Genetics: Published Articles Ahead of Print, published on August 10, 2009 as 10.1534/.109.106559

Effects of chromosomal rearrangements on transvection at the yellow of Drosophila melanogaster

Sharon A. Ou*, Elaine Chang†, Szexian Lee†, Katherine So†, C.-ting Wu*1, James R. Morris*†1

*Department of Genetics, Harvard Medical School, Boston, MA 02115, †Department of Biology, Brandeis University, Waltham, MA 02453

1Corresponding authors

1

Running head: Somatic homologue pairing in Drosophila

Key words or phrases: , homology, nuclear organization, somatic pairing, transvection

1Corresponding authors:

C.-ting Wu Department of Genetics 77 Avenue Louis Pasteur, NRB 264 Boston, MA 02115 (617) 432-4431 Office (617) 432-7663 FAX [email protected]

James R. Morris Department of Biology Brandeis University Waltham, MA 02453 (781) 736-3119 Office (781) 736-3107 FAX [email protected]

2

ABSTRACT

Homologous chromosomes are paired in somatic cells of Drosophila melanogaster.

This pairing can lead to transvection, which is a process by which the proximity of homologous can lead to a change in gene expression. At the yellow gene, transvection is the basis for several examples of intragenic complementation involving the enhancers of one acting in trans on the of a paired second allele.

Using complementation as our assay, we explored the chromosomal requirements for pairing and transvection at yellow. Following a protocol established by ED LEWIS, we generated and characterized chromosomal rearrangements to define a region in cis to yellow that must remain intact in order for complementation to occur. Our data indicate that homologue pairing at yellow is efficient, as complementation was disrupted only in the presence of chromosomal rearrangements that break ≤650 kbp from yellow. We also found that three telomerically placed chromosomal duplications, containing ~700 kbp or more of the yellow genomic region, are able to alter complementation at yellow, presumably through competitive pairing interactions. These results provide a formal demonstration of the pairing-dependent nature of yellow transvection and suggest that yellow pairing, as measured by transvection, reflects the extent of contiguous homology flanking the locus.

3 INTRODUCTION

Cytological studies of a wide variety of systems are revealing the strategies by which a large amount of DNA can be organized into an extraordinarily small volume yet still be accurately expressed, replicated, and passed through cell divisions. In the somatic cells of Drosophila and other Dipteran insects, a striking feature of nuclear organization is the extensive amount of pairing that occurs between homologous chromosomes. This pairing was first noted by NETTIE STEVENS (STEVENS 1908) and CHARLES METZ (METZ

1916) through the examination of mitotic nuclei. Somatic pairing of homologous chromosomes has now been observed in Drosophila interphase nuclei using DNA as well as RNA in situ hybridization techniques (reviewed by MCKEE 2004; for example, CSINK and HENIKOFF 1998; FUNG et al. 1998; GEMKOW et al. 1998; HIRAOKA et al. 1993;

KOPCZYNSKI and MUSKAVITCH 1992; SASS and HENIKOFF 1999; BANTIGNIES et al. 2003;

RONSHAUGEN and LEVINE 2004; WILLIAMS et al. 2007; HARTL et al. 2008), assessment of the frequency of site-specific FLP-mediated recombination (GOLIC and GOLIC 1996a), and methods that mark chromosomes with protein tags (VAZQUEZ et al. 2001; 2002;

VAZQUEZ and SEDAT 2006). Here, we present our studies using transvection as our phenotypic assay for chromosomal pairing in Drosophila.

Transvection is a process by which the pairing of homologous genes results in a change in expression, in some situations causing gene activation and in other situations causing gene repression (reviewed by PIRROTTA 1999; WU and MORRIS 1999; DUNCAN

2002; KENNISON and SOUTHWORTH 2002). Because it depends on pairing, transvection can be used as a powerful assay for the paired state of genes. In this study, we used the protocol designed by ED LEWIS during his defining studies of transvection at the

4 Ultrabithorax (Ubx) gene (LEWIS 1954). LEWIS began his analyses with the observation that certain pairs of Ubx support intragenic complementation. He predicted that this complementation depends on the physical pairing of participating alleles and then confirmed this prediction through the generation and analysis of chromosomal rearrangements that disrupted complementation. Interestingly, the vast majority of the rearrangements had at least one breakpoint in a large chromosomal region between the centromere and Ubx, on the order of 12 Mbp in size and covering about half the arm. This region was named the “critical region” and was interpreted as the segment of the chromosome whose integrity is important for homologue pairing, and hence transvection, at Ubx. Based on the location of this large critical region between the centromere and Ubx, LEWIS suggested that somatic pairing might initiate at the centromere and proceed distally toward the telomere.

A number of genes have now been reported to show transvection in Drosophila, and application of the LEWIS method to some of these genes has demonstrated that critical regions can vary greatly in size (reviewed by DUNCAN 2002; KENNISON and

SOUTHWORTH 2002). Like transvection at Ubx, transvection at decapentaplegic (dpp)

(GELBART 1982), eyes absent (eya) (LEISERSON et al. 1994), vestigial (vg) (COULTHARD et al. 2005), and one type of transvection at Abdominal-B (Abd-B) (SIPOS et al. 1998) are associated with relatively large critical regions, spanning up to a third of a chromosome arm or more and many megabases proximal to the gene. Consistent with the large size of these critical regions, it is generally not difficult to isolate rearrangements that disrupt transvection at these loci. For example, studies of transvection at Ubx (LEWIS 1954), dpp

(GELBART 1982), eya (LEISERSON et al. 1994), and Abd-B (SIPOS et al. 1998) showed that

5 0.2 - 0.8% and possibly more of chromosomes that had been exposed to 4,000-4,500 rads of X-rays carried a transvection-disrupting rearrangement. In contrast, transvection at white (w) (GUBB et al. 1997; JACK and JUDD 1979; SMOLIK-UTLAUT and GELBART 1987) and a second example of transvection at Abd-B (HENDRICKSON and SAKONJU 1995;

HOPMANN et al. 1995) are associated with small critical regions; only chromosomal rearrangements that break very close to white or Abd-B are able to disrupt transvection.

Why is transvection more difficult to disrupt for some genes than for others? A number of explanations have been suggested (reviewed in Duncan 2002; Kennison and

Southworth 2002; also see LEWIS 1954; GELBART 1982; SMOLIK-UTLAUT and GELBART

1987; WU 1993; LEISERSON et al. 1994; HENDRICKSON and SAKONJU 1995; HOPMANN et al. 1995; DERNBURG et al. 1996; GOLIC and GOLIC 1996b; GUBB et al. 1997; FUNG et al.

1998; GEMKOW et al. 1998; SIPOS et al. 1998; COULTHARD et al. 2005). For example, if homologue pairing is mediated by pairing sites and if pairing near such sites is difficult to disrupt, then a gene located in the vicinity of a pairing site would be expected to have a small critical region. The sizes of critical regions may also reflect gene- or cell-specific aspects of pairing or transvection. For instance, if pairing is enhanced by an increased amount of time in which homologous regions have to find each other, then the pairing of genes which are expressed in cells that are dividing slowly, if at all, would be expected to be more tolerant of chromosome rearrangements, again leading to small critical regions.

A small critical region would also be expected for genes for which the effects of pairing can be imprinted or for which complementation requires only minimal or transient homologue interactions (SABL and LAIRD 1992; WU 1993).

6 Our studies use transvection at the yellow (y) gene of Drosophila to address the chromosomal requirements for somatic pairing. The yellow gene, located at the end of the X chromosome, is ideal for studies of transvection because it is structurally simple and has an easily assayed phenotype of dark cuticular pigmentation (GEYER and CORCES

1987; MARTIN et al. 1989). It is controlled by several tissue-specific enhancers, including the wing and body enhancers lying upstream of the transcription start site and the bristle contained within the single intron (GEYER and CORCES 1987).

Transvection can occur through two mechanisms at yellow, both of which result in intragenic complementation. In one mechanism, the enhancers of one allele activate the promoter of a second allele in trans when pairing brings the two alleles close together (GEYER et al. 1990; MORRIS et al. 1999a). According to the other mechanism, enhancers are believed to bypass a chromatin through a pairing-mediated topological change in gene structure (MORRIS et al. 1998). Here, we describe our work using yellow alleles that support enhancer action in trans.

The assumption that yellow transvection requires homologue pairing has rested on a number of studies, the most important of which addresses the ability of ectopically located yellow genes to support transvection (GEYER et al. 1990; CHEN et al. 2002). In particular, while complementation is observed for pairs of yellow transgenes inserted into allelic chromosomal sites, it is not observed between yellow transgenes at nonallelic sites or between an ectopic yellow transgene and yellow at its natural chromosomal location.

In addition, it was found that while a yellow allele that has been translocated to the Y chromosome can support complementation in X/Y flies, it does not do so in X/X/Y flies.

7 Presumably, in the latter case, preferential pairing between the two yellow alleles on the X chromosomes precludes their pairing with the yellow allele on the Y (GEYER et al. 1990).

In this paper, we use the method of LEWIS (1954) to demonstrate formally that yellow enhancer action in trans depends on homologue pairing and, in doing so, further elucidate the chromosomal requirements for pairing at the yellow locus. We find that yellow transvection is associated with a small critical region; only chromosomal breaks very close to the yellow locus are able to disrupt complementation. Our data also suggest that transvection at the natural location of yellow can be disrupted by competitive pairing interactions in the presence of duplications of the tip of the X chromosome containing the yellow locus. Taken together, our findings suggest that somatic pairing of yellow, as assayed by transvection-mediated complementation, requires on the order of ~700 kbp or less of flanking homology.

MATERIALS AND METHODS

Drosophila stocks: The y2, y1#8, y82f29, and y1 alleles have been described

- - elsewhere (GEYER et al. 1990; MORRIS et al. 1998, 1999a). Df(1) refers to Df(1) y ac w1118, which carries a deficiency of the yellow and achaete genes and a mutation in the

- - white gene (GEYER and CORCES 1987). Stocks carrying P[enh ] or P[pro ] were provided by P. GEYER (CHEN et al. 2002) and made isogenic for the chromosome carrying the transgene.

Culture conditions: Flies were cultured at 25 ± 1°C on standard Drosophila cornmeal, yeast, sugar, and agar medium with p-hydroxybenzoic acid methyl ester added

8 as a mold inhibitor. In general, three females were mated to three males, and crosses were transferred every 1-2 days to avoid crowding.

Pigmentation scores: Flies were immobilized by chilling in a vial submerged in an ice water bath and examined on a cold stage as previously described (WU and HOWE

1995). Pigmentation was scored in 1-3-day-old flies on a scale of 1 to 5, where 1 represents the null or nearly null pigmentation level, and 5 represents the wild-type or nearly wild-type level. At least 30 flies of the relevant genotype from each of two different crosses were scored to determine each phenotypic score.

Mutageneses: Four screens (A-D) were carried out to determine the critical region for yellow transvection at its natural location. In Screen A, y2/Y males were aged

2-4 days at 25° ± 1°C, irradiated with 4,000 or 4,185 rads of X-rays using a Phillips X- ray machine at a dose of 465 rads/minute and mated to y1#8/y1#8 females in bottles. Flies were transferred to fresh bottles daily for four days, after which the males were discarded.

Approximately 54,000 y2*/ y1#8 female progeny were screened. Screen B followed a similar protocol, except that the y2/Y males were irradiated with 4,500 rads of γ-rays from a cesium-137 source at a dose of ~350 rads/minute. Approximately 24,000 y2*/ y1#8 female progeny were screened.

Screen C allowed us to screen simultaneously for rearrangements that disrupt transvection at yellow (located at polytene chromosome position 1B1-2 on the X chromosome; http://flybase.org), those that disrupt transvection at white (located at polytene position 3C1-2 proximal to yellow; http://flybase.org), or those that disrupt transvection at both loci. Transvection at white can be observed in a zeste1 (z1) background, which does not affect unpaired white genes but causes paired white genes to

9 be repressed, producing flies with eyes that are yellow in color instead of wild-type red

(JACK and JUDD 1979; reviewed by PIRROTTA 1991). Interestingly, mutations at zeste have also been shown to disrupt transvection-associated phenotypes at dpp (GELBART and

WU 1982), eya (LEISERSON et al. 1994), and Ubx (LEWIS 1954). Although we have not observed an effect of zeste mutations on yellow transvection (J. MORRIS, unpublished), a disruptive effect has been reported by GEYER et al. (1990). Therefore, incorporation of the z1 mutation into Screen C had the potential of revealing a critical region that would differ from those obtained in Screens A and B.

In Screen C, y2 z1/Y males were irradiated with 4,500 rads of γ-rays and crossed to y1#8 z1/ y1#8 z1 females. Approximately 17,000 (y2 z1)*/ y1#8 z1 female progeny were screened both for reduced pigmentation of the wing and/or body caused by disruption of yellow complementation, as well as for red eyes caused by disruption of z1-mediated repression of white. We found no females with a phenotype consistent with the disruption of transvection at both yellow and white. In contrast, we found six yellow- eyed females with reduced levels of pigmentation in both the wing and body (Table 1).

Of these six, two were sterile and the remaining four are described in Results. We also found 16 females showing normal levels of complementation but red eyes. Of these, 10 were sterile or had too few progeny with which to establish lines. Four of the six remaining females produced white-eyed y2 z1*/ Y progeny, indicating that the white gene of the mutagenized chromosome had been disrupted. This interpretation is consistent with the red-eyed phenotype of y2 z1*/ y1#8 z1 females being due to a structural disruption or deletion of white that can prevent z1-mediated repression. The remaining two red-eyed females produced red-eyed y2 z1*/ Y progeny and may have resulted from white

10 transvection-disrupting rearrangements or trans-acting dominant suppressors of the z1 phenotype (reviewed in PIRROTTA 1991).

Screen D differed from Screens A, B, and C in that rearrangements were induced on a chromosome carrying y1#8 instead of y2. Males of the genotype y1#8/Y were irradiated with 4,500 rads of γ−rays and crossed to y2 w1118/y2 w1118 females, where w1118 was used to distinguish y2 w1118/ y2 w1118 (white-eyed) lightly pigmented females from y1#8*/y2 w1118 (red-eyed) non-complementing females arising from non-disjunction.

Approximately 20,000 y1#8*/ y2 w1118 female progeny were screened.

We carried out two types of screens to determine whether transvection at ectopic loci is also associated with a small critical region. In the first type, Df(1)/Y; P[enh-

]47A/P[enh-]47A and Df(1)/Y; P[enh-]92A/P[enh-]92A males homozygous for an enhancerless yellow transgene were irradiated with 4,500 rads of γ-rays and crossed to

Df(1)/Df(1); P[pro-]47A/CyO and Df(1)/Df(1); P[pro-]92A/TM3 females, respectively, carrying a promoterless yellow transgene. Flies were transferred to fresh bottles daily for four days, after which the males were discarded. Approximately 10,000 and 26,000 flies carrying the 47A and 92A transgenes, respectively, were screened.

The second type of screen was similar, except that we used X-rays instead of γ- rays and transvection at Ubx, manifested as wing-to-haltere transformation, as a positive control for the generation of rearrangements. In this screen, Df(1)/Y; Cbx Ubx gl3/P[enh-

]92A males were irradiated with 4,500 rads of X-rays and crossed to Df(1)/Df(1); P[pro-

]92A/TM3 females. Flies were again transferred to bottles and brooded daily for four days, after which the males were discarded. Progeny flies carrying P[enh-]92A and

P[pro-]92A were screened for reduced yellow complementation, and progeny flies

11 carrying Cbx Ubx but not TM3 were screened for reduced wing-to-haltere transformation.

Approximately 15,000 flies of each genotype were screened.

For all screens, exceptional flies were backcrossed to confirm the original phenotype, after which mutagenized chromosomes believed to carry informative changes were isolated and put into stock using standard genetic techniques.

Southern analyses: Genomic DNA for Southern analyses was isolated from adult flies as described by M. ASHBURNER (ASHBURNER 1989) or as described by E.

REHM (REHM 2003). GeneScreen hybridization membranes were used according to the recommendation of the manufacturer (NEN) and probes were labeled with the Roche

Random-Primed DNA Labeling Kit. To determine the integrity of the yellow locus, genomic DNA was digested in two separate reactions, one using HindIII and BamHI and the other using PstI. The digested DNA was separated on a 0.8% agarose gel and probed with a 7.7 kbp SalI fragment encompassing the entire yellow gene, including the upstream wing and body enhancers and a region 3’ of the transcribed region.

Cytology and genomic distances: Salivary glands of third instar larvae heterozygous for the rearranged y2 chromosome and an otherwise structurally normal chromosome bearing y1#8 were dissected in 45% acetic acid and stained with 2% orcein in equal parts lactic acid and glacial acetic acid. Polytene chromosomes were analyzed with phase optics. The estimate of ~650 kbp or less for the critical region of yellow was determined by considering the YTD breakpoints falling between the polytene location of yellow at 1B1-2 and polytene subdivisions 1B-D (Table 3), assignment of yellow and the band in polytene subdivision 1D that is most distant from yellow, 1D4, to sequences

240,542-255,278 and 884,615-884,935, respectively (http://flybase.org), and then

12 determining the distance between yellow and 1D4 in terms of base pairs. The estimates of the sizes of Dp(1;2)y2A, Dp(1;4)y2B, and Dp(1;4)y2C to be ≤ 700 kbp, 2.5 Mbp, and 1.1

Mbp, respectively, were determined by considering the breakpoints of these duplications in polytene subdivisions 1B-C, 3A, and 1E, respectively, and the assignment of the bands in each of these regions that are most distant from yellow to ~700,000, ~2,500,000, and

~1,100,000 base pairs on the genome map, respectively (http://flybase.org).

Tentative new orders of yellow transvection-disrupting rearrangements (YTDs):

YTD1: 1A – 1B | 78A – 100;

61 – 77D| 1C – 20

YTD2: *1C – 1A | (100A7 – 100C1) | 1D – 20;

61 – 100A7 | 100C1 – 100F

* = unknown telomeric sequence

YTD3: 1A – 1B | 40F/41B – 36D | 5D – 1C | 40F/41B – 60;

21 – 36B | 5E – 20

YTD4: 1A – 1B | 28D – 21D | 11C – 1C | 28F – 60;

21A – 21C| 99D – 61

100 – 99D | 11D – 20;

YTD5: 1A – 1B | 80B – 98F | 80A – 61;

21 – 39E | 1C – 20;

60 – 39F | 98F – 100

RESULTS

13 Our study involved the generation and characterization of chromosomal rearrangements that disrupt complementation between the y2 and y1#8 alleles (Figure 1A).

The y2 allele is caused by the insertion of a gypsy retrotransposon, which carries a chromatin insulator that blocks the wing and body enhancers from interacting with the promoter, and therefore flies homozygous or hemizygous for y2 are lightly pigmented

1#8 specifically in the wings and body (HARRISON et al. 1989). The y allele is a true null.

It is caused by a deletion of the promoter region, and flies homozygous or hemizygous for y1#8 show completely mutant yellow pigmentation in all tissues. Importantly, although neither y2 nor y1#8 is able to direct significant pigmentation of the wings or body, y2/y1#8 flies show nearly wild-type pigmentation levels due to action of the y1#8 wing and

2 body enhancers on the y promoter in trans (GEYER et al. 1990; MORRIS et al. 1999a).

Conveniently, because the insulator of y2 does not prevent the bristle enhancer from interacting with its promoter, the bristles of y2 flies are darkly pigmented and can be used in our crosses as a phenotypic marker for the y2 allele.

Transvection at yellow is associated with a small critical region: Modeling our experiments on those carried out by LEWIS (1954) at Ubx, we began with the idea that yellow complementation depends on pairing of the yellow alleles (GEYER et al. 1990).

We then tested this idea by generating mutations that disrupt complementation and, subsequently, determining whether the mutations are, in fact, chromosomal rearrangements that have breakpoints on the X chromosome. To score wing and body pigmentation, we used a five-point scale with 1 representing the null or nearly null phenotype and 5 representing the wild-type or nearly wild-type phenotype. According to this scale, y2/y2 flies and y1#8/y1#8 flies give mutant scores of (1, 1+) and (1, 1),

14 respectively, while y2/y1#8 flies give a nearly wild-type score of (4, 4), where the first number refers to the level of wing pigmentation and the second to body pigmentation. In our screens, we selected exceptional y2/y1#8 progeny whose pigmentation scores were less than (4, 4). In general, because yellow is on the X chromosome, only female flies can support yellow complementation. Male flies, with only a single X, usually do not show complementation, but are able to do so when they carry an appropriate duplication of yellow (GEYER et al. 1990) or when complementation occurs between two yellow transgenes that are located at allelic positions on an autosome (CHEN et al. 2002).

Four screens were carried out (Table 1). In Screens A and B, y2/Y males were irradiated with X-rays and γ-rays, respectively, to induce chromosomal rearrangements and then mated to y1#8/y1#8 females. In Screen C, y2 z1/Y males were irradiated with γ-rays and crossed to y1#8 z1/y1#8 z1 females. Finally, in Screen D, y1#8/Y males were irradiated

2 1118 2 1118 with γ-rays and then mated to y w /y w females. (See MATERIALS AND METHODS for further description of all four screens.)

Based on the frequencies with which transvection-disrupting rearrangements have been recovered for other loci, we anticipated that if yellow were associated with a large critical region, we should recover YTDs at a frequency of two to eight for every 1,000 females. To ensure that our screen would be informative even if we failed to recover disruptive rearrangements, we aimed to screen tens of thousands of females as well as confirm the effectiveness of irradiation through the isolation of other types of mutations.

We screened approximately 54,000 females in Screen A, 24,000 females in Screen B, and

17,000 females in Screen C, and obtained a total of 34 exceptional females with lighter wing and/or body pigmentation (Table 1; Screen D is discussed below). Importantly,

15 these females were able to produce dark bristles, indicating that the coding region of the y2 allele on the mutagenized chromosome was functional. Thirteen of the exceptional females were sterile or gave too few progeny to allow further studies. Lines were established from the remaining 21 females.

The mutagenized X chromosomes of all lines were then analyzed in two ways.

First, we determined whether the X or any other chromosome had been rearranged through the cytological analyses of larval polytene chromosomes (Figure 2). Second, we determined the structural integrity of the yellow locus by carrying out Southern analysis using genomic DNA and a probe covering the entire yellow locus (data not shown).

These cytological and structural analyses placed the 21 lines into five classes (Table 2).

The 18 lines falling into Classes I through IV are discussed directly below, while the three lines of Class V are described in a later section.

Class I is defined by five lines (all from Screen A) which carry chromosomal rearrangements involving the X chromosome, as revealed by polytene chromosome spreads, and unaltered y2 alleles, as demonstrated by Southern analyses (Table 2; Figure

2). These lines reduce pigmentation from a score of (4, 4) to as low as (1, 1) in our complementation assays (Table 3). Significantly, the rearrangements in these lines all have a breakpoint in the 1B-D polytene region very close to yellow located at 1B1-2 and about 110 kbp from the telomere of the X chromosome (Figure 2). In this way, these rearrangements appear to define a region in cis to yellow that is important for transvection at yellow. As the simplest interpretation of these rearrangements is that they disrupt yellow complementation by disrupting pairing of the yellow genomic region, these X- linked rearrangements were designated as YTDs (YTD1-5; Table 3). It should be noted

16 that YTD3 and YTD5 place yellow in the vicinity of centric heterochromatin, making it possible that the reduced complementation associated with these two rearrangements is due at least in part to position-effect variegation (see new orders for these chromosomes in MATERIALS AND METHODS). Consistent with this interpretation, YTD5 is associated with a variegated pattern of bristle pigmentation (Table 3).

We placed 13 lines into Classes II, III, and IV (Table 2). Class II consists of five lines (two from Screen A, two from Screen B, and one from Screen C), all of which have a cytologically visible rearrangement breakpoint within the 1B-C region of the X but are also altered in the yellow genomic region (Table 4). For example, PCR analysis showed that the X chromosome of Line 10 has a breakpoint within the 250 bp 3’ untranslated region (3’UTR) of yellow (MATERIALS AND METHODS). The location of chromosomal disruptions so close to yellow in these Class II lines precluded our ability to determine whether these lines compromise complementation by disrupting pairing or by disrupting the integrity of the yellow gene itself.

Class III consists of five lines (all from Screen A) which have no obvious chromosomal rearrangements but give abnormal Southern patterns (Table 2). As was the case with the Class II lines, these alterations of the yellow genomic region obscured our ability to determine whether loss of complementation resulted from disruption yellow pairing.

Class IV includes three lines (two from Screen A and one from Screen C) that are structurally normal by both cytological examination and Southern analyses. The inability of these lines to support complementation may be due to rearrangements that are not detectable in polytene spreads or to mutations at the yellow locus that are not detectable

17 by Southern analyses. It is also possible that one or more of the Class IV lines harbor trans-acting mutations in genes that are important for pairing and/or transvection at yellow.

Our fourth screen, Screen D, was designed to determine whether disruption of yellow complementation is sensitive to the particular allele of yellow carried on the rearranged chromosome. This screen resembled Screens A, B, and C in that it was designed to recover disruptors of complementation between y2 and y1#8, but differed from them in that it targeted the y1#8 rather than the y2 chromosome for irradiation.

Approximately 20,000 progeny females were screened for light wing and/or body pigmentation and seven exceptional females were recovered (Table 1). Four of these females were sterile or gave too few progeny for further studies. The remaining three were used to generate mutant lines, each of which was subsequently shown by Southern analysis to be structurally altered in the yellow genomic region. Although we did not characterize these lines by cytology, our Southern analyses indicate that they fall into either Class II or III and, like the other lines in these classes, may be unable to support complementation due to disruption of the yellow locus.

Considering all four screens together, it may be noteworthy that all five YTDs were found in Screen A, the only screen in which males were irradiated with X-rays instead of γ-rays. However, as γ-rays may be somewhat less mutagenic than X-rays

(ASHBURNER 1989), it may not be surprising that, given our small sample size of five

YTDs, we did not recover YTDs using γ-rays. This view is consistent with the observation of LEWIS (1954), who found γ-rays less effective than X-rays at generating transvection-disrupting rearrangements, even though γ-rays are capable of generating

18 such rearrangements (SMOLIK-UTLAUT and GELBART 1987, HENDRICKSON et al. 1995;

GUBB 1997).

One way to confirm the effectiveness of both X-rays and γ-rays in our studies is to assess the recovery of other classes of exceptions, especially those associated with gross chromosomal rearrangements. In particular, Table 2 shows that Class II mutants were recovered in both Screen A, which used X-rays, as well as in Screens B and C, which used γ-rays. Another class of exceptions that may serve as a measure for the efficacy of

X-rays and γ-rays is summarized in Table 1. These exceptions were isolated as darkly pigmented sterile males and may have resulted from irradiation-induced rearrangements which attached the tip of the irradiated X chromosome, carrying y2 or y1#8, to an autosome, followed by loss of the remainder of the X. In this scenario, dark pigmentation would result from complementation between y2 or y1#8 carried on the translocated X tip and y1#8 or y2, respectively, on the maternally-derived X chromosome, producing XO flies, which have male characteristics but are sterile due to lack of a Y chromosome.

Alternatively, these exceptional males may have been caused by large irradiation-induced intrachromosomal deletions of the X, which would produce free duplications of the X carrying y2 or y1#8, noted as Dp(1;f)y2 or Dp(1;f)y1#8. Here, dark pigmentation would be explained by pairing of the yellow allele on the Dp(1;f)y2 or Dp(1;f)y1#8 chromosome with the yellow allele on the maternal X, the male characteristics and sterility resulting from deletion of X material and absence of a Y chromosome.

The frequencies with which Class II mutants and darkly pigmented sterile males were generated can be used to determine the relative effectiveness of X-rays (Screen A) and γ-rays (Screens B, C, and D) in our hands. Screen A produced two Class II mutants

19 among 54,000 females (0.004%), while Screens B and C together produced three Class II mutants among 41,000 females (0.007%), and Screen A produced 28 dark sterile males

(0.05%) while Screens B, C, and D together produced 35 such males among 61,000 females (0.06%). Based on these observations, the frequencies of generating rearrangements in our screens are comparable whether X-rays or γ-rays are used.

To summarize, our screens of 115,000 females yielded five lines that belong to

Class I and are YTDs (Tables 1-3; Figure 2). This frequency for the generation of putative transvection-disrupting rearrangements (0.004%) is much less than that observed for loci associated with large critical regions and indicates that the critical region for yellow transvection is small, on the order of ~650 kbp or less (MATERIALS and

METHODS). One explanation for the small size of the yellow critical region may be that pairing of yellow alleles depends on a pairing site within ~650 kbp of yellow. In this view, only rearrangements that break between yellow and the pairing site would be able to disrupt transvection. Alternatively, proximity to a telomere may enhance pairing

(PANDITA et al. 2007) and, if so, the small critical region of yellow may be the consequence of the telomeric location of yellow. Finally, our data indicate that yellow alleles may require up to ~650 kbp of contiguous flanking homology in order to achieve the level of somatic pairing necessary for an observable degree of complementation..

Transvection at yellow transgenes may also be associated with a small critical region: We tested whether the yellow critical region reflects the presence of a pairing site, proximity to a telomere, or the requirement for a certain amount of contiguous flanking homology by applying the LEWIS (1954) protocol to yellow transgenes which support transvection at ectopic sites in the genome (CHEN et al. 2002). We reasoned that

20 if the critical region for yellow is determined by the distance between yellow and the nearest pairing site, then the critical regions for the transgenes would likely differ in size from each other and from that of yellow at its natural location, unless the transgene insertion sites were each within ~650 of a pairing site. Alternatively, if the critical region for yellow at its natural location is determined by proximity to a telomere, then the critical regions of transgenes located far from telomeres should be considerably larger and differ from each other. Finally, if the critical region for yellow reflects a requirement for a minimum amount of flanking homology, then the critical regions of transgenes should be small, resembling that of yellow at its natural location.

The transgenes we used were generated in an earlier study from a construct that had been inserted into eight ectopic sites (CHEN et al. 2002). This construct carried a yellow gene that had been modified by target sites for the Cre and FLP recombinases such that separate expression of the recombinases produced two derivatives at each insertion site, one lacking the wing and body enhancers (P[enh-]) and another lacking the

- promoter (P[pro ]) (Figure 1B; CHEN et al. 2002). Importantly, complementation was observed between pairs of allelic P[enh-] and P[pro-] derivatives at all eight ectopic sites, indicating that the genome is generally permissive of yellow transvection.

In our screens for transvection-disrupting rearrangements, we chose two pairs of complementing transgenes, one located in the middle of the right arm of chromosome 2 at polytene position 47A and a second located in the middle of the right arm of chromosome 3 at polytene position 92A (Figure 1B). Transgene pairs at these two sites, compared to the other six sites, were chosen because they supported relatively good viability and, furthermore, were predicted to give a significant difference in pigmentation

21 between complementing and noncomplementing flies. Specifically, the transgenes at position 47A and 92A produced strong complementing levels of pigmentation, (3+, 4) and

(4, 4), respectively, while flies carrying just the P[enh-] transgene at these two sites

+ produced relatively low levels of pigmentation, (2, 2 ) and (2, 2), respectively (CHEN et al. 2002).

To find rearrangements that disrupt transvection, males carrying P[enh-] at 47A or 92A and a deletion of the natural yellow gene, Df(1), were irradiated with γ-rays and crossed to Df(1)/Df(1) females carrying P[pro-] at 47A or 92A, respectively, and the resulting progeny were screened for flies with a level of wing and body pigmentation lower than that expected of a complementing genotype (see MATERIALS AND METHODS for full description of Df(1) and screen). We chose to irradiate flies carrying P[enh-] rather than P[pro-] because P[enh-] retains the bristle enhancer and, by providing a dark bristle phenotype, provided a convenient marker with which we could follow the irradiated chromosomes in our crosses. Our strategy also permitted us to screen for disruptive rearrangements in both males and females as the location of the transgenes on the autosomes permitted complementation in both sexes.

We screened 10,000 and 26,000 flies carrying the 47A and 92A transgenes, respectively, and found no dark-bristled non-complementing flies. We believe that γ- irradiation was effective in our screens because we did recover several flies lacking dark bristle pigmentation, indicating that the P[enh-] transgene had likely been disrupted.

Furthermore, because our screen involving the 92A transgenes made use of the TM3 third chromosome balancer, which carries a recessive mutation of the ebony gene, we were able to recover four apparent mutations of ebony. These findings indicate that the

22 frequency of the recovery of transvection-disrupting rearrangements for yellow transgenes at 47A and 92A as determined by our complementation assay is on the order of less than 0.01% (1/10,000) and 0.004% (1/26,000), respectively, and predict that the critical regions for these transgenes is small, as is the critical region for yellow at its natural location.

As no transvection-disrupting rearrangements were found in the screens using

P[enh-] and P[pro-] transgenes at 47A and 92A, we did a subsequent screen using X-rays instead of γ-rays because X-rays were the mutagen used in Screen A where YTDs were obtained. This screen was also designed to simultaneously recover rearrangements that disrupt transvection at Ubx, giving us a positive control for the generation of rearrangements. Specifically, we used Cbx Ubx transvection, where Cbx Ubx / + + flies show wing-to-haltere transformation due to the action of the gain-of-function Cbx mutation on the wild-type Ubx+ allele in trans; rearrangements can disrupt this interaction, resulting in flies with wings showing less wing-to-haltere transformation

(reviewed in DUNCAN 2002). In this screen, we irradiated Df(1)/Y males carrying Cbx

Ubx on one third chromosome and the enhancerless transgene at 92A on the other, with

X-rays. These flies were then crossed to Df(1)/Df(1) females carrying the promoterless transgene at 92A. In this way, we were able to screen for disruption of transvection at yellow by looking for flies that carried the enhancerless transgene and the promoterless transgene but showed reduced wing and or body pigmentation and, simultaneously, screen for disruption of transvection at Ubx by looking for flies that carried Cbx Ubx but showed reduced wing-to-haltere transformation.

23 We screened 15,000 flies for reduced cuticular pigmentation and another 15,000 for reduced wing-to-haltere transformation. We found two flies showing reduced levels of pigmentation, but they did not transmit this phenotype. We also found 92 flies showing reduced wing-to-haltere transformation, giving a ~0.6% (92/15,000) frequency of recovery, comparable to that found in other studies (LEWIS 1954). Seven of these were analyzed cytologically, and each was found to have a rearrangement between the centromere and Ubx on the right arm of chromosome 3 (Table 5).

Our ability to generate rearrangements that disrupt transvection at Ubx but none that disrupt transvection between ectopic yellow transgenes suggests that the X-rays effectively generated rearrangements and that the critical region for yellow transvection at ectopic sites is small, similar to that for the endogenous yellow gene. Taken together, our results are most consistent with yellow transvection, as determined by complementing levels of pigmentation, requiring a minimum amount of contiguous flanking homology.

Duplications of y2 can affect yellow complementation: During the course of these studies, we recovered three duplications of the tip of the X chromosome containing y2 (Table 2; Class V). The presence of a duplication was recognized through crosses of exceptional y2*/y1#8 females to y1#8/Y males that produced apparent complementing male progeny as well as three classes of female progeny – complementing, partially complementing, and non-complementing. These four classes of progeny indicated that a y2 allele was segregating independently of the X chromosome, and cytological studies confirmed the presence of duplications (Figure 3). Importantly, the ability of these duplications to affect complementation suggested that the y2 duplication can interact with

24 yellow at its natural position on the X chromosome. We therefore used these three duplications as a tool to better understand how yellow genes pair.

One duplication, called Dp(1;2)y2A, reduces the level of complementation normally observed with y2/y1#8 females from a score of (4, 4) to (3, 3) (Table 6). It consists of the tip of the X chromosome through polytene subdivision 1B-C attached to the tip of the right arm of the second chromosome and is ≤ 700 kbp in size (MATERIALS

AND METHODS). We reasoned that the reduction in complementation caused by

Dp(1;2)y2A might be due to its ability to disrupt the productive interaction of y2 and y1#8 at the natural yellow location, permitting y2 carried by Dp(1;2)y2A to pair with either the y2 or y1#8 allele on the X, being productive only in the latter case (Figure 4, A and C).

To further test whether pairing of Dp(1;2)y2A with y1#8 can be productive with respect to pigmentation, we determined the effect of Dp(1;2)y2A on the homozygous y1#8/y1#8 as well as the heterozygous y1/y1#8 genotypes. In contrast to y2/y1#8, neither of these genotypes supports complementation, giving us an opportunity to detect productive interactions between the y2 of Dp(1;2)y2A and y1#8 as increases in pigmentation.

Consistent with our model, we found that the presence of Dp(1;2)y2A increased the pigmentation scores of both y1#8/y1#8 as well as y1/y1#8 flies from (1, 1) to (2, 2) (Table 6;

Figure 4, B and D).

Finally, we determined the effect of Dp(1;2)y2A when it is not competing with another yellow allele in pairing interactions by placing it in females carrying a yellow allele at the natural yellow locus in trans to Df(1), or in males, which carry only a single

X chromosome and are therefore hemizygous for the yellow locus. Remarkably,

Dp(1;2)y2A increases the pigmentation score of Df(1)/y1#8 flies from (1, 1) to (4, 3+).

25 Similarly, male y1#8/Y flies carrying Dp(1;2)y2A also show dark pigmentation.

Interestingly, however, male y1#8/Y flies with two copies of Dp(1;2)y2A have light pigmentation (Table 6). These results suggest that, when present in only one copy, a y2 allele carried by Dp(1;2)y2A can pair well with an unpaired yellow allele on the X and compete effectively for paired alleles on the X. However, when homozygous,

Dp(1;2)y2A becomes much less effective in pairing with X-linked yellow alleles.

The two other duplications carrying y2, called Dp(1;4)y2B and Dp(1;4)y2C, were also tested for their ability to affect complementation. These duplications are both attached to the fourth chromosome and are much larger than Dp(1;2)y2A (Figure 3).

Dp(1;4)y2B attaches the tip of the X chromosome through polytene subdivision 3A to the fourth chromosome at polytene subdivision 102F, and Dp(1;4)y2C attaches the tip of the

X through polytene subdivision 1E to the fourth chromosome at 102C. Based on the

Drosophila genome sequence, Dp(1;4)y2B and Dp(1;4)y2C are ≤ 2.5 Mbp and ≤ 1.1 Mbp in size, respectively (MATERIALS AND METHODS).

We find that the pigmentation scores of y2/y1#8 flies are reduced from (4, 4) to (2,

1) by both Dp(1;4)y2B and Dp(1;4)y2C (Table 6; Figure 4, A and E). This reduction in pigmentation is in contrast to the higher score of (3, 3) produced by y2/y1#8 females carrying Dp(1;2)y2A, suggesting that Dp(1;4)y2B and Dp(1;4)y2C are relatively more effective at disrupting pairing at the natural yellow locus. Consistent with this interpretation, we find that the pigmentation of y1#8/y1#8 flies is increased from (1, 1) to

(3, 3) in the presence of Dp(1;4)y2B or Dp(1;4)y2C, in contrast to the lower (2, 2) score of y1#8/y1#8 flies carrying Dp(1;2)y2A (Table 6; Figure 4, B and F). Although our data rest on just three duplications, they suggest that duplication location and/or size can influence

26 pairing competition. More specifically, the greater ability of Dp(1;4)y2B and Dp(1;4)y2C compared to Dp(1;2)y2A to compete for pairing with alleles on the X may be the result of their attachment to the fourth rather than the second chromosome or the larger sizes of

Dp(1;4)y2B and Dp(1;4)y2C.

DISCUSSION

Our studies were aimed toward a better understanding of homologue pairing at yellow. Using a technique devised by LEWIS (1954), we show that yellow transvection, as assayed by intragenic complementation between y2 and y1#8, is dependent on pairing. In addition, we find that this transvection is extremely difficult to disrupt by chromosomal rearrangements; only five YTDs were recovered from 115,000 female flies screened in our studies. As these rearrangements all have at least one breakpoint close to yellow, our data further suggest that the critical region for yellow is small. We assume from this that all rearrangements that broke the X chromosome further from yellow allowed enough pairing to produce flies with complementing levels of pigmentation and, therefore, were not recovered in our screens.

Cytological analysis showed that all five YTDs had breakpoints less than ~650 kbp from yellow (Figure 2; MATERIALS AND METHODS). Because the chance of having all five breakpoints fall, at random, within a ~650 kbp interval is 1 x 10-9 [(650 kbp/40

Mbp)5],assuming that the X chromosome contains approximately one fifth of the genome, or ~40 Mbp (BOSCO et al. 2007), we believe that ~650 kbp is a good estimate of the critical region for yellow transvection as assayed by complementation between y2 and y1#8.

27 In addition to the five YTDs, we also recovered three duplications of the tip of X chromosome containing the y2 allele (Figure 3). These duplications were identified by their ability to disrupt complementation at the natural yellow locus and are likely exerting their influence by acting as competitive pairing partners (Figure 4). As the smallest of these duplications is ≤700 kbp in size, it appears that this amount of yellow genomic region is sufficient to support a level of homology searching and pairing that permits yellow enhancer action in trans. This interpretation is consistent with our estimate of

~650 kbp as an upper limit for the size of the yellow critical region. Interestingly, these duplications differ from each other when tested for their impact on yellow transvection, suggesting that duplication location and/or size can influence the extent to which duplications compete for pairing partners. These findings are in agreement with those of

2 GEYER et al. (1990), who studied a duplication of the yellow genomic region attaching y to the Y chromosome.

The critical regions for yellow are not consistent with a role for pairing sites:

The small size of the critical region we found for transvection at the natural location of yellow was reinforced by our studies using yellow transgenes. Although we found no rearrangements that disrupted transvection at the two ectopic locations tested, the number of flies screened, 10,000 for transgenes at 47A and 41,000 for transgenes at 92A, is informative. In particular, for genes with large critical regions spanning up to a third of a chromosome arm or more, the percentage of irradiated chromosomes found to carry transvection-disrupting rearrangements is comparatively large, ranging from 0.2% to

0.8% or more when chromosomes are treated with 4,000 - 4,500 rads of X-rays (LEWIS

1954; GELBART 1982; LEISERSON et al. 1994; SIPOS et al. 1998) and, thus, if transvection

28 at the two ectopic locations of yellow were associated with large critical regions, we should have recovered on the order of 20-80+ YTDs for transvection at 47A and on the order of 82-328+ YTDs for transvection at 92A. Instead, we failed to recover a YTD for transvection at these ectopic locations, even though we obtained disruptors of transvection at Ubx at a frequency of ~0.6%. These data suggest that the critical regions for ectopic yellow transvection are small and similar in size to that for transvection at the natural yellow locus.

Our findings argue against the yellow critical region being determined by proximity to a telomere or distance to a nearby hypothetical pairing site, as both transgene pairs were located far from any telomere and it is unlikely that both would have coincidentally inserted very near pairing sites. Instead, our data are consistent with yellow requiring ≤700 kbp of contiguous flanking homology in order to support a level of pairing-mediated complementation that can be detected in our visual assays. It is possible that the location of the 47A and 92A transgenes in the middle of chromosomal arms contributed to our inability to recover transvection-disrupting rearrangements because a mid-chromosome location would, a priori, permit pairing to be established or stabilized either proximally or distally to the transgene. In this situation, disruption of transgene complementation could require that the transgenes be flanked by two chromosomal breakpoints, each within 650 kbp of the yellow transgene. Such events would be very rare.

Our analysis of yellow transgenes is consistent with a previous study demonstrating that the size of the critical region associated with white gene transvection also does not depend on the chromosomal location of white (SMOLIK-UTLAUT and

29 1 1 GELBART 1987). Transvection at white occurs in a zeste (z ) background, where the mutant zeste protein leads to suppression of the paired w+ genes. Using insertional

+ translocations that placed a w gene within the large dpp critical region, SMOLIK-UTLAUT and GELBART (1987) found that rearrangements which had breakpoints proximal to both white and dpp and which disrupted dpp transvection did not disrupt white transvection. A similar outcome was found using a P-element insertion of w+ in the large critical region for Ubx. That is, the critical region for white was small regardless of its location within the large critical region of another gene, suggesting that pairing of white, as assayed by

1 the z phenotype, is not determined by a pairing site (SMOLIK-UTLAUT and GELBART

1987). Instead, these observations, together with our findings for yellow, suggest that the critical regions of at least some genes may reflect gene-specific features, such as the tissue in which a gene is expressed and the cell cycle length of that tissue, the degree or amount of pairing that is required for a gene to respond to pairing, the capacity of a gene to remember the paired state, and/or the strength of the interaction between the promoter of a gene and its enhancers (for a more in-depth review, see DUNCAN 2002; also see

LEWIS 1954; GELBART 1982; SMOLIK-UTLAUT and GELBART 1987; WU 1993; LEISERSON et al. 1994; HENDRICKSON and SAKONJU 1995; HOPMANN et al. 1995; DERNBURG et al.

1996; GOLIC and GOLIC 1996b; GUBB et al. 1997; FUNG et al. 1998; GEMKOW et al.

1998; SIPOS et al. 1998; MCKEE et al. 2000; COULTHARD et al. 2005). For example, the critical region of yellow may be small because the yellow wing and body enhancers may be especially capable of long distance, productive, and/or prolonged interactions.

Another determinant of the size of a critical region may be the sensitivity of a specific transvection-mediated phenotype to transcript levels (reviewed by DUNCAN

30 2002, also see SMOLIK-UTLAUT and GELBART 1987; SIPOS et al. 1998; MORRIS et al.

1999b). For example, a phenotype that can be achieved with low transcript levels may remain unaltered by a pairing-disruptive rearrangement if the rearrangement does not completely abolish pairing. In this case, a low but productive amount of pairing resulting from transient homologue interactions in each cell or from the cumulative amount of pairing across a cell population may be sufficient to produce a phenotype that approximates that of a fully paired genotype. In contrast, a phenotype that requires high transcript levels may be sensitive to all rearrangement breakpoints that reduce gene expression, even if by just a small amount, and consequently be associated with a large critical region. Transvection at yellow may be particularly difficult to disrupt because wild-type pigmentation can be achieved with only ~33% of the normal transcript level, as assayed in whole organisms (MORRIS et al. 2004; LEE et al. 2006), limiting visual assays to the recovery of only those breakpoints close enough to yellow to reduce transcript levels below this threshold.

Finally, the small size of some critical regions may reflect a means by which two alleles become paired and/or are maintained in a paired state as a consequence, at least in part, of their being individually localized to a specific nuclear address or nuclear compartment, such as a transcription factory. This potential mechanism of homologue

1 association has been proposed for both z -mediated repression of white (DAVIDSON et al.

1985; WU 1993; COOK 1997; XU and COOK 2008) as well as PcG-mediated silencing

(MATEOS-LANGERAK and CAVALLI 2008). Here, we suggest that the smaller size of some critical regions may reflect a correspondingly larger contribution of this mechanism to pairing and transvection. If the element being targeted to a nuclear location lies close to

31 the gene, then only breakpoints falling near enough to the gene to separate it from such an element would be able to disrupt transvection. On the other hand, if the targeted element is the gene itself, then it may be that transvection can only be disrupted by rearrangement breakpoints that are near enough to the gene to overcome the localization of the gene to the correct nuclear compartment. Alternatively, a rearrangement could introduce a foreign element that pulls the gene to another nuclear location. Note that, as some nuclear compartments are formed by the coalescence of multiple chromosomal regions, a gene targeted to such a compartment may have numerous opportunities to enter the compartment, making transvection at such a gene relatively more permissive of rearrangements and, therefore, more likely to be associated with a small critical region.

In contrast, a gene targeted to only a single nuclear address would have limited opportunities to co-localize with its homologue, be less tolerant of rearrangements, and have a larger critical region. In light of these arguments, nuclear compartmentalization in organisms where extensive homologue pairing has not been observed may be the equivalent of somatic pairing (WU 1993; COOK 1997; XU and COOK 2008).

In addition to the different sizes of critical regions, researchers have observed a propensity of the chromosomal breakpoints that disrupt transvection to fall proximal to the gene (reviewed in DUNCAN 2002; KENNISON and SOUTHWORTH 2002). Again, arguments that consider chromosomal positioning may be relevant. For example, a proximal location of the breakpoints would be expected if co-localization of homologous chromosomes to the same nuclear territory facilitates homology-searching or the targeting of alleles to the same nuclear position; rearrangements that reattach a gene to another centromere could antagonize pairing by placing that gene in a nuclear territory

32 that is different from that of its homologue, as has also been suggested by COULTHARD et al. 2005. In light of this interpretation, it is noteworthy that the y2 alleles of Dp(1;2)y2A,

Dp(1;2)y2B, and Dp(1;2)y2C seem able to pair with a yellow allele on the X chromosome even though they are attached to autosomal centromeres. Such pairing may reflect the ability of telomeric sequences to more easily find their homologues, as compared to sequences located more centrally in chromosomes, either because of the greater capacity of telomeres to traverse nuclear space and/or because the Rabl configuration of chromosomes after cell division promotes the interaction of telomeres (PANDITA et al.

2007).

The proximal location of rearrangement breakpoints has also been interpreted to indicate that pairing progresses in a centromere to telomere direction, with the proximal endpoint of the critical region marking the location of a pairing site (LEWIS 1954). In contrast, FISH analyses of developing embryos indicate that pairing initiates at multiple sites along a chromosome in random order (CSINK and HENIKOFF 1998; FUNG et al. 1998;

GEMKOW et al. 1998; VAZQUEZ et al. 2001). This difference in interpretation may result from differences between the experimental approaches, the first relying on phenotypic outcomes in the adult fly of changes in gene expression caused by chromosomal rearrangements, and the second using direct labeling of chromosomes to reveal the initiation of pairing in embryonic nuclei. Alternatively, it may indicate that the level of pairing necessary for transvection requires multiple steps and that different experimental approaches address different steps. For example, pairing may involve homology sensing followed by homologue alignment, pairing initiation, pairing establishment, pairing maintenance, and perhaps even a refinement or intensification of pairing prior to the

33 event of transvection. If so, FISH analyses of embryonic nuclei may be addressing pairing initiation while rearrangement studies may be defining the parameters required for later stages of pairing. In this light, the proximal location of rearrangement breakpoints for some genes may correspond to a step of pairing, perhaps establishment, that progresses in a centromere to telomere direction.

We thank P. GEYER for many years of close collaboration and fly stocks, J.

BATEMAN for suggesting the simultaneous screen for disruption of yellow and Ubx transvection, the WU lab for insightful discussion and support, W. BENDER, J. LEE, N.

PERRIMON, R. PRUITT, L. RAFTERY, and F. WINSTON for thoughtful input, A. MORAN for generous technical assistance, and the Bloomington Stock Center for fly stocks. This study was supported by GM085169 and GM61936 from the National Institutes of Health to C.-t. W. and by DUE-0736995 from the National Science Foundation to J.R.M. and S.

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41 TABLE 1

Summary of screens for transvection-disrupting rearrangements at yellow

Screen A Screen B Screen C Screen D

Type of irradiation X-ray γ−ray γ−ray γ−ray

2 2 2 1 1#8 a y * y * y z * y * Genotype of F1 females y1#8 y1#8 y1#8 z1 y2 w1118

# of females screened 54,000 24,000 17,000 20,000

Exceptional femalesb 23 5 6 7

Sterile 9 2 2 4

Fertile 14 3 4 3

Darkly pigmented malesc 28 4 13 18

aThe mutagenized chromosome is indicated by * bFemales with lighter wing and/or body pigmentation cSterile males with dark wing and body pigmentation

42 TABLE 2

Classes of mutant lines generated in Screens A, B, and C

Class Polytene Southern Screen A Screen B Screen C

IR+500

IIRR221

III + R 5 0 0

IV++201

V Duplication n/a 0 1 2

Total 14 3 4

Mutant lines were characterized and placed into four classes (I - IV) based on the cytological appearance of their X chromosomes, as determined by polytene analysis, and the integrity of the yellow gene, as determined by Southern analysis. Class V lines carry a duplication of the tip of the X chromosome. R: rearranged, +: normal. Pigmentation scores of y2*/y1#8 Class I and II females are provided in Tables 3 and 4. y2*/y1#8 pigmentation scores for the five Class III females are (1-2, 2), (4, 2), (4, 2), (2, 2), and (1, 1), and for the three Class IV females are (3, 2), (2, 1), and (2, 3).

43

TABLE 3

Class I lines: yellow transvection-disrupting rearrangements (YTDs)

Name Cytology Pigmentation scores (W, B)

YTD1 T(1;3) 1B-C; 77D-78A (1, 1)

YTD2 In(1) 1A; 1C-D + Tp(3;1) 100A7; 100C1; 1C-D (2, 2)

YTD3 T(1;2) 1B-C; 5D-E; 36B-D; 40F-41B (2, 1)

YTD4 T(1;2;3) 1B-C; 11C-D; 21C-D; 28D-F;99D (3, 1)

YTD5 T(1;2;3) 1B-C; 39E-F; 80A-B; 98F (1, 1)a

Each of these five YTDs carries a structurally normal y2 gene and a rearrangement breakpoint on the X chromosome near yellow. (W, B) refers to wing and body pigmentation of females on a scale of 1 to 5, with 5 being wild-type (MATERIALS and METHODS). Pigmentation scores refer to YTD/y1#8 flies. aThe bristle pigmentation of these genotypes is variegated, ranging from dark to light, and is consistent with an intact yellow gene whose expression is not uniform.

44

TABLE 4

Class II lines

Name Cytology Pigmentation scores (W, B)

Line 6 T(1;4) 1B-C; 102D-F (4, 1)

Line 7 In(1) 1B-C; 9B; 20 (1, 1)

Line 8 T(1;2) 1B-C; 11-12; 20; 51 (1, 1)

Line 9 T(1;2;3) 1B-C; 40-41A; 98C-E (1, 3)a

Line 10 T(1;2) 1B-C; 28C (1, 1)

Each of these five Class II lines carry a structurally disrupted yellow gene as well as a breakpoint on the X chromosome near yellow. Pigmentation scale is as described for Table 3. Lines 6 and 7 are from Screen A, line 8 and 9 are from Screen B, and line 10 is from Screen C. aThe bristle pigmentation of these genotypes is variegated, ranging from dark to light, and is consistent with an intact yellow gene whose expression is not uniform.

45 TABLE 5

Bithorax transvection-disrupting rearrangements (BTDs)

Name Location of breakpoint on 3R

BTD1 82A-B

BTD2 87E-F

BTD3 84E

BTD4 82C-D

BTD5 88A

BTD6 82F

BTD7 83C

Seven lines of flies showing reduced wing-to-haltere transformation were analyzed by cytology. The locations of the breakpoint on the right arm of chromosome 3 are indicated above. Other breakpoints are not indicated.

46 TABLE 6

Pairing competition in the presence of y2 duplications

Duplication Genotype Pigmentation scores (W, B)

None y2/ y2 (1, 1+)

y1#8/ y1#8 (1, 1)

y2/ y1#8 (4, 4)

y1/ y1#8 (1, 1)

Df(1)/ y1#8 (1, 1)

y1#8/ Y (1, 1)

Dp(1;2)y2A y1#8/ y1#8; +/ Dp(1;2)y2A (2, 2)

y2/ y1#8 ; +/ Dp(1;2)y2A (3, 3)

y1/ y1#8; +/ Dp(1;2)y2A (2, 2)

Df(1)/ y1#8 ; +/ Dp(1;2)y2A (4, 3+)

y1#8/ Y; +/ Dp(1;2)y2A dark pigmentation*

y1#8/ Y; Dp(1;2)y2A/Dp(1;2)y2A light pigmentation*

Dp(1;4)y2B y1#8/y1#8; +/Dp(1;4)y2B (3, 3)

y2/ y1#8; +/ Dp(1;4)y2B (2, 1)

Dp(1;4)y2C y1#8/y1#8; +/ Dp(1;4)y2C (3, 3)

y2/ y1#8; +/ Dp(1;4)y2C (2, 1)

*Pigmentation scale is only used to score female wing and body pigmentation. The Df(1)y- ac- w1118 chromosome is indicated as Df(1).

47

FIGURE 1.–Transvection at yellow at endogenous (A) and ectopic (B) locations. (A)

The y2 enhancers are blocked from the promoter by a gypsy insulator. y1#8 has a deletion of the promoter. y2/y1#8 flies show nearly wild-type wing and body pigmentation due to the action of the wing and body enhancers in trans on the intact promoter. W, wing enhancer; B, body enhancer; Br, bristle enhancer. (B) P[enh-] and P[pro-] are derived from P[yellowCF] (CHEN et al. 2002). P[enh-] is a deletion of the wing and body enhancers. P[pro-] has a deletion removing the promoter. White triangles, loxP sites used to generate the P[enh-]; striped triangles, FRT sites used to generate P[pro-].

Brackets indicate deleted sequences. 47A is on the right arm the second chromosome;

92A is on the right arm of the third chromosome. Gray box, P[enh-]; white box, P[pro-].

Figure not to scale.

FIGURE 2.–Locations of chromosomal breaks. (A) Polytene sections 1A-1F are labeled on the image of the tip of the X chromosome (SAURA et al. 1997). Locations of breakpoints of the five YTDs as determined by cytology are indicated below the figure.

(B) Images of unpairing at the tip of the X chromosomes due to chromosomal rearrangements in the five YTDs. For reference, the prominent band 1B3 is indicated on the non-rearranged X chromosome.

FIGURE 3.–(A) Sizes of duplications. Dp(1;2)y2A, Dp(1;4)y2B, and Dp(1;4)y2C are duplications of the tip of the X chromosome that are able to complement a yellow allele on the non-rearranged X chromosome. Breakpoints are indicated above the image of the

48 tip of the X chromosome (BRIDGES 1938). The duplications are ≤700 kbp (breakpoint at

1B-C), ≤2.5 Mbp (breakpoint at 3A), and ≤1.1 Mbp (breakpoint at 1E) in size, respectively. (B) Images of duplications. Dp(1;2)y2A is attached to the tip of the right arm of the second chromosome, while Dp(1;4)y2B and Dp(1;4)y2C are attached to the fourth chromosome.

FIGURE 4.–Model for pairing competition in the presence of duplications of y2. Gray diamonds represent y2 and white diamonds represent y1#8. Arrow thickness corresponds to strength of pairing interactions. Numbers indicate pigmentation scores of wing and body, respectively. (A) In the absence of a duplication, y2 and y1#8 in their normal location on the X chromosome are able to pair and give a pigmentation score of (4, 4).

(B) In the absence of a duplication, two y1#8 alleles in their normal location pair but do not complement, giving a pigmentation score of (1, 1). (C) Presence of Dp(1;2)y2A disrupts pairing of y2 and y1#8 on the X chromosome, reducing the pigmentation score to

(3, 3). (D) Presence of Dp(1;2)y2A disrupts pairing of the two noncomplementing y1#8 alleles on the X chromosome, permitting pairing between the y2 allele of Dp(1;2)y2A and y1#8 and, therefore, complementation corresponding to a pigmentation score of (2, 2). (E)

Presence of Dp(1;4)y2B or Dp(1;4)y2C, which appear to be stronger competitors of pairing compared to Dp(1;2)y2A, disrupts pairing of y2 and y1#8 on the X chromosome to a relatively high degree, reducing the pigmentation score to (2, 1). (F) Presence of

Dp(1;4)y2B or Dp(1;4)y2C disrupts pairing of the two noncomplementing y1#8 alleles on the X chromosome, resulting in a relatively high level of pairing between the y2 allele of

49 the duplications and y1#8 and, therefore, a relatively high level of complementation, corresponding to a pigmentation score of (3, 3).

50 Figure 1

A gypsy insulator

y2 W B Br

y1#8 W B Br

B P[yellowCF] 5’ W B Br 3’

P[enh-] 5’ [ ] Br 3’

P[pro-] 5’ W B [ ] 3’

II 47A

or III 92A Figure 2

yellow 1B1-2 1B3

YTD1 YTD2 YTD3 YTD4 YTD5

1B3 1B3

YTD1 YTD4

1B3 1B3

YTD2 YTD5

1B3

YTD3 Figure 3

A

Dp(1;2)y2A Dp(1;4)y2B Dp(1;4)y2C

B

1E

1B10 3A4 Dp(1;2)y2A Dp(1;4)y2B Dp(1;4)y2C Figure 4

No duplication AB

(4, 4) (1, 1) Dp(1;2)y2A, small duplication

CD

(3, 3) (2, 2) Dp(1;4)y2B or Dp(1;4)y2C, large duplication

EF

(2, 1) (3, 3)

= y2 = y1#8