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Identification of target genes regulated by homeotic proteins in Drosophila melanogaster through genetic selection of Ultrabithorax protein-binding sites in yeast.

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Copyright 0 1995 by the Genetics Society of America

Identification

of

Target Genes Regulated

by

Homeotic Proteins

in

Drosophila melanogaster

Through

Genetic Selection

of Ultrabithorax

Protein-Binding Sites

in

Yeast

Grant S. Mastick,

'** Renee McKay,

Thomas Oligino, Katya Donovan

and

A. Javier Lopez

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213

Manuscript received June 21, 1994 Accepted for publication October 4, 1994

ABSTRACT

A method based on the transcriptional activation of a selectable reporter in yeast cells was used to identify genes regulated by the Ultrabithorax homeoproteins in Drosophila melanogaster. Fifty-three DNA fragments that can mediate activation by UBX isoform Ia in this test were recovered after screening 15% of the Drosophila genome. Half of these fragments represent singlecopy sequences in the genome. Six single-copy fragments were investigated in detail, and each was found to reside near a transcription unit whose expression in the embryo is segmentally modulated as expected for targets of homeotic genes. Four of these putative target genes are expressed in patterns that suggest roles in the development of regional specializations within mesoderm derivatives; in three cases these expression patterns depend on Ultrabithorax function. Extrapolation from this pilot study indicates that 85-170 candidate target genes can be identified by screening the entire Drosophila genome with UBX isoform Ia. With appropriate modifications, this approach should be applicable to other transcriptional regulators in diverse organ- isms.

M"'

genes that control developmental processes encode transcriptional regulators of unknown target genes. Important examples include the eight ho- meotic genes of the Antennapedia and bithorax com- plexes (HOM-C genes) , whose function determines segment-specific cell fates and behavior in the epider- mis, mesoderm, endoderm and nervous system of Dro- sophila melanogaster (reviewed in: DUNCAN 1987; PEIFER et al. 1987; KAUFMAN et al. 1990; MCGINNIS and KRUM- LAUF 1992). The HOM genes encode proteins charac- terized by a homeodomain DNA-binding motif, and sev- eral lines of evidence have shown that these proteins mediate autoregulatory and cross-regulatory interac- tions and also control the transcription of hierarchically downstream genes (reviewed in SCOTT et al. 1989; HAY-

ASHI and SCOTT 1990; ANDREW and SCOTT 1992; BOTAS 1993). The identity of these downstream genes is an important problem because they must mediate the function of HOM genes to control cell differentiation and morphogenesis. With a few exceptions, however, the number, nature and specific functions of these tar- gets have remained elusive.

Most currently known targets were identified through

Corresponding authm: A.Javier Lbpez, Department of Biological Sci- ences, Carnegie Mellon University, 4400 Fifth Ave., Pittsburgh, PA

15213.

'

Shared first authorship.

'

Present address: Department of Biology, University of Michigan, Ann Arbor, MI 48109.

other criteria but exhibit developmental expression pat- terns that suggest regulation by one or more HOM proteins. Such targets include decapentaplegzc, wingless, distalless, apterous and teashirt. These genes encode ei- ther transcriptional regulators ( distalless, apterous, teash-

irt) or secreted factors that mediate cell interactions

(decapentaplegzc, wingless) (reviewed by ANDREW and SCOTT 1992; BOTAS 1993). This bias may indicate that HOM proteins act primarily to modulate the expression of other regulatory molecules. Alternatively, the trend among known targets may simply reflect the discrete mutant phenotypes (in some cases involving alterations of regional cell identity) that led to the identification and detailed investigation of these particular genes. The observation that ,8-3 tubulin expression appears to be under direct control by HOM genes ( HINZ et al. 1992) suggests that the targets may be a diverse group comprising both regulatory and structural components of the cellular machinery required for morphogenesis.

Systematic attempts to identify target genes have in- volved enhancer trap screens and immunoprecipitation of chromatin fragments with antibodies against particu- lar homeoproteins. In a search for targets of Anten- napedia ( Antp) , examination of 550 enhancer trap lines led to the identification of 16 insertions whose expres- sion differs between leg and antennal imaginal discs of third instar larvae (WAGNER-BERNHOLZ et al. 1991 )

.

One of these enhancer traps appears to reflect the di- rect control by Antp of a nearby transcription unit, spalt

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350 G. S. Mastick et al.

majur, that encodes a protein with zinc-finger DNA- binding motifs ( WAGNER-BERNHOLZ et al. 1991 )

.

Immu- noprecipitation of chromatin fragments with antibodies against the proteins encoded by Ultrabithorax ( Ubx) has led to the identification of connectin as a target ( GOULD et al. 1990; GOULD and WHITE 1992), This gene encodes a leucine-rich cell-surface protein that may play a role in recognition of target muscles by motoneurons (NOSE

et al. 1992). Antibodies against the UBX homeopro-

teins have also been used to immunoprecipitate geno- mic DNA fragments after protein-DNA crosslinking with ultraviolet light in the developing embryo ( GFUBA et al. 1992). These experiments identified scabrous as a putative target. The scabrous gene encodes a protein with similarities to fibrinogen that may play a role in cell interactions during development of larval and imaginal sense organs (BAKER et al. 1990; MLODZIK et al. 1990).

Although these methods have identified several can- didate target genes, they have various shortcomings, and the number of targets reported is still small. En- hancer trap screens are laborious and are equally likely to detect direct targets and indirect downstream genes. The frequency of P element insertion varies greatly among different loci, and the expression of an en- hancer trap line need not correspond perfectly to that of the endogenous gene (WILSON et al. 1990). In princi- ple, chromatin immunoprecipitation screens should enrich for direct targets, but published accounts have not provided estimates of their efficiency or of the num- ber of accessible targets. Immunoprecipitation screens require appropriate antibodies and the construction of a separate DNA library for each protein for which tar- gets are sought. The construction of such libraries from DNA immunoprecipitated after crosslinking is ham- pered by low cloning efficiency ( GRABA et al. 1992)

.

In addition, it might be difficult to identify targets that interact with the regulatory protein only in a few cells or during brief periods in development.

Because of these limitations, we sought a more versa- tile and efficient method that would complement ex- isting techniques for identification and cloning of tar- get genes regulated by HOM proteins. It has been proposed that yeast cells might be used to identify target genes of specific transcriptional regulators by selecting DNA fragments that contain binding sites recognized by such proteins (WILSON et al. 1991; LIU et al. 1993). Using the selectable reporter plasmid pHR307a (WIL-

SON et al. 1991 ) , we performed a screen in the yeast

Saccharomyces cmmisiae to isolate fragments from the Drosophila genome that contain binding sites recog- nized by one of the UBX protein isoforms. We expected that a subset of these sites would be associated with target genes controlled by Ubx. Targets of other HOM genes were also anticipated because of the overlap in

i n vitro DNA-binding specifities of different homeopro-

teins ( HAYASHI and SCOTT 1990). The results of our pilot study suggest that 50% of the clones identified with UBX isoform Ia are associated with target genes and that 170 such genes, of which 85 or more may be regulated by Ubx itself, can be isolated from the Drosophila genome. With appropriate modifications this approach should be applicable to other transcrip- tional regulators that control development in diverse organisms.

MATERIALS AND METHODS

Yeast and Drosophila strains: Saccharomyses cereuisiae strain JM749 (h4ATa/ MATa, ura3-52/ ura?-52, trpl-AlOl/ trpl-

A l o l , lys2-801/ lys2-801, leu2-A1/ leu2-A1, his3-A200/ his3 A200) was used as the host for the selection experiments. This strain was chosen empirically for its ability to grow effi- ciently while expressing UBX proteins on galactose medium. Wild-type Drosophila melanogaster was strain Oregon-R from our collection. The mutant allele U6x".2" balanced over TMI, produces only truncated UBX proteins that lack the homeo- domain and C-terminal sequences because of a deletion and associated frameshift ( WEINZIERL et al. 1987) .

Growth media and conditions: YEPD and synthetic media lacking tryptophan, uracil or histidine were prepared as de- scribed (ROSE et aZ., 1990). 5-fluoro-orotic acid (5-FOA) (PCR, Gainesville, FL) was added to synthetic medium at a concentration of 0.1%. Expression of UBX protein was in- duced to low levels on media containing 20 g galactose plus 0.25 g glucose per liter. Yeast strains were grown at 30". Dro- sophila strains were reared on standard medium (LEWIS

1960) at 25".

Construction of activator plasmids and expression of UBX proteins in yeast: BamHI fragments containing the complete coding regions of type Ia or type IVa U6x cDNAs were excised from plasmids pASIa or pASIVa (LOPEZ and HOGNESS 1991 )

and ligated into the BamHI site of pBM258T ( a URA3, ARSl,

CEN4 plasmid carrying the GALl promoter) (C. KAO and A. BERK, unpublished results) (see Figure 1 ) , downstream of the GALl promoter and upstream of the CYC transcriptional terminator. Plasmids carrying the cDNA inserts in the sense orientation were identified by digestion with PstI. The re- sulting expression constructs, pBM258T-UbxIa or pBM258T- Ubxwd, were transformed into Saccharomyces cervisiae JM749. Media containing 20 g galactose and 0.25 g glucose per liter were used to induce low levels of UBX expression in all experi- ments. Under these conditions the full-length UBX protein wasjust detectable in cell extracts by Western irnrnunoblotting with a rabbit polyclonal antiserum and alkaline phosphatase- conjugated secondary antibodies, using the chromogenic sub-

strate nitro-blue tetrazolium as described ( L ~ P E Z and HOG NESS 1991 ) (data not shown).

Construction of reporter plasmids: Constructs to test the ability of UBX-Ia to activate the HIS? gene on plasmid pHR307a ( WIISON et al. 1991 ) (see Figure 1 ) were generated by inserting one of two fragments containing known UBX- binding sites ( BEACHY et al. 1988) between the EcoRI sites of the reporter plasmid: a 2-kb EcoRI fragment containing the H472 UBX binding-site clusters from the Antennapedia PI promoter or a 1-kb EcoRI/ StuI fragment containing the bind- ing-site clusters U-A and U-B from the U6x promoter. The U-

A / B fragment was prepared by digesting plasmid ~ 4 3 1 0 2 8

(BENDER et aZ., 1983) at the StuI site at -690 bp relative to

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Targets of Homeotic Genes 351

ers onto the resulting blunt ends. Subsequent digestion with

EcoFU trimmed the linkers and released the desired fragment by cleavage at +358 bp. The U-A/ B fragment was then ligated into the EcoRI site of pHR307a. Both orientations were iso- lated for each fragment. The UBX-binding sites are located -400 bp upstream of the transcription start site in the (

+

)

orientation, and 1 kb (UA/B) or 2 kb (H472) upstream in the ( - ) orientation.

Construction of a Drosophila reporter library: Genomic DNA from Drosophila melunoguster strain ORE-R was digested partially with Sau3AI so that half the DNA was <1 kb long. Fragments between 100 and 1000 bp were selected by elec- trophoresis through a 1% agarose gel followed by ortho- gonal electrophoresis onto NA-45 paper (SCHLEICHER and SCHUELL) . Following elution in 50 mM Tris pH 8.0,2 M LiCl, 10 mM EDTA and precipitation with ethanol, the purified fragments were ligated to BamHI-digested, dephosphorylated pHR307a. Aliquots of the ligation mixture were electropor- ated into Eschen'chin coli DH5a, resulting in separate pools of

6000 colonies and a total library of 120,000 plasmids. Plasmid DNA for transformation of yeast was prepared directly from the pooled bacterial colonies.

Screening protocol: Pools of 6000 library clones each were transformed into JM749/ pBM258T-UbxIa using lithium ace- tate ( ITO et d . 1983), and the cells were plated on glucose synthetic medium lacking uracil and tryptophan. The trans- formants were replica plated to synthetic medium lacking histidine and containing 20 g galactose plus 0.25 g glucose per liter (gal-his medium) . After 3 days at 30", the His + colo- nies were replica plated to gal-his medium with or without 0.1 % 5-FOA those colonies that were His+ only in the absence of 5-FOA were picked and retested on gal-his, gal-his+5-FOA and glucose-his media. Strains that were His+ only on galac- tose medium lacking 5-FOA were chosen for further analysis. These strains were also checked for the Leu ~ and Lys- pheno-

types expected for JM749.

Sequencing URE fragments Plasmids were isolated from yeast cells using a rapid protocol (HOFFMAN and WINSTON

1987) and electroporated into E. coli DH5a. The nucleotide sequences of the URE clones were determined using Seque- nase (U. S. Biochemicals). Single-stranded sequencing tem- plates were generated by asymmetric PCR (&LARD et 01.

1991 ) using opposing primers HR1 ( 5 '-CTAATCGCAmA- TCATCCTA3 ' ) and HR2 ( 5 'ATAGGCGTATCACGAGGC- CC3'). HR1 hybridizes between the polylinker and the GALl

promoter in pHR307a; HR2 hybridizes just upstream of the polylinker. The GenBank and EMBL databases were searched for sequences matching the URE fragments using the FMTA

( PEARSON and LIPMAN 1988) and BLASTn (ALTSCHUL et al. 1990) algorithms.

In situ hybridization: Hybridizations to salivary gland poly- tene chromosomes were performed as described ( ASHBURNER 1989) . Probes labeled with digoxigenindUTP were prepared from the URE clones by PCR amplification (LO et al. 1990) using primers HRI and HR2 described above. Embryos were hybridized as whole mounts with digoxigenin-labeled probes as described (TAUTZ and PFEIFFLE 1989). The double- stranded hybridization probes were prepared by extension of random hexamers with DNA polymerase I using as template fragments of genomic DNA or cDNA subcloned into pKS (Stratagene). Hybridized RNA was detected with alkaline phosphatase-conjugated antibody against digoxigenin (Boe- hringer-Manheim) . The hybridized embryos were dehydrated through an increasing ethanol series, substituted into xylene, and mounted in a 1:l mixture of xylene and Permount. The

embryos were examined and photographed under Nomarski

Northern blot hybridization: Poly ( A )

+

RNA was prepared from developmentally-staged embryo collections as described

( KORNFELD et al. 1989). Four micrograms of poly ( A )

+

RNA

from various stages were fractionated on a 1% agarose gel containing 6.7% formaldehyde, transferred to Nytran mem- brane ( SCHLEICHER and SCHUELL) , hybridized and washed under stringent conditions ( SAMBROOK et al. 1989), The 32P- labeled probes were generated by random hexamer primer extension on purified subfragments of genomic DNA clones or cDNAs. As loading controls, the blots were also hybridized with a random-primed probe against the RNA encoding ribo- somal protein 49 ( O'CONNEIL and ROSBASH 1984).

Other methods: Screening of genomic and cDNA libraries, Southern blot hybridization, nucleotide sequencing and other molecular biology techniques were performed using standard protocols ( SAMBROOK et al., 1989).

optics.

RESULTS

Screening strategy: The selection of UBX-binding- site clones from the Drosophila genome was based on their ability to confer UBXdependent activation on a minimal promoter in yeast cells. Two plasmids were used: an activator to provide expression of UBX protein and a reporter in which the test DNA fragments were inserted upstream of a minimal promoter driving ex- pression of a selectable gene (Figure 1A)

.

The reporter constructs were generated in plasmid pHR307a (WIL

SON et al. 1991 ) , which carries the HZS3coding se- quence under the control of an inactive minimal promoter. It was expected that UBX-dependent tran- scription of HIS3would allow a his3- host carrying both plasmids to grow on medium lacking histidine. The activator plasmid used for the screen expressed UBX isoform Ia, which is found predominantly in the epider- mis and mesoderm of Drosophila embryos (LOPEZ and HOGNESS 1991; ARTERO et al. 1992). Conditional ex-

pression of UBX-Ia was achieved by placing the corre- sponding Ubx cDNA under the control of the GALl promoter in plasmid pBM258T as described under MA-

Previous work has shown that UBX proteins can func- tion as transcriptional activators in S. cereuisiae (SAMSON et al. 1989). Before initiating the screen, we tested whether UBX-Ia can activate the HZS3 reporter gene in pHR307a via fragments containing known UBX-binding sites and thus support galactosedependent growth in the absence of histidine. For this purpose a set of reporter constructs was generated by inserting one of two fragments of Dro-

sophila DNA between the EcoRI sites of pHR307a: a 2-

kb EcoRI fragment containing the H472 UBX-binding- site clusters from the Antennupedia P1 promoter and a 1-kb EcoRI/StuI fragment containing the binding site clusters U-A and U-B from the Ubx promoter. In both cases the binding sites consist of TAA repeats ( BEACHY et al. 1988). Both orientations of each insert were isolated,

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G . S. Mastick f / nl. 3.52

A

B

Ubx cDNA

JM749IpBM258T-Ubxla Transform

with reporter library

Glucose -Urn -Trp

0

80 $ Transforments

0. 8 0 carrying both plasmids

Repllce plate to select His+ colonies

Galactose -His

Replica plate to Identify UBXdependent His+ colonies

Glucose -His Galactose -HIS

UBX-lndependent

Galactose -HIS +CFOA

UBX-lndependent

FIGI.RE: 1.-Selection of yeast-bearing reporter clones activated by UBX. ( A ) The activator and reporter plasmids. Expression of URX-la is indt~ccd on galactose me dim^ antl repressed on glucose medium; the lJRA3 marker allows selection lor or against pB”L.58T-UDXla on medium lacking uracil or containing .i-FOA, respectively. Activation of transcription of the HIS3 reporter gene mediated by a UBX-binding site inserted upstream of the minimal promoter in pHR30ia allows a hr3- host to grow on medium lacking histidine. ( B ) Screening strategy. Most His+ colonies are expected to contain clones in which HIS3 transcription is activated hy endogenous yeast factors ( M ‘ I I . . ; ~ ~ e/ rrl. 1991 )

.

His’ colonies with reporter clones that are activated specifically

hv UBX-Ia (enclosed hv sguarcs) are identified hv their His- Dhenotvpe on glucose medium antl on galactose medium containing

placing the binding sites -400 bp upstream of the re- porter transcription start site in the (

+

) orientation and 1 kb ( U A / R ) or 2 kb (H472) upstream in the ( - ) orientation. Each fragment, when present in either ori- entation, mediated URX-dependent activation of the HIS3 reporter and growth of strain JM749 on medium lacking histidine (not shown).

Identification and sequence characterization of UBX

response element clones: A reporter library was con- structed by inserting partial Snu3AIfragments from the Drosophila genome into the BnmHI site upstream of the minimal promoter in pHRSOia, as described in MA- TER1AI.S A N D S1ETI~IOI)S. The screening strategy illus- trated in Figure 1B was used to identi9 plasmids car- rying inserts that mediate activation of HIS3 by URX-Ia (YRX response element clones or UREs)

.

M‘e identified 53 URE clones after screening 30,000 plasmids representing 15% of the Drosophila genome.

The nucleotide sequences of 42 URE fragments were determined and compared against entries in the EMRI, and GenRank databases, as described in MATERIAIS AND METHODS. M7e were concerned that repetitive DNA might dominate the collection o f URE clones for two reasons. First, UBX proteins can bind to relatively sim- ple AT-rich sequences ( REACHY p/ nl. 1988; EKKER p/ nl.

1991, 1992) such as are found in some satellite DNA. Second, ftlnctional binding sites for homeoproteins have been identified in some Drosophila retrotranspo- sons ( CAVAREC and HEIDMANN 1993), and transcrip tion of retrotransposon 412 may be regulated by the HOM gene nl)dominnl-A (nl)d-A) in the mesoderm

( BROOKMAN Pf nl. 1992). However, only 19 of the 42

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Targets of Homeotic Genes 3.58

ila main band DNA is estimated at 43% ( L A I R D and MCCARTHY 1969). Two pairs of duplicates were identi- fied in this group. Seventeen distinct clones in this group were mapped by in situ hybridization to polytene chromosomes, and all were observed to hybridize to single sites, as expected for unique sequences (Table 2 and data not shown).

Previous studies employing random sequence oligo- nucleotides have defined the sequence 5 '-T-T-A-A-T- G>T-G>A-GGS' as the optimal binding site for the UBX homeodomain ( EKKER et nl. 1991, 1992). Within this motif the sequence 5 '-T-A-A-T-3' was found to make the strongest contribution to binding affinity, consistent with its presence in the core of many binding sites defined for UBX and related homeodomains ( EK-

KER et al. 1991, 1992). All but five of the 23 character- ized unique sequence URE fragments contain one or more TAAT sequences, and in 1 1 of these clones one or more TAAT cores are found in contexts that closely match the optimum UBX binding site, for a total of 16 sites where at least seven nucleotides match this motif (Figure 2 ) . The sequence TAATGG, which consists of the strongest preferences within the optimum site con- sensus (EKKER Pt nl. 1991, 1992), is significantly over- represented: the probability of obtaining the seven sites observed is P = 0.021, calculated from the Poisson dis- tribution where P( x ) = e""( m x / x ! ) , x is the number of sites observed, n is the total number of nucleotides analyzed (5558 bp) and m = 2 ~ ~ ( 0 . 2 3 5 ~ ) ( 0 . 2 6 5 " ) . Footprinting experiments on clones CS.4 and C9.5 con- firmed UBX binding to these consensus motifs in vitro (data not shown )

.

To examine the possibility that addi- tional features play a role in UBX binding or function, we compared the sequences flanking the TAAT cores in the unique sequence URE clones. Because most clones contain several TAAT cores but not all of these need play equal roles, we chose for comparison those TAAT motifs that were most likely to represent UBX binding sites in vivo: those matching the optimal binding site TTAATGGCC at seven or more positions and those from URE clones with only one TAAT motif (Figure 2 ) . Alignment of these URE sequences revealed significant preferences for specific nucleotides at nine positions flanking the optimum binding-site consensus (Figure 2 and Table 1 )

.

Most of these positions are on the 5' side of the TAAT motif and at a distance greater than that analyzed by the in vitro-selection experiments that defined the optimal binding site (EWER et al. 1991, 1992). Even when all TAAT sites are aligned, the same sequence bias can still be detected at five of these nine positions (Figure 2 and Table 1 )

.

It remains to be deter- mined whether these nucleotides actually influence UBX binding or function in yeast or Drosophila cells and, if so, whether they make contact$ with UBX pro- tein or act in some other way.

C O N S E N S U S

.r.

.T.

.am..

.

.m..

. .

. . .

.

.

-w.

A 2 . 2 4 A 7 . 1

A 7 . 2

85.1

89.2

89.4

c7.4

C 8 . 4

C 8 . 5

c9.5 c9.7

C 9 . 1 2

014.1

D 1 9 . 3 D 1 9 . 4

E 1 6 . 1 E 1 6 . 4

E Z O . l

92- 30- 217- 77- 209- 88- 254- 16- 119- 555- 560- 532- 119- 16-

5 5 5 -

560- 532- 77- 99- 100- 150- 12- 231- 51- 276- 303- 39- 30- 217- 77- 113- 192- 165- 72- 172- Zll- 110- 407- 446- 411- 418- 163- 81- 91- 24-

C~?AA~"~CCATCTCC GCAAATTGATGT - 5 5

A T T A A A A A A ~ G -48

T C A A A T G T A A C A A T G C A C T T G A G C T C C G

G C G T C T C G G T T G G A C G T C C T C T G A T A -254

A T T C G A C A A T G A G A

C T T A C C A T C T T G A A T A G G T A T A G A T T -51

A C G G T C A A A A C T -246

T T A C T C G G T C A G T C T T T A T G G A G G C G A C

G C C G G C G T G T T G - 2 9 1

A C A A C T T T T T T A T G

C A A G A A C C T T T T -53

C C A A A G G T C C G C - 1 6 7

A A A A T T A C T T T A T A A T A T T T G A T T T C T G T - 5 18

A T T A G A A C G G A G -68

. . . . . .

T A T G C A A A A T T A C T A A T T T T T A T T T G A T T

-

52 3

A A A A T T A A A T T A T A T T T A T G G A G G C G A C

T G C A T A A G A A T T T C A - 5 6 9

A C A A C T T T T T T A T G T G C C C A A A G G T C C G C - 1 6 7

A A A A T T A C T T T A T A A T A T G C A A A A T T A C T

T T A T T T G A T T T C F G T - 5 1 8

A A A A T T A A A T T A T A A

A T T T T T A T T T G A T T - 52 3

C T A A G C T C G C G A T G G

G C A T A A G A A T T T C A - 5 6 9

G G G A A C C T T C C A C A -40

T C A A C G T T G T A C A A C A A G C T C G C G A T G G A - 6 2

(~JGCAAGAACCTTTT -53

T G T T G A A G T G C C A C T T G G A C G C G A A T C C A G T C G A GCTTGAGCCC,GGCACGA T G T T A A C A T T T C T T T C A A A T G T A A C A A T G C A C T T G A G C T C C G G C G T C T C G G T T G G A

A C T C G A A G A G G T T T T T T T T T A T G A T G - 1 5 0

A T G T A G G A T T A A C G C T T C G A C T G C A A - 1 5 5

A G G T G T C A T T A T A G C G A C A T G T T G T T A T G A - 2 0 2

. . . -. . .

A T T A T C G A C T G G A - 2 6 8

A T T A C A A G T G G C A - 2 3 9

GATTCCGACTTGG - 3 4 8

G G T T C T C G T A A G - 2 A T T A G A A C G G A G - 6 8

A T T A A A A A A T T G -40

C G T C C T C T G A T A - 2 5 4

A T C A T T C T G A A C T G T A T A T T T A A T C G A C T T - 4 4 4

A A A A G T C G A T T A A A T A T T C T G A A C T G T A C T A

T A C A G T T C A G A A T G - 4 0 9

A T C G A C T T T T A G - 4 4 8

C T G T A C T A A T T A A T A T T T A G G G C T A T T -455

T C A G T G T T C G T A C C C C G C A G C A A G T T C A A T

T T A T T T T A A A T C -126

T C G G C G A A C A C A - 4 3

A C C T T T G T G T T T C C C A

G T T A C A A T T T G A A C G A T A C G T A G A C C G - 1 2 8

C T G G C G T T G C C G -61

FIGURE 2.-UBX-binding-site motifs in unique sequence URE clones. Portions of the URE sequences flanking the ho- meodomain binding site core motif 5'-TAAT-8' are shown. The name of each URE clone is indicated on the extreme left. Numbers indicate the positions within the URE fragment of the first and last nucleotides for each sequence interval, in order of increasing distance from the HRl primer site; differ- ent sequence intervals within a given clone are listed in this same order. The appropriate strand is shown so that the core appears as 5'-TAAT-3'. Clones B9.2 and B9.4 overlap but are distinct fragments; B7.5 and C7.5 are identical but were isolated independently. Dark shading, nucleotides matching the optimal binding site for the UBX homeodomain, 5'-TTA-

ATGGCCS' ( EKKER PI al. 1992). including secondary prefer- ences at positions G and 7 ( T and A, respectively) ( EWER

d nl. 1992). Light shading, flanking sequence preferences deduced from URE clones containing a single TAAT core motif and from sites matching the optimal URX binding site at seven or more nucleotides ( * , sequence intervals) ; the shaded nucleotides are those that occur at frequencies with probabilities < 0.03 (calculated from the binomial distribu- tion) at positions exhibiting a sequence bias at a level of significance P < 0.01 (chi square test) within this subset of clones (see Table 1 ) .

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354 G . S. Mastick d nl.

TABLE 1

Sequence preferences flanking UBX-binding-site motifs in unique sequence URE clones

TAAT sites used to define consensus

( N = 16) All TAAT sites (N = 45)

x'

X'

Position" Nucleotide Frequency Probability" probability Frequency Probability" probability

-16 C 8 0.01 4 P < 0.01 15 0.04 P < 0.2

-13 T 8 0.026 P < 0.01 I6 0.051 P < 0.2

-10 A 9 0.0086 P < 0.01 18 0.017 P < 0.05

-9 G 9 0.0038 P < 0.01 15 0.04 P < 0.2

-8 T 8 0.026 P < 0.01 23 0.00025 P < 0.001

-7 T 11 0.00077 I'< 0.001 22 0.00071 P < 0.001

-2 T 12 0.000063 P < 0.001 24 0.000085 P < 0.001

+15 T 8 0.026 P < 0.01 17 0.031 P < 0.1

+ I 6 T 11 0.00077 P < 0.001 19 0.0092 I'< 0.02

" Negative coordinates are relative to the 5' nucleotide in the TAAT core; positive coordinates are relative to the 3' nucleotide

"Calculated from the binomial distribution with overall nucleotide frequencies A = T = 0.265, G = C = 0.235. in the core.

with transcription units that might be regulated by Ubx

or other HOM genes, six of these fragments were picked randomly and used as probes to screen Drosoph- ila genomic libraries in lambda vectors. The locations of the UREs within the genomic clones were mapped by Southern blot hybridization, and subfragments from the genomic clones were then used as probes for North- ern blot hybridization against poly ( A )

+

RNA from dif- ferent embryonic stages. In all six cases the genomic clones contained sequences belonging to a zygotically expressed transcription unit (Figure 3 and Table 2 ) . In all but one case the

UREs

were located within 6 kb or less of the closest transcribed sequences (Figure 3 ) . Although some of the RNA patterns were complex, Northern blot analyses with different genomic frag- ments indicated that in each case the multiple bands are derived from a single transcription unit, because nonoverlapping subfragments recognized the same set of RNAs (data not shown )

.

The expression patterns of the transcription units associated with the URE clones were analyzed by in silu

hybridization to whole mount embryos using digoxi- genin-labeled probes made from the corresponding ge- nomic DNA fragments. The expression patterns of T-CS.12 and T-D14.1 were also analyzed by in situ hy- bridization with corresponding cDNA probes. All six transcription units exhibit complex, developmentally regulated expression patterns (Table 2 ) . In every case the expression pattern in the epidermis or mesoderm suggests regulation by one or more HOM genes because it is segmentally modulated and correlates positively or negatively with the expression of the corresponding homeoproteins. Detailed descriptions of these six can- didate target genes, their expression patterns and regu- lation will be presented elsewhere; the following sec-

tions concentrate primarily on those aspects of their expression that relate to control by Ubx.

Expression of

T-A7.1

in the mesoderm: T-A7.1 is ex- pressed in the mesoderm of the trunk region beginning at stage 11 (stages as defined by CAMPOS-ORTEGA and HARTENSTEIN 1985). By stage 13 this expression re- solves into two clusters of mesodermal cells in most trunk segments (Figure 4 ) . T h e more ventral cluster exhibits strong expression in the thoracic segments,

A7.1

F

F

F F

87.2

89.3

B9.4

D l 4.1

-

3Kb

FIGURE 3.-Maps of genomic regions surrounding six URE fragments. Restriction enzyme cleavage sites are indicated: B,

(7)

Targcts o f Homeotic Genes 3.5.5

TABLE 2

Transcription units associated with six URE clones

Gene Imcation mRNAs (kh) Emhryonic expression pattern

T-127. I X9B 1 4.2, 4.6 Mesoderm (segmentally modulated" T437.2 79<: 2.5 Epidermis (segmentally modulated)"

T-R9.3 91FI 9, 7, 6 Mesectoderm

Epidermis (segmentally modulatetl)"

Visceral mesoderm (segmentally modulated)" Endoderm (segmentally modrllatetl)"

Gonadal mesotlerm"

Salivary gland

CNS

T-R9.4 .5.5Cl 11, 7.4, 4.5, 2..5 Epidermis (segmentally modrllatcd)"

T-C9.12 96D 8, 7, 4 Epidermis (segmentally motlulatetl)"

Visceral mesoderm (segmentally modulated)" Endoderm (segmentally modulated)"

CNS (segmentally moduIatetI)"

Gonaclal mesoderm (persisten t ) "

CNS

T-D 14.1 94D 4, 2 Ubiquitous (transient)

" Descrihed in text.

"Strong expression in specific midline and lateral cells in the late emhlvo is restricted to T1, T2, T3.

whereas abdominal segments A1 through A6 exhibit T3 but weaker in Al-A6. Many of the cells in these weak expression in the corresponding cells. The more clusters contribute to the fat body, in which T-Ai.1 ex- dorsal cluster is absent from T1 but stretches diagonally pression can still be detected during late stages (see from the anterior to the posterior edge of the other Figure 5 ) , hut it is not known whether all the cells share segments. Expression in this cluster is strong in T2 and this fate. This segmentally-modulated pattern ofT-Ai.l

A

C

B

D

FIGCWI:. 4 . - C h regulates expression of T-A7.1 in the lateral mesoderm. Transcript distrihutions were detected hy in si/u hybridization with digoxigenin-laheled prohes as described in MATERIAIS ANI) MEI"I-ODS. Embryos are oriented with the anterior e n d to the left in all panels and are staged according to CAMPOS-ORTEGA and HARTENSTEIN ( 198.5). Abdominal segments are numbered. (A and R ) Side views focused on the mesoderm of wild-type emhryos (A) or U I X - emhryos ( R ) a t stage 13. ( C and

D ) Ventral views fc>cusctl on the mesoderm of wild-type ( C ) or IJhx- ( I ) ) emhryos at stage IS. Two clusters o f T-Ai.1-expressing cells are ohsenred in the mesoderm of each trunk segment except T I , which lacks the more dorsal of the two clusters. In wild-

(8)

356 G. S. Mastick rt nl.

A

B

.. .

.**%cA

."-

%

.e

. T e ,;;.l)w W" :, . ,*- * r . ~

<- .&. 4 ',,. Av;\: 4

.-

: .G t &?*

/. .:

M V

4

C

D

*>

e,

e&

-PC.,'

9

?*;

,@".V

' .~

-

* = ' . ? . p-: .; - 4 ~

J "*k

I -~

, .. -

FIGCKI;. .?.--Expression of TA7.1 in the dorsal mesoderm. ( A and R ) Dorsal views focused o n the dorsal mesoderm of stage

14 emhryos. Strong expression extends from T1 to anterior T3 in the dorsal mesoderm of wild-type embryos (arrowheads in

A ) . In U / J . X - emhryos a second domain of strong expression appears within the ahdominal region (arrowheads in €3). ( C and

D ) Dorsal views focused o n the dorsal vessel and lymph glands of stage 16 embryos. In wild-type emhryos ( C ) strong expression

is ohserved onlv in the lymph gland (arrowheads). In U/JX embryos ( D ) ectopic tissue resemhling the lvmph gland forms along

the dorsal vessel in the abdominal region (arrowheads in D ) and expresses TA7.1 strongly. Icxpression in the dorsal arm of the

fat body can also he ohsenwl in A-I).

expression is consistent with negative regulation by UDX, which is expressed from A1 through A7 in the mesoderm (WHITE and WII.C:OX 1985). In Uhx- em- bryos T-A7.1 is derepressed strongly in both clusters within A1 and A2 and more weakly in A3 through A6, confirming that this transcription unit is downregulated by UBX (Figure 4). This pattern of regulation is also consistent with the role of Uhx in establishing regional morphological differences within the fat body (RIXKI

and RIZKI 1978). The failure to derepress T-A7.1 ex- pression completely in segments posterior to A2 in Uhx-

mutants may be due to the overlapping expression and function of nMA in the mesoderm of these segments

( MAc:fAs pt nl. 1990)

.

During stage 14 T-A7.1 expression appears in the dorsal edge of the mesoderm, which gives rise to com- ponents of the circulatory system (reviewed by BATE

1993). A group of dorsal mesoderm cells located within the thoracic segments expresses high levels of T-A7.1 (Figure 5A) and eventually forms the lymph gland, which continues to express T-A7.1 late in development (Figure 5C). These cells are confluent with more poste- rior cells that exhibit low o r n o expression of T-A7.1 and differentiate into the pericardial cells that flank the dorsal vessel. In Ufm- embryos a second domain of strong T-A7.1 expression appears in the dorsal edge of the mesoderm within the abdominal region (Figure 5 B ) , indicating that T-A7.1 is normally repressed by UBX in these cells. Furthermore, in the mutant em-

bryos these cells assume a clustered organization like that of lymph gland cells, instead of forming rows like pericardial cells (Figure 5D).

Expression of T-B9.3 and T-C9.12 in the developing

guk Although other aspects of their expression are un- related, T-BS.3 and TC9.12 are both expressed in spe- cific domains within the visceral mesoderm and endo- derm that coincide with the expression of particular homeotic genes. Both are expressed in the mesoderm and endoderm at the level of PS7, a domain that coin- cides with the expression of Uhx in the visceral meso- derm ( TREMML and DIENX 1989) and labialin the endo- derm (IMMERCI.CJCK et nl. 1990) and lies immediately anterior to the site of formation of the second midgut constriction (shown at preconstriction stages in Figure 6 ) . Expression of T-B9.3 and T-C9.12 in PS7 becomes pronounced during germ band retraction (stage 12) but declines as the second midgut constriction forms during stage 15. T h e expression of both genes in this domain is strongly reduced but not eliminated in Uhx-

embryos in both the mesoderm and endoderm (Figure 6 ) , indicating that these genes are positively regulated by UBX in the midgut. This regulation must be indirect in the endoderm but may involve the Labial protein, whose expression at high levels and concentration in nuclei requires Ufm function in the overlying visceral mesoderm ( IMMERGLCJCK ~t nl. 1990).

(9)

Targets of Homeotic Genes 357

FIGURE 6."Ubxdependent expression of T-B9.3 and TC9.12 in the midgut. ( A and B) Ventral views focused on the midgut of stage 13 embryos hybridized with a digoxigenin-labeled probe for T-B9.3. Prominent expression is seen at the level of PS7

within the visceral mesoderm (arrowheads) and endoderm (arrows) of wild-type embryos ( A ) , but in Ubx- embryos ( B ) this expression is strongly reduced. ( C and D ) Ventral views focused on the midgut of stage 13 embryos hybridized with a digoxigenin- labeled probe for TC9.12. Prominent expression is seen at the level of PS7 within the visceral mesoderm (arrowheads) and endoderm (arrows) of wild-type embryos. In Ubx- embryos ( D ) this expression is strongly reduced.

tric caeca (Figure 7A). This domain coincides with the expression of the homeotic gene Sex combs redutxd ( Scr)

in the visceral mesoderm (TREMML and BIENZ 19S9), suggesting that T-C9.12 may also be positively regulated by

Scr.

Unlike its expression in PS7, TC9.12 is present in this domain at very low levels during stage 13, but its abundance increases and continues at high levels until late in embryogenesis. T-B9.3 is also expressed in this region (Figure 7B), but at low levels that decrease even further after stage 13. In favorably stained older embryos, T-B9.3 appears also to extend more anteriorly, over the developing gastric caeca (not shown)

.

Finally, T-B9.3 is expressed in a band of cells at the posterior boundary of the foregut (visible in Figure 6 A and B; Figure 7 B ) , resembling the expression de- scribed for the homeotic gene poboscipedia ( p b ) ( PULTZ

et al. 1988). Like the pkxpressing cells, the T-B9.3- expressing cells later spread over the esophagus and proventriculus (not shown).

Expression of T-B9.3 and T-D14.1 in the gonadal mesoderm: T-B9.3 is expressed throughout the go- nadal mesoderm beginning at stage 12, but by stage 16 it has become restricted to mesodermal cells at the anterior end of the gonad (Figure SA). T-D14.1 is ex- pressed ubiquitously during early and mid-embryogene- sis but has particularly strong and persistent expression in the gonadal mesoderm. By stage 16 it is restricted to the mesodermal interstitial cells of the gonad, in addi- tion to the central nervous system (Figure SB)

.

Because

differentiation of the gonadal mesoderm and gonad formation depend on the expression and function of

abd-A and Abd-B ( BROOKMAN d al. 1992; CUMBERLEDGE

et al. 1992), T-B9.3 and T-D14.1 may also be under positive regulation by these HOM genes.

Expression patterns in head and tail segments: Four transcription units associated with URE clones exhibit complex expression patterns in the head and tail seg- ments that are also consistent with regulation by HOM genes. The most striking example is T-B9.4, which is expressed exclusively in the ectoderm of head and tail segments between stages 12 and 14 (Figure 9 ) . In the head region this transcription unit is expressed in the pregnathal and gnathal segments, including the dorsal ridge (which is believed to represent the fused dorsal regions of the mandibular, maxillary and labial seg- ments)

.

This coincides with the domains of expression of the homeotic genes labial, D@med

( D

f d ) and p b

and with the anterior part of the Scr domain (reviewed by JORGENS and HARTENSTEIN 1993). In the tail region

T-B9.4 is expressed in AS, which coincides with the posterior part of the AbdB m domain, and in A9/A10, which coincides with the expression of the AbdB r pro- tein ( reviewed by JCJRGENS and HARTENSTEIN 1993).

(10)

358 G . S. Mastick et al.

FIGURE 7.-Expression of T-B9.3 and TC9.12 in the ante-

rior region of the midgut. ( A ) Ventral view focused on the midgut of a stage 14 wild-type embryo, showing expression of

TC9.12 in the visceral mesoderm just posterior to the sites of formation of the gastric caeca (arrowheads). ( B ) Ventral view focused on the midgut of a stage 13 wild-type embryo, showing expression of T-B9.3 in the visceral mesoderm at the

level of PS4 (arrowheads). Expression in parasegment 7 can also be observed in both cases.

expression in the region of the primordia of the ante- rior spiracles and the dorsal imaginal disc of T1. T- C9.12 is expressed strongly in the clypeolabrum and in the labial, maxillary and mandibular segments from stage 12 onward (partially visible in Figure 6, C and D and Figure 7A), and T-B9.3 is expressed strongly in the labial and maxillary segments during the same period (partially visible in Figure 6, A and B and Figure 7B).

As described above, T-B9.3 and T-Cg.12 also exhibit mesodermal expression that is clearly correlated with that of UBX, thus providing an explanation for their isolation in this screen, but the expression of T-B7.2 and T-B9.4 does not show an obvious direct correlation with UBX in any tissue. The latter two genes may have been isolated by promiscuous binding of UBX-Ia to sites that normally mediate regulation by other homeopro- teins. Alternatively, Ubx and other homeotic genes such as Antp and abd-A may play an active role in repressing these putative target genes in the trunk segments. Be- cause of the cross-regulatory interactions among the HOM genes themselves, detailed analyses of the expres- sion of these target genes in single and multiple home- otic mutant backgrounds will be required to distinguish between these possibilities.

DISCUSSION

A genetic method for identification of target genes: Previous studies have shown that activation of

the selectable reporter gene HIS3 in yeast can be used to identify recognition sequences for specific DNA- binding proteins, and it has been proposed that this might provide a method to identify target genes con- trolled by such factors (WILSON et al. 1991; LIU et al. 1993). Our results demonstrate that this approach can be used efficiently to identify candidate target genes controlled by a specific transcriptional regulator in h e

sojhhih mehnogaster. About half of the clones obtained by screening a random sample of the Drosophila ge- nome with UBX-Ia represent single copy DNA, and among these the six clones that were tested all proved to be near or within transcription units whose expression patterns are consistent with regulation by HOM pro- teins. At least three of these transcription units appear to be regulated by UBX itself. Although the method relies on activation of transcription mediated by bind- ing sites in yeast, we note that the candidate target genes identified include transcription units under posi- tive ( e.g., T-B9.3, T-C9.12) and negative ( e.g., T-A7.1) regulation by UBX in the Drosophila embryo. On the basis of this pilot study, we estimate that the Drosophila genome contains about 340 URE fragments that can be identified with UBX-Ia. Among these, 50% may be associated with HOM target genes, whereas 25-50% may be regulated by Ubx itself. Although this suggests a minimum estimate of 80-90 targets for U b x in the genome, we do not know what fraction of the actual targets are accessible by this method. Additional URE clones and target genes might also be isolated by screen- ing with different UBX isoforms.

(11)

Targets of H

A

. - - , 8 .

fl e:,?: ..

.

b * *

B

FIGURE 8.-Expression of T-B9.S and T-D14.1 in the go- nadal mesoderm. Dorsal views focused on the gonads of stage

16 embryos hybridized with digoxigenin-labeled probes. ( A )

T-B9.S is expressed in mesodermal cells at the anterior end of the gonads (arrowheads)

. (

B ) T-D14.1 is expressed in mesodermally-derived interstitial cells throughout the gonad (arrowheads )

.

a significant enrichment for transcription units whose expression is modulated by HOM proteins, in particular for putative targets of UBX.

Compared with other systematic approaches that have been used to identify target genes regulated by HOM proteins, the method described here is techni- cally simple but highly effective. It does not require immunoprecipitating antibodies, purified proteins, subtractive hybridization or the construction of special- ized libraries from small amounts of DNA, and it is expected to enrich for direct targets. In addition, a potential advantage of this method should be the ability to identify genes, such as T-B9.3 and TC9.12, that are regulated by a specific factor only in a small number of cells or during brief periods in development. Such targets may be associated with the regulatory factor only in a small proportion of nuclei, making them difficult to identify by immunoprecipitation of chromatin. With appropriate modifications this approach should be a p plicable to many other transcriptional regulators. For example, the DNA-binding domains of factors that do not themselves activate transcription in yeast can be fused to the activation domain of a protein that does (WIISON et nl. 1991; LIU et nl. 1993), whereas factors that do not bind DNA on their own can be coexpressed with the necessary partner. Obviously, the type and rela- tive frequency of biologically irrelevant or uninteresting positives will depend on the sequence specificity of the

'omeotic Genes 359

protein and the characteristics of the genome and may vary considerably in other cases. However, if they can be eliminated easily (as in this case), the frequency of such positives that correspond to repetitive DNA is less important than the fraction of single copy positives that correspond to authentic target genes.

UBX

specificity and the structure of URE clones: UBX proteins can bind in vitro to relatively simple AT- rich sequences ( BEACHY pt nl. 1988; HAYASHI and S C O ~

1990; EKKER et nl. 1991), so the Drosophila genome is expected to contain tens of thousands of sites poten- tially recognized by UBX-Ia. Most of these sites are un- likely to have functional significance. Thus, Ultrnbithornx

presents a particularly demanding test case for this a p proach, and it is remarkable that the proportion of relevant URE clones is so high. We maintained low levels of UBX-Ia expression in our experiments, and this may have favored selective interaction with types of sites that are functionally associated with target genes. In addition, UBX-binding specificity in yeast nuclei might resemble that in Drosophila more closely than

in nitro conditions. For example, nucleosomes might restrict the range of sequences bound by homeopro- teins ( KISSINCER et al. 1990). Another possibility is that binding to certain sequences induces a conformational change in the homeoprotein that leads to functional interaction with the transcription machinery ( HAYASHI and Scorn 1990).

(12)

360 G. S. Mastick r! nl.

A

r

D

V

.

.

.._

,e-

* "

' e

J

A

A

B Y

J

E

;#

-1-

I.'

;f*

-:.- %.,?

, 3'

z

A

C

' F

s 4 .

. +.

%*',,

'9)

I

FIGURE 9,"Expression of T-B9.4 in head and tail segmel.,. Embryos are oriented with the anterior end to the right. ( A )

Dorsal view of a late stage 12 emhryo showing expression of T-B9.4 in A8 a n d A9 (arrowheads). ( B ) Side view of a stage 14

emhryo showing expression of T-B9.4 in the posterior spiracles ( A 8 ) (arrowheads), clvpeolahrum (short arrow) and gnathal segments (long arrow)

.

( C ) Side view of same embryo focused on the surface, showing expression in the dorsal ridge (arrow- h e a d ) . ( D a n d E ) Dorsal views of the same emhryo at different focal planes showing expression in the clypeolabrum (arrow in D) , dorsal ridge (arrowheads in D and E ) and labial segment (arrow in E ) . ( F ) Ventral view of a late embryo showing strong expression in the salivary glands, which are derived from the ectoderm of the labial segment.

high affinity sites, from many low affinity sites o r from intermediate combinations, and the spatial relation- ships among interacting sites might be highly variable ( BEACHY et nl. 1993). Previously characterized natural binding sites with UBXdependent regulatory function in Drosophila d o exhibit a wide range of structural fea- tures and organization. A regulatory element that medi- ates repression of the Ant/) P2 promoter by UBX and ABD-A proteins in tracheal cells appears to involve 30 suboptimal sites distributed throughout a region of 2.3 kb ( APPEI. and SAKONIU 1993). In contrast, the dpp303 enhancer contains one optimal and four suboptimal binding sites distributed throughout a region of 303 bp

(CAPOVIIJA rt nl. 1994); this structure resembles that of many URE clones (Figure 2 and data not shown).

In Drosophila, cooperative interactions between

UBX and the homeodomain protein encoded by extra-

dmtick may also influence binding-site selectivity (PEIFER and WIESCHAUS 1990; RAUSKOLR et al. 1993; CHAN et nl. 1994; VANDI.JK and MURRE 1994). Thus, it is interesting that 11 unique sequence URE clones also contain one or more close matches to the 9-bp consen- sus extradentick protein binding site (5"ATCAATCAA- 3') (VAN DI,JK and MURRE 1994). URE C9.12, for exam- ple, contains both an exact match and a site that differs by two nucleotides. It will be interesting to determine whether the URE clones can act as UBXdependent regulatory elements in Drosophila and, if so, what struc- tural features contribute to their function.

(13)

Targets of Homeotic Genes 361

investigating the molecular mechanisms by which HOM genes control morphogenesis in these tissues and or- gans.

At least some of the trunk mesoderm cells in which T-

A7.1 is expressed give rise to the fat body, which exhibits morphological features that are controlled by Ubx ( & Z W and

Rrzm

1978). The dorsal mesoderm cells in which it is expressed give rise to the lymph gland, a regional specialization of the circulatory apparatus whose restriction to the anterior end of the dorsal vessel appears to depend on Ubx function (this study). The expression of T-A7.1 in fat body precursors (Figure 4 ) and in the gastrulating embryo (not shown) are very similar to those described for RNAs encoding Abf, a member of the GATA family of transcription factors identified by its ability to bind regulatory sequences from the Alcohol dehydrogenase gene (ABEL et al. 1993). Like T-A7.1, Abf cDNAs map to cytological loca- tion 89B1. Side-by-side comparisons using T-A7. l geno- mic probes and an Abf cDNA probe (courtesy of Ted Abel ) have revealed that their expression patterns are identical, including expression in the dorsal mesoderm and mature lymph gland not previously described for Abf (T. OLICINO and A. J. LOPEZ, unpublished results)

.

Experiments are in progress to determine whether T- A7.1 and Abf are the same or closely related genes. If this is the case, T-A7.1 may control the expression of further downstream genes required for the morpho- genesis or function of the fat body and lymph gland.

In the visceral mesodem Ubx is required to induce high expression and nuclear concentration of the labial

protein in underlying endoderm cells and for the for- mation of the second midgut constriction at the bound- ary between PS7 and PS8 ( BIENZ and TREMML 1988; IMMERGLUCK et al. 1990; PANGANIBAN et al. 1990; REUTER

et al. 1990). These functions are mediated at least in part by the decapentaplepc gene product ( BIENZ and 'TREMML 1988; IMMERGLUCK et al. 1990; PANGANIBAN et

al. 1990; REUTER et al. 1990; CAPOVILLA et al. 1994), a secreted protein belonging to the transforming growth factor

p

family (PADGETT et al. 1987). T-B9.3 and T- C9.12 may encode additional components of the cell- signaling mechanisms involved in these processes. The expression of these genes in both the mesoderm and endoderm at the level of PS7 suggests that they might ]participate in mutual interactions between different cell types required for morphogenesis or regional special- lization within the midgut. The nature of the T-(3.12 ]product is consistent with these ideas. Sequence analysis of T-C9.12 c D N h ( R . McKAY and A. J. LOPEZ, unpub- llished results) reveals that this gene is identical to Dfurl

'( d I U I P - I ) , which also maps to cytological location 96D and encodes a set of kexin-like protease isoforms

HAYFL FLICK

et al. 1992; ROEBROEK et al. 1992, 1993). Members of this protease family in yeast and mammals

are involved in processing various proproteins to re- lease peptides that mediate cell interactions (reviewed in ROEBROEK et al. 1993). The expression of T-C9.12 and T-B9.3 in an anterior domain of the mesoderm that coincides with the expression of Scr suggests that they are also regulated by this homeotic gene and may mediate its control of cell interactions that lead to for- mation of the gastric caeca ( REUTER et al. 1990).

The expression of T-D14.1 throughout the gonadal mesoderm suggests that this gene might play a role in general organization of the gonad or in interactions between germ cells and mesoderm. In contrast, the ex- pression of T-B9.3 becomes restricted to mesodermal cells at the anterior end of the gonad, suggesting that it plays a role in regional differentiation within this organ.

We thank ELIZABETH W. JONES, TRACY RIPMASTER andJoHN WOOL FORD for discussions and technical advice on yeast genetics and molec- ular biology. G.M. was supported by National Institutes of Health predoctoral Lraineeship. R D . was a participant in a Summer Research Experience for Undergraduates program funded by the National Sci- ence Foundation and the Howard Hughes Foundation. This work was supported by a Basil O'Connor Starter Scholar Award from the March of Dimes Birth Defects Foundation to A.J.L. and aided by grant IRG58-31 from the American Cancer Society.

LITERATURE CITED

ABEL, T., A. M. MICHELSON and T. MANIATIS, 1993 A Drosophila GATA family member that binds to Adh regulatory sequences is expressed in the developing fat body. Development 119: 623-

633.

ALw,M. W., D. L. ELISWORTH and R. L. HONEYCWT, 1991 The production of single-stranded DNA suitable for sequencing using the polymerase chain reaction. BioTechniques 10: 24-26. ALTSCHUL, S. F., W. GISH, W. MIILER, E. W. MYERS and D. J. LIPW,

1990 Basic local alignment search tool. J. Mol. Biol. 215: 403- 410.

ANDREW, D. J., and M. P. S c o n , 1992 Downstream of the homeotic

genes. New Biol. 4: 5-15.

ARTERO, R. D., M. AKAM and M. PEREZ-ALONSO, 1992 Oligonucleo- tide probes detect splicing variants in situ in Drosophila embryos. Nucleic Acids Res. 20: 5687-5690.

ASHBURNER, M., 1989 Drosophila: A Laboratoy Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

BAKER, N. E., MLODZIK, M. and G. M. RUBIN, 1990 Spacing differen- tiation in the developing Drosophila eye: a fibrinogen-related lateral inhibitor encoded by scabrous. Science 250: 1370-1377. BATE, M., 1993 The mesoderm and its derivatives, pp. 1013-1090

in The Deuelopmat of Drosophila mdanogaster, edited by M. BATE and A. MARTINEZ-ARIAS. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

BEACHY, P. A,, M. A. KRASNOW, E. R. GAvIS and D. S. HOGNESS, 1988

An Ultrabithorax protein binds sequences near its own and the Antennapedia P1 promoters. Cell 55: 1069-1081.

BEACHY, P. A,, J. V ~ YK. E. , YOUNG, D. P. VON KESSI.ER, B. I. SUN ~t al., 1993 Cooperative binding of an Ultrabithorax homeodomain protein to nearby and distant DNA sites. Mol. Cell. Biol. 13:

6941-6956.

BENDER, W., M. AKAM, F. KARCH, P. A. BEACHY, M. PEIFER et al., 1983 Molecular genetics of the bithorax complex in Lhosophila mlanogaster. Science 221: 23-29.

BIENZ, M., and G. TREMMI., 1988 Domain of Ultrabithoraxexpression sion. Nature 333: 576-578.

Figure

FIGURE 2.-UBX-binding-site URE clones. Portions of the URE sequences flanking  the ho-
TABLE 1 Sequence  preferences flanking  UBX-binding-site motifs in  unique sequence URE clones
Figure 5), hut it is not known whether all the cells share this  fate. This segmentally-modulated  pattern ofT-Ai.l
FIGURE 6."Ubxdependent expression within the visceral mesoderm (arrowheads)  and  endoderm  (arrows) of  wild-type embryos labeled probe for expression is strongly reduced
+4

References

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