Proc. Natl. Acad. Sci. USAVol. 90, pp. 11376-11380, December 1993Genetics

The enhancer of position-effect variegation of Drosophila, E(var)3-93D,codes for a chromatin protein containing a conserved domaincommon to several transcriptional regulators

(position-effect variegation/transcription factor/imprinting)

R. DoRN*, V. KRAuss*, G. REUTER*, AND H. SAUMWEBERt*Institut fur Genetik, Martin-Luther-Universitat, 06108 Halle, Germany; and tInstitut fur Entwicklungsbiologie, Universitat zu Koin, 50923 Cologne, GermanyCommunicated by C. Nusslein-Volhard, August 20, 1993

ABSTRACT In Drosophila modifying mutations of posi-tion-effect variegation have been successfully used to geneti-cally dissect chromatin components. The enhancer of position-effect variegation E(var)3-93D [formerly E-var(3)3J encodesproteins containing a domain common to the transcriptionalregulators tramtrack and the products of the Broad complex.It interacts with a number of chromatin genes that suppressposition-effect variegation. Mutations in E(var)3-93D exhibitan imprinting-like effect on the Y chromosome. This effect istransmitted paternally over several generations. Homeotictransformations in E(var)3-93D mutants indicate an involve-ment of the gene products in regulation of homeotic genecomplexes. An antiserum raised against E(var)3-93D proteindetects this chromosomal protein in a large subset of sites inpolytene chromosomes. Our genetic and molecular data sug-gest that the proteins of E(var)3-93D are generally involved inestablishing and/or maintaining an open chromatin confor-mation.

The state of chromatin condensation plays an important rolein the control of gene activity. To identify genes causallyconnected with the regulation of chromatin structure, dom-inant modifier mutations of position-effect variegation (PEV)have been isolated in Drosophila (1-4). These mutationsstrongly suppress or enhance the variegated expression ofeuchromatic genes in rearrangements subject to positioneffects. In PEV rearrangements, euchromatic regions areplaced in the immediate vicinity of centromeric heterochro-matin (5). Spreading of the heterochromatic structure acrossthe newly established junction (heterochromatinization)leads to the inactivation of euchromatic loci. This inactiva-tion is clonally inherited during development and becomesvisible as the mosaic expression of the mutant phenotype ofthe affected loci. In PEV, therefore, a local chromatincondensation is connected with gene inactivation, and dom-inant modifier mutations of PEV are thought to identify lociencoding structural or regulatory chromatin components (6,7).The In(l)wm4h rearrangement has frequently been used for

the isolation and genetic characterization of PEV-modifyingmutations. This paracentric inversion juxtaposes the whitelocus to centric X heterochromatin. All wm4h flies express astrong white mottled eye phenotype. Suppressor mutationslead to an almost wild-type red-eye phenotype, whereasenhancer mutations result in a nearly completely white-eyemutant phenotype.A number of suppressor genes have been cloned, and the

molecular data are consistent with their proposed role incondensation of chromatin (8-10). The products of enhancerloci are suggested to be involved in decondensation of

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

chromatin. This is an important step in the regulation ofgeneactivity. We recently identified by mutagenesis >50 PEVenhancer loci in Drosophila (11). Several of these mutationswere induced by P transposon insertions, and E(var)3-93Dtis the first enhancer locus that we have isolated molecularly. §

MATERIALS AND METHODSFlies were raised at 25°C on standard Drosophila mediumusing Tegosept M as a mold inhibitor. The pUChsneory+-induced enhancer mutation E(var)3_93Dneol29 is described inDorn et al. (11). All other strains used in this study aredescribed in Lindsley and Zimm (12).Pigment concentration was determined by extraction ofred

eye pigments and measurement at 480 nm as described (1).The homeotic transformation of the fifth abdominal seg-

ment into the fourth in E(var)3-93D flies was quantified byclassifying a minimum of 50 offspring males ofeach genotypeinto seven groups (group 0, no transformation; group 1-3,small areas are transformed; group 4, half of the segmenttransformed; group 5, most parts ofthe segment transformed;group 6, complete transformation).DNA from Drosophila melanogaster was prepared as

described by Jowett (13), and plasmid rescue has been doneas described (11). Genomic clones were isolated from awild-type library in EMBL4, and cDNA clones have beenisolated from a 3- to 12-hr embryonic library (14). Libraryscreening, DNA restriction and labeling, and Southern anal-ysis were done by standard procedures (15). DNA wassequenced by the dideoxynucleotide chain-terminationmethod using a T7 sequencing kit.The expression vector was constructed by introduction of

a BamHI site to the start site of translation in cDNA clone129gt-23 by PCR and cloning the resulting BamHI fragmentcontaining the putative stop signal to the expression vectorpGEX 2T (16). This procedure allowed the expression of a91,000 Mr fusion protein, slightly larger than the calculatedMr [26,000 Mr glutathione-S-transferase and 58,000 MrE(var)3-93D], which was enriched as inclusion bodies byserial urea extraction (17). The methods for immunizationand preparation of antisera after injection of this enrichedfraction were as described (18).

Indirect immunofluorescence on polytene chromosomeswas as described (18, 19).

RESULTSE(var)3-93D Interacts with Strong Suppressors of PEV.

E(var)3-93D is located in region 93D of chromosome arm 3R.

Abbreviations: PEV, position-effect variegation; BAL, balancer.tE(var)3-93D is the changed name for E-var(3)3 (11), according toLindsley and Zimm (12).§The sequences reported in this paper have been deposited in theGenBank data base (accession nos. X75498 and X75499).

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Table 1. Interaction of E(var)3-93DneoI29 in trans-heterozygoteswith strong suppressors of PEV

Pigment concentration in offspring males,

Su(var) mutation A40 unit[crossed to SuIE or Enhancer

E(var) males] Su/+* Su/+;E/+ effecttSu(var)2-11 0.194 ± 0.007 0.033 ± 0.003 0.17Su(var)2-131 0.109 ± 0.009 0.004 ± 0.002 0.04Su(var)2-54 0.221 ± 0.002 0.010 ± 0.002 0.05Su(var)2-55 0.247 ± 0.002 0.043 ± 0.008 0.17Su(var)3-310 0.192 ± 0.008 0.012 ± 0.003 0.06Su(var)3-96 0.183 ± 0.002 0.079 ± 0.008 0.43Wi4h;Su(var)Ba1 females were crossed to w.. hIlY;E(var)3-93Dneo/

Bal males, and pigment concentration of Su E offspring males wasdetermined by extraction of red eye pigment.*Control Su(var) males were obtained by a cross of W4h;Su(var)/Bal females to wPu4h/Y;+/+ males without the enhancer mutation.tReduction of suppressor effect by E(var)3-93D"lo is represented bythe ratio of Su E males to Su control males.

Mutants for this locus display a strong dominant enhancereffect on white variegation in w 4h. The insertional mutationE(var)3-93Dneol29 is a cold-sensitive lethal. It is semilethal at29°C, and homozygotes are female sterile. Viable and fertileEnhancer+ revertants ofE(var)3-93Dneol29 have been isolatedthat are precise excisions of the pUChsneory+ mutatorelement. This result proves that all mutant effects observedare from the insertion of the transposon.The E(var)3-93Dneol29 mutation suppresses the strong dom-

inant suppressor effect of the mutations Su(var)2-11, Su-(var)2-54, Su(var)2-55, Su(var)3-3'0, and Su(var)3-96 (Table1), indicating that the products of these genes interact. Many

In(l)vm4h +Pateail ; =cross Irk(1)v4h +

In(l)vm4h1. =

ln(l)v4h

Il(l)Vm4h2. =

In(l)Vm4h

+

Ir(l)vm4h BIx - - =

E-var

:OffsprieggnerationsI

In(l)vm4h Belx

w^ w,"I

+>' +

+ t In(l)v4h

+.

Il(l)vm4h +

3In(l)vm4h +

11. Generation

+

+

+

+

I-lw

In(1)v44 +=%%%% % J

+-w

Table 2. Homeotic effect of E(var)3-93D1eI129 on transformationof abdominal segment A5-A4 and its interaction with a deficiencyof trithorax, a positive regulator of Bithorax complex

A5-A4Genotype of males transformation

E(var)3-93DiIeol29ITM3, Sb Ser 0.06 (106)E(var)3-93D"lol29IE(var)3-93Dweol29 2.43 (46)Dft3R)red'"2/TM3, Sb Ser* 0.34 (104)Df(3R)redls2/E(var)3-93D1neo]29* 1.96 (122)

Flies were classified into seven groups, according to the degree oftransformation ofA5 (group 0, no transformation; group 6, completetransformation); number of flies scored is shown in parentheses.*Flies were generated by crossing Ptf3R)red"P52/TM3, Serfemales tow?l4h;E(var)3_93Dneo°29/TM3, Sb Ser males.

of the known suppressor loci encode chromatin proteins.Su(var)2-11 affects histone H4 deacetylation, and mutationsof this locus display a recessive lethal interaction with theheterochromatic Y chromosome (20). Su(var)2-5 encodesHP1, a structural component ofheterochromatin (21), and theSu(var)3-9 protein may belong to the same class of proteins(B. Tschiersch, personal communication). The observationthat E(var)3-93D mutations interact with all suppressor mu-tations tested suggests that the products of E(var)3-93D arealso chromatin proteins.

E(var)3-93D is a Positive Regulator of Bithorax Complex. IfE(var)3-93D protein is involved in chromatin decondensa-tion, we would predict a positive influence of this protein onthe activity ofgene complexes. This influence is, in fact, whatwe observed. Homozygous E(var)3-93Dnteo29 males showeda significant transformation of the fifth into the fourth ab-dominal segment, whereas the transformation in heterozy-

Test crosses: The enhancer effect aquired by theY chromosome was quantified in the presence of dominantsuppressor mutations of PEV

Suppressor Enhancer effect of the Y chromosomeallele in the Red eye pigments in Su/+ offspringtest cross males*

Su/+ Su/+ (test- Reduction of(control) cross) Su-effect

1. Su-var(2)101 0.194±0.007 0.108±0.003 56%

2. Su-var(2)101 0.194±0.007 0.114±0.013 59%

3. Su-var(2)101 0.194±0.007 0.109±0.003 56%

1 1. Generation

Su-var(2)10 1 0.194±0.007 0.113±0.010 58%

Su-var(2)1301 0.109±0.009 0.010±0.004 90/0

Su-var(3)31 ° 0.192±0.008 0.054±0.009 28%

Su-var(3)908 0.183±0.002 0.108±0.010 59%

FIG. 1. Y-dependent paternal effect of E(var)3-93D"01o29. After the paternal cross the imprinted Y chromosome (hatched chromosome) waspropagated by 11 successive offspring generations, as indicated at left. After crossing offspring males of successive generations towPWh;Su(var)JBa1 (CyO or TM3) females the enhancer effect attained by the Y chromosome was tested. The results obtained by measuring theextracted red eye pigments (1) ofthe male progeny ofthese test crosses are shown in the table at right. The effect ofthe imprintedY chromosomeof wl4h/Y;Su(var)2-1l/+; +/+ males on white variegation (test cross) was compared with that of males of a control cross of the samechromosomal constitution, whose male ancestors had not been in contact with the E(var)3-93D mutation (100o; control). Note that the enhancereffect of the imprinted Y chromosome significantly reduces the suppressor effect of several Su(var) mutations and that it is stably inherited overat least 11 generations.

Genetics: Dom et al.

:IV0

in(,)Vln4hx C..

.............2P, )

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AV Exon 1

TCAAAGAACTCGGACGCGTTCTGCGTGTCGGCCGCGCTAGCAAAAAACTCTGGCTTTAGT 60

V Exon 2TAGTTATTTTATTGGAAAAATATTTAGTCAAGAGCCAACAAACGCATAGATACAGAAAAG 120

IM A D D E Q F S L C W N N FTATTGATTTTCGTCCAAGATGGCGGACGACGAGCAATTCAGCTTGTGCTGGAACAACTTC

N T N L S A G F H E S L C R G D L V D VAACACGAATTTGTCGGCCGGCTTCCACGAGTCGCTATGCCGCGGCGACCTGGTGGACGTC

S L A A E G Q I V K A H R L V L S V C STCGCTGGCCGCCGAGGGCCAAATAGTGAAGGCCCACCGATTGGTGTTATCCGTCTGCTCG

14

180

34240

54300

V Exon 3P F F R K M F T Q M P S N T H A I V F L 74CCCTTCTTCCGCAAGATGTTCACTCAGATGCCGTCGAACACCCACGCTATCGTATTCCTG 360

N N V S H S A L K D L I Q F M Y C G E V 94AACAACGTCAGCCACTCGGCGCTGAAGGACCTCATCCAATTCATGTACTGCGGCGAGGTC 420

N V K Q D A L P A F I S T A E S L Q I K 114AACGTGAAGCAGGACGCCCTGCCCGCGTTTATTAGCACCGCGGAATCGCTGCAAATCAAG 480

V Exon 4G L T D ]N D P A P Q P P Q E S S P P P A 134GGGCTAACGGATAACGATCCTGCGCCTCAGCCGCCGCAGGAATCCAGCCCGCCGCCAGCA 540

A P H V 0.0.0.0 . 0....Q 0.0...P .R 154GCACCTCATGTACAACAACAGCAAATACCGGCTCAGCGGGTGCAGCGCCAGCAGCCTCGC 600

.A.VX A ..XK , E,Y .G....L,GD....,K Q$. 174GCCTCCGCCCGCTACAAGATCGAGACCGTGGACGACGGCCTGGGCGACGAGAAACAGAGC 660

.T...T .....Q.I.... ... .I T.T....A A R.Q.A .. .. QQ... 194ACCACCCAGATTGTCATCCAGACCACGGCCGCACCCCAAGCCACCATTGTACAACAGCAG 720

Q... QZ. Qa I.Q0.jQ IQ . T. 214CAACCCCAACAGGCGGCGCAGCAGATCCAATCGCAGCAGCTGCAGACGGGAACCACGACG 780

.T....J .V.L . /5.... &.. ....I5 K . ..2.. .S.... 234ACGGCGACGCTGGTGTCGACAAACAAACGCTCTGCCCAGCGCTCCTCCCTGACCCCCGCC 840

.AG....V.K...J*B. . K.T.... .T.S...A ....J4.V..,.M,,, 12....A , 254TCCTCGAGCGCCGGCGTGAAGCGCTCCAAGACAAGCACCTCCGCAAATGTTATGGACCCT 900

L .....T....G.... 1.......TTA...rO A A Q L V PQQI274CTCGACTCGACCACTGAAACTGGAGCCACGACAACCGCCCAACTTGTGCCGCAACAGATC 960

T V Q T S V V S A A E A K L H Q Q S P Q 294ACCGTGCAAACCTCTGTGGTCTCTGCGGCAGAAGCTAAGCTCCACCAGCAATCGCCGCAG 1020

Q V R Q E E A E Y D D L P M E L P T K S 314CAGGTTCGACAGGAGGAAGCCGAGTACATTGATCTCCCCATGGAATTGCCCACGAAATCT 1080

E P D Y S E D H G D A A G D A E G T Y V 334

GAACCTGACTACTCTGAAGACCATGGCGACGCCGCCGGCGATGCCGAGGGCACCTATGTG 1140

E D D T Y G D M R Y D D S Y F T E N E D 354GAGGACGATACTTACGGAGACATGCGCTACGACGACAGCTACTTCACTGAAAACGAGGAT 1200

A G N Q T A A N T S G G G V T A T T S K 374GCTGGCAACCAGACTGCGGCAAATACGAGCGGTGGCGGAGTGACGGCGACCACTTCCAAG 1260

A V V K Q Q S Q N Y S E S S F V D T S G 394GCTGTGGTCAAGCAACAGTCCCAGAACTACAGTGAATCATCGTTTGTCGACACCAGCGGG 1320

D Q G N T E A 401GATCAGGGCAACACAGAGGCT 1341

BQ D G P S K D T A I P K P A E H P R K P 421CAAGATGGTCCAAGCAAGGACACTGCCATCCCGAAGCCCGCGGAGCATCCAAGGAAACCA 1401

A T D S V Q K S P R D A D A I P L F D G 441GCAACTGATAGCGTGCAGAAGTCCCCTAGAGATGCTGACGCTATCCCCTTGTTCGATGGC 1461

S R V F V S K V A L A K A Y I P M P M I 461AGCCGGGTCTTTGTGTCCAAGGTGGCTCTGGCCAAGGCGTATATCCCCATGCCGATGATA 1521

Y T C R V M D L V I G K D K L V R I A Q 481TATACATGCCGTGTGATGGATCTGGTGATTGGTAAAGACAAGCTGGTGCGCATCGCCCAG 1581

H E E T T D K D L I Q D I I T H V C K V 501CACGAGGAGACCACGGACAAGGACCTGATCCAGGACATCATAACCCATGTGTGTAAAGTG 1641

F A L R G N Q L T P S A V Q E F I D H K 521TTTGCCCTGCGCGGCAACCAGCTGACCCCGTCCGCAGTACAGGAGTTCATTGACCACAAG 1701

L S T L K L M P I K E G K 534CTGTCTACTCTTAAACTTATGCCTATAAAGGAGGGGAAATAGTTTAAATTGATTGG 1757

FIG. 2. (Figure continues in next column.)

Proc. Natl. Acad. Sci. USA 90 (1993)

CQ A A T S A S A T K I P P R K R G R P K 421CAGGCCGCCACATCCGCATCTGCAACTAAGATTCCACCACGTAAACGCGGCAGACCGAAA 1401

T K V E D Q T P K P K L L E K L Q A A T 441ACAAAGGTTGAGGATCAGACGCCCAAGCCGAAGCTCCTGGAAAAACTACAGGCGGCTACC 1461

L N E E A S E P A V Y A S T T K G G V K 461CTGAACGAGGAGGCCAGCGAGCCTGCGGTGTACGCCTCCACCACGAAGGGCGGTGTCAAG 1521

L I F N G H L F K F S F R K A D Y S V F 481CTAATCTTCAATGGTCACCTGTTCAAGTTCTCGTTCCGCAAGGCGGACTATTCCGTGTTT 1581

Q C C Y R E H G E E C K V R V V C D Q K 501CAGTGCTGCTACCGTGAGCACGGCGAGGAGTGCAAGGTGCGGGTGGTATGCGACCAGAAA 1641

R V F P Y K G E H V H F M Q A S D K S C 521AGAGTCTTTCCTTACAAGGGCGAGCACGTGCACTTCATGCAGGCCAGCGACAAAAGCTGC 1701

L P S Q F M P G E S G V I S S L S P S K 541CTACCCTCTCAATTCATGCCTGGCGAATCGGGAGTCATATCCTCGCTGTCGCCCAGCAAG 1761

E L L M K N T T K L E E A D D K F.D E D 561GAGCTGCTGATGAAGAATACCACCAAGCTCGAGGAAGCCGACGACAAGGAGGATGAAGAT 1821

F E E F E I 0 E I D E I E L D E P E K T 581TTCGAGGAGTTTGAGATTCAGGAGATCGACGAGATTGAATTGGACGAACCGGAGAAGACG 1881

P A K E E E V D P N D F R E K I K R R L 601CCCGCCAAGGAGGAAGAAGTGGACCCCAATGATTTCCGTGAAAAAATCAAGCGACGACTG 1941

Q K A L Q N K K K 610CAAAAAGCACTGCAGAACAAGAAGAAGTGACACGGAATATCCCTTAGATCTTCAAACTAC 2001

ACCTTTTACTATATAAACTTAACTAAGATCGAGAAGGCTCAAGAAGGCCCAACAGTACAA 2061

GATCTGAGCTGCCTAAGGCTTCACATTATACTTTCTATATCCATTTGGGTCTTAAATAGC 2121

CCTAATAATTCTAAATAATTAAGTTTTTCGTAATAAAACAGTGGCAATAAATC polyA (130)

FIG. 2. Nucleotide and predicted amino acid sequences ofE(var)3-93D cDNA clones. Nucleotide positions are numbered attop; amino acid residues are numbered at bottom. (A) Exon/intronstructure of the common 5' part of all cDNA clones sequenced,including exon four. The conserved N-terminal protein domain isboxed, the glutamine/threonine-rich domain is underlined by dots.The two alternative cDNA sequences after exon four are shown incDNA clone 129gt-23 (B) and cDNA clone 129gt-38 (C). The acidicstretch in the protein deduced from cDNA clone 129gt-38 is under-lined; the potential polyadenylylation signals are double-underlined.

gous E(var)3_93Dneol29 males used as a control was very weak(Table 2). Moreover, we found additive effects of this ho-meotic transformation in trans-heterozygotes of E(var)3-93Dneol29 with the mutation Dft3R)redP52 (Table 2), whichwas shown to be a positive regulator ofBXC (22). This resultindicates that E(var)3-93D like trx is a positive regulator forBithorax complex.

Alleles of E(var)3-93D Display a Stable Paternal Effect onPEV. We find a strong paternal enhancer effect of E(var)3-93Dneol29 on PEV. The w m4h/Y;+/+ male offspring of Wm4h/Y;E/Bal fathers express an enhanced phenotype, althoughthe enhancer mutation is not present. Such an effect is notfound in the w n4h/Y;+/+ males when the mutation ismaternally inherited. Genetic crosses indicate that the Ychromosome is responsible for this paternal enhancer effect.The Y chromosome from an E(var)3-93Dneo male can betransferred into a completely unrelated genetic backgroundby consecutive outcrossing without losing its acquired abilityto enhance white variegation in wm4h males (Fig. 1). There-fore, our results indicate that the Y chromosome in w7#4h/Y;E/Bal males is genetically imprinted by the E(var)3-93Dneol29 mutation. This imprinting is stable for at least 11generations, the maximal number of generations tested.

Cloning ofE(var)3-93D Locus by Transposon Tagging. Clon-ing of the genomic region containing the E(var)3-93D locuswas initiated by plasmid rescue of E(var)3_93Dneo129 (11), andseveral cDNA clones have been isolated. Sequence analysisof genomic and cDNA clones demonstrated that thesecDNAs represent partially overlapping transcripts, whichresult from alternative splicing. Abundant transcripts in thesize of 2.0 and 2.3 kb were detected on Northern blots (datanot shown). The sequence deduced from two cDNA clones

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Genetics: Dorn et al. Proc. Natl. Acad. Sci. USA 90 (1993) 11379

E-var(3)3 Q F SLLC[W NN F N rN L 1nC 7 H I , |L )L

tramtrack R P C L R W N N H Q S N_ L V HFCTLHA

BRcore H F C L R W N N NY Q S S I T KA F NL_RE LLID tA F

[Consensus - F A W N N F

V D V S L A A H G Q I V K A H R L V L S V C S P F F R K MN 7T DV T L A V G Q H L K A 1M V L S A SP Y F L V

V7D V T L A CE G R I K A H S V V L S A C S F L LKD V -L A E C K ANH

Q M P S N T H A V F FL N N V S H S A DK L i Q F M Y C C EH P HHK P I V I L K DV F Y S M LKS L CD F M Y R G E

T P C - NAH P IV I L L Q DV N F MD H A L V E F I Y H G E

L - P - VL V L F Y

) A P

V D QE R L TA LR V A ES LR I KG L T E V X P

H K S [L S L K T A V N_V L T -" Q A F f) T

v- v -C L F A K C __r_

FiG. 3. Comparison of the N-terminal part of proteins from E(var)3-93D, Tramtrack, and Broad core (BRcore) of the Broad complex. Theprotein sequence of E(var)3-93D and BRcore starts with aa 6, whereas the Tramtrack protein sequence starts with aa 5. Identical amino acidpositions are shown as shadowed boxes. The consensus sequence of all three proteins is shown in the open box at bottom.

is shown in Fig. 2. The part common to both cDNAs is shownin Fig. 2A. Downstream of exon 4 the cDNAs differ insequence, and both differing sequences are shown in Fig. 2B and C, respectively. The insertion site of the transposon inthe E(var)3-93Dneol29 mutation is in the third intron, 121 bpdownstream to the 5' splice junction, and phenotypic rever-tants show precise excisions of the transposon, confirmingthat this transcription unit identifies the E(var)3-93D gene.The Putative E(var)3-93D Proteins Contain a Conserved

N-Terminal Domain. The two cDNAs contain open readingframes encoding proteins identical in their N-terminal regionbut different at their C termini (Fig. 2). The common region

contains stretches of glutamine and threonine that render ithydrophilic in character. The hydrophobic N-terminal 120 aashow a striking homology to the Broad core sequence of theBroad complex (23) and to the N terminus of the Tramtrackproteins (Fig. 3; ref. 24). Our preliminary studies indicate theexistence of >12 genes in the Drosophila genome containingthis conserved N-terminal domain. Within this region theresidues between aa 33-57 (68% identity and 96% homology)and aa 88-104 (53% identity and 89%o homology) are moststrongly conserved between these proteins. In contrast to theTramtrack and Broad complex proteins, no zinc-finger motifhas been identified in E(var)3-93D proteins. The C terminus

FIG. 4. Distribution of E(var)3-93D proteins on polytene chromosomes (a, c, and e) DNA staining with Hoechst 33258 dye. (b, d, andf)Indirect immunofluorescence using the E(var)3-93D antiserum; in c-f, the chromosomes are oriented distal toward the left; location ofANTCis indicated by arrowheads in c and d; that of BXC is indicated by arrowheads in e and f; note that several bands are stained at BXC by theantiserum. [Bar = 12 ,um (a and b) and 1.5 ,gm (c-f).]

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of the protein E(var)3-93D-A (Fig. 2B) is rich in chargedamino acids. Stretches of acidic and basic amino acids arepresent at the C terminus ofE(var)3-93D-B protein (Fig. 2C).Such stretches are typically found in proteins that bind DNA(e.g., nucleolin, high-mobility group proteins, and nucleo-plasmin) but do not display marked sequence specificity (25).Our observation that E(var)3-93D mutations display ho-

meotic effects similar to those ofthe trx-group genes supportsthe view that E(var)3-93D proteins are involved in geneactivation. An antiserum raised against a full-length E(var)3-93D-A protein detects a nuclear protein in Drosophila tissueculture cells and embryos both in indirect immunofluores-cence and on immunoblots (data not shown). The corre-sponding preimmune serum did not show any reaction. Onpolytene salivary gland chromosomes this protein is found ata large number ofloci including the BXC- and the ANTC-genecomplexes (Fig. 4) and the white locus (data not shown).Many of these sites can be correlated with less condensedinterbands, although not all interbands are stained (compareFig. 4 c and d with e andf).

DISCUSSIONWe have cloned an enhancer of PEV and demonstrate thatE(var)3-93D codes for chromosomal proteins found in a largenumber of sites on polytene chromosomes. The E(var)3-93Dproteins are generated from alternatively spliced transcriptsand contain a conserved N-terminal domain common to othernuclear proteins like Tramtrack and Broad Complex pro-teins. The latter are presumed to function as gene-specificregulators of transcription (23, 24). In the case of theTramtrack protein, the zinc-finger motif has been demon-strated as essential for binding to the fushi tarazu and evenskipped promoter sequences (24). However, sequence-specific binding may not be required for the E(var)3-93Dproteins. The acidic C termini may be important for theinteraction with DNA, and the conserved hydrophobic do-main could be involved in protein-protein interactions toestablish an open chromatin conformation. This result issupported by our finding that E(var)3-93D mutations displayhomeotic effects similar to those of mutations in the trx-groupgenes (G.R., unpublished work). In preliminary studies wefound strong interactions between several mutations in trxgroup genes and E(var)3-93Dneo129, resulting in inappropriateexpression of homeotic genes. This result is in accordancewith the presence of E(var)3-93D proteins at the Antenna-pedia Complex and Bithorax Complex loci. The presence ofE(var)3-93D proteins in a large number of sites in polytenechromosomes supports the hypothesis that these proteins aregenerally involved in establishing and/or maintaining an openchromatin conformation as a prerequisite for transcription.The imprinting-like effect of E(var)3-93Dneol29 suggests thatthe proteins encoded by this gene also play an active role intransmitting chromatin conformations between successivegenerations. The Y chromosome, stably imprinted by theE(var)3-93Dneol29 mutation, modifies the expressivity of sev-

eral Su(var) mutations (Fig. 1), indicating that such paternaleffects are molecularly complex. Further molecular andgenetic analysis of E(var)3-93D and interacting chromatingenes could uncover the molecular mechanism of imprintingin Drosophila, and in perspective Drosophila could be asuitable model system for a detailed explanation ofimprintingphenomena observed in mammals.

We are grateful for the technical assistance by B. Habbig. Theproject was supported by a grant from the Deutsche Forschungsge-meinschaft (Sa 338/3-1; Do 407/1-1).

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