Nonclassical Regulation of Transcription: Interchromosomal ... · in gene regulation across a wide variety of species. With the availability of a wide array of genetic tools, and

INVESTIGATION

Nonclassical Regulation of TranscriptionInterchromosomal Interactions at the Malic enzyme

Locus of Drosophila melanogasterThomas E Lum and Thomas J S Merritt1

Department of Chemistry and Biochemistry Laurentian University Sudbury Ontario P3E 2C6 Canada

ABSTRACT Regulation of transcription can be a complex process in which many cis- and trans-interactions determine the final patternof expression Among these interactions are trans-interactions mediated by the pairing of homologous chromosomes These trans-effects are wide ranging affecting gene regulation in many species and creating complex possibilities in gene regulation Here wedescribe a novel case of trans-interaction between alleles of the Malic enzyme (Men) locus in Drosophila melanogaster that results inallele-specific non-additive gene expression Using both empirical biochemical and predictive bioinformatic approaches we show thatthe regulatory elements of one allele are capable of interacting in trans with and modifying the expression of the second alleleFurthermore we show that nonlocal factorsmdashdifferent genetic backgroundsmdashare capable of significant interactions with individualMen alleles suggesting that these trans-effects can be modified by both locally and distantly acting elements In sum these resultsemphasize the complexity of gene regulation and the need to understand both small- and large-scale interactions as more completemodels of the role of trans-interactions in gene regulation are developed

THE regulation of gene expression is a complex processoften involving many levels of organization In a simple

model gene expression is determined by intragenic inter-actions (eg enhancerndashpromoter interactions occurring incis on the same chromosome) In more complex modelsof regulation expression is also influenced by the three-dimensional genomic structure and organization of chro-mosomes In the latter more realistic models nuclearorganization governs interactions between neighboringgenetic elements such as transcription factories hetero-chromatin homologous chromosomes and genes that arecapable of acting in trans to modulate gene expression(reviewed in Henikoff and Comai 1998 Wu and Morris1999 Lanctot et al 2007 Xu and Cook 2008) These trans-interactions create levels of complexity in gene regulationinvolving interphase chromatin structuring its impact onpairing of homologs and the potential exchange of transcrip-

tional or regulatory proteins between homologs One suchtrans-interaction transvection is the modification of geneactivity through interactions between the regulatory elementsof one allele and its homolog on the homologous chromo-some (reviewed by Pirrotta 1999 Wu and Morris 1999Duncan 2002) Here we describe a case of trans-interactionat the Malic enzyme locus (Men) (Merritt et al 2005) po-tentially transvection and examine interactions between pu-tative regulatory elements on homologous chromosomes

The term ldquotransvectionrdquo was first coined by E B Lewis(1954) to describe complementation and trans-interactionsbetween two Ultrabithorax (Ubx) alleles in DrosophilaLewis found that certain Ubx alleles were able to comple-ment each other and that this complementation could beinterrupted by chromosomal rearrangements that disruptedlocal homolog pairing (Lewis 1954) Since this initial de-scription the term transvection has generally been used todescribe interallelic interactions at a single locus in whichtwo mutant alleles on paired homologous chromosomes in-teract leading to either higher or lower gene expressionthan would be predicted from each allele independently[eg enhancer elements acting in trans to promote geneexpression or zeste-mediated silencing reducing gene expres-sion in paired alleles (reviewed in Duncan 2002 Southworth

Copyright copy 2011 by the Genetics Society of Americadoi 101534genetics111133231Manuscript received July 25 2011 accepted for publication August 29 2011Supporting information is available online at httpwwwgeneticsorgcontentsuppl20110907genetics111133231DC11Corresponding author Department of Chemistry and Biochemistry LaurentianUniversity 935 Ramsey Lake Rd Sudbury Ontario P3E 2C6 CanadaE-mail tmerrittlaurentianca

Genetics Vol 189 837ndash849 November 2011 837

and Kennison 2002)] Transvection effects have been de-scribed for more than a dozen Drosophila melanogastergenes (eg Gelbart and Wu 1982 Davison et al 1985 Babuet al 1987 Geyer et al 1990 Leiserson et al 1994 Gindhartand Kaufman 1995 Hopmann et al 1995 Morris et al1999 Southworth and Kennison 2002 Coulthard et al2005 Merritt et al 2005 Gohl et al 2008 Ou et al 2009)and in other species including humans (eg Koeman et al2008) Several models for the molecular mechanisms oftransvection have been described including trans enhancerndashpromoter interactions insulator bypassing and pairing-dependent silencing (reviewed in Duncan 1987 Henikoff1997 Henikoff and Comai 1998 Southworth and Kennison2002) Of particular interest for the study presented hereare enhancerndashpromoter interactions occurring in transmdashthetype of interactions that our work suggests are responsiblefor the trans-interactions that we observe at the Men locus

In Drosophila and many other dipteran species homolo-gous chromosomes are extensively paired throughout thenucleus of interphase somatic cells (Stevens 1908 Metz1916 Fung et al 1998 reviewed in McKee 2004) Thispairing facilitates a wide array of homology effects includingtransvection that have remarkable roles in gene regulation(Wu and Morris 1999) Similar pairing and transvection-likeeffects have also been found in plants (reviewed in Chandlerand Stam 2004 Grant-Downton and Dickinson 2004 Stam2009) mammals (Thatcher et al 2005 Bacher et al 2006Xu et al 2006) and fungi (reviewed in Shiu et al 2006 Vyaset al 2006) emphasizing the importance of trans-interactionsin gene regulation across a wide variety of species With theavailability of a wide array of genetic tools and because ex-tensive pairing occurs between homologous chromosomesDmelanogaster is an excellent system in which to study theseinterallelic pairing effects on gene expression

Cytosolic malic enzyme oxidizes malate to pyruvate andis one of four enzymes primarily responsible for the re-duction of the cofactor NADP+ to NADPH (Wise and Ball1964) In earlier work using D melanogaster Men knockoutalleles to study the interaction between the NADPH enzymesunexpectedly high non-additive amounts of MEN activitywere observed when small deletion knockout alleles wereheterozygous with wild-type alleles (Merritt et al 20052009) The high level of MEN activity was shown to bedependent on the specific deletion creating the knockoutallele and not simply on physiological up-regulation sug-gesting transvection or a similar type of trans-interactionbetween the functional and nonfunctional Men alleles (Merrittet al 2005)

Here we characterize these trans-interactions betweenfunctional and nonfunctional alleles at the Men locus Usinga suite of 19 Men null activity (knockout) alleles (MenExi2)that differ only in their genomic lesions we tested MENenzyme activity and Men gene expression in MenExi2MenExi+ heterozygotes using two different wild-type alleles(MenExi+) We found significant differences in MEN activityacross these heterozygotes that are dependent upon specific

characteristics of each deletion The presence or absence ofputative regulatory elements based upon computationalprediction was correlated with differences in trans-interac-tion across the five sets of knockout alleles with significantlydifferent levels of trans-interaction In addition we exam-ined the impact of five different genetic backgrounds on thetrans-interaction effects finding significant interactions be-tween specific deletions and specific backgrounds Thesesignificant interactions illustrate the importance of large-and small-scale interactions between the functional andnonfunctional alleles in trans-interactions

Materials and Methods

Stock lines

Isothird chromosome lines (VT26 HFL53 JFL12 CT21MD76 and MD5) were a subset of nonlethal third chromo-somes extracted from isofemale lines (Duvernell and Eanes2000 Merritt et al 2005) Inbred lines used as commongenetic backgrounds were obtained either from Blooming-ton Drosophila Stock Center at Indiana University (BDSCBloomington IN line no 6326) or from the Eanes lab(VT83) (Merritt et al 2005) The P-element line used forthe P-element-mediated deletion EP(3)0517 was obtainedfrom the Szeged Drosophila Stock Center (Szeged Hungary)The line used as the source of transposase (stock 2030)and a line containing a deficiency that covers the entire Mengene Df(3R)kar31 (stock 6160) were obtained from theBDSC Three P-element-derived excision alleles for the Mengene initially described in an earlier articlemdashMenEx3+(wildtype) MenEx92 (knockout) and MenEx152 (knockout)(Merritt et al 2005)mdashwere also included in both the en-zyme activity and quantitative PCR (qPCR) analysis

Culture conditions

Flies were maintained on a standard cornmeal media at 25with a 12-hr12-hr photocycle All enzyme activity andqPCR assays were conducted on male flies aged for 4ndash6 daysafter emergence

Mutagenesis

We performed P-element-directed mutagenesis (Rorth1996) to create small deletion alleles of the Men locus usingEP(3)0517 The P-element construct carries the eye colormarker gene mini-white (w+mC) and is inserted 473 bases59 to the Men start codon (Merritt et al 2005) The EP(3)0517 element was mobilized by crossing virgin females tomales carrying the D2-3 source of transposase Dysgenicmales EP(3)517D2-3TM3 were crossed to w2 6326DrTM8 Sb females and w2 Sb Dr+ males ie malescontaining a copy of the EP(3)517 chromosome with theP-element excised were collected Candidate excision maleswere again crossed with TM8 for complete third chromo-some isolation and male and female progeny were back-crossed to establish each line The excision chromosomesgenerated are essentially isogenic differing only in a small

838 T E Lum and T J S Merritt

region at the point of P-element excision Recovered excisionchromosomes were screened for MEN activity Flies contain-ing chromosomes showing no MEN activity ie knockoutallele candidates were placed in a common X and secondchromosome background using simple crosses (w11186326)(Merritt et al 2005 2009) Seventeen new alleles with noapparent MEN activity (knockout lines) were generated(Table 1)

For each new allele 15 kb of genomic sequence sur-rounding the transcription start site (TSS) was characterizedusing a series of overlapping PCR amplifications Primerpairs were designed to amplify 1-kb regions that over-lapped with the next primer pair (Supporting InformationFigure S1) Regions that did not amplify in comparisonwith a wild-type positive control indicated regions of theexcision site Primers that flanked the excision site wereused to directly amplify and sequence the excision breakpoints

Fly homogenizations

Flies were homogenized for enzyme activity determinationin 100 ml of grinding buffer (50 mM TrisndashHCl pH 74) perfly In general five flies per sample were homogenized how-ever if there were insufficient flies fewer were assayed andthe homogenate volume was adjusted accordingly Eachsample was centrifuged at 20000 middot g for 5 min at 4 topellet and remove all insoluble residues

Enzyme kinetic assay

Malic enzyme activity was measured using 10 ml of whole-fly homogenate in 100 ml of assay solution in a SpectraMax384Plus 96-well plate spectrophotometer (Molecular Devi-ces) Absorbance at 320 nm was measured every 9 sec for

3 min at 25 and activity was quantified as the slope of thisline Each sample was assayed three times and the meanwas used for statistical analysis The assay solution consistedof 100 mM TrisndashHCl 034 mM NADP+ 50 mM MnCl2 and50 mM malate (pH 74)

Quantitative RT-PCR

Total RNAwas extracted and purified from four groups of fivemale flies for each genotype using the RNeasy kit (QIAGEN)One microgram of total RNA was reverse-transcribed usingrandom hexamers and High Capacity cDNA Reverse Tran-scription Kits with RNase Inhibitor (Applied Biosystems) ThePCR reaction consisted of 2 ml of undiluted cDNA template04 mM of each primer and 02-mM probe and QuantitectProbe PCR Master Mix (QIAGEN) in a total volume of25 ml cDNA synthesis of samples lacking reverse transcrip-tase were used to ensure that there was no genomic DNAcontamination and ldquono-templaterdquo blanks were used to ensurethat there was no contamination within our reagents Theprimers and probe flank the intron between exon 2 andexon 3 [3R 8540309ndash8540365 exon 3 and 4 respectivelyas annotated by FlyBase forward (GTATTGCCAACCTGTGCC)reverse (AGC TTGTGTTCGGTGAGT) and probe (56-FAMATGGTGGATAGCCGTGGTGTCA3IABkFQ)] FlyBase anno-tates four exons with two transcript variants that differ inthe genomic location of the first exon for Men However onthe basis of the examination of expressed sequence tags(ESTs) (Figure S2) and annotation by the Paired-End Anal-ysis of TSS (PEAT) project (Ni et al 2010) we suspect thatthe true TSS is downstream of this predicted first exon andthus this first ldquoexonrdquo was ignored in our analysis Two qPCRreactions per template were performed in parallel usinga Mastercycler ep realplex Thermal Cycler (Eppendorf) All

Table 1 Detailed summary of MenExi2 alleles

ExcisionDeletionsize (bp) Insertion

5 deletionfrom TSS

3 deletionfrom TSS

MenExi2MenEx3+

activity

MenEx8 1651 TCATCATCATAACATAAAG 21347 304 07249 6 00397MenEx9 4080 NA 22203 1877 06876 6 00313MenEx12 2500 TTAATA 21635 865 07422 6 00397MenEx15 2414 NA 22306 108 07560 6 00364MenEx30 NA Not sequenced NA NA 07922 6 00374MenEx43 2682 NA 22044 638 06639 6 00352MenEx48 3581 NA 22014 1567 07553 6 00345MenEx52 3058 TAAACAGACATT 21623 1435 06619 6 00361MenEx55 16231 NA 210245 5986 05414 6 00356MenEx57 1379 GATATATAG 21304 75 07454 6 00462MenEx58 535 AACAATTCGCAGAGTCCT 2215 320 08400 6 00401MenEx60 646 CATGATGAAATAATAAATAATAATA 2213 433 10569 6 00490MenEx76 669 CATGATGAAATAACATAA 2215 454 09097 6 00443MenEx77 2070 TAAATAA 21404 666 07648 6 00440MenEx81 2765 NA 21759 1006 06670 6 00300MenEx86 2239 NA 22147 92 07665 6 00391MenEx109 3551 NA 22635 916 07523 6 00521MenEx119 1379 GATATATAG 21304 75 07663 6 00451MenEx125 2378 GTT 21513 865 07032 6 00317

MenExi2 lines are partitioned into overlapping groups by a Tukeyrsquos HSD test of differences in transvection-influenced enzyme activity MenExi2 alleles that do not sharea letter code are significantly different

Trans-Interactions at the D melanogaster Men Locus 839

sample expression results were normalized to RpL32 [(for-ward CCATTTGTGCGACAGCTT) (reverse ATACAGGCCCAAGATCGT) and (probe 56-FAMACCAAGCACTTCATCCGCCAC3IABlk_FQ)] and quantifiedreported relative toMenEx3+ using the DDCT method (Livak and Schmittgen2001)

Data analysis

Flies of specific genotypes were generated by mating fivemales and five virgin females in single vials Each cross wasdone in six separate vials (replicates) to allow for statisticalanalysis All samples were weighed prior to homogeniza-tions and this wet weight was used as a covariance tostandardize the MEN activity for differences in fly sizebetween individuals Multivariate analysis of variance testswere conducted to ascertain possible significant differencesin MEN activity across heterozygote individuals for Menknockouts Tukeyrsquos honestly significant difference (HSD)multiple comparison tests were conducted to group excisionlines for similarity in MEN activity

Phylogenetic footprinting

Approximately 15 kb of genomic sequence around theMen locus 9 kb upstream of the TSS and 6 kb downstreamwas aligned and the degree of sequence divergence wasquantified across 10 Drosophila species (D melanogasterD simulans D sechellia D yakuba D erecta D ananassaeD pseudoobscura D persimilis D mojavensis and D virilis)D melanogaster genomic sequence was obtained from Fly-Base (httpwwwflybaseorg) while the other Drosophilasequences were obtained from the assemblyalignmentannotation of 12 related Drosophila species project (ranalblgovdrosophila the corresponding genomic sequencecould not be reliably aligned from D grimshawi and D wil-listoni so these two species were not included in our phylo-genetic analysis) BigFoot a Bayesian alignment andphylogenetic footprinting software was used to align geno-mic sequences and score regions for the degree of conserva-tion with settings of 1 million burn-in cycles and 5 millionsamples (Satija et al 2009) MatInspector was used to ex-amine aligned sequences for potential transcription factor-binding sites (TFBS) across the same 10 species with anoptimized core matrix similarity of 075 (Cartharius et al2005) The combination of BigFoot and MatInspector anal-ysis was used to determine regions and potential regulatoryelements at the Men loci that affect trans-interactions

Results

P-element excision-derived Men alleles

We generated a series of excision-based Men knockoutalleles that varied in the size and location of the genomicexcisions Using P-element-mediated dysgenesis (Tsubotaand Schedl 1986 Salz et al 1987 Rorth 1996) with theEP(3)0517 fly stock line we generated 17 novel knockoutalleles (denoted as MenExi2) (Table1) Two other knockout

alleles were previously reported (MenEx92 and MenEx152)(Merritt et al 2005) Regions flanking the deletion siteswere amplified and sequenced (summarized in Table 1)We were unable to amplify the excision site of MenEx302suggesting that this allele has a more complex excision sitetherefore this allele was not considered further in this studyAll 18 (ie not including MenEx302) deletions removeregions of the TSS annotated by the PEAT project (Ni et al2010) and examination of Men ESTs (Figure S2) and assuch are considered to be promoter deficient All deletionswere found to excise some portion of the protein-codingregion and upstream nonprotein-coding genomic regions(Figure 1) MenEx552 was the largest deletion generatedand has a 16321-bp excision that removes a majority ofthe Men coding region as well as a substantial portionupstream of the TSS MenEx602 and MenEx582 have thetwo smallest deletions 646 and 535 bp respectively whichremove a portion of the first exon including the TSS Theremainder of the deletion alleles have 13- to 4-kb dele-tions that are roughly centered around the EP(3)0517 in-sertion site All the deletions are homozygous viable but ashomozygotes show no MEN enzyme activity P-elementmutagenesis largely modifies only the site of insertionexcision (Cooley et al 1988 Spradling et al 1995) Sinceall alleles are placed into a common genetic background(w6326MenExi2) we expect that the only differences ingenetic architecture between the lines are at the Men locusexcision site although it is possible that other changescould exist elsewhere on the third chromosome

Trans-interaction and MEN activity in heterozygotes isdependent on the deletion of the knockout allele

Transvection is the modulation of gene expression due tointeractions between paired homologous chromosomes(reviewed in Duncan 2002 and Southworth and Kennison2002) and our results are consistent with the high levels ofexpression that we observe being driven by transvection Atpaired loci enhancer elements have been shown to act ona trans-promoter when its cis-promoter is deficient increas-ing overall gene expression (Geyer et al 1990 Morris et al1998 1999 2004 Lee and Wu 2006) In the specific case ofthe Men locus and MEN enzyme activity we expect thatexcisionwild-type heterozygotes would have 50 wild-typeactivity if the MenExi2 allele contributed no activity InsteadMenExi2 heterozygotes have shown greater-than-expectedlevels of MEN activity that are dependent on the deletionof the MenExi2 allele a phenomena that has been pre-viously attributed to transvection (Merritt et al 20052009) Previous examination of MenEx92 and MenEx152

suggested that the level of trans-interaction might be de-pendent on the specific deletion allele (data not shown)To test for variation in up-regulation between deletionalleles MEN enzyme activity in MenExi2 allele heterozy-gotes was quantified using two different wild-type thirdchromosome lines (w6326VT83iMenExi2HFL53 andw6326VT83iMenExi2VT26) The average MEN enzyme


activity between both sets of crosses was used in the analysis(Figure 2 the activities of the excision alleles in either back-ground are shown individually in Figure S3 and Figure S4)While all homozygousMenExi2 alleles have no MEN activitywe found that the amount of MEN enzyme activity inMenExi2 heterozygotes varied significantly depending onthe specific MenExi2 deletion in each cross (F18222 =188916 P 00001) Tukeyrsquos HSD test placed the linesinto five overlapping bins on the basis of their activity(Figure 2)

Trans-interactions are transcriptional phenomena andwe expected that the up-regulated levels of MEN enzymeactivity observed resulted from elevated transcription fromthe functional Men copy in the heterozygotes due to trans-interactions with the promoter-deficient MenExi2 alleles Totest if the up-regulation of MEN activity that we observedtruly was a transcriptional phenomenon Men gene expres-

sion was measured using quantitative RT-PCR (qRT-PCR)from heterozygotes from each Tukeyrsquos HSD test bin ofMenExi2VT26 (Figure 3A) As predicted we found a signif-icant effect on gene expression of the specific deletionalleles similar to the allele-specific differences in enzymeactivity (F7 53 = 30665 P = 00098) MEN enzyme activityand Men gene activity were significantly correlated withR2 = 0700 (Figure 3B) strongly suggesting that the observeddifferences in enzyme activity are transcriptionally drivenMenEx552 the largest deletion shows no up-regulationwith heterozygotes expressing essentially 50 of wild-typegene and enzyme activity Similarly MenEx602 heterozy-gotes have essentially wild-type levels of gene expressionand enzyme activity even though one allele is completelynonfunctional suggesting very strong up-regulation Thelack of exact fit in the comparison of MEN enzyme activityand Men gene expression may simply reflect the greater

Figure 2 Heterozygote MEN enzyme activitygraphed as a ratio of wild-type activity acrosstwo isochromosomal backgrounds HFL53 andVT26 ie heterozygote activity was calculatedas the average activity of MenExi2HFL53 andMenExi2VT26 All MenExi2 lines are groupedinto overlapping bins on the basis of TukeyrsquosHSD classification For each excision n frac14 6and samples were measured in triplicate Errorbars represent standard error

Figure 1 Genomic map of MenExi2 excisions PredictedMen TSS is at ldquo0rdquo The bars indicate excised regions


error associated with measuring gene expression rather thanprotein activity a technical limitation of qRT-PCR

Given the nature of the knockout alleles (removing theTSS) and the general model for trans-interactions weexpected that all observed gene expression (and proteinactivity) was from the wild-type functional allele To testthis expectation we crossed a subset of the deletion allelesto a wild-type allele that could be distinguished by a singleSNP site in the coding region The Men gene has a GCpolymorphism at position 338 (Sezgin et al 2004) all ofthe MenExi2 alleles have a G at this position ThreeMenGExi2 lines (MenEx552 MenEx432 and MenEx602)were crossed to a MenC line MD5 to create MenGC hetero-zygotes (w6326VT83iMenExi2MenGMD5MenC) TotalRNA was extracted from male MenExi2MD5 heterozygotescDNA was synthesized and the polymorphism-containingregion of the Men gene was amplified and sequencedMenEx552 acts as a negative control since the deletion re-moves this polymorphic site BothMenEx432 andMenEx602

have this site intact If both alleles were actively being

transcribed the sequence of the polymorphic site wouldhave appeared as a double GC peak Only MenC transcriptwas found upon analysis of the sequence indicating thattranscription occurs only on the functional allele and not onthe excision allele (Figure S5)

Phylogenetic footprinting and the prediction ofregulatory elements

Our results indicated that the amount of trans-interaction isdependent on the excision allele present suggesting that thesize location or sequencemdashor a combination of all threemdashof the deletion modulates the interactions In general wefound a broad correlation between deletion size and trans-interactions the largest deletion (MenEx552) is associatedwith no up-regulation midsize deletions have moderate lev-els of up-regulation and small deletions have high levels ofup-regulation A regression analysis of activity and deletionsize however found no significant relationship between ac-tivity and deletion size (Figure S6) In general there is nocorrelation between deletion size and amount of trans-interaction (up-regulation) across the midsize deletions inthe data set This lack of correlation could be a function ofthe low amount of variation in trans-interaction betweenmidsize deletions and the relatively low accuracy of ourgene expression assay However it seems more likely giventhe lack of pattern across all 19 alleles that this lack ofsimple correlation with deletion size indicates that deletionsize alone does not fully explain the impact of each alleleon the trans-interactions Instead interactions appear to bea function of the presence or absence of specific genomicsequences in each allele suggesting that specific regulatoryelements are present or absent in the different excisionalleles

To identify such potential elements we used bothphylogenetic footprinting and regulatory element predictionanalysis In general regulatory elements are likely associ-ated with regions of high phylogenetic conservation (Tagleet al 1988 Blanchette et al 2002) while consensus sequen-ces can be used to predict specific elements Regulatory el-ement prediction analysis within these regions allowed us toidentify consensus regulatory sites We attempted to identifypotential modifying elements by comparing this in silico reg-ulatory element discovery with genomic regions that differbetween alleles that cause different amounts of trans-inter-actions (heterozygous MEN activity)

We examined 15 kb of genomic sequence across 10Drosophila species for conservation using BigFoot a com-bined statistical alignment and phylogenetic footprintingsoftware that uses a Markov chain Monte Carlo samplingmethod to detect conserved functional elements withoutassuming a fixed alignment (Satija et al 2009) The 15-kbregion encompasses the entire Men coding region as well as86 kb upstream of the TSS covering all of the deletionsites in the alleles used We identified three potentially in-formative regions of high conservation that may impacttrans-interactions and Men expression regions indashiii (Figure 4)

Figure 3 (A) The averageMen gene expression measured fromMenExi2HFL53 and MenExi2VT26 heterozygotes relative to MenEx3+HFL53 andMenEx3+VT26 respectively Expression was normalized to RpL32 Rela-tive expression was calculated using the ΔΔCT method (Livak andSchmittgen 2001) (B) MEN enzyme activity plotted against Men geneexpression


The first region of interest is an 650-bp region that cen-ters on the Men TSS and spans both the MenEx602 andMenEx582 deletions (Figure 5) Regions ii and iii are con-served regions that are absent in Tukeyrsquos HSD test groupsCD D and DE (Figure S7) All three of these regions are richwith predicted regulatory binding elements and provide alist (Table S1) of potential regions that impact overall Mengene regulation and transvection Although we limited oursearch to regions of high conservation we recognize that itis possible for D melanogaster to have functional elementsthat are not conserved across all the species examined Insome cases however predicted TFBS do coincide very wellwith regions of high conservation For example knirps found920 bp downstream of ATG within region iii (Table S1Figure S7) was predicted well within a peak of high con-servation This approach of associating quantified trans-interactions between MenExi2 and wild-type alleles and anin silico discovery of regulatory elements provides a step inidentifying potential regions in the genomic architecture thatimpact trans-interactions

Different genetic backgrounds and their impact ondifferent excisions

Overall examination of the 19 excision alleles heterozygouswith the VT26 and HFL53 third chromosomes indicatedthat different excision alleles differentially impact trans-interactions and suggested a general grouping of the allelesinto sets with similar impact on trans-interactions Howevera linear regression between the VT26 (Figure S3) and theHFL53 (Figure S4) heterozygote activities suggested thatsome excisions have a different impact in different back-groundsmdasha background by MenExi2 allele interaction effect(Figure 6) To further examine this possibility we selecteda subset of the excision alleles and crossed these to fivedifferent third chromosome genetic backgrounds (VT83HFL53 JFL12 CT21 and MD76) (Figure 7) We selectedan allele that deviated from the correlation (MenEx82)alleles that followed the correlation (MenEx432 MenEx862and MenEx762) and low- and high-activity alleles (MenEx552

and MenEx602) Importantly we found similar levels ofMEN enzyme activity across the five background lines to

Figure 4 The degree of conservation within the Men ge-nomic region measured across 10 Drosophila genomes(gray lines left y-axis) and average relative activity of theexcision heterozygotes (height of bars representing exci-sion groups) Tukeyrsquos HSD groups (overlapping groups AndashE) were determined by relative MEN enzyme activity (righty-axis) Red lines show total deletion size within that binand the black lines indicate common deletion size per binDeletion groups without a black line are made up of onlyone MenExi2 allele Regions of interest have a bracketabove and are denoted i ii and iii

Figure 5 MenEx58 and MenEx60 mapped above the degree of conservation (Figure 4) and the predicted regulatory elements The degree ofconservation is measured from 0 to 1 in which 0 represents no conservation and 1 represents high conservation Overlapping predicted regulatoryelements are shown in different shades Regulatory elements in the top row are in the forward direction whereas regulatory elements in the bottom roware in the reverse direction


that observed in the first two backgrounds studied confirm-ing the impact of individual excision alleles and genomicregions on the observed trans-interactions For exampleMenEx602 heterozygotes consistently had high levels of ac-tivity across all five isogenic backgrounds while MenEx552

consistently had low levels of activity We standardized allcrosses by both excision allele and genetic background andlooked for outliers to determine if any excision allele bygenetic background effects deviated from that average (Fig-ure 8) Genetic backgrounds (third chromosomes) differfrom each other in MEN activity consistent with previousstudies of this locus (Merritt et al 2005 2009) We there-fore standardized all crosses to a given third chromosometo the average activity of that chromosome (Figure S8)Similarly all crosses to a specific MenExi2 allele were stan-dardized to the average activity of that excision allele Bystandardizing for both background and excision alleles ifno interactions are present then the individual crosses will

not significantly differ from zero With the exception ofMenEx552 we found very specific interactions between ge-netic backgrounds and specific genetic lesions indicatingthat genetic backgrounds play a substantial role in trans-vection with their ability to complement each lesion inde-pendently The lack of interaction with Me0nEx552 is notsurprising this is the largest deletion in our set and the onlydeletion that does not show evidence for trans-interactions(up-regulation) Specific cases in which interactions maydiffer between MenExi2 alleles and genetic backgroundsappear to be highly dependent on variables that affecttrans-regulation such as transcription factor expression ina particular genetic background and the presence or absenceof a binding site for that transcription factor on the MenExi2

allele

Discussion

Here we characterize interallelic trans-interactions in generegulation at the Men locus in D melanogaster Our resultsare consistent with a specific type of trans-interactionmdashtransvectionmdashdriving high levels of gene expression at theMen locus Using small deletions in the 59 region of thegene we identified genomic regions putative local regula-tory elements which may be involved in interallelic trans-interactions Our approach identifies regulatory elementsthat are capable of acting in trans elements that are stillpoorly understood and begins to annotate the regulatory re-gion of Men a region largely uninvestigated to date In addi-tion we determined the impact of larger-scale variation (theentire third chromosome) on these trans-interactions to de-termine the impact of nonlocal genetic factors on local trans-interactions at the Men locus Although other transvectionsystemsmdasheg the yellow gene (Morris et al 1998 19992004 Lee and Wu 2006) Ubx (Lewis 1954 Qian et al1991) and the white gene (Benson and Pirrotta 1988Pirrotta 1999)mdashhave better-characterized regulatory regionsthe strength of the Men system is the ability to accuratelyquantify the trans-interactions using simple assays for bothgene expression and enzyme activity Enzyme activity can be

Figure 6 The correlation between MenExi2HFL53 and MenExi2VT26heterozygote MEN enzyme activity Certain alleles deviate away fromthe regression suggesting that there may be a genetic background bythe MenExi2 allele interaction effect Alleles tested across multiple back-grounds (Figure 7 Figure 8 Figure S8) are indicated by an open circle

Figure 7 Heterozygote MEN enzymeactivity measured across five third iso-chromosomal backgrounds Third chro-mosomes were CT21 HFL53 JFL12MD76 and VT26 For each data pointn frac14 4 and samples were measured intriplicate Error bars represent standarderror


quantified with much more sensitivity and accuracy thanmost morphological changes These assays therefore al-low us to very accurately determine the role of differentgenomic regions on the observed trans-interactions

While transvection is often studied through the comple-mentation between two mutant alleles (eg Lewis 1954Geyer et al 1990 Morris et al 1998 2004 Lee and Wu2006) we used interaction between mutant and wild-typealleles to identify elements that modify trans-interactionsWe identified these putative regulatory regions by creatinga matched suite of syntheticMen null activity alleles (denotedMenExi2) and by quantifying differences in trans-regulation(up-regulation of Men expression and MEN activity) acrossthese lines (Table 1) By placing the knockout alleles in aheterozygous condition with wild-type third chromosomeswe found that different MenExi2 alleles resulted in signifi-cantly different amounts of MEN activity (Figure 2) Becausegenetic background is controlled for these differences canbe directly attributed to differences at the excision sitesHeterozygotes of all but one excision allele have signifi-cantly higher activity than the expected 50 wild-type ac-tivity this up-regulation is attributed to trans-interactions(Merritt et al 2005) The significant differences in the amountof up-regulation between alleles appears to be a function ofthe different genomic elements present or absent in differentdeletionsmdashie the size location of the lesions or a combina-tion of both Our results could also be explained by changes inthe gene topology of the functional allele driven by the exci-sion allele (Morris et al 1999) In this model potential neg-ative regulators are ldquolooped-outrdquo of the functional allelecausing the observed up-regulation Although this type ofinteraction has been observed in other transvection systems(Morris et al 1998 1999) given that no potential negativeregulators were identified in the region of this gene and thevariation seen between interactions of the different excisionalleles regulatory elements acting in trans appears to bea simpler and more likely explanation of our observations

The up-regulation that we observe at the Men locusappears to be a function of trans-interactions between the

functional and nonfunctional alleles and is consistent witha specific type of trans-interactionmdashtransvection (Figure 9)Our results and those of other labs and systems supportthis model Often the definitive case for transvection is thedisruption of trans-interaction by chromosomal rearrange-ments In our case we have not created rearrangementsbut we do show that a large deletion (MenEx552) (Figure 2)abolishes the observed up-regulation in a similar way as wespeculate rearrangements would supporting our claim thatthe observed phenomena is transvection This effect of thelarge deletion is not however definitive proof of transvec-tion as the deletion removes regulatory elements as well aspotentially disrupting pairing and our results are thereforeconsistent with forms of trans-interaction other than trans-vection Alteration of the observed effects by chromosomalrearrangements would be strong evidence for transvection

Figure 8 Heterozygote MEN enzymeactivity measured across five isochromo-somal backgrounds (Figure 7) Withineach excision group activities are stan-dardized by both average excision alleleand third chromosome activities Starsindicate lines that are significantly differ-ent from the standardized average ata 095 threshold ie significant exci-sion alleles by genetic backgroundinteractions

Figure 9 (A) Proper gene expression occurs in wild-type flies in whichregulatory elements drive the expression on the cis-promoter for eachindividual allele (B) In our current model of transvection MenExi2 alleleslack a promoter When MenExi2 alleles are paired with a wild-type alleleenhancer elements of the null activity allele are capable of acting in transand driving gene expression on the functional promoter of the wild-typeallele


and is the subject of ongoing research in the Merritt labora-tory Specific genomic regions have been shown to modulatetransvection at other loci At the yellow locus for examplespecific wing and body enhancer elements are capable of act-ing in trans to drive gene expression (Morris et al 19981999 2004) These examples are dependent on the allelesbeing cis-promoter deficient and our genomic analysis sug-gests that the MenExi2 alleles are similarly promoter de-ficient (Figure 4) We hypothesize that the regulatoryelements of the nonfunctional alleles act in trans to up-regulate the activity of the functional allele The differencesin MenExi2 heterozygote activity then result from the dif-ferences between the deletions different size and locationof deletions remove different regulatory elements that arecapable of interacting in trans to modulate transcription onthe promoter-competent allele The difference in MEN activityand deletion size between MenEx552 and MenEx602 heter-ozygotes strongly supports this model As homozygotesboth alleles have no MEN activity (the case for all the knock-out alleles used) As heterozygotes however the two alleleshave very different activities The MenEx602 allele hetero-zygotes have essentially wild-type levels of MEN activitywhile the MenEx552 alleles have essentially 50 wild-typeactivity (no up-regulation) The MenEx552 allele has an16-kb deletion the largest in this study while theMenEx602 allele has one of the smallest deletions in thisstudy The lack of trans-interaction effects in MenEx552 het-erozygotes is consistent with removal of all the genomicelements required for trans-interactions Presumably theMenEx552 deletion has removed all or nearly all regulatoryelements capable of acting in trans The pronounced up-regulation of the MenEx602 allele is consistent with thisallele retaining a large number of elements capable of actingin trans to modify the activity of the intact allele

Given the difference in deletion size and activity betweenMenEx602 and MenEx552 heterozygotes a reasonable andsimple model to explain the differences in trans-interactionbetween the deletion alleles is that the size of the dele-tion influences the amount of activity In general we foundan overall trend in which the largest deletion shows noup-regulation midsize deletions have moderate levels ofup-regulation and small deletions have high levels of up-regulation The correlation between size and activity how-ever is low (Figure S6) although this may be due to therelatively large number of the midsize deletions with littlevariation in transvection levels between them The lack of anexact fit in the midsize deletions does suggest that the pres-ence or absence of specific genomic regionsmdashputative regu-latory elementsmdashis driving the trans-interactions observedPhylogenetic footprinting in combination with the TukeyrsquosHSD-groupedMenExi2 allele sets and their specific deletions(Figure 4) identified three potential genomic regions ofinterest that are conserved across 10 Drosophila species(identified as indashiii in Figure 4) These three regions differen-tiate between the deletions of the MenExi2 bins (discussedfurther below) and the high degree of evolutionary conser-

vation in these regions suggests that they may be of functionalimportance Furthermore motif prediction (Cartharius et al2005) identified potential elements within these regionsnamely TFBS and their associated transcription factors thatmay interact in trans (Table S1)

In at least one case deletion of a putative elementappears to increase up-regulation at this locus We predictedthat larger deletions would decrease the possibilities oftrans-interactions between the nonfunctional and the wild-type allele by decreasing available binding sites for regula-tory proteins capable of trans-interactions leading to loweractivity in heterozygotes (ie larger deletions would resultin less transvection and lower activity as heterozygotes)Counter to this expectationMenEx602 with a 252-bp largerdeletion region than MenEx582 has more Men expressionand enzyme activity as heterozygotes Motif prediction(Cartharius et al 2005) identifies potential motif ten ele-ment (MTE) 290 bases downstream of the predictedMen TSS MTEs are core promoter elements generally foundwithin 20ndash30 bases of the TSS (Lim et al 2004) MenEx602

and MenEx582 differ by only this region (Figure 5)MenEx602 which lacks the MTE sequence shows signifi-cantly greater MEN enzyme activity than MenEx582 (Figure2) ie trans-interactions between the knockout and func-tional allele are increased when this region is removed Leeand Wu (2006) demonstrated that regulatory elements havea greater preference for cis- than for trans-promoter inter-actions Although we predict that all of the MenExi2 allelesare promoter deficient core cis-promoter sequences that stillexist may have an affinity to some regulatory elements suchaffinity could restrict their ability to interact in trans Thedeletion of such an element would then lead to an increasein trans-interaction as seen in the comparison of MenEx602

and MenEx582 MTEs have previously been shown to func-tion across tens not hundreds of base pairs and this pre-dicted element is almost 300 bp from the TSS This distancecould suggest that the sequence is not truly a MTE or couldsuggest a novel longer distance function for MTEs Onlyexperimental testing will resolve this uncertainty This isin fact true of all of our in silico results they present in-teresting possibilities that all require experimental testingfor verification

Transvection at many loci including w decapentaplegicand Ubx is dependent on zeste expression (reviewed byDuncan 2002) At the Men locus we found a predictedzeste-binding site with high matrix identity within a regionof high conservation near the Men TSS (Figure 5 and TableS1) Zeste proteins may facilitate trans-interactions by bind-ing specific DNA sites and forming multimers bringing transand long-distance cis sequences into close proximity (Bensonand Pirrotta 1988 Bickel and Pirrotta 1990) Therefore locilocated near zeste-binding sites may be susceptible to zeste-dependent transvection At the Men locus only a singlezeste-binding site was predicted within the region of phylo-genetic footprinting However allMenExi2 deletions removethe predicted zeste-binding site making it unlikely that


transvection at the Men locus is zeste dependent (given thatall but one of the alleles show the up-regulation that we areattributing to transvection) We cannot rule out howevera model of hemizygous zeste-dependent transvection inwhich a single binding site on the functional allele allowstransvection

The transcription factor-binding sites that we identifiedin regulatory regions that differentiate between low andhigh trans-interaction MenExi2 alleles are also required forproper gene expression at other loci that show transvectioneffects We found putative binding sites for knirps hunchbackand tailless three transcription factors that are also requiredfor proper expression of the Drosophila homeotic geneUltrabithorax (White and Lehmann 1986 Irish et al 1989Qian et al 1991) As mentioned transvection has been pre-viously documented at the Ubx locus Given that these TFBSare required for gene expression at another loci that alsoexhibits transvection effects aside from proper gene regula-tion these transcription factors may have a role in trans-vection Interestingly and indicative of how widespreadtransvection effects may be transvection has also beendocumented at the knirps locus (Lunde et al 1998) showingthat regulation of an element that appears to play a rolein modulating transvection may itself be a function oftransvection

Previous results (T Merritt unpublished) as well as thevariation in results between VT26MenExi2 and HFL53MenExi2 heterozygotes (Figure 6) suggested that geneticbackground can modify the effects of specific individualMenExi2 alleles ie genomic background and specificdeletions may interact to determine the magnitude oftrans-interactions We examined this possibility by creatingheterozygotes using a subset of the MenExi2 alleles and fivedifferent third chromosome genetic backgrounds Strikinglycertain MenExi2 and genetic background combinations ex-hibit significantly different interactions (Figure 8) support-ing the model of trans-interaction (transvection) beinga product of the interaction of local and nonlocal factorsThese interactions might be a result of nonlocal geneticfactors (such as the expression of individual transcriptionfactors required for the proper expression of Men) thecapability of certain MenExi2 alleles to pair with specificgenetic backgrounds or a combination of both For exam-ple the smaller deletion alleles MenEx602 (646bp) andMenEx762 (669bp) show significantly less MEN activitywith HFL53 than the group averages However two largerdeletion alleles MenEx82 and MenEx432 (165 and 268 kbrespectively) show significantly greater MEN activity withHFL53 than the group averages The smaller deletions mayhave a factor that interacts significantly with HFL53 to re-press trans-interaction at the Men locus Consistent with theabove result (Figure 5) the removal of this region couldallow for greater transvection We expected that MenEx862having a deletion size between MenEx82 and MenEx432would also follow this pattern and show significantly greaterinteraction with HFL53 However MenEx862 does not show

any significant interaction with HFL53 suggesting thatthe interactions are more complex than our simple modelMenEx552 one the largest deletions did not show any sig-nificant differences in activity across the five tested geneticbackgrounds supporting our previous hypothesis that thedeletion has removed nearly all regulatory elements capableof trans-interaction Possible future research then wouldbe to examine these interactions with more genetic back-grounds and MenExi2 alleles

Given the widespread occurrence of transvection andother forms of trans-interaction effects across many speciesan understanding of these phenomena and their mecha-nisms is fundamental to a complete understanding of generegulation Extensive homologous pairing that likely medi-ates or facilitates many of these trans-interactions is foundacross Diptera and in Drosophila specifically making thisa rich system for the study of these trans-effects Althoughother species do not necessarily undergo such extensivepairing similar pairing-related phenomena have been welldocumented in non-dipterans (reviewed in Wu and Morris1999)

Examining the Men genomic region using bioinformatictools is a ldquofirst-passrdquo analysis very little is known aboutregulation of this gene in this species While this is only aninitial study of regulatory elements we are able to makeinteresting predictions A strength in this gene system isour ability to quantify both gene expression and enzymeactivity with high accuracy a feature unique among systemsused to study transvection The ultimate strength of thissystem will be in comparing genomic regions of other genesthat do and do not show the trans-effects that we docu-ment here Here we have shown large trans-interactioneffects at the Men locus In some cases the interactionsbetween functional and nonfunctional alleles produce geneexpression and enzyme activity as high as the expression oftwo functional alleles Similar deletion heterozygotes inother NADPH enzymes (eg Idh and G6pd) have been pre-viously examined and limited or no evidence of transvec-tion has been found in these other cases (Merritt et al2009) suggesting that these interactions are not a functionof deletionwild-type heterozygosity and likely involve morecomplex interactions or genomic requirements The Men lo-cus for example has a relatively large 59 untranscribed re-gion with many potential regions for trans-regulation It ispossible that this region contains a large number of regula-tory elements or is packaged or not packaged in such a wayto facilitate these trans-interactions Genomic comparisonsof loci that do and do not show such pronounced trans-interactions are ongoing and are expected to shed more lighton this type of gene regulation Further differentiating be-tween regions and regulatory factors that are capable ofinteracting in trans will also shed light on specificities oftrans-interactions as well as why some loci are capableof or susceptible to trans-interactions while others arenot In addition we have shown that local trans-interactionscan be influenced by nonlocal genetic background factors


although it is still unclear what specific factors are involvedFuture research elucidating specific factors capable or in-volved in trans-interactions between two alleles will beimportant in understanding and further exploring the mech-anisms of this pairing phenomenon

Acknowledgments

The authors thank Joe Lachance Luciano Matzkin EricGauthier Amadeo Parissenti and two anonymous reviewersfor constructive comments on earlier versions of this manu-script and Rahul Satija for assistance with the BigFootsoftware We thank the Bloomington and Szeged stockcenters for stocks This study was supported by the NaturalSciences and Engineering Research Council of Canada(NSERC) Discovery (3414-07) and Research Tools andInstruments (346023-07) grants a Canadian Foundationfor Innovation Leaders Opportunity Fund grant (16729)a Canada Research Chair (950-215763) to TJSM and aNSERC Undergraduate Student Research Award to TEL

Literature Cited

Babu P K S Selvakumar and S Bhosekar 1987 Studies ontransvection at the bithorax complex in Drosophila mela-nogaster Mol Gen Genet 210 557ndash563

Bacher C P M Guggiari B Brors S Augui P Clerc et al2006 Transient colocalization of X-inactivation centres accom-panies the initiation of X inactivation Nat Cell Biol 8 293ndash299

Benson M and V Pirrotta 1988 The Drosophila zeste proteinbinds cooperatively to sites in many gene regulatory regionsimplications for transvection and gene regulation EMBO J 73907ndash3915

Bickel S and V Pirrotta 1990 Self-association of the Drosophilazeste protein is responsible for transvection effects EMBO J 92959ndash2967

Blanchette M B Schwikowski and M Tompa 2002 Algorithmsfor phylogenetic footprinting J Comput Biol 9 211ndash223

Cartharius K K Frech K Grote B Klocke M Haltmeier et al2005 MatInspector and beyond promoter analysis based ontranscription factor binding sites Bioinformatics 21 2933ndash2942

Chandler V L and M Stam 2004 Chromatin conversationsmechanisms and implications of paramutation Nat Rev Genet5 532ndash544

Cooley L R Kelley and A Spradling 1988 Insertional mutagen-esis of the Drosophila genome with single P elements Science239 1121ndash1128

Coulthard A B N Nolan J B Bell and A J Hilliker2005 Transvection at the vestigial locus of Drosophila mela-nogaster Genetics 170 1711ndash1721

Davison D C H Chapman C Wedeen and P M Bingham1985 Genetic and physical studies of a portion of the whitelocus participating in transcriptional regulation and in synapsis-dependent interactions in Drosophila adult tissues Genetics110 479ndash494

Duncan I 1987 The bithorax complex Annu Rev Genet 21285ndash319

Duncan I W 2002 Transvection effects in Drosophila AnnuRev Genet 36 521ndash556

Duvernell D D and W F Eanes 2000 Contrasting molecularpopulation genetics of four hexokinases in Drosophila mela-nogaster D simulans and D yakuba Genetics 156 1191ndash1201

Fung J C W F Marshall A Dernburg D A Agard and J WSedat 1998 Homologous chromosome pairing in Drosophilamelanogaster proceeds through multiple independent initia-tions J Cell Biol 141 5ndash20

Gelbart W M and C T Wu 1982 Interactions of zeste muta-tions with loci exhibiting transvection effects in Drosophila mel-anogaster Genetics 102 179ndash189

Geyer P K M M Green and V G Corces 1990 Tissue-specifictranscriptional enhancers may act in trans on the gene locatedin the homologous chromosome the molecular basis of trans-vection in Drosophila EMBO J 9 2247ndash2256

Gindhart J G Jr and T C Kaufman 1995 Identification of Poly-comb and trithorax group responsive elements in the regulatoryregion of the Drosophila homeotic gene Sex combs reducedGenetics 139 797ndash814

Gohl D M Muller V Pirrotta M Affolter and P Schedl2008 Enhancer blocking and transvection at the Drosophilaapterous locus Genetics 178 127ndash143

Grant-Downton R T and H G Dickinson 2004 Plants pairingand phenotypes Tworsquos company Trends Genet 20 188ndash195

Henikoff S 1997 Nuclear organization and gene expression ho-mologous pairing and long-range interactions Curr Opin CellBiol 9 388ndash395

Henikoff S and L Comai 1998 Trans-sensing effects the upsand downs of being together Cell 93 329ndash332

Hopmann R D Duncan and I Duncan 1995 Transvection in theiab-567 region of the bithorax complex of Drosophila homologyindependent interactions in trans Genetics 139 815ndash833

Irish V F A Martinez-Arias and M Akam 1989 Spatial regu-lation of the Antennapedia and Ultrabithorax homeotic genesduring Drosophila early development EMBO J 8 1527ndash1537

Koeman J M R C Russell M H Tan D Petillo M Westphalet al 2008 Somatic pairing of chromosome 19 in renal onco-cytoma is associated with deregulated EGLN2-mediated [cor-rected] oxygen-sensing response PLoS Genet 4 e1000176

Lanctot C T Cheutin M Cremer G Cavalli and T Cremer2007 Dynamic genome architecture in the nuclear space regu-lation of gene expression in three dimensions Nat Rev Genet 8104ndash115

Lee A M and C T Wu 2006 Enhancer-promoter communica-tion at the yellow gene of Drosophila melanogaster diverse pro-moters participate in and regulate trans interactions Genetics174 1867ndash1880

Leiserson W M N M Bonini and S Benzer 1994 Transvection atthe eyes absent gene of Drosophila Genetics 138 1171ndash1179

Lewis E B 1954 The theory and application of a new method ofdetecting chromosomal rearrangements in Drosophila mela-nogaster Am Nat 89 73ndash89

Lim C Y B Santoso T Boulay E Dong U Ohler et al2004 The MTE a new core promoter element for transcriptionby RNA polymerase II Genes Dev 18 1606ndash1617

Livak K J and T D Schmittgen 2001 Analysis of relative geneexpression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Methods 25 402ndash408

Lunde K B Biehs U Nauber and E Bier 1998 The knirps andknirps-related genes organize development of the second wingvein in Drosophila Development 125 4145ndash4154

McKee B D 2004 Homologous pairing and chromosome dy-namics in meiosis and mitosis Biochim Biophys Acta 1677165ndash180

Merritt T J D Duvernell and W F Eanes 2005 Natural andsynthetic alleles provide complementary insights into the natureof selection acting on the Men polymorphism of Drosophila mel-anogaster Genetics 171 1707ndash1718

Merritt T J C Kuczynski E Sezgin C T Zhu S Kumagai et al2009 Quantifying interactions within the NADP(H) enzymenetwork in Drosophila melanogaster Genetics 182 565ndash574


Metz C W 1916 Chromosome studies on the Diptera II Thepaired association of chromosomes in the Diptera and its sig-nificance J Exp Zool 21 213ndash279

Morris J R J L Chen P K Geyer and C T Wu 1998 Twomodes of transvection enhancer action in trans and bypass ofa chromatin insulator in cis Proc Natl Acad Sci USA 9510740ndash10745

Morris J R J Chen S T Filandrinos R C Dunn R Fisk et al1999 An analysis of transvection at the yellow locus of Dro-sophila melanogaster Genetics 151 633ndash651

Morris J R D A Petrov A M Lee and C T Wu2004 Enhancer choice in cis and in trans in Drosophila mela-nogaster role of the promoter Genetics 167 1739ndash1747

Ni T D L Corcoran E A Rach S Song E P Spana et al2010 A paired-end sequencing strategy to map the complexlandscape of transcription initiation Nat Methods 7 521ndash527

Ou S A E Chang S Lee K So C T Wu et al 2009 Effects ofchromosomal rearrangements on transvection at the yellow geneof Drosophila melanogaster Genetics 183 483ndash496

Pirrotta V 1999 Transvection and chromosomal trans-interac-tion effects Biochim Biophys Acta 1424 M1ndashM8

Qian S M Capovilla and V Pirrotta 1991 The bx region en-hancer a distant cis-control element of the Drosophila Ubx geneand its regulation by hunchback and other segmentation genesEMBO J 10 1415ndash1425

Rorth P 1996 A modular misexpression screen in Drosophiladetecting tissue-specific phenotypes Proc Natl Acad Sci USA93 12418ndash12422

Salz H K T W Cline and P Schedl 1987 Functional changesassociated with structural alterations induced by mobilization ofa P element inserted in the Sex-lethal gene of Drosophila Ge-netics 117 221ndash231

Satija R A Novak I Miklos R Lyngso and J Hein2009 BigFoot Bayesian alignment and phylogenetic footprint-ing with MCMC BMC Evol Biol 9 217

Sezgin E D D Duvernell L M Matzkin Y Duan C T Zhu et al2004 Single-locus latitudinal clines and their relationship totemperate adaptation in metabolic genes and derived allelesin Drosophila melanogaster Genetics 168 923ndash931

Shiu P K D Zickler N B Raju G Ruprich-Robert and R LMetzenberg 2006 SAD-2 is required for meiotic silencingby unpaired DNA and perinuclear localization of SAD-1 RNA-directed RNA polymerase Proc Natl Acad Sci USA 1032243ndash2248

Southworth J W and J A Kennison 2002 Transvection andsilencing of the Scr homeotic gene of Drosophila melanogasterGenetics 161 733ndash746

Spradling A C D M Stern I Kiss J Roote T Laverty et al1995 Gene disruptions using P transposable elements an in-tegral component of the Drosophila genome project Proc NatlAcad Sci USA 92 10824ndash10830

Stam M 2009 Paramutation a heritable change in gene expres-sion by allelic interactions in trans Mol Plant 2 578ndash588

Stevens N M 1908 A study of the germ cells of certain dipterawith reference to the heterochromosomes and the phenomenaof synapsis J Exp Zool 5 359ndash374

Tagle D A B F Koop M Goodman J L Slightom D L Hesset al 1988 Embryonic epsilon and gamma globin genes ofa prosimian primate (Galago crassicaudatus) nucleotide andamino acid sequences developmental regulation and phyloge-netic footprints J Mol Biol 203 439ndash455

Thatcher K N S Peddada D H Yasui and J M Lasalle2005 Homologous pairing of 15q11-13 imprinted domains inbrain is developmentally regulated but deficient in Rett andautism samples Hum Mol Genet 14 785ndash797

Tsubota S and P Schedl 1986 Hybrid dysgenesis-induced re-vertants of insertions at the 5 end of the rudimentary gene inDrosophila melanogaster transposon-induced control mutationsGenetics 114 165ndash182

Vyas M C Ravindran and D P Kasbekar 2006 Chromosomesegment duplications in Neurospora crassa and their effects onrepeat-induced point mutation and meiotic silencing by un-paired DNA Genetics 172 1511ndash1519

White R A and R Lehmann 1986 A gap gene hunchbackregulates the spatial expression of Ultrabithorax Cell 47 311ndash321

Wise E M Jr and E G Ball 1964 Malic enzyme and lipogenesisProc Natl Acad Sci USA 52 1255ndash1263

Wu C T and J R Morris 1999 Transvection and other homol-ogy effects Curr Opin Genet Dev 9 237ndash246

Xu M and P R Cook 2008 The role of specialized transcriptionfactories in chromosome pairing Biochim Biophys Acta 17832155ndash2160

Xu N C L Tsai and J T Lee 2006 Transient homologouschromosome pairing marks the onset of X inactivation Science311 1149ndash1152

Communicating editor T C-t Wu


GENETICSSupporting Information

httpwwwgeneticsorgcontentsuppl20110907genetics111133231DC1


Locus of Drosophila melanogasterThomas E Lum and Thomas J S Merritt

Copyright copy 2011 by the Genetics Society of AmericaDOI 101534genetics111133231

TELumandTJSMerritt2SI

FigureS1SchematicdiagramofPCRcharacterizationoftheexcisionsiteonMenExi‐allelesForwardandreversearrowheadsrepresentforwardandreverseprimersrespectivelyandareorganizedintopairsBlackblocksrepresentampliconsfortheircorrespondingprimerpairsPrimerpairsweredesignedtoamplifypartiallyoverlapping~1kbregionstoensurefullamplificationcoverage(A)Awild‐typecontrolshowingamplificationacrossthegenomicregion(B)HypotheticalexcisioninwhichamplificationwithintheexcisionsitedoesnotproduceanampliconthroughPCRamplificationPrimersthatflanktheexcisionsite(circledinred)wereusedtoamplifyaroundtheexcisionandtheproductwassequenced

TELumandTJSMerritt 3SI

FigureS2AvisualalignmentofESTsofDmelanogasterMalicenzymeThemajorityofESTsbeginat3R8545514(indicatedbytheblueverticalline)thetranscriptionstartsite(TSS)predictedthroughthePEATproject(NIetal2010)AllofourMenEx‐allelesaremissingsomeportionofsequenceflankingthisTSSandareconsideredtobepromoterdeficient


FigureS3MenExi‐VT26heterozygoteMENenzymeactivitygraphedastheratioofwild‐typeactivity(MenEx3VT26)Errorbarsrepresentstandarderrorandwithineachdatapointn=6


FigureS4MenExi‐HFL53heterozygoteMENenzymeactivitygraphedastheratioofwild‐typeactivity(MenEx3VT26)Errorbarsrepresentstandarderrorandwithineachdatapointn=6


FigureS5ChromatogramsfromthesequencingofMencDNAfromMenEx60‐MenEx43‐andMenEx55‐TheMengenehasaGCpolymorphismatposition338(indicatedbytheblackarrow)MenGEx‐lineswerecrossedtoaMenClineMD5IncaseswherebothallelesarebeingexpressedaGCdoublepeekisobserved(highlightedbytheredcircle)IntheMenGEx‐MenCheterozygotesonlyasinglepeakisobservedindicatingthatonlythewild‐typealleleisbeingexpressedNotethatbecausesequencingwasdonewiththereverseprimernucleotidesneedtobereversed(egGrepresentsaC)


FigureS6MENenzymeactivityplottedagainstdeletionsizeNotethelowcorrelationbetweenMenExi‐deletionsizeandMENenzymeactivitysuggeststhatanothercharacteristicofeachexcisionnotsimplythesizeofthedeletionisimpactingtransinteractionswiththewild‐typeallele


FigureS7Thedegreeofconservationmeasuredbetween0and1inregionsiiandiii(Figure4)andregulatoryelementpredictionRegionscloserto1representhighconservationwhereas0representsnoconservationOverlappingpredictedregulatoryelementsareshownindifferentshadesRegulatoryelementsinthetoprowareintheforwarddirectionwhereasregulatoryelementsinthebottomrowareinthereversedirection


FigureS8TodeterminethedifferencesinbackgroundspecificMENactivityacrossallgeneticbackgroundstheaverageactivitywastakenacrossallstandardizedexcisiongroupsforeachgeneticbackground


TableS1SummaryofregulatoryelementspredictedbyMatInspector

DetailedFamilyInformation Startpos Endpos Strand

Core

sim Matrixsim Sequence

Iroquoisgroupof

transcriptionfactors ‐1944 ‐1936 ‐ 1 0974 atattAACA

Drosophilaforkheadfactors ‐1942 ‐1926 ‐ 1 0981

accttcaTAAAt

attaa

Transcriptionfactorswith

POU‐domain‐N‐terminalto

homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g

Drosophilasnailprotein ‐1908 ‐1898 ‐ 0868 0931 cacACATgttt

Drosophilabasichelix‐loop‐

helixtranscriptionfactors ‐1907 ‐1897 + 1 0991 aacaTGTGtga

DrosophilaT‐box

transcriptionfactors ‐1906 ‐1892 ‐ 0893 0905

atatatcACACa

tgt

Drosophilasegmentation

genetailless ‐1868 ‐1860 ‐ 0897 0938 gaaagTAAA

Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g

Drosophilabroad‐complexfor

ecdysonesteroidresponse ‐1860 ‐1842 ‐ 1 0905

aattattacACA

Aaaattg

Drosophilagianttranscription

factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt

Drosophilahomeoproteins ‐1851 ‐1837 ‐ 1 0978

tattcaATTAtta

ca

Drosophilahomeoproteins ‐1850 ‐1836 + 1 096

gtaaTAATtgaa

tat

DrosophilaT‐cellfactor ‐1839 ‐1827 ‐ 1 0897 ctgtTTGAtatat

DrosophilaOVOtranscription

factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga

GAGAelementbindingsites

forproteinsofthetrithorax

group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca

Zestetransvectiongene

product ‐48 ‐38 ‐ 1 0948 attcGAGTgtg

DrosopohilaOVO

transcriptionfactor 121 137 + 1 0923

ctgccGTTAtcgt

tatc

DNAreplication‐related 125 135 ‐ 1 092 taaCGATaacg


elementfactor

Boundaryelementassociated

factor 126 138 ‐ 1 094

ggataaCGATa

ac

DNAreplication‐related

elementfactor 128 138 + 075 0802 tatCGTTatcc

Drosophilaneuronalcis

elementbindingfactor 259 275 ‐ 1 0879

gaatcgGGTTtg

ctcat

Corepromotermotiften

elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt

DrosophilaCEBPlikebZIP

transcriptionfactors 322 334 ‐ 1 0911

cATTGtcaccag

t

Drosophilasnailprotein 765 775 + 1 1 gccACCTgcta

Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox

transcriptionfactorwithCUT

domain 808 816 ‐ 1 0935 tatGATTtg

DrosophilaAbd‐Bgroup 810 820 + 1 096 aatcATAAata

Drosophilahomeoproteins 825 839 ‐ 1 0901

cgccTAATagtt

att

DrosophilaDorsalVentral

Factor 863 873 ‐ 1 093 ttttTTTCgct

Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof

transcriptionfactors 992 1000 ‐ 1 0997 acaaaAACA

Corepromoterinitiator

elements 998 1008 + 1 0952 tgTCAGttttt

Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa


elements 1021 1031 + 0969 0949 ttTCATttttt


elementfactor 1051 1061 ‐ 1 0801 tatCGATtttc


factor 1051 1063 + 1 088

gaaaatCGATa

ga


elementfactor 1054 1064 + 1 0936 aatCGATagac

DrosophilaAbd‐Bgroup 1068 1078 ‐ 1 0947 aattATAAaag



gcctcaATTAta

aaa

Drosophilahomeoproteins 1070 1084 + 1 0976

tttaTAATtgagg

ca


transcriptionfactors 1075 1087 + 1 0914

aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt


helixtranscriptionfactors 1097 1107 ‐ 1 0957 ggcaTGTGcca

TGIF(TG‐interactingfactor)‐

Exd(extradenticle)group 1129 1135 ‐ 1 1 TGTCaac


ecdysonesteroidresponse 1151 1169 ‐ 1 094

taaatttgTAAA

cgaaatc


geneknirps 1183 1195 + 1 0917 tttcaaGTTCaat


Factor 1232 1242 + 1 0944 ctttTTTCcca

Drosophilasupressorof

Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat

StartandEndpositionarerelativetotheTSS

and Kennison 2002)] Transvection effects have been de-scribed for more than a dozen Drosophila melanogastergenes (eg Gelbart and Wu 1982 Davison et al 1985 Babuet al 1987 Geyer et al 1990 Leiserson et al 1994 Gindhartand Kaufman 1995 Hopmann et al 1995 Morris et al1999 Southworth and Kennison 2002 Coulthard et al2005 Merritt et al 2005 Gohl et al 2008 Ou et al 2009)and in other species including humans (eg Koeman et al2008) Several models for the molecular mechanisms oftransvection have been described including trans enhancerndashpromoter interactions insulator bypassing and pairing-dependent silencing (reviewed in Duncan 1987 Henikoff1997 Henikoff and Comai 1998 Southworth and Kennison2002) Of particular interest for the study presented hereare enhancerndashpromoter interactions occurring in transmdashthetype of interactions that our work suggests are responsiblefor the trans-interactions that we observe at the Men locus

In Drosophila and many other dipteran species homolo-gous chromosomes are extensively paired throughout thenucleus of interphase somatic cells (Stevens 1908 Metz1916 Fung et al 1998 reviewed in McKee 2004) Thispairing facilitates a wide array of homology effects includingtransvection that have remarkable roles in gene regulation(Wu and Morris 1999) Similar pairing and transvection-likeeffects have also been found in plants (reviewed in Chandlerand Stam 2004 Grant-Downton and Dickinson 2004 Stam2009) mammals (Thatcher et al 2005 Bacher et al 2006Xu et al 2006) and fungi (reviewed in Shiu et al 2006 Vyaset al 2006) emphasizing the importance of trans-interactionsin gene regulation across a wide variety of species With theavailability of a wide array of genetic tools and because ex-tensive pairing occurs between homologous chromosomesDmelanogaster is an excellent system in which to study theseinterallelic pairing effects on gene expression

Cytosolic malic enzyme oxidizes malate to pyruvate andis one of four enzymes primarily responsible for the re-duction of the cofactor NADP+ to NADPH (Wise and Ball1964) In earlier work using D melanogaster Men knockoutalleles to study the interaction between the NADPH enzymesunexpectedly high non-additive amounts of MEN activitywere observed when small deletion knockout alleles wereheterozygous with wild-type alleles (Merritt et al 20052009) The high level of MEN activity was shown to bedependent on the specific deletion creating the knockoutallele and not simply on physiological up-regulation sug-gesting transvection or a similar type of trans-interactionbetween the functional and nonfunctional Men alleles (Merrittet al 2005)

Here we characterize these trans-interactions betweenfunctional and nonfunctional alleles at the Men locus Usinga suite of 19 Men null activity (knockout) alleles (MenExi2)that differ only in their genomic lesions we tested MENenzyme activity and Men gene expression in MenExi2MenExi+ heterozygotes using two different wild-type alleles(MenExi+) We found significant differences in MEN activityacross these heterozygotes that are dependent upon specific

characteristics of each deletion The presence or absence ofputative regulatory elements based upon computationalprediction was correlated with differences in trans-interac-tion across the five sets of knockout alleles with significantlydifferent levels of trans-interaction In addition we exam-ined the impact of five different genetic backgrounds on thetrans-interaction effects finding significant interactions be-tween specific deletions and specific backgrounds Thesesignificant interactions illustrate the importance of large-and small-scale interactions between the functional andnonfunctional alleles in trans-interactions

Materials and Methods

Stock lines

Isothird chromosome lines (VT26 HFL53 JFL12 CT21MD76 and MD5) were a subset of nonlethal third chromo-somes extracted from isofemale lines (Duvernell and Eanes2000 Merritt et al 2005) Inbred lines used as commongenetic backgrounds were obtained either from Blooming-ton Drosophila Stock Center at Indiana University (BDSCBloomington IN line no 6326) or from the Eanes lab(VT83) (Merritt et al 2005) The P-element line used forthe P-element-mediated deletion EP(3)0517 was obtainedfrom the Szeged Drosophila Stock Center (Szeged Hungary)The line used as the source of transposase (stock 2030)and a line containing a deficiency that covers the entire Mengene Df(3R)kar31 (stock 6160) were obtained from theBDSC Three P-element-derived excision alleles for the Mengene initially described in an earlier articlemdashMenEx3+(wildtype) MenEx92 (knockout) and MenEx152 (knockout)(Merritt et al 2005)mdashwere also included in both the en-zyme activity and quantitative PCR (qPCR) analysis

Culture conditions

Flies were maintained on a standard cornmeal media at 25with a 12-hr12-hr photocycle All enzyme activity andqPCR assays were conducted on male flies aged for 4ndash6 daysafter emergence

Mutagenesis

We performed P-element-directed mutagenesis (Rorth1996) to create small deletion alleles of the Men locus usingEP(3)0517 The P-element construct carries the eye colormarker gene mini-white (w+mC) and is inserted 473 bases59 to the Men start codon (Merritt et al 2005) The EP(3)0517 element was mobilized by crossing virgin females tomales carrying the D2-3 source of transposase Dysgenicmales EP(3)517D2-3TM3 were crossed to w2 6326DrTM8 Sb females and w2 Sb Dr+ males ie malescontaining a copy of the EP(3)517 chromosome with theP-element excised were collected Candidate excision maleswere again crossed with TM8 for complete third chromo-some isolation and male and female progeny were back-crossed to establish each line The excision chromosomesgenerated are essentially isogenic differing only in a small




Fly homogenizations





Quantitative RT-PCR




5 deletionfrom TSS

3 deletionfrom TSS

MenExi2MenEx3+

activity





Data analysis




Results


































allele

Discussion




























Acknowledgments


Literature Cited
























































































Core


Iroquoisgroupof



accttcaTAAAt

attaa



homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g




DrosophilaT‐box


atatatcACACa

tgt



Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g



aattattacACA

Aaaattg


factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt


tattcaATTAtta

ca


gtaaTAATtgaa

tat



factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga



group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca



DrosopohilaOVO


ctgccGTTAtcgt

tatc



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat




Fly homogenizations





Quantitative RT-PCR




5 deletionfrom TSS

3 deletionfrom TSS

MenExi2MenEx3+

activity





Data analysis




Results


































allele

Discussion




























Acknowledgments


Literature Cited
























































































Core


Iroquoisgroupof



accttcaTAAAt

attaa



homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g




DrosophilaT‐box


atatatcACACa

tgt



Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g



aattattacACA

Aaaattg


factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt


tattcaATTAtta

ca


gtaaTAATtgaa

tat



factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga



group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca



DrosopohilaOVO


ctgccGTTAtcgt

tatc



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat



Data analysis




Results


































allele

Discussion




























Acknowledgments


Literature Cited
























































































Core


Iroquoisgroupof



accttcaTAAAt

attaa



homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g




DrosophilaT‐box


atatatcACACa

tgt



Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g



aattattacACA

Aaaattg


factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt


tattcaATTAtta

ca


gtaaTAATtgaa

tat



factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga



group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca



DrosopohilaOVO


ctgccGTTAtcgt

tatc



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat




























allele

Discussion




























Acknowledgments


Literature Cited
























































































Core


Iroquoisgroupof



accttcaTAAAt

attaa



homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g




DrosophilaT‐box


atatatcACACa

tgt



Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g



aattattacACA

Aaaattg


factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt


tattcaATTAtta

ca


gtaaTAATtgaa

tat



factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga



group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca



DrosopohilaOVO


ctgccGTTAtcgt

tatc



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat






















allele

Discussion




























Acknowledgments


Literature Cited
























































































Core


Iroquoisgroupof



accttcaTAAAt

attaa



homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g




DrosophilaT‐box


atatatcACACa

tgt



Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g



aattattacACA

Aaaattg


factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt


tattcaATTAtta

ca


gtaaTAATtgaa

tat



factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga



group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca



DrosopohilaOVO


ctgccGTTAtcgt

tatc



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat












allele

Discussion




























Acknowledgments


Literature Cited
























































































Core


Iroquoisgroupof



accttcaTAAAt

attaa



homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g




DrosophilaT‐box


atatatcACACa

tgt



Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g



aattattacACA

Aaaattg


factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt


tattcaATTAtta

ca


gtaaTAATtgaa

tat



factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga



group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca



DrosopohilaOVO


ctgccGTTAtcgt

tatc



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat





allele

Discussion




























Acknowledgments


Literature Cited
























































































Core


Iroquoisgroupof



accttcaTAAAt

attaa



homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g




DrosophilaT‐box


atatatcACACa

tgt



Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g



aattattacACA

Aaaattg


factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt


tattcaATTAtta

ca


gtaaTAATtgaa

tat



factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga



group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca



DrosopohilaOVO


ctgccGTTAtcgt

tatc



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat

























Acknowledgments


Literature Cited
























































































Core


Iroquoisgroupof



accttcaTAAAt

attaa



homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g




DrosophilaT‐box


atatatcACACa

tgt



Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g



aattattacACA

Aaaattg


factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt


tattcaATTAtta

ca


gtaaTAATtgaa

tat



factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga



group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca



DrosopohilaOVO


ctgccGTTAtcgt

tatc



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat


















Acknowledgments


Literature Cited
























































































Core


Iroquoisgroupof



accttcaTAAAt

attaa



homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g




DrosophilaT‐box


atatatcACACa

tgt



Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g



aattattacACA

Aaaattg


factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt


tattcaATTAtta

ca


gtaaTAATtgaa

tat



factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga



group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca



DrosopohilaOVO


ctgccGTTAtcgt

tatc



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat










Acknowledgments


Literature Cited
























































































Core


Iroquoisgroupof



accttcaTAAAt

attaa



homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g




DrosophilaT‐box


atatatcACACa

tgt



Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g



aattattacACA

Aaaattg


factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt


tattcaATTAtta

ca


gtaaTAATtgaa

tat



factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga



group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca



DrosopohilaOVO


ctgccGTTAtcgt

tatc



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat



Acknowledgments


Literature Cited
























































































Core


Iroquoisgroupof



accttcaTAAAt

attaa



homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g




DrosophilaT‐box


atatatcACACa

tgt



Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g



aattattacACA

Aaaattg


factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt


tattcaATTAtta

ca


gtaaTAATtgaa

tat



factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga



group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca



DrosopohilaOVO


ctgccGTTAtcgt

tatc



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat






















































Core


Iroquoisgroupof



accttcaTAAAt

attaa



homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g




DrosophilaT‐box


atatatcACACa

tgt



Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g



aattattacACA

Aaaattg


factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt


tattcaATTAtta

ca


gtaaTAATtgaa

tat



factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga



group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca



DrosopohilaOVO


ctgccGTTAtcgt

tatc



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat


























Core


Iroquoisgroupof



accttcaTAAAt

attaa



homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g




DrosophilaT‐box


atatatcACACa

tgt



Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g



aattattacACA

Aaaattg


factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt


tattcaATTAtta

ca


gtaaTAATtgaa

tat



factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga



group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca



DrosopohilaOVO


ctgccGTTAtcgt

tatc



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat





















Core


Iroquoisgroupof



accttcaTAAAt

attaa



homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g




DrosophilaT‐box


atatatcACACa

tgt



Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g



aattattacACA

Aaaattg


factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt


tattcaATTAtta

ca


gtaaTAATtgaa

tat



factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga



group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca



DrosopohilaOVO


ctgccGTTAtcgt

tatc



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat



















Core


Iroquoisgroupof



accttcaTAAAt

attaa



homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g




DrosophilaT‐box


atatatcACACa

tgt



Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g



aattattacACA

Aaaattg


factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt


tattcaATTAtta

ca


gtaaTAATtgaa

tat



factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga



group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca



DrosopohilaOVO


ctgccGTTAtcgt

tatc



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat

















Core


Iroquoisgroupof



accttcaTAAAt

attaa



homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g




DrosophilaT‐box


atatatcACACa

tgt



Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g



aattattacACA

Aaaattg


factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt


tattcaATTAtta

ca


gtaaTAATtgaa

tat



factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga



group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca



DrosopohilaOVO


ctgccGTTAtcgt

tatc



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat















Core


Iroquoisgroupof



accttcaTAAAt

attaa



homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g




DrosophilaT‐box


atatatcACACa

tgt



Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g



aattattacACA

Aaaattg


factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt


tattcaATTAtta

ca


gtaaTAATtgaa

tat



factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga



group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca



DrosopohilaOVO


ctgccGTTAtcgt

tatc



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat













Core


Iroquoisgroupof



accttcaTAAAt

attaa



homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g




DrosophilaT‐box


atatatcACACa

tgt



Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g



aattattacACA

Aaaattg


factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt


tattcaATTAtta

ca


gtaaTAATtgaa

tat



factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga



group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca



DrosopohilaOVO


ctgccGTTAtcgt

tatc



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat











Core


Iroquoisgroupof



accttcaTAAAt

attaa



homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g




DrosophilaT‐box


atatatcACACa

tgt



Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g



aattattacACA

Aaaattg


factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt


tattcaATTAtta

ca


gtaaTAATtgaa

tat



factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga



group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca



DrosopohilaOVO


ctgccGTTAtcgt

tatc



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat









Core


Iroquoisgroupof



accttcaTAAAt

attaa



homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g




DrosophilaT‐box


atatatcACACa

tgt



Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g



aattattacACA

Aaaattg


factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt


tattcaATTAtta

ca


gtaaTAATtgaa

tat



factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga



group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca



DrosopohilaOVO


ctgccGTTAtcgt

tatc



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat







Core


Iroquoisgroupof



accttcaTAAAt

attaa



homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g




DrosophilaT‐box


atatatcACACa

tgt



Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g



aattattacACA

Aaaattg


factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt


tattcaATTAtta

ca


gtaaTAATtgaa

tat



factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga



group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca



DrosopohilaOVO


ctgccGTTAtcgt

tatc



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat





Core


Iroquoisgroupof



accttcaTAAAt

attaa



homeoboxdomain ‐1940 ‐1928 + 0889 0931

aatatttATGAa

g




DrosophilaT‐box


atatatcACACa

tgt



Drosophilagapgene

hunchback ‐1860 ‐1848 ‐ 1 0984

tacacAAAAatt

g



aattattacACA

Aaaattg


factor ‐1856 ‐1842 + 1 0981

ttttgtGTAAtaa

tt


tattcaATTAtta

ca


gtaaTAATtgaa

tat



factor ‐162 ‐146 + 1 0917

cggctGTTAcac

gaaga



group(trxG) ‐114 ‐100 + 1 0978

tgagagAGAGa

gtca



DrosopohilaOVO


ctgccGTTAtcgt

tatc



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat



elementfactor


factor 126 138 ‐ 1 094

ggataaCGATa

ac





gaatcgGGTTtg

ctcat


elements 295 315 + 0875 0773

cagcgcgATCGc

ctgggcctt



cATTGtcaccag

t


Drosophilagapgene

hunchback 793 805 + 1 1

agaatAAAAaa

at

Drosophilahomeobox





cgccTAATagtt

att



Drosophilaproneural

repressor 958 972 + 1 0945

ggcaCACGcgcc

act

Iroquoisgroupof




Drosophilagapgene

hunchback 1004 1016 ‐ 1 0986

gcaacAAAAaa

aa






factor 1051 1063 + 1 088

gaaaatCGATa

ga






gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat




gcctcaATTAta

aaa


tttaTAATtgagg

ca



aATTGaggcaat

t


cagcTAATtgcc

tca


gaggcaATTAgc

tgt







taaatttgTAAA

cgaaatc






Hairless 1234 1246 ‐ 1 0923

tgcGTGGgaaa

aa


factor 1271 1285 ‐ 088 0887

tattttGTGAaac

aa



group(trxG) 1314 1328 + 1 0988

gaagagAGAGc

gcgc


elements 1329 1349 ‐ 0938 0771

ataaacaAACG

caaagcccat


Documents

Nonclassical Regulation of Transcription: Interchromosomal ... · in gene regulation across a wide variety of species. With the availability of a wide array of genetic tools, and