iab elements on iabYh* abd-A instead, and abd-A substitutes for Abd-B. We have chromosomes would be able to function. Why then directly tested this hypothesis by inducing abd-A muta- don't these iab elements function in cis or trans when tions on an i~b-7~"~chromosome. We find that these there is a break in the iab7 region? We consider three abd-A mutations do indeed result in loss of male-specific possible models. pigment from A5, as if under control of the iab5 ele- In the first model iab7&* chromosomes may impose ment. some physical constraints for iab elements to interact That abd-A can substitute for AM-B seems surprising with Abd-B. It is not clear, however, what these con- because the abd-A and Abd-B proteins share only 38 straints may be because our data suggest that iab ele- out of 61 residues in the homeodomain (SCOTTet al. ments can interact with an AM-B promoter anywhere 1989) and have no amino acid sequence similarity out- in the genome. In the second model iab7 breakpoint side the homeodomain (DELORENZIet al. 1988;CEL chromosomes may inactivate iab regions to the left of NIKER et al. 1989; ZAVORTINK and SAKONJU 1989; &RCH the break and thus prevent them from regulating Abd- et al. 1990).Furthermore, experimentswith heat-shock B. Nearby iab elements could be inactivated if the constructs show that these two proteins, when ectopi- breakpoint mutations juxtapose the iab regions to new cally expressed, confer different larval phenotypes chromatin domains that inactivate the adjoining ele- (LAMKAet al. 1992; KUZIORA1993; SANCHEZ-HERRERO ments. We consider this possibility unlikelybecause the et al. 1994). In the adult,however, abd-A and Abd-Bmay different iab7 breaks we used behave similarly with re- specify the formation of some of the same morphologi- 846 J. E. andHendrickson S. Sakonju cal features. For instance, Abd-B specifies the expression balance between the two, resulting in ectopic activation of dark pigment in a male-specific pattern on the ter- of abd-A in A4. gites. abd-A may also be able to specify dark pigment Evolutionary considerations: Trans regulation ap on the tergite because in abdominal segments where pears to be a general phenomenon in the bithorax abd-A is expressed but Abd-B is not expressed (A2-A4), complex. Trans regulation between the regulatory re- dark pigment appears, although only in a stripe at the gions for Ubx and the Ubx promoter, referred to as posterior edge of the tergite. If abd-A were to come transvection, has been known for a long time (LEWIS under the control of iab5, it may be expressed in the 1954, 1955). More recently, trans interactions between pattern that would allow it to confer the formation of iab elements and the abd-A promoter have also been dark pigment over the entire tergite. abd-A could then found (JIJAKLIand GHBEN 1992). We have discovered substitute for Abd-B by acting on its own downstream the existence of ITR, a trans regulation of the Abd-B genes in an Abd-B pattern. promoter by the iab regulatory elements. Because allof Implicitin this model is the assumption that iab5 these examples of trans regulation are so far seen only would prefer to interact with the Abd-B promoter and in mutant combinations, we can only speculate on the only acts on the abd-A promoter when it cannot find the wild-type function of trans regulation. We propose that AM-B promoter. Our data supports the validity of this trans regulation may be a byproduct of extremely high assumption becauseabd-A mutations on an i~b7~~chro- affinities of the regulatory elements in the bithorax mosome result in loss of pigment, whereas abd-A muta- complex for their promoters. These high affinities be- tions on an intact Abd-B- chromosome do not. This in- tween regulatory elements and promoters could have terpretation may also explain other unexpected resulted from promoter competition during the evolu- complementation patterns in Abd-B mutants. For in- tion of the bithorax complex, as described below. stance, adult escapers of flies homozygousfor the muta- It has been suggested that the bithoraxcomplex and tions Abd-BD14and Fab7R9 show male-specificpigmenta- other HOM-C complexes arose through gene duplica- tion on the posterior abdominal segments ( CELNIKERet tion (LEWIS1978). Further evolution could also arise al. 1990; J. E. HENDFUCKSONand S. SAKONJU, unpub from changes in the regulation of these genes. For ex- lished results) . These mutations also exhibit weaker mu- ample, AKAMet al. (1988) have suggested that, in the tant phenotypes when heterozygous with iab7b""k muta- ancestral state, abd-A controlled the specification of ab- tions (see Table 1 legend). Abd-BDI4and Fab7R9 have dominal segments, whereas Abd-B was used exclusively been molecularly characterized and have a deletion and in the terminalia. The interaction of iab elements with an insertion, respectively, within the promoter region abd-A may reflect this ancestral state. Abd-Bm function for the class A transcript, which codes for the Abd-BI in the abdominal segments could arise from the cre- protein ( ~AVORTINK and SAKONJU 1989; GWRKOVICSet ation of a new promoter able to recruit iab functions al. 1990). Oneexplanation for these results is that muta- away from abd-A. Such a promoter competition may tions in the promoter region prevent iab5 from inter- result in higher and higher affinities of promoters for acting with Abd-B and thus free it to look for another their regulatory elements. Trans regulation may result suitable target, namely abd-A, that then substitutes for from these very high affhities between promoters and AM-B. enhancers. It is intriguing to think that the cases of i~b-7~"chromosomes over DfP9 or over Fab7R59 trans regulation that are more difficult to disrupt, such show patches of dark pigmentation in A5-A7, which as that at Abd-B, reflect the higher affinity interactions we have explained as being the result of the regulation of more recently evolved promoters. of one copy of abd-A by iab5. These flies also show We thank HENRIKGWRKOVICS, FRANCOIS KARCH, ROBERT WHITTLE, patches of dark pigmentationin A4. We havenot inves- CHAO-TINGWu and ANA BUSTURJAfor sharing their mutants withus and ANA BUSTURIA,ERNESTO SANCHEZ-HERRERO,KENT GOLIC, IAN tigated the cause of this pigmentation. It could result DUNCANand members of the Sakonju lab for insightful discussions. from very low levels of ectopic Abd-B expression from We are especially grateful toSORAYA PEW for communicating infor- the iab7-& chromosomes in this segment ( CROSBYet mation published in her doctoral thesis about Abd-B alleles she has al. 1993).As an alternative explanation, we hypothesize sequenced. Wethank ANNE BOULET,CHARLES SACKERSON,STEVE that it may be a result of the inactivation of silencer HENIKOFF,and members of the Sakonju lab, especiallyANA BUSTURIA and DANIELCIMBORA, for critical reading of the manuscript. We also elements in the iab regions. The loss of posterior iden- acknowledge the contribution of ANNE BOUI.ETwho first noticed tity from A5 to A7 is undoubtedly due to the inability unexpected complementation in the adult phenotype and in embry- of the enhancer elements in iab5-iab7 that activate onic protein expression between iab7 and Abd-B alleles. Finally, we Abd-B in those segments. Similarly, the gain of posterior thank KEVIN KOZAKand MAJID GOLAFSHANI for technical assistance. J.E.H. was supported in part by a National Cancer Institute predoc- identity in A4 may be due to the inability of silencer toral fellowship to the University of Utah. elements in iab5-iab7 to repress abd-A or Abd-B in A4. Perhaps the disruption of the intact iab region in iab LITERATURECITED AKAM,M., I. DAWSONand G. Tm, 1988 Homeotic genes and the 7be* chromosomes alters the correct interactions of si- control of segment diversity. Development 104 (Suppl.) : 123- lencer and enhancer elements in a way that upsets the 133. Trans Regulation in the BX-C 847

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