interplay of developmentally regulated gene expression and ... · interplay of developmentally...

Copyright � 2008 by the Genetics Society of AmericaDOI: 10.1534/genetics.107.083105

Interplay of Developmentally Regulated Gene Expression andHeterochromatic Silencing in Trans in Drosophila

Brian T. Sage,1 Michael D. Wu and Amy K. Csink2

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

Manuscript received October 8, 2007Accepted for publication November 30, 2007

ABSTRACT

The brownDominant (bwD) allele of Drosophila contains a heterochromatic block that causes the locus tointeract with centric heterochromatin. This association silences bw1 in heterozygotes (trans-inactivation)and is dependent on nuclear organizational changes later in development, suggesting that trans-inactivation may not be possible until later in development. To study this, a P element containing anupstream activating sequence (UAS)–GFP reporter was inserted 5 kb from the bwD insertion site. Sevendifferent GAL4 driver lines were used and GFP fluorescence was compared in the presence or the absenceof bwD. We measured silencing in different tissues and stages of development and found variable silencingof GFP expression driven by the same driver. When UAS–GFP was not expressed until differentiation inthe eye imaginal disc it was more easily trans-inactivated than when it was expressed earlier in undif-ferentiated cells. In contrast to some studies by other workers on silencing in cis, we did not find con-sistent correlation of silencing with level of expression or evidence of relaxation of silencing with terminaldifferentiation. We suggest that such contrasting results may be attributed to a potentially different roleplayed by nuclear organization in cis and trans position-effect variegation.

POSITION-EFFECT variegation (PEV) is the silenc-ing of gene expression by nearby heterochroma-

tin. The phenomenon was first described in Drosophilaand has long served as a model for the downregulationof expression by chromatin compaction and changes inhigher-order chromatin organization. Classic cis-actingPEV results from a chromosomal break and rejoiningthat juxtaposes a euchromatic gene and a block of con-stitutive heterochromatin. In contrast to earlier ideasconcerning the continuous, straightforward linear spread-ing of the silent state, a collection of recent studies havefound that the phenomenon is more complex. Manyfactors influence the susceptibility or resistance of agene to silencing by heterochromatin in cis, such as thesize, composition, and distance of the heterochromaticblock and, most importantly, the specifics of the ex-pression properties of the gene being silenced (forreview see Talbert and Henikoff 2006).

While these studies addressed cis-acting PEV, there is asimilar, related phenomenon in flies of silencing byheterochromatin in trans. In trans-acting PEV, or ‘‘trans-inactivation,’’ the block of heterochromatin and thesilenced gene are not necessarily on the same DNAmolecule, but are brought close to each other within thespace of the interphase nucleus. It is probable that this

type of PEV is similar to downregulation of a number ofnormal genes in higher eukaryotes, as increasing evi-dence finds developmentally silenced loci that moveinto association with large blocks of heterochromatin(for example, see Su et al. 2004). A well-studied exampleof trans-inactivation is the brownDominant (bwD) allele ofthe Drosophila melanogaster brown (bw) eye-color gene.The bwD allele contains an insertion of �1.6 Mbp ofheterochromatin into the brown coding sequence (Platero

et al. 1998). Except for a few small distinct spots, eyes ofbwD /bw1 flies totally lack red eye pigment. The trans-inactivation of the bw1 gene on the homologous chro-mosome is a consequence of the repositioning of bwD

into the pericentric heterochromatic compartment (Csink

and Henikoff 1996; Harmon and Sedat 2005; Thakar

et al. 2006) through interactions of heterochromaticbinding proteins (Sage and Csink 2003). In bwD/bw1

heterozygotes somatic pairing of the homologs causesthe bw1 chromosomal region to associate with pericen-tric heterochromatin along with the bwD heterochro-matic insertion. Hence, the wild-type bw gene is silenceddue to its localization in a neighborhood of the nucleusinhibitory to its transcription.

In parallel to the work on cis PEV, studies have foundthat some genes are resistant to trans-inactivation.Martin-Morris et al. (1997) have demonstrated thatsome mini-white transgenes can be trans-inactivatedwhile full-length white cannot. Additionally, earlier workof ours demonstrated that transgenes with differentpromoters have different susceptibilities to silencing by

1Present address: Brown University, Providence, RI 02912.2Corresponding author: University of Washington, Department of

Biology, 24 Kincaid Hall, Box 351800, Seattle, WA 98195-1800.E-mail: [email protected]

Genetics 178: 749–759 (February 2008)

heterochromatin in trans (Csink et al. 2002). One factorthat differed between these promoters was the develop-mental time of expression. This is intriguing, as studieson cis-acting PEV have shown that development plays arole in silencing (Lu et al. 1996; Weiler and Wakimoto

1998; Ahmad and Henikoff 2001). The studies on cis-acting PEV and trans-inactivation have provided in-triguing results and suggest additional questions to beaddressed. For example, does gene expression play thesame role in the ability of a gene to be silenced in cisand in trans? In the circumstance of cis silencing thesilenced gene is always located near heterochromatin,while in trans-inactivation the heterochromatic associa-tion required for silencing changes during develop-ment (Thakar and Csink 2005). Because of this, onemay predict that a gene with the same pattern of ex-pression could have different susceptibilities to cis andtrans silencing.

Here we study the role that temporal, spatial, andquantitative differences in gene expression play in thesusceptibility of a gene to trans-inactivation. We use anupstream activating sequence (UAS)–GFP transgenenear the brown locus in combination with lines activatingGAL4 in specific patterns of expression (Brand andPerrimon 1993). This allows an examination of trans-inactivation when the reporter is expressed in differenttimes of development and in different tissues and celltypes. We find that transgenes whose expression is de-layed until differentiation are more likely to be silenced,while those with earlier expression are more resistant tosilencing. Additionally, we found no general trendtoward increased or decreased silencing of transgenesas adult flies aged, except that there was a bit less

silencing in freshly emerged adults. We also did not finda tendency for silencing established in earlier develop-mental stages to be lost upon differentiation. Examin-ing the level of silencing in different tissues showed thateven the same gene with the same driver was differen-tially susceptible to trans-inactivation in the various tissues.Finally, we were unable to find a consistent correlationbetween ability to be trans-inactivated and level of wild-type expression. Combined, these results demonstratethe diversity of outcomes of heterochromatin in transand the strong influence of developmental expressionpattern in determining the sensitivity of a gene tochromatin-mediated gene silencing.

MATERIALS AND METHODS

Fly culture: All experimental crosses were done at 21�. Flieswere reared on standard yeast–cornmeal–molasses medium.Starting Drosophila stocks were either obtained from theBloomington Stock Center or previously generated in our lab(P{hsp-w-hsp26-pt-T}chrwab28) (Csink et al. 2002). In this articlewe refer to the driver lines with abbreviations, and here weindicate the full names: P{GawB}C155, P{GawB}167Y,P{GawB}179Y, and P{GawB}c355 are denoted elav, 167Y,179Y, and c355, respectively. P{w1mW.hs¼GAL4-arm.S}4aP{w1mW.hs ¼GAL4-arm.S}4b is denoted arm. P{w1mC¼Act5C-GAL4}25FO1 is denoted act. P{w1mC ¼GAL4-ninaE.GMR} isdenoted as GMR. The expression pattern of each line isdescribed in Table 1. Lines were selected to show a variety ofdifferent expression patterns both quantitatively andqualitatively.

The cross generating flies and larvae in which to measureexpression and trans-inactivation of lines with the driver on theX chromosome (c355, GMR, elav, 179Y, and 167Y ) is shown inFigure 1A. To examine expression and trans-inactivation of

TABLE 1

Expression of UAS–GFP at 59E controlled by various driver lines in the absence (wild-type expression) or presence(trans-inactivation) of bwD

act arm c355 GRM elav 179Y 167Y

Wild-type expression3- to 6-hr embryo D D None None None None None16- to 24-hr embryo D D None None C U, S SFirst instar D C, I S None C C, I, S SSecond instar D C, I U, S, C, E None C U, S, C, I SThird instar D S, C, E U, S, C, E S, C, ED S, C, ED U, S, C, ED U, S, CPupae D ND A ND E, Y U, S SAdult D H A, G, O, T ND B, Y O, T ND

Trans-inactivationThird instar eye disc — — — 1 1 1 NDThird instar CNS — — — — — 1 —Third instar salivary — 1 — — ND — —Whole adult 1/� 1 1/� ND �, early; 1, late 1, f; 1/�, m ND

A, abdomen; B, adult brain; C, larval CNS; D, diffuse general; E, eye discs (entire); ED, eye disc differentiated cells; G, gut; H,adult head; I, imaginal discs (unidentified, not eye); O, adult ovary; S, salivary glands; T, adult testes; U, unidentified additionalregions or structures; Y, eye; ND, expression not determined. In the larval and later stages if an above-mentioned tissue is not listedit means it was examined and no GFP expression was seen.�, no significant trans-inactivation; 1, significant trans-inactivation;�/1,trans-inactivation differs depending on age of adults; f, female; m, male.

750 B. T. Sage, M. D. Wu and A. K. Csink

UAS–GFP driven by act, we first recombined P{A5CGAL} ontothe P{UAS–EGFP}chrw bsf36 chromosome and then we per-formed the cross shown in Figure 1B. The cross used toexamine expression and trans-inactivation of UAS–GFP drivenby arm came from the cross shown in Figure 1C.

P-element replacement: The cross performed to replaceP{hsp-w-hsp26-pt-T}chrw with P{UAS-EGFP} was similar to thosedescribed by Csink et al. (2002). For the replacement wescreened for loss of hsp-w (red eye color) and mobilization ofwmc1 (orange eye color) from the X chromosome to anautosome. Of 1892 chromosomes screened, 30 (1.59%)contained an excision of P{hsp-w-hsp26-pt-T} and transpositionof P{UAS–EGFP} to an autosome. Of these, 15 (0.79%)contained a wmc1 gene that segregated with the secondchromosome. One of these was a precise replacement intothe chrw locus, as determined by PCR using primers specific for

P{hsp-w-hsp26-pt-T}, P{UAS-EGFP}, and the flanking genomicsequence. The mini-white gene in P{UAS-EGFP} is almostcompletely silenced when over the bwD chromosome.

Microscopic examination and measurement of GFPexpression: Eye imaginal discs, salivary glands, and the centralnervous system were examined for GFP expression and trans-inactivation. Wandering third-instar male larvae were usedand 3–13 tissues of each genotype were examined. Tissueswere from at least three different larvae. Occasionally one of apair of discs or glands was unusable due to damage duringdissection; hence odd numbers of these tissues were some-times used (Table 2). Two bwD and two bw1 larvae weredissected in tandem in PBS for 5 min. Tissues were placed infix mix (made immediately before use: 100 ml 37% para-formaldehyde, 900 ml PBS, 1 ml of 1 mg/ml DAPI) for 10 min.Tissues were further dissected during this 10 min, rinsed once

Figure 1.—Crosses to obtain bothlarvae and adult flies to test for transsilencing of P{UAS–EGFP}chrwbsf36

driven by selected lines producingGal4 in a variety of patterns. Bc is amarker visible in larvae and Elp is amarker visible in adults, allowingthe same cross to be used for bothlife stages. (A) The cross used whenP{driver-Gal4} was on the X chromo-some (lines c355, GMR, elav, 179Y,and 167Y ). (B) The cross used to testfor trans-inactivation when Gal4 wasdriven by the Act5C promoter. (C)The cross used to examine silencingof arm-driven GFP expression.

Gene Silencing During Development 751

with PBS, and then placed in permeabilization mix (madeimmediately before use: 665 ml PBS, 2 ml Triton-X, 0.67 ml of1 mg/ml DAPI) for 3 min. Tissues were rinsed twice with PBS.Tissues were attached to a polylysine-coated printed-well slidein a drop of PBS. All tissues from the four larvae were placedon the same slide, with bwD and bw1 tissues juxtaposed, andcovered by a 18 3 18-mm coverslip. Tissues were examinedimmediately after the slide was made. Images were taken usinga 43 objective so that multiple tissues could be visualized inone image. Microscopy used a Deltavision system (AppliedPrecision) built around an Olympus IX70 microscope. Theimages were gathered with a cooled CCD camera (Micromax350; Photometrics, Tucson, AZ). Quantification utilized Soft-Worx software (Applied Precision) and was performed bymeasuring the total GFP fluorescence in the tissue andstandardized by dividing by total DAPI staining in the tissue.This was performed for bwD and bw1 tissues from each of thelines, as well as a negative control for each cross (see Figure 1)lacking UAS–EFGP. Background fluorescence was removed bysubtracting the lowest value obtained from the negativecontrol line from each of the driver lines. These correctedvalues were averaged and are shown with their 95% confidenceintervals in Figures 3–5. All statistical analysis and graphingwere done using the Statview program (Abacus Concepts,Berkeley, CA). In all statistical analysis significance was testedby a two-tailed, unpaired Student’s t-test. P-values ,0.05 aremarked as significant in Table 2.

Fluorometric measurement of GFP expression in adults:Flies were aged for 0–24 hr, 3–5 days, 8–12 days, 18–22 days, or30–35 days (denoted day 1, 5, 12, 22, or 35 in Figure 6 andTable 3) before freezing at �80�. For each assay, 200 ml GFPassay buffer ½50 mm NaH2PO4, 10 mm Tris-HCl (pH 8.0), 200mm NaCl, pH 8.0� were added to a 1.5-ml microfuge tubecontaining five frozen adult flies. The sample was manually

homogenized and an additional 100 ml GFP assay buffer wasadded while rinsing the pestle. After homogenization, thesample was centrifuged for 10 sec in a microcentrifuge and thesupernatant immediately measured for GFP fluorescence.Measurements were taken of the sample in a capillary tube.The assay was performed with six different samples (of fiveflies each) for each genotype and sex. Measurements weremade using a Photon Technology International (Santa Clara,CA) QuantaMaster spectrofluorometer with the followingsettings: excitation 480, emission 513, excitation and emissionmonochromator slits of 2.5 mm, 2-sec readings were takenover 6 sec, and the average value was used as one reading. Foreach driver, fluorescence was measured from the four differ-ent genotypes as shown in Figure 1. The average value from thesiblings without the GFP reporter was subtracted from eachindividual sample of the animals that carried the reporter tocorrect for background fluorescence. These corrected valueswere averaged and are shown with their 95% confidenceintervals in Figure 6. All statistical analysis and graphing wasdone using the Statview program (Abacus Concepts). In allstatistical analysis significance was tested by a two-tailed, un-paired Student’s t-test. P-values ,0.05 are marked as signifi-cant in Table 3.

TABLE 2

Trans-inactivation of UAS–GFP by bwD in larval tissue

Driver Tissuea bw1 meanb nc bwD mean n bw1:bwD P-valued

elav E 0.170 6 0.078 10 2.18 0.0003179Y E 0.060 4 0.015 5 4.00 0.0020act E 0.840 7 0.794 7 1.06 0.5726arm E 0.014 7 0.013 13 1.08 0.9735c355 E 0.239 7 0.206 6 1.16 0.2182GRM E 0.236 7 0.141 7 1.67 0.0120elav C 0.432 3 0.460 4 0.94 0.7697167Y C 0.067 4 0.056 3 1.20 0.5667179Y C 0.406 3 0.180 4 2.26 0.0179act C 0.960 4 0.910 3 1.05 0.8215arm C 0.173 6 0.081 6 2.14 0.1645c355 C 0.756 5 0.719 5 1.05 0.5126GMR C 0.036 4 0.019 3 1.89 0.5867167Y S 3.238 9 3.723 9 0.87 0.2027179Y S 2.867 8 2.680 5 1.07 0.6638act S 3.485 3 4.018 3 0.87 0.2538arm S 0.230 9 0.055 9 4.18 0.0147c355 S 2.607 10 2.572 8 1.01 0.8482GMR S 0.138 6 0.101 6 1.37 0.5381

a C, central nervous system; E, eye disc (entire); S, salivarygland.

b Mean GFP fluorescence/DAPI fluorescence in each tissue.c The number of tissues examined.d Underlined numbers are ,0.05 and are considered signif-

icant.

TABLE 3

Trans-inactivation of UAS–GFP by bwD in whole adults

Driver Day bw1 meanb nc bwD mean n bw1:bwD P-valued

179Y (f)a 1 5,400 6 3,824 6 1.41 0.04165 5,360 6 2,301 6 2.33 ,0.0001

12 3,569 6 1,732 6 2.06 0.046022 3,461 6 1,232 6 2.81 0.006435 2,115 6 984 6 2.15 0.0018

179Y 1 22,984 6 18,339 6 1.25 0.00025 10,393 6 7,586 6 1.37 0.1070

12 5,213 6 3,272 6 1.59 0.000222 7,252 6 5,802 6 1.25 0.051935 7,576 6 4,359 6 1.74 0.0004

Act 1 36,950 6 29,376 6 1.26 0.00575 15,469 6 11,438 6 1.35 0.0025

12 9,268 6 6,686 6 1.39 ,0.000122 14,648 6 12,196 6 1.20 0.076435 14,879 6 8,732 6 1.70 0.0002

arm 1 7,693 6 1,250 6 6.16 ,0.00015 6,286 6 699 6 8.99 0.0003

12 5,576 6 382 6 14.61 ,0.000122 8,869 6 1,388 6 6.39 ,0.0001

elav 1 5,884 6 4,822 6 1.22 0.11605 5,765 6 2,507 6 2.30 0.0106

12 5,732 6 2,625 6 2.18 0.004622 6,291 6 3,025 6 2.08 0.003135 4,704 6 1,818 5 2.59 ,0.0001

c355 1 8,462 6 7,522 6 1.13 0.19165 11,586 6 9,670 6 1.20 0.0563

12 14,903 6 14,901 6 1.00 0.998522 20,356 6 16,690 6 1.22 0.121335 19,942 6 13,300 6 1.50 ,0.0001

a All data are from males except the 179Y (f), which arefrom females.

b Mean GFP fluorescence.c The number of samples (of five flies each).d Underlined numbers are ,0.05 and are considered signif-

icant.


RESULTS

To address the importance of developmental timingof expression in gene silencing we placed a GFP reportergene containing a variably inducible promoter near thebw gene. We used a reporter controlled by the yeast UASsequence so that available collections of GAL4-producingdriver lines could be used (Brand and Perrimon 1993;Duffy 2002). The advantage of this system is that theone reporter in 59E is driven by a number of GAL4-expressing lines in various developmental and tissue-specific patterns and at varying intensities (Figure 2).Tests for trans-inactivation by bwD are done without chang-ing the location of the reporter construct, yet allow forquantitative and qualitative changes of gene expression.A similar scheme has been used to measure the effect ofvarying gene expression on silencing by heterochroma-tin in cis (Ahmad and Henikoff 2001).

To examine trans-inactivation by bwD we needed togenerate a fly line that had P{UAS–EGFP} located veryclose to the bwD insertion site, but on the wild-typehomolog (Figure 2). To accomplish this we performed aP-element replacement of P{hsp-w-hsp26-pt-T}chrwab28

with P{UAS–EGFP}. The site of this transposon insertionis �5 Kbp from where the heterochromatic insertion isfound on the bwD chromosome. P{UAS-EGFP} also con-tains a mini-white reporter gene that gives the eye anorange color. As with other transgenes we have studiedcontaining the mini-white reporter gene in this samelocation (Csink et al. 2002; Sage et al. 2005), the ex-pression from this reporter is very strongly repressedwhen heterozygous with a bwD chromosome.

We selected seven driver lines to use in this study onthe basis of two criteria: pattern of expression as reportedin FlyBase and the previous examination of silencing byheterochromatin in cis (Ahmad and Henikoff 2001).To confirm their pattern of expression, we reexaminedthe expression of these drivers lines throughout de-velopment using our P{UAS–GFP}59E reporter hetero-zygous to a wild-type chromosome. The embryos, larvae,pupae, adults, and some dissected tissues were exam-

ined under a fluorescent dissecting scope and/or astandard compound fluorescent microscope. Our find-ings are summarized in Table 1 and generally confirmedthe previous descriptions of these various driver lines inFlyBase (Crosby et al. 2007).

Lines were initially selected to test a number ofspecific hypotheses concerning trans-inactivation. Forinstance, lines c355 and 167Y were chosen to test thepossibility that lack of expression in the embryo pro-moted silencing. arm and act were used to test both theimportance of very early transcription and the strengthof the expression. Lines GMR, elav, and 179Y were usedto test for the role of differentiation in trans-inactivation.

Trans-inactivation in differentiated and undifferenti-ated cells of the eye imaginal disc: The eye imaginaldiscs of third-instar larvae are bisected by the morpho-genetic furrow with cells posterior to the furrow un-dergoing differentiation and cells anterior beingundifferentiated. Previous results from our lab haveshown that there are profound changes in both nuclearorganization and chromatin dynamics that accompanythis differentiation event in the eye imaginal disc(Thakar and Csink 2005; Thakar et al. 2006). Todetermine if these changes were correlated with changesin specific aspects of gene expression, we examined theeye discs of wandering third-instar larvae for trans-inactivation of UAS–GFP. Our selection of driver linesallowed us to compare trans-inactivation in differenti-ated and undifferentiated cells. Three of the lines (act,arm, and c355) expressed throughout the disc, whilethree others (elav, 179Y, and GMR) expressed GFP onlyin the differentiated cells posterior to the morphoge-netic furrow. The trans-inactivation of the lines expressingin the eye discs is shown in Figure 3A and quantificationis shown in Figure 3B and Table 2.

The lines that show trans-inactivation are only thosewhere expression is not activated until after differenti-ation. In none of the lines with GAL4-driven GFP ex-pression in undifferentiated cells do we see silencingby bwD heterochromatin in trans, including the weaklyexpressing arm and moderately expressing c355 drivers.While this resistance to silencing appears uninfluencedby the strength of expression of the transgene in theundifferentiated cells, careful examination of the later-expressing lines (three rightmost bar graph sets inFigure 3B) reveals that the level of silencing (decreasein the bwD bars in Figure 3B) is slightly weaker in themore strongly expressed lines. It should also be notedthat expression during embryogenesis does not seem tobe necessary to confer resistance to silencing. Line c355does not show expression in eye discs until the secondinstar, but is still not trans-inactivated.

Tissue-specific differences in trans-inactivation: Inour study, the amount and timing of GAL4 expression insome of the driver lines are different in different tissues.To determine if silencing would vary depending on the

Figure 2.—Scheme to use the GAL4–UAS binary system totest for the differential silencing of the GFP reporter by bwD

heterochromatin under various expression regimes. This dia-gram shows the P element with the GAL4 driver on the Xchromosome and the reporter transgene at 59E. Euchromaticchromosome arms are indicated by open boxes, heterochro-matin (including the heterochromatic block in bwD at the dis-tal end of 2R) is indicated by solid boxes, and centromeres areindicated by solid circles.


tissue examined we examined trans-inactivation in twoadditional third-instar larval tissues: the central nervoussystem (CNS) (Figure 4) and the salivary glands (Figure5). The only line to display significant trans-inactivationin the CNS was line 179Y. The only line to display trans-inactivation in the salivary glands was line arm. Therefore,a single driver is differentially susceptible to trans-inactivation in different tissues (Table 2). For instance,arm is silenced in salivary glands, but not in the eye disc.The elav expression is silenced in differentiated eye disccells, but not in the CNS. This tissue specificity mayresult from differences in the state of the nucleus as weare comparing diploid, polytene, and differentiatedcells and is further discussed below.

Silencing in adults: We examined whole adult flies fortrans-inactivation of UAS–GFP activated by five of thedriver lines. Adult flies contain more differentiatedtissue than larvae, so one may expect a bit more trans-inactivation. Additionally, we were interested in findingout if the level of silencing changed in a general di-rection as the adults aged. Therefore, we examined fliesat five different times after eclosion. Figure 6 shows thelevels of expression in the bw1 male flies in the top graphand the level of trans-inactivation in the bottom graph.

In general, there is a modest level of silencing in all linesat least at some age. This silencing seems mostly unaf-fected by the age of the adult and there is no obviouscorrelation between overall expression and silencing.However, there does seem to be a bit less silencing in thenewly emerged flies (1 day, 0–24 hr after eclosion) usingthe 179Y (females), elav, and arm drivers and in no linesis the strongest trans-inactivation seen in this earliestsample (Table 3).

Strong trans-inactivation is seen for GFP driven by theelav promoter in all but the youngest adults. The elavregulatory region would drive GAL4 expression mostlyin differentiating or differentiated neurons, not in theirneuroblast precursors (Robinow and White 1991). Addi-tionally, very strong silencing is found for the ubiqui-tous, but weakly expressed arm driver. Silencing is notseen for elav in the larval CNS and is seen only for arm inthe larval salivary gland. These two lines highlight thefact that we often see different levels of trans-inactivationin different tissues and stages of development.

Fluorescence was measured separately in adult malesand females. With one exception the trend was the samein both sexes (data not shown). The one line that had adifference was 179Y, where there was substantial silenc-

Figure 3.—(A) GFP expression and trans-inac-tivation in the third-larval-instar eye disc. Green isGFP, blue is DAPI staining. The gains have beenadjusted in these photographs in the more weaklyexpressing lines so that the GFP expression isreadily visible in the composite. (B) Bar graphof data comparing expression of UAS–GFP con-trolled by various driver lines and in the presenceor the absence of bwD in the third-larval-instar eyeimaginal disc. Error bars show the 95% confi-dence interval. An asterisk indicates a significantdifference in GFP fluorescence between bwD andbw1 samples (P , 0.05 using Student’s t-test). Then of each sample and the exact P-values for thesedata are in Table 2. Below each driver name is asummary of the wild-type expression of thatdriver in earlier developmental stages. 1, expres-sion; �, no expression.


ing in females, but less in males (Figure 6 and Table 3).This line drives expression in the ovary and the testis(Table 1), so it is probable that this difference arisesfrom a different degree of silencing in the gonads.Alternatively it could be due to the large number of eggswithin the female, which have a different developmentalstate from the rest of the adult fly.

DISCUSSION

Previous studies have examined the influence ofterminal differentiation and developmental expressionon silencing by heterochromatin in cis. The work of Lu

et al. (1996) and Weiler and Wakimoto (1998) hasshown that silencing occurs early in development andthat there is a relaxation of silencing with terminaldifferentiation. While these studies agree, a more recentstudy emphasized the importance of the strength of theexpression of the target gene in counteracting its si-lencing (Ahmad and Henikoff 2001). The researchersdemonstrated differences in silencing on the basis ofthe level of expression of the transgene; when driversare expressed at similar developmental times a strongerdriver is less likely to be silenced than a weaker driver. Inthis study, when the transgene was turned on earlier indevelopment it was less likely to be silenced than when itwas turned on later in development. In contrast to the

results of Lu et al. (1996) and Weiler and Wakimoto

(1998), the majority of lines examined by Ahmad andHenikoff (2001) did not show complete silencing inpredifferentiated cells and a relaxation of silencingconcurrent with differentiation. Only when the trans-gene had low levels of expression did they see completesilencing and then relaxation with differentiation. Infact, strong early-acting drivers displayed anti-silencingevents spontaneously in postmitotic cells that had begundifferentiation and in mitotic cells.

The commonality between these studies is the dem-onstration that the expression pattern of a gene affectsits ability to be silenced. While these studies addressedcis-acting PEV, we wanted to address the role of ex-pression pattern in trans-inactivation. Previous resultsfrom our lab have demonstrated that different trans-genes have different abilities to be trans-inactivated.Additionally, we have shown that enhancer trap trans-genes located near the brown locus are unable to betrans-inactivated. This implies that the expression pat-tern of the enhancers near the brown locus renders themresistant to trans-inactivation (Csink et al. 2002; Sage

et al. 2005).In this study, we determined that transgenes not

expressed until differentiation are more likely to be trans-inactivated than those expressed earlier. This result con-curs with the cis-acting PEVresults. In the study of Ahmad

and Henikoff (2001), when the transgene was turned on

Figure 4.—(A) Trans-inactivation in the third-instar larval CNS of GFP expression driven by the179Y-GAL4 driver line. Green is GFP, blue is DAPIstaining. (B) Bar graph of data comparing ex-pression of UAS–GFP controlled by various driverlines and in the presence or the absence of bwD inthe larval CNS. Error bars show the 95% confi-dence interval. The asterisk indicates a significantdifference in GFP fluorescence between bwD andbw1 samples (P , 0.05 based on a Student’s t-test). The n of each sample and the exact P-valuesfor these data are in Table 2. Below each drivername is a summary of the wild-type expressionof that driver in earlier developmental stages.1, expression; �, no expression.


earlier in development it was less likely to be silencedthan when it was turned on later in development. As wesee a similar trend in cis and trans silencing, this may be afundamental feature of silencing and not particular to cisor trans mechanisms. Additionally, we determined thatthe level of trans-inactivation varies depending on thestage of development in which it is examined (Table 1).This result further supports the hypothesis that when agene is expressed in development affects the ability ofthis gene to be silenced.

We examined the overall amount of trans-inactivationin adults to determine if there was a general relaxation

of silencing with differentiation. Such relaxation wouldpredict that lines whose expression was inhibited by bwD

in an earlier developmental stage (larvae or undiffer-entiated cells in the eye disc) would become active laterin development (adult or differentiated cells in the eyedisc). Indeed, it would predict little silencing in adulttissue, as the bulk of the adult is terminally differenti-ated. This is not what we find. Indeed, we find the exactopposite in the eye imaginal discs, with silencing seenonly in differentiated tissue and no silencing in un-differentiated tissue. We also find that the 179Y-drivenexpression is silenced in eye imaginal discs and CNS inlarvae and is still mostly trans-inactivated in older adults.Additionally, the highest level of silencing in this study isseen for the GFP expression driven by arm in adults,even though it is not seen at all in some larval tissues. Wealso find that GFP expression driven by the elav, arm, and179Y (females) regulatory regions (Figure 6) shows anoverall increase in silencing in the older flies comparedto the day-1 flies. This may indicate that heterochroma-tin formation is stable during aging and that the abilityof genes to escape from silencing decreases as an organ-ism matures. Alternatively, it is possible that in newlyeclosed adults there is perdurance of GFP from the un-differentiated state that is degraded as the fly ages.

It is not surprising that there are changes in silencingwith development, as there are large-scale changes inheterochromatin during development (Arney andFisher 2004). In Drosophila, heterochromatin is firstvisible (Mahowald and Hardy 1985; Vlassova et al.1991), and heterochromatin protein 1 (HP1) firstbegins to be concentrated in heterochromatin ( James

et al. 1989), in syncytial blastoderm. Heterochromatinstays in a condensed state and replicates late in the cellcycle, beginning with the 14th cell cycle (Foe et al. 1993;Hiraoka et al. 1993). Additionally, recent results fromour group have found that bwD associations are stabilizedand promoted during differentiation. This is correlatedwith a general decrease in the movement of euchro-matic loci within the nuclei of the differentiated cells ofthe eye imaginal disc (Thakar et al. 2006).

The experiments we present in this article find verydifferent levels of trans-inactivation in different tissues(Table 1). Among other things, this could be due to thedifferent organization of the nucleus in these differenttissues. The CNS, eye discs, and salivary glands wereexamined in a third-instar larva, which allowed us toexamine trans-inactivation in diploid tissue, tissue pre-and postdifferentiation, and polytene tissues, respec-tively. The differences seen pre- and postdifferentiationrelate to time of developmental expression and are dis-cussed above. An unanticipated result was to find trans-inactivation in salivary glands. This was unexpected assalivary glands contain polytene chromosomes, whichmostly lack the heterochromatic association betweenbwD and pericentric heterochromatin that promotesbwD trans-inactivation (Talbert et al. 1994; Csink and

Figure 5.—(A) Trans-inactivation in the third-instar larvalsalivary gland of GFP expression driven by the arm driver line.Green is GFP, blue is DAPI staining. (B) Bar graph of datacomparing expression of UAS–GFP controlled by variousdriver lines and in the presence or the absence of bwD inthe larval salivary gland. Error bars show the 95% confidenceinterval. The asterisk indicates a significant difference in GFPfluorescence between bwD and bw1 samples (P , 0.05 based ona Student’s t-test). The n of each sample and the exact P-valuesfor these data are in Table 2. Below each driver name is a sum-mary of the wild-type expression of that driver in earlier de-velopmental stages. 1, expression; �, no expression.


Henikoff 1996). In this special circumstance, however,inactivation by bwD may not require heterochromaticassociation for the transgene to be located near a largeconcentration of heterochromatin. Silencing could bedue to the juxtaposition of the transgene and hetero-chromatin found in the bwD allele. In polytene tissuesthe DNA strands are copied thousands of times; how-ever, the pericentric heterochromatin does not have acorrespondingly high copy number. Interestingly, bwD

heterochromatin does appear to have this high copynumber. In these polytene tissues bwD represents a largefraction of heterochromatin and strongly concentratesHP1 (Platero et al. 1998). This results in a large con-centration of heterochromatic proteins, which couldsilence the euchromatic transgene located nearby. Inthis special circumstance, bwD may be acting as a hetero-chromatic compartment to silence the transgene. There-fore, although the transgene is in trans to the bwD allele,in this circumstance heterochromatic association may

not be required and may be more similar to cis-PEV. Cor-respondingly, the only driver silenced in the polytenetissues was a very weak driver line, arm.

In contrast to the results on cis silencing (Ahmad andHenikoff 2001), we were unable to find a consistentcorrelation between ability to be trans-inactivated andlevel of expression. On the one hand, there are certainaspects of our study that fit this trend. For instance, inthe three lines where GPF is driven and silenced only inthe differentiated cells of the eye disc (GMR, elav, and179Y ) we find that the degree of silencing is inverselyrelated to the level of wild-type expression. Additionally,arm, the most strongly silenced line in the adult is alsoone of the normally weakly expressing lines. However,there are clear data in this study that are inconsistentwith the level of expression being of primary importanceto susceptibility to trans-inactivation. Neither arm-drivenexpression in eye discs, nor 167Y-driven expression inCNS, nor GMR expression in the salivary glands is si-

Figure 6.—Trans-inactivation inwhole adult flies of GFP expressiondriven by the five different GAL4driver lines at five different timepoints after eclosion. (A) Box plotsshowing the total expression in fliescontaining the various GAL4 driverlines and the UAS–GFP reporter at59E over a wild-type chromosome(top) or a bwD chromosome (bot-tom). Except for 179Y all data arefrom males. Error bars show the95% confidence interval. (B) Boxplots showing the ratio of GFP fluo-rescence of bw1 flies divided by thatfrom bwD flies. The dotted line high-lights the ratio of one, which indi-cates no trans-inactivation. Eachbw1, bwD pair was tested for signifi-cant differences. An asterisk indi-cates a significant difference in GFPfluorescence between bwD and bw1

samples (P , 0.05 based on a Stu-dent’s t-test). The n of each sampleand the exact P-values for these dataare in Table 3. A detailed summary ofthe developmental and tissue-specificexpression of each driver line is inTable 1.


lenced, despite their very weak expression in those tis-sues. Second, 179Y is nicely silenced in the CNS despitehaving more expression than 167Y and a similar level toelav, neither of which are trans-inactivated. We believethat our data do not support a primary role for level ofexpression in susceptibility to trans-inactivation. How-ever, it is possible that level of expression plays a role insilencing once the locus comes close to the heterochro-matic compartment and this could account for theinconsistencies. That is, if the locus is unassociated withcentric heterochromatin and being expressed, the levelof expression is irrelevant because silencing cannotoccur when the UAS–GFP is not close enough to theheterochromatic compartment. However, once it re-sides in this compartment the level of silencing may besubject to similar influences that modify cis heterochro-matic silencing, such as the overall concentration of theGAL4 transcription factor (Ahmad and Henikoff 2001).

The above results indicate that there may be funda-mental differences between cis and trans silencing.Other results that may also be due to such differencesinvolve the behavior of two lines used both in this studyand in the work by Ahmad and Henikoff (2001). Thoseauthors found variegated silencing in cis of arm- and act-driven expression both before and after the morphoge-netic furrow, while we find no silencing by bwD in thosesame two lines in the eye disc either before or after themorphogenetic furrow. These contrasting results maybe due to differences in how silencing is brought aboutand maintained in the different types of PEV (Figure 7).In the circumstance of cis silencing the silenced gene isalways located near heterochromatin, while in trans-

inactivation the heterochromatic association requiredshould change during development. Close associationof the gene in cis with a large block of heterochromatinearly in development would allow the effect of hetero-chromatic proteins to modify the early organization ofthe regulatory region and may limit the influence ofearly-acting transcription factors.

Our data indicate that early transcription interfereswith trans-inactivation, although it should be pointedout that it is not certain if this interference is stable or ifa gene that is associated with the centric heterochro-matin will eventually be silenced. Such a possibility isseen for UAS–GFP expression driven by the arm driver.This expression is not silenced in some of the larvaltissue examined, but is very strongly silenced in theadult. One way to interpret this is that while early-expressed genes are initially resistant to silencing, theycan eventually be silenced. We do not believe that earlytranscription prevents or disrupts association of a locuswith the heterochromatic compartment. Earlier studiesof ours examined the results of upregulating a trans-inactivated heat-shock promoter and found no tran-sient or permanent change in the large-scale nuclearlocation of the region containing the upregulated gene.However, it should be pointed out that the study did nothave the resolution to detect a subtle disengagement ofthe locus from the surface of the heterochromaticcompartment (Csink et al. 2002). We speculate thatwhen expression is activated before heterochromaticassociation, the formation of active transcription com-plexes is not influenced by heterochromatin. It appearsthat subsequent association of the locus with centric

Figure 7.—Differencesincis silencingand trans-inac-tivation. Heterochromatin isrepresented in black, whilewhite boxes represent eu-chromatin. Silencing factorsare represented by blackcircles. An arrow representstranscription, while an X ad-jacent to an arrow representssilencing. In trans-inactiva-tion the statusof heterochro-matic association is depictedwith schematics of inter-phase nuclei. In the circum-stance of cis silencing, thesilenced gene is always lo-cated near the silencing fac-tors present in pericentricheterochromatin. In trans-inactivation the heterochro-matic association requiredfor localization near the si-lencing factors present inpericentric heterochroma-tin shouldchange during de-velopment and cell cycle.


heterochromatin has little immediate consequence fortranscription. However, it is possible that silencingoccurs after a period of association. The abundance ofthe appropriate transcription factor may influence thelength of this period of resistance. A higher localconcentration of transcription factor may allow longermaintenance of expression, or perhaps total resistance,despite close association of the locus with the centricheterochromatin. On the other hand, a gene thatattempts to begin transcription later in development,when it finds itself in a heterochromatic environment,may have more difficulty recruiting the necessary factorsto initiate transcription. Perhaps such initiation factorsare few or absent within the heterochromatic compart-ment. Another possibility is that an untranscribed genelocated near the heterochromatic compartment takeson the inhibitory chromatin structure typical of hetero-chromatin, making the initiation and maintenance oftranscription difficult. Studies are presently underwayinvestigating these possibilities.

Combined, our results demonstrate that the patternof expression of a gene affects its ability to be trans-inactivated and emphasize the importance of the in-teraction of nuclear organization, chromatin structure,and temporal and quantitative variation in transcriptionfactor abundance in determining the final expressionstate of a locus.

We thank the Bloomington Stock Center for fly lines, Lauren Ernstfor instructing us on use of the fluorometer, and Wei Tang fortechnical assistance. This work was supported by an American CancerSociety grant (RSG-00-073-04-DDC) to Amy Csink.

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Communicating editor: J. Tamkun


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