The Drosophila Homeotic Mutation Nasobemia (Antpns)And Its Revertants: an Analysis of Mutational Reversion

The Drosophila Homeotic Mutation Nasobemia (AntPNS)and Its Revertants: An Analysis of Mutational Reversion

Paul B. Talbert' and Richard L. Garbe8 University of Washington, Department of Genetics, Seattle, Washington 981 95 Manuscript received November 18, 1992 Accepted for publication July 19, 1994

ABSTRACT The homeotic gene Antennapedia (Antp) controls determination of many different cell types in the thorax and abdomen ofDrosophila melanogaster. The spontaneous mutant allele Nasobemia (AntpNs)and its revertants have been widely used to infer normal Antp gene function but have not themselves been thoroughly characterized. Our analysis reveals that AntpNsconsists ofan internal 25-kb partial duplication of the Antp gene as well as a complex insertion of >4O kb of new DNAincluding two roo transposons. The duplication gives the mutant gene three Antp promoters, and transcripts from each of these are correctly processed to yield functional ANTP proteins. At least two of the promoters are ectopically active inthe eye-antennaimaginal discs,leading to homeotic transformation of the adult head. A molecular and genetic description of several AntpN' revertants shows them to be diverse in structure and activity, including a restoration of the wild type, rearrangements separating two of the AntpN' promoters from the coding sequences, and protein nulls and hypomorphs affecting expression from all three of the promoters. Finally, one revertant has a suppressing lesion in the osa locus far away from Antp. These features explain the unusual homozygous viable nature of AntpNs,suggest a mechanism by which its homeotic transformation occurs, and exempllfy the diversity of ways in which mutational reversion can take place.

HE homeotic mutationsofDrosophila melanogaster development are then directedto be made in abnormal T transform structures of one body segment to those locations such as the eye-antenna imaginal discs which of another. Extensive study of these mutations has de- giverise tothe adult head (FRISCHERet al. 1986; fined two homeotic gene complexes that control the JORGENSEN and GARBER1987; SCHNEWLYet al. 1987; segment-specific gene expression along the anterior- STROEHERet al. 1988). The resulting dominant alleles posterior body axis: the bithorax complex (BX-C; LEWIS typically cause adult antennae to be replacedby meso- 1978) controls the identities of the posterior thoracic thoracic legs. Most dominant alleles, however, are also and abdominal segments, and the Antennapedia com- recessive lethals because needed Antp gene functions in plex (ANT-C; KAUFMAN et al. 1980) controls the identi- thethorax fail tooccur in the event of promoter ties of the cephalic and thoracic segments. The protein replacement. products of these genes are thought to actas transcrip- The dominant allele Nasobemia (AntpNs)arose spon- tion factors that regulate the developmental programs taneously and is not associated with a visible chromo- of other genes in a segment-specific manner (for recent some rearrangement (GEHRING1966). It causes an reviews, see DUNCAN1987; MAHAFFEYand KAUFMAN 1988; antenna-to-leg transformation similar to other domi- et al. 1990). KAUFMAN nant Antp alleles, but differs in that the homozygous The Antennapedia (Antp)gene of the ANT4 is nec- larva is viable and has a wild-type morphology essary for correct thoracic development. Homozygous (WAKIMOTOand KAUFMAN 1981). The gain-of-function loss-of-function Antp mutants typically suffer lethality as (or neomorphic) nature of the AntpNstransformation embryos or larvae and have features of the thoracic cu- ticle transformed into structures normal to more ante- was demonstrated by reverting this phenotype to generate purely recessive lossof-function alleles and defi- rior thorax or to the head (WAKIMOTOand KAUFMAN 1981; STRUHL1983; SATOet al. 1985; SCHNEUWLYand ciencies of Antp (DENELL1972; DUNCANand KAUFMAN GEHRING1985; MARTINEEARIAS 1986; JORGENSEN 1989). 1975; TOTTOLI1977; STRUHL1981). Genetic studies uti- Occasionally a chromosomerearrangement removes lizing these revertants have revealed an embryonic le- normal Antp promoter elements and brings new DNA thality for nullalleles and a larval or pupal lethal period sequences into the gene causing gain-of-function: the for hypomorphic alleles retaining some Antp function normal Antp geneproducts that promote thorax (DENELLet al. 1981). The lethal phenotypes of AntpNs revertants have helped define the normal domains of ' Present address: Universityof Washington, Department of Botany KEb-15, Antp activity in the embryonic thorax (STRUHL1981, Seattle, Washington 98195. * Present address: PathoGenesis Corporation,201 Elliott Avenue West, Suite 1983; MARTINEZ-ARIAS 1986; F~EUTERand SCOTT1990). 150, Seattle, Washington 98119. The phenotypes of adult cell clones homozygous for

Genetics 138 709-720 (November, 1994) 710 P. B. Talbert and R. L. Garber

AntpN' revertants have demonstrated that Ant9 is not Antp mutantsdefective in P1 function or p2 function required in the head (DENELLet al. 1981; STRUHL1981), (Antpl and Antp"). The character of test alleles is inferred but it is required in all three thoracic segments for nor- from the ability of the latter two alleles to complement the mal adult development (STRUHL1981, 1982). former but not each other (ABBOTT and KAUFMAN 1986), from the fact that these alleles lackdetectable ANTP proteins in the These genetic inferences about Antp function have spatial domain of one Antp promoter or the other (R. L. been corroboratedand extendedby the molecular char- Gmm, unpublished results) and from molecular data on acterization of the normal Antp gene. It is at least 102 their gene structures (AntpI7 = AntprL':ABBOT and KAUFMAN kilobases in length (GARBERet al. 1983; SCOTTet al. 1986; Antp23 = Ant?": Scorn et al. 1983; LAUGHON et al. 1983) with two promoters, P1 and P2, located over 60 kb 1986). Antp' (= AntprA58)is altered between the Hind111 apart (Figure 1A).Each promoter generatesa set of tran- site immediatelyupstream of exon 3 and the EcoN site within exon 3, probably due to an insertion event (P. B. TALBERT, scripts with promoter-specific noncoding exons spliced unpublished results). to common protein coding exons(LAUGHON et al. 1986; To test for complementation, 5-15 virgin females heterozy- SCHNEUWLYet al. 1986; STROEHERet al. 1986). The al- gous for Antp", Antp' or Antp" and 5-10 males heterozygous ternative splicing of coding exons generates messages for one of the AnlpNsrevertants under study were mated in for four distinct ANTP proteins (BERMINGHAMand Scorn half-pint bottles.The Antp genotypes of 100 or more eclosing 1988; STROEHERet al. 1988). The two Antp promoters progeny from each cross were inferred from markers on the balancer chromosomes. The heteroallelic mutant combina- direct transcription in distinct spatial patterns in the tho- tions were classified as lethal if no such adults were recovered rax and abdomen (JORGENSEN and GARBER1987; BOULET and as fully viable theif number of heteroallelic mutant adults and Scorn 1988; IRISHet al. 1989; JORGENSEN 1989; was 295% of the number of sibs heterozygous for one of the BERMINCHAMet al. 1990). Two mutually complementing testalleles. Tests for lethality of AntpNs-m'with osa alleles classes of recessive lethal Antp alleles, P1- and P2-, have followed analogous procedures. Wild-typeDNA clones: The genomic DNA fragments from been identified (UUFMANand ABBOTT 1984;ABBOTT and an Antp chromosomal walk (GARBERet al. 1983) were sub KAUFMAN 1986) that lack expression from the P1 pro- cloned into plasmid vectors. Plasmid p66-71contains a 4.5-kb moter or the P2 promoter, respectively (BOULETand ClaI-BamHI fragment from the Antp map coordinates 66-71. SCOTT1988; JORGENSEN 1989). A third class of Antp mu- The inserts of plasmids p44-49 and p70-73 are shown in Fig- tations consists of null alleles, and these fail to comple- ure 2. The Antp exon 1 plasmid used for in situhybridization ment both P1- and P2- alleles (ABBOTT and KAUFMAN was described by FRISCHER et al. (1986). The exon 3 plasmid 1986). used for in situ hybridization contains a I.6kb genomic DNA fragment beginning 100 bp 5' to exon 3 and terminating at the In contrast to thewild-type gene, themolecular prop PstI site 3' to exon 3. The exon 3 plasmid used in RNase pro- erties of AntpN' and its numerous revertants have not tection was described by JORGENSEN and GARBER(1987). The been fully elucidated. The unique genetic features of exon 8 plasmid used in RNase protections contains a l&kb AntpNs and the central roles that its revertants have XhoI-BanzHI fragment including 206 bp of the 5' end of played in understanding Antp function and dominant exon 8. Southern analysis Southern blot analysis was used to de- homeosis prompted us to investigate the genestructures termine the molecular structure of several Antp alleles. and transcriptional properties of these alleles. We have Genomic DNAwas prepared from adult flies asin POTTERet al. found anunusual and surprisingly complex DNA struc- (1980). Restriction endonucleases were obtained from Pro- ture forAntpN' which involvesa partial gene duplication mega Biotecand used in the supplied buffers with the addition and a large insertion. Because of this complexity, rever- of spermidine to 1 mM to aid in obtaining complete enzyme sion of the dominant phenotypehas been able to occur digestion. Electrophoresis was in 0.2% Seakem GTG agarose for higher molecular weight DNA fragments (>lo kb) or in by a wide variety of molecular mechanisms. In this report 0.8% BFU electrophoresis grade agarose for smaller fragments we present a structure-function analysis of AntpNsand (Fly stocks and culture conditions: Fly stocks were raised at males. Since D/(3R)Antpl7 deletes the Antp locus (DUNCAN 23" on standard medium containing cornmeal, molasses, agar, and KAUFMAN 1975), the AntpNs/Df(3R)Antpl7 progeny of yeast extract and tegosept. Lethal Antp alleles were maintained each cross are hemizygous for the single AntpN' allele they over the third chromosome balancers TM3, TM6 or TM6B. received from their father. DNA was extracted from 25-30 of AntpN"m86is a homozygous viable revertant that arose spon- these hemizygous adult progenyfrom four sublines for taneously in our th st AntpNscu stock. All other mutations used Southern analysis. in this work were described in LINDSLEYand ZIMM (1992). Polytenechromosome cytology and hybridization: Chro- The proper cytological information on three alleles was ob mosome squashes for phase contrast microscopy were pre- tained from prior publications: AntpN""' is In(3R}81F;90BC, pared by dissecting larval salivary glands in 45% acetic acid, AntpN"m2is T(3;4)84BI-3;102F (DUNCANand KAUFMAN 1975) fixing them 2-5 min in lactic acid/water/acetic acid (1:2:3) and AntpNs-m72is In(3R)8483;84D (LEWISet al. 1980). and squashing. For in situ localization of cloned sequences, Complementation tests: Crosses were performed to assess AntpNschromosome squashes were hybridized with plasmid the ability of AntpN' revertants to complement the lethality of DNA labeled with digoxigenindUTP according to the Genius Anfp" and Its Revertants 71 1 A Antp' P1- P2- promoters splicing exons

1 2 3 6 567 6 protein coding

I I I I I -8 I 0 10 20 30 40 *%" 60 r70 80 90 100 kb

B AntpNS lambda clones ANslO3 hK518 FI(;URF.1.-The structure of Anfp+.(A) The wild-type Antp gene has two promoters, P1 and P2. which initiate transcription at exons 1 and 3, respectively. Thin lines indicate the patterns of splicing that are found. Exons 1-4 are noncoding; 5-8 encode protein. Coordinates are in kilobases. (B) Diagram of two bacteriophage A clones isolated from Antp" genomic DNA libraries. ANsl03 and AIC518 originate from coordinates 46 and 71, respectively. Both differ from wild-type Anfpsequences by including fragments of roo retrotransposons (black boxes). Also indicated are the long terminal repeat.. (vertical white lines interrupting the boxes) and the direction of transcription of each roo (arrow below the element).

Kit manufacturer (Boehringer Mannheim). After denatur- (The homozygous Antpv'/AntpVs genotype is here ation for 2 min at loo", 10-12 pl of hybridization solution abbreviated Ns.) Each described clones containing the X Denhardt's solution, mw NaCI, .50 mM Na,HPO,, 5 (1 600 insertion in the Antp gene of a transposable element of mw MSI,, 0.1 pg/pl sonicated herring sperm DNA and 2.5 ng/pl tligoxigenin-labeled probe) were added per slide and the family roo or R104 (MEYEROWrZ and HOGNFS 1982; overlaid with a coverslip. Aftersealing with rubber cement the SCHERER et al. 1982) and suggested this as the cause of slide was incubated in a moist chamber for 12 hr at 55". Washes the dominantNs phenotype. Scol-r et nl. (1983) isolated were in 2 X SSC three times for 20 min at 53" and 2 X SSC for the cloneAK518, which cames a truncated roo element 10 min at room temperature. Detection was according to the Genius Kit protocol. (designated roo") in opposite transcriptional orienta- RNase protection: The exon composition of ectopic tran- tion to the Antp intron sequences it adjoins at map po- scripts in Anfp"'/Anfji.''and Anfp'\"."'''/+ eye-antenna imagi- sition 71 (Figure IB). A. KUROWA (SCHNEWTY et nl. nal discswas determined by their protection of antisense RNA 1986; A. KUROIWA,personal communication) isolated probes. Isolation of RNA, preparation of ['*P]CTP antisense the clone ANsl03, which contains the half of a roo RNA probes, and protection from digestion with RNase A and 5' R%se T, were as described in STROEHERef al. (1988). RNA transposon (designated roo') in opposite transcrip from 120-130 eve-antenna discs was used for each lane. tional orientation to adjoining Anfp sequences from Tissue in situ hybridization: To localize Anfp P1 and P2 map position 46 (Figure IB). Two other clones encom- transcripts, imaginal discs were removedfrom third instar lar- passing map position 46 from the KUROIWANs library vae as clusters (to minimize false signals occurring in tissue damaged by dissection) and fixed 10 minin phosphate- have wild-typerestriction patterns uninterruptedby roo, buffered saline (PBS)containing 4% paraformaldehyde and as do clones from map position 71 (not shown). Con- 10 min more in PDS containing 4% paraformaldehvde, 0.1% sidering the clones from these libraries together, there sodium deoxycholate and 0.1% Triton X-100 at room tem- are two distinct Antp map positions at which both wild- perature. Discs were washedtwo times for 5 min in025% Tween type clones (lacking roo) and mutant clones (bearing 20 in PBS and dehydrated. Probe preparation and hvbridiration were performed as in RILEY ef al. (1991). True hybrid- roo) have been isolated from Ns DNA. These findings bation was indicated by a strong dot in each nucleus which led us to carry outfurther characterization of IVS appears to derive from hybridization to nascent transcripts lo- genomic DNA. calized at the Anfpgenes of the synapsed third chromosomes Genomic DNA mapping: To determine the origin of (SIIERMOEN O'FARRELLand 1991; KOWWSKI and MUSKA\ITCH 1992). the variable genomic DNA clones, Southern blot analv- Protein detection: The spatial distribution of ANTP prw sis was camed out ongenomic DNAs prepared from a teins was determined by immunocytochemistry using an anti- wild-type Canton4 stock, from a standardNs stock, and ANTP monoclonal antibody (CONDIE pf al. 1991). Imaginal from four Antp'" genetic sublines. Each of the sublines discs were fixed as for in situ hvbridiration. Embryos were was derived from a single Antp"' chromosome to ensure prepared according to TAV~.and PFFJFLE(1989). that no population variation existed (see MATERIALS AND RESULTS METHODS). The probes were wild-type DNA sequences from both the 46- and 71-kb map regions (p44-49 and Structure ectopic of AntpN' and expression p70-73 in Figure 2). The results confirmed that Ns DNA Two groups (Scorr et nl. 1983; SCHNEUWLY et al. 1986) yields two patterns of restriction fragments at both previously investigated the DNA structure of Antp"'. positions 46 and 71, one corresponding to wild type 712 P. B. Talbert and R. L. Garber

E E BH E E

E 10.6 kb EcoRI 4 Fragment 1An@”

r. B HBH E HE S ES

Probe

11.3 1.6

4.8 9 *

FIGURE 2.4rrespondence of Anfpv’cloneswith duplicatedAnfp”’genomic sequences.The restriction enzyme maps ofAnff’ genomic DNA clones were compared to those of wild-type and An@‘ genomic DNAs by Southern mapping. (A and R) Probes used to map the duplicated sequences atpositions 51 (A) and 46 (€3) are shown djacent to the wild-type maps. Also shown are maps of the roebearingAnfp” clones, AK518 (A) and ANslOS (B). as well as of a standard roo retrotransposon (center). For Ad, only sitesin roo are shown, and for unclonedAnfp‘“ genomic DNA only the Avnl site closest to the prohe-homologous sequences (which could be verified in Southern blots) is shown. (A) Comparison of the AK.518 map with the Southernmaps of wild type and of a 10.6-kb EcoRl genomic DNA fragment containingrooA from An@’. AK.518 contains partof a second tandem rooelement that shares a long terminal repeat with roo’. (B) Comparison of the ANslOS map with the Southernmaps ofwildtype and of an 11.3kb EcoRI genomic DNA fragment containing roo” from Anfp”. (C) Blot showing the detection of a single EroRI fragment (corresponding to the probe) in wild-type DNA and of two fragments in Anf DNA Probe p44-49 hybridized to a 1.8kb band in DNA from Anfp’/Dj(3R)Anfp/ 7fliesand to I l.Sand 4.8kb bandsin Anf$/Anfp’;flies. Similarly, p7O-53 hybridized to a 2.8kbEroRI fragment in wild-type DNA, but to IO.6-and 2.8kb bandsin Anfp”/Anfp”’and AnfpS‘/Df(3R)Anlp/ 7animals. A, Avnl; B, RamHI; E, /animals. However, Pz-derived transcripts ac- (Antp’ and Anyz3)alleles (see MATERIALSAND METHODS) 714 P. B. Talbert and R. L. Garber

~66-71 p44-49 A mm + Ns + Ns

56 40.5 38.4 29.9 23.1

17.1

10.1 11 (9.4)

If I1 \I 71) Antp+ B ;O6 U P44-49 p66-71

- 56 kb cc l1kb c \I AntPs D 46A 71A c 718' data hK5 1 8 ANslO3 FIGURE Q.P^uthern blot mapping the size of the insertion in An@". Probes that hybridize to each sideof the duplicated region were hybridized to Clnl digests of wild-type and Ns genomic DNA to determine the size of the region altered in Anfp"'. (A) Probe pM-71 (mixed with a bacteriophage A DNA probe to detect the size markers) hybridized to an 11 kb fragment in wild-type DNA and to both the same size fragment and a 56kb fragment in Anfp"' DNA. Rehybridization of the same blot with probe p44-49 allowed precise alignment of bands detected bv these probes using the residual signal from hybridizationof p66-71 to the 11-kb band. Probe p44-49 detected a 14kb fragment in wild-type DNA and both this fragment and a 56kb band in Antp"' DNA. The latter band is indistinguishable in size fromthe 56kb band detected by p66-71, but the relative intensityof signal clearly indicates that this band is not due to residual signal from hybridization to p66-il. No hybridization was seen to the 11 kb band when Clnl-digested genomic DNA was hvbridized with p44-49 alone (not shown). (B) Map of the wild-type AnfP gene indicating the relevant CInl sites and the fragments that are detected with a p44-49 probe (solid line) and a p66-71 probe (dotted line). (C) Map for the simplest model of the An@' duplication, which predicts a novel 31-kb Clnl fragment when probing across the duplicated region. (D) Map representing the data from the Southern blot shown in (A). Probes homologous to sequencesat either end of the transposons detected 56kb fragments. Extra material (shown as a hatched region) is inserted between or in the two roo elements. and found that they fell into several classes. Two rever- scribed below. Three revertants (AntjlN""'2,Antp"*.m" tan& (Antp""'" and AntpS"mRd)were completely viable and Antpy"n'i2) were lethal over the P1- test allele but over the P1- and P2- test alleles. This indicates that in completely viable over the P2- alleles, meaning that re- both revertants the P1 and P2 promoters are functional, version had destroyed P1 activity and ectopic P2 expres and their ectopic activities have been reduced or elimi- sion while leaving normal P2 function intact. Six rever- nated. Differences between these two revertants are de- tant.. ~~~p.Vs-nK3. ~~tp.Vs-n1(.'4, ~~lp.Vs.ntC5, Antp" and Its Revertants 715

FIGLIRE4.-Ectopic expression in Antjr". Eye- antenna discs from third instar larvae were hybridized with digoxigenin-labeled probesto detect ectopic Anlp transcripts. The upper and lower halves of the discs develop into adult antenna and eye, respectively. Discs are oriented showing the ventral columnar epithelium with anterior at the top and the medial edge at the left. (A) Using a promoter PBspecific probe (exon 3), some non- specificstaining was observed in wild-typeeye- antenna discs at the edges and in the macrophage- like cells adhering to the center of the antenna disc. Nuclear transcripts were not detected. See M,\TTERIAIS AND METHOI)~for distinguishing signal from background staining. (B) Ectopic P1 transcripts (small dots indicated by arrowhead) were detected in Ns discswith anexon 1 probe. (C) Ectopic P2 transcripts were detected in Ns discs with the exon 3 probe. The disc shown has low background staining; the dark staining is signal from cytoplasmic transcript.. . Nuclear transcripts (dot..) of the antennal region are in the plane of focus. (D) Ectopic ANTP proteins were detected withanti-ANTP antibody in Ns eye-antenna discs.

P1- P2A - P2B -

A AntpNS

46A 71A 71 468 B 1, 0 rb 20 30 40 50A 60A 78A (50B 60B 708 80 90 100 kb

B AntpNS ,,,,,,.,,,,O,,I,I.,OI.II.I.II.,.I. t tt t Revertants rvll rv72 rv2 rvC8 rvC4 FIGURE5.-Summary map of the structures of An@'' and five revertants. (A) The >160-kb AntpNsgene has three promoters. The putative pattern of RNA splicing is shown. (B) The sequences affected in each of the revertants studied are indicated. Anlp"-"'~' deletes the domain A duplicated region, the first transposon roo", and an undetermined amount of upstream sequences. The breakpoints of AntpVS-n!2,Anlps%-n?72, AntpVI-d:4and AntpV%.Wc:8 are indicated by arrows.

Antp"s-ntC9and Antp"s-n'c'2)were apparently null since function in AntpA's"''"llsince rare viable "escapers" are they were lethal over both P1- and P2- alleles. found for certainP1- and P2- genotypes (ARROTT1984; F~~~ revertants (AntpV~.n~C2, A ntpVwd:6, ~~,~pY'-nd.'R ABBOTT and KAUFMAN 1986; JORGENSEN 1989; P. B. and ~~tpX-n!Cll) had partial viability over some of the TALBERT,unpublished observations). It is therefore dif- test alleles as shownin Table1. The survivors frequently ficult to determine whetherAntpNs-mC1l is different from exhibited defects in the thorax. All of the survivors car- a null allele, but Antp"s""c2, AntpNs.n'c6and A~~~NwvCX rying Antp" had defects of the anterior mesonotum clearly have partial P2 function. The differentviabilities similar to those previously described for Antp2'/AntpIi of the latter three alleles over An@ compared with and other viable P1- "escapers" (ARROTTand KAUFMAN AntpZ3probably reflect interactions between other loci 1986). The survivors carrying a P2- allele often had de- on the mutant third chromosomes, since neither P2- fects of the humeral calli, wings and legs which will be allele makes any detectableANTP proteins from P2 (R. described in another report. Antp'r'~n'"Rand Antprs-n'cll L. GARBER,unpublished results) or is consistently more have previously beeninterpreted as null mutations lethal (Table 1). (STRUI-IL1981; ABBOTT and KAUFMAN 1986; MARTINEZ- Clearly thevarious revertants differin the amount and ARIAS 1986). The two surviving AntpVs~n'C"/Antpliindi- type of their residual Antp function. We next investi- viduals do not necessarily indicate the presence of P1 gated their molecular structures and found these also 716 B. P.and Talbert R. L. Garber

TABLE 1 corresponding thefrom borderfragment of the B Partial complementation of Antp promoter mutants by some domain, and a probe from the borderregion detects two AntpNs revertants distinct bands on Southern blots. The same enzyme digest ofrevertant DNA generates thesame two distinct A r&no- P1- and B border fragments unless one of them is disrupted Allele AntpI7 Antp' AntpZ3 by a reversion breakpoint, in which case a novel fragment is produced. We chose a restriction enzyme for 1/64 (2)' 216/736 (28) 157/344 (45) each revertant DNA that generated a fragment includ- 4/65 (6) 188/318 (59) 10/14 (71) 0/21 (0) 65/101 (64) 12/109 (11) ing both the5' (or 3') border of the duplication and the 2/64 (3) 0/72 (0) Not tested revertant breakpoint. The domain containing thebreak- AntpNSrevertants were tested for their abilities to complement point could then be identified because its border frag- the lethalities of Antp alleles lacking P1 function (Pl-: Antp") or ment was disrupted relative to the corresponding border lacking P2 function (P2-: Antp' and AntpZ3) by crossinghetero- zygous adults. For each combination, the number of heteroallelic fragment of AntpNs.The revertant breakpoint positions mutant adults is followed by the number of adult siblings heterozy- are summarized in Figure 5B. gous for the test allele and a balancer chromosome. The inversion breakpoint of AntbY"mc4was found to 'Percent viability, estimated as the ratio of these two numbers, is listed in parentheses. be on the 3' side of exon 3B between 66B and 71B, while the inversion breakpoint of AntpNs-N72is in the uniden- showed major differences, representing four distinct tified material inserted between the roo elements. The modes of reversion. translocation AntpNs""C8has breakpoints between posi- Reversion to wild type: AntpNs-N86is a spontaneous tions 53B-55B and between positions 57B-59.5B. The se- revertant thathas no Antp mutant phenotypewhen ho- quences between these positions are deleted (data not mozygous or hemizygous over the Antp deletion Df(3R) shown). AntpNS""'contains a breakpoint ina 6-kb region Antpli'. ANTP proteins are absent from AntpNs-Ns6/ centered at position 46B, the junction between rooB and the B domain. The cytologically normal revertant AntPNs-m86eye-antenna imaginal discs (not shown). Southern blot analysis of homozygous AntpNs-m86DNA AntpNs-m"was found to be deleted for rooA and the en- with probes collectively spanning positions 44 through tire A domain, but notfor rooB or the3' half ofthe Antp 73 failed to detect either the roeinsertions or the du- gene. Since AntpNS-""fails to complementP1- mutants, plication characteristic of AntpNs,although the threere- the deletionmust extend intosequences essential for P1 striction site polymorphisms characteristic of the parent transcription (Figure 5B). AntpNschromosome were found. AntpNs-m86the refore The null allele AntpNs-mC4has a rearrangement that restores the wild-type gene structure, proteinexpression separates all three promoters from the coding exons, pattern andcuticle morphology. The reversion probably while AntpNs-Nz, AntpNs-N'l, AntpNs-w72and AntpNs-NC8 occurred by unequal homologous exchange between separate P1, P2A and rooA from P2B and the protein- the duplicatedP2 regions of AntpNs,resulting in the loss coding exons (Figure 5B). This suggests that theectopic of the duplication/insertion. The concomitant loss of P2 activity ofAntpNs is associated with P2A, which can no ectopic gene expression and mutant phenotypeis strong longer produce ANTP proteins in the rearranged re- evidence thatthe duplication/insertion found in vertants. In contrast, P2B must provide normal P2 ac- AntpNsis directly responsible for ectopic expression in tivity in AntpNs-Nz,AntpNS""" and AntpNs""72since these the head. three revertants are completely viableover the P2- Revertantswith disrupted transcription units Five re- test alleles. P2B must also be partially functional in cessive lethal revertants were found tohave transcription AntpNs-NC8,which has reduced viabilitywith the P2- units disrupted by rearrangements. The rearrangement alleles (Table 1). P2B must not direct ectopic transcrip- breakpoints for AntpNs-wC4,AntpNs""C8 and AntpNs-m72 tion in the eye-antenna discs of these four revertants, had previously been mapped (GARBERet al. 1983; Scorn however, because such transcription would yield ANTP et al. 1983) to what we have foundhere to be the proteins and the mutant Ns phenotype. duplication/insertion region. We wished to determine If ectopic transcription occurs from P2A and notfrom if the revertant breakpoints fell in duplication domain P2B, then AntpNs-m72should generate transcripts in the A or B. Southern blot analysis of these revertant chro- head containing exon 3A but not exon 8 (since these mosomes detected novel restriction fragments (relative exons are no longer contiguous). We used an RNase to both AntpNsand the balancer chromosomes) that protection assay to test for the presence of exon 3 and were inferredto contain the rearrangement break- exon 8 in the eye-antenna discs ofwandering thirdinstar points. Each breakpoint was assigned to the A or B do- larvae. As shown in Figure 6, Antp transcripts in Ns eye- main by linking the breakpoint to uniquesequences at antenna RNA contain both exon 3 and exon 8, but in either the 5' or 3' border of the duplication as follows. AntpNs-m72/+larvae only exon 3 is present ineye- For any enzyme digest of Ns DNA, the fragment con- antenna transcripts. The size of the protected exon 3 taining the 5' (or 3') border of the A domain differs fragment is appropriate for transcripts initiating from AntpN' and Its Revertants 717

Exon 3 Exon 8

182

162 . -165

149

126- 124

112 u *-

A B FIGURE 6.-Ectopic Antp transcription in AntpN'"''72.Anti- sense probes from exon 3 (A) or exon 8 (B) were separately hybridized to RNA from yeast, wild-type embryos, and third instar eye-antenna (EA) discs from wild-type (+), An@"/ AntpN' (Ns),or Antp'vS-N72/+(~72) larvae. Protection of a part of the probefrom RNase digestion indicated the presence of mRNA complementary to the protected fragment. A fragment of 126 bp from exon 3 and a fragment of 165 bp from exon 8 were protected by Antp RNA in wild-type embryos, but Antp RNAwas absent in wild-typeeye-antenna discs. The exon 3 probe was protected by RNA from both Ns and AntpNs"''72/+ eye-antennadiscs (A). The fragment from exon 8 was protected by RNA only from Ns discs (B). Size markers are shown on the left.

FIGURE7.-Antp expression in four revertants. (A-D) ANTP P2, and cannot bederived from primary transcripts ini- proteins were detected with an anti-ANTP antibody in rever- tiating from P1. Thus we conclude thatP2A but notP2B tant eye-antenna discs and embryos. (A) Protein staining in is ectopically active in AntpNssm72.This ectopic expres- ~~tp'v'-mG5/+ was similar to AntpNs (compare Figure 4D). sion is not specific to AntpNs-"", since nonfunctional P2 (B) ANTP proteins were undetectable in Antp'r'-n'C3/+. transcripts attributable toP2A have been detectedin the (C)An Antphr-wc2/AntpNr-wC29-1 1-hr embryo showed reduced protein staining compared with (D) its wild-type sib. Tissuein eye-antenna discs ofAntpN'-"C8/+ larvae by tissue in situ situ hybridization revealedthat theP2 transcripts found in (E) hybridization (P. B. TALBERT,unpublished results). an Antp""/ + eye-antenna disc werestrongly reduced in (F) the Revertants altered in effective protein level: Three eye-antenna disc of its Antp'"'/AntpN5."'' sib. cytologically normal revertants were investigated that were disrupted in normal P1 and P2 functions as well insertion. For all three revertants the spatial patterns of as the ectopic P2 function: AntpNs-Nc2,AntpNs""C3 and ectopic P2 transcripts detectable by in situhybridization AntpNs-"C6.Based on genetic studies AntpN""'" appears in third instar eye-antenna discs wereidentical to that of to be a null mutation. AntpNs-wC2and AntpN'""C6are likely AntpN' (not shown). Ectopic ANTP protein accumula- to be protein hypomorphsbecause they showpartial sur- tion in AntpNS-Nc6/+eye-antenna discs was not distin- vival in combination with both P1- and P2- test alleles, guishable from that in AntpNs,but ANTP proteins were and because weak antenna-to-leg transformations were undetectable in AntpNS-NC3/+eye-antenna discs (Figure observedin 3 of274 AntpN'-mC2/+and 10 of213 7, A and B). ANTP protein detection was reduced in AntpN'-"'C6/+ adults. Thus, both normal and ectopic AntpNs-n'c2/+eye-antenna discs (not shown), and this Antp functions are partially reduced. At the DNA level reduction was also found in about one quarter of the all three alleles appeared by Southern blot analysis to be 5-1 1 hr embryos of AntpNs-n'c2/+parents (Figure 7C). indistinguishable from AntpN" in theduplication/ The reduction does not appearto be tissue- or promoter- 718 P. B. Talbert and R. L. Garber specific, since careful examination reveals at least Some GARBER1987) with no indication of aberrant processing. ANTP protein even in such weakly staining regions of The primary P1 transcripts, however, must be at least 165 Antp expression as the peripheral nervous system. kb, including an intron of approximately 115 kb be- Reversion by second-sitesuppression: AntpNs-m‘is tween exons 2 and 4. The primary transcripts of P2A and a partial revertantretaining normal Antp functions P2B are processed into indistinguishable RNAs, but the and partial ectopic function.It is associated with separate activities of these two promoters can be in- In(3R)81F;90BC (DUNCAN andKAUFMAN 1975). Since ferred because the P2A and P2B transcripts in AntpN’ the inversion does not break in Antp and Southernblot revertants bearingchromosome rearrangements are analysis detected no alterations in theduplication/ structurally and functionally distinct. Nonfunctional ec- insertion, we suspected that this revertant might have a topic transcripts containing exon 3A initiate from P2A lesion inthe om locus, which is a known dosage- in at least two ofthese revertants and functional thoracic dependent modifier of AntpNs located at 90B1-Dl transcripts originate fromP2B. For AntpNswe infer that (KENNISON and TAMKUN1988). This hypothesis was con- functional ectopic transcripts arise from P2A and that firmed: the AntpNs-mlchromosome was lethal over both P2B provides normal function in the thorax. It seems osa’ and osa2.The AntpNs-m’lesion in osa was implicated likely that P2A is also activein the thoraxand P2B could as the cause of partial reversion by recombining itaway be ectopically active since AntpNs,unlike the revertants, from AntpN’ using another inversion with similarbreak- still has P2B in a cis relation to the A domain and points, In(3R) UbxlZ5,and thereby restoring theNs phe- inserted sequences. notype. In(3R)AntpN’-m’, AntpNs+ osa +/In(3R)UbxfZ5, What causes the ectopic activation in AntpNs?The ec- + Ubx + e females were crossed to In(3R) UbxZz5,f Ubx topic transcription from P2A indicates that all of the + e/TM3, Sb e males, and two ebony recombinants were sequences on the “B” side of the AntpNs-m72inversion recovered with strong antenna-to-leg transformations, breakpoint, including the entire B domain, are unnec- normal halteres and stubbly bristles.These were shown essary for ectopic activity (Figure 5A). This activity must be AntpNs+ + e/TM3, Sb e by their ability to comple- depend either on activating elements in the inserted ment osa mutations. AntpN’-mlsuppressed the dominant sequences on the “Aside of the inversion, on the sepa- phenotype of AntPNsin AntpNs/AntpNs-mlflies, and this ration of P2A from cis-acting negative regulatory sites suppression was shown to take place at the level of located 3’ to position 71, or both. The P1 promoter is ectopic transcript accumulation by tissue in situ probably weakly ectopically activated by the same hybridization (Figure 7F). mechanism that activates P2A. It is unlikely that P1 is indirectly activated by ectopic P2derived ANTP pro- DISCUSSION teins, because other dominant Antp alleles expressing DENELL(1973) suggested that AntpNsdiffers in its ho- ectopic ANTP proteins do not activate P1 in the head: mozygousviability and complementationproperties Antpwuis an exampleof a dominant homeoticallele that from other Antp dominant alleles either because it produces a stable P1 transcript (in the thorax) which is alone gains neomorphic activity without loss of normal not activated in eye-antenna discs by the P2derived function, or because it is a gene duplication with one ANTP proteins there( JORGENSEN and GARBER1987). Fur- gene providing wild-type function and the other pro- thermore, since Antpw” has an inversion breakpoint at viding the new function. We have demonstrated molecu- position 46-49 (SCOTTet al. 1983) that separates P1 larly that An@’ has featuresof both of DENELL’Sideas. from the 3’ half of the locus and does not ectopically Unlike other Antp dominant alleles that gain ectopic activate P1,it is unlikely that ectopic P1 activityin AntpN’ function and lose P1 function by physically removing results solely from the displacement of a negative regu- promoter P1 from the locus, AntpNsretains all parts of latory site located 3’ to position 71. Thus both P1 and the locus and duplicates a large portion of the gene, P2A are probably ectopically activated by non-Antp se- including theP2 promoter. Between the duplicatedpor- quences located 3‘ to these promoters in the AntpN‘in- tions of the Antp gene lies a pair of roo transposons that sertion sequences. This inference parallels the conclu- are likely to haveplayed a role in theduplication/ sion of ABBOTT and UUFMAN(1986) that ectopic insertion event that created Antp. Inserted into or be- activation of P2 in other dominantAntp alleles depends tween the roo elements is about 25 kb of uncharacterized on non-Antp sequences located 5‘ to this promoter. DNA, the natureofwhich cannot be determinedwithout Although foreign activators may be necessary for ec- additional genomic DNA clones. This DNA isprobably not topic activation, the large Antp regulatory regions seem derived from Antp, since probes collectively spanning Antp to be readily susceptible to such external influences. Ex- map positions 44-73 failed to hybridize to the inserted periments with germline transformants carrying re- sequences and two genomic libraries yielded onlywild-type porter constructs and portions of the Antp P1 or P2 clones from other parts of the Antp gene. regulatory regions (BOULETand SCOW1988; ZINKet al. P1 and P2 transcripts of normal sizeshave been 1991) imply that extensive cis-regulatory sequences are detected in AntpNs eye-antenna discs (JORGENSEN and required to maintain repression of Antp in the head. AntpN” and Its Revertants 719

ZINK et al. (1991) havesuggested that the ectopic adult survivors havebeen observed onlyfor allelic com- expressioncommonly seen in theseAntp promoter binations involvingthree P1- alleles (Antp’, Antp”and constructs occurs because they do not carry sufficient AntPCB)that do not separate the P1 regulatory se- elements insulating them fromnearby enhancers. quences in the first intron away from the P2 promoter “Insulatorelements” have been described for a number by chromosomal rearrangement (ABBOIT and ~UFMAN of genes, and serve to isolate a gene regulatory region 1986). from external enhancing or repressing factors,perhaps The differential survival and other diverse properties by defining a separate chromatin domain (reviewed by of the mutants we have investigated emphasizethe need EISSENBERGand ELGIN 1991; WOLFFE1994). Among to distinguish between various kindsof possible rever- homeotic genes, for example, PEIFXRet al. (1987) pro- tants when using them to help understand the normal posed that the regulatory regionsof the BX-C consist of functions of a gene. Promoter mutations that affect the large chromatin domains that canassume either an tissue localizationof gene products can eliminatepart of open active chromatin conformation or a condensed the normal (or ectopic) distribution of proteins ema- inactive chromatin conformation. Boundary regions be- nating from the gene. The more complicated the pro- tween pairs of such domains have been proposed to moter, the more diverse the altered distributions pos- serve as insulators allowingthe domains to be activated sible. Mutations that alter the accumulation or effkacy independently (GWRKOVICSet al. 1990; SANCHEZ- of proteins, but not their distribution, can be found as HERRERO1991). When theseboundaries are deleted, the hypomorphic partial reversionsor nulls. Finally, second- active state ofone domain appears to spread to the other site suppressorsmay help identify proteins that regulate domain. or interact with the original mutation. Similarly, we propose that in An@‘’ eye-antenna disc We thank A. BOULET,H. BROCK,R E. DENELL,T. C. KAUFMAN,J. A. cells an activating element in the uncharacterized ma- KENNISON M. P. Scorn,J. W. TAMKUNand the Bloomington Stock Center terial of the AntpNsinsertion causes anopen chromatin for providing stocks, A. KUROIWAand M. P. SCOTT for sharing clones, conformation in the insertion which spreads into the and D. BROWERfor supplyingantibodies. We thank D. CLARK,L. MARTIN- adjacent regulatory domain of P2A unimpeded by any MORRISand J. SABLfor critical reading of the manuscript. This work was insulator, subjecting P2A to misactivation. A good can- supported by U.S. Public Health Service training grant T32-GM07735 (to P.B.T.) and National Science Foundation grant DMJ3-8803324 didate for a trans-acting factorthat helps to mediate this (to R.L.G.). misactivation is the product of the osa locus. Mutations in the osa gene specifically suppress An@‘’,but do not LITERATURE CITED sect other Antp alleles (P. B. TALBERT,unpublished results). Such allele-specific suppressorsare expected if ABBOTT, M. K., 1984 The relationship between the structure and function of the Antennapedia locus of Drosophila melanogaster. Antp ectopic activation is dependent on foreign regu- Ph.D. Dissertation, Indiana University, Bloomington. lators rather than simply on the displacement of ABBOTT, M. K., andT. C. KAUFMAN,1986 The relationship between the negative regulatory sites common to all Antp alleles. functional complexity and the molecular organization of the Antennapedia locus of Drosophila melanogaster. Genetics 114 Since Antp P1 and P2 are regulated independently 919-942. (JORGENSEN and GARBER1987; BOULXTand SCOTT1988; BERMINCHAM,J. R, JR., and M. P. SCOTT,1988 Developmentally regulated alternative splicing of transcripts from the Drosophila ho- IRISH et al. 1989; JORGENSEN 1989; BERMINGHAMet al. meotic gene Antennapedia can produce four different proteins. 1990), some kind of insulator can be expected to sepa- EMBO J. 7: 3211-3222. rate their regulatory regions.The weak activation of P1 BERMINGHAM,J. R., JR., A. MARTINEZ-ARIAS,M. G. PETITTand M. P. Scorn, by the AntpN” insertion over 71 kb away may imply that 1990 Different patterns of transcription from the two Anten- napedia promoters during Drosophila embryogenesis. Develop the Antp insulating sequences sometimes fail tothe stop ment 109: 553-566. spread of the activating change in chromatin confor- BOULET,A., and M. P. Scorn, 1988 Controlelements of the P2 promoter of the Antennapedia gene. Genes Dev. 2: mation. 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