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Molecular and Cellular Biology, July 2007, p. 4991-5001, Vol. 27, No. 13
0270-7306/07/$08.00+0     doi:10.1128/MCB.00515-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Transcriptional Activity of the Telomeric Retrotransposon HeT-A in Drosophila melanogaster Is Stimulated as a Consequence of Subterminal Deficiencies at Homologous and Nonhomologous Telomeres{triangledown}

Radmila Capkova Frydrychova,1 Harald Biessmann,2 Alexander Y. Konev,1,{dagger} Mikhail D. Golubovsky,1,{ddagger} Jessica Johnson,1 Trevor K. Archer,3 and James M. Mason1*

Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709,1 Developmental Biology Center, University of California, Irvine, California 92697,2 Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 277093

Received 24 March 2007/ Accepted 13 April 2007


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ABSTRACT
 
Drosophila melanogaster telomeres have two DNA domains: a terminal array of retrotransposons and a subterminal repetitive telomere-associated sequence (TAS), a source of telomere position effect (TPE). We reported previously that deletion of the 2L TAS array leads to dominant suppression of TPE by stimulating in trans expression of a telomeric transgene. Here, we compared the transcript activities of a w transgene inserted between the retrotransposon and TAS arrays at the 2L telomere in genotypes with different lengths of the 2L TAS. In contrast to individuals bearing a wild-type 2L homologue, flies with a TAS deficiency showed a significant increase in the level of telomeric w transcript during development, especially in pupae. Moreover, we identified a read-through w transcript initiated from a retrotransposon promoter in the terminal array. Read-through transcript levels also significantly increased with the presence of a 2L TAS deficiency in trans, indicating a stimulating force of the TAS deficiency on retrotransposon promoter activity. The read-through transcript contributes to total w transcript, although most w transcript originates at the w promoter. While silencing of transgenes in nonhomologous telomeres is suppressed by 2L TAS deficiencies, suggesting a global effect, the overall level of HeT-A transcripts is not increased under similar conditions.


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INTRODUCTION
 
Telomeres, nucleoprotein structures at chromosome ends, play two crucial roles in chromosome biology: they provide end protection by "capping" chromosome ends, and they maintain chromosome length by adding telomeric sequence and thus solve the end replication problem (8). There are several known mechanisms of telomere maintenance, including telomerase and homologous recombination extending short canonical telomere repeats in humans, homologous recombination extending tandem arrays of more complex repeats in lower Diptera and alliaceous plants, and targeted transposition in Drosophila melanogaster (5, 6, 43). Three non-long-terminal-repeat retrotransposable elements, HeT-A, TART, and TAHRE, which maintain Drosophila telomere length by transposition onto chromosome ends, are the only known retroelements with such a cellular function. The telomeric retroelements contain unusually long 3' untranslated regions (UTRs), which carry a promoter (14, 27, 40) to transcribe the elements downstream in the terminal array. Occasional long read-through transcripts encompassing more than one retroelement or homologous recombination may give rise to simultaneous addition of multiple elements (4). Specific targeting of the elements by their 3' oligo(A) tails to chromosome ends probably occurs via target-primed reverse transcription (34) and does not depend on the DNA sequence at the terminus (4, 5). Incomplete replication, combined with transposition, leads to mixed tandem arrays of complete and 5'-truncated elements on the ends of Drosophila chromosomes (1, 21, 35, 44). The level of retrotransposon transcription is correlated with cell proliferation, with the highest level of transcript being found in proliferating imaginal disc cells (20, 53) and in adult gonads, the primary location of cell proliferation during adult life. Recent evidence (48) showed a role for the RNA interference (RNAi) machinery in the transposition of telomeric retroelements, especially of TART, to chromosome ends and thus a possible role for RNAi in the control of Drosophila telomere length.

Immediately proximal to the terminal HeT-A, TART, TAHRE (HTT) array, Drosophila telomeres contain 15 to 26 kb of complex subterminal telomere-associated sequence (TAS) (1, 28, 33, 54). TAS arrays in Drosophila vary in sequence among telomeres but share sequence motifs among chromosome ends (28, 54). Similar structural motifs are also found in TAS regions of other eukaryotes (45). Genetically marked transposable elements that have inserted into TASs show repressed and variegated expression of the reporter gene (11, 19, 22, 24, 28, 32), referred to as telomeric silencing or telomere position effect (TPE). Evidence that the TAS is responsible for TPE comes from studies showing that a TAS adjacent to a reporter gene at nontelomeric positions also reduces reporter expression (9, 30). These observations are consistent with the immunostaining results showing that TASs, either at telomeres or in ectopic locations, bind Polycomb group proteins (2, 9). HTT arrays, however, bind proteins associated with open chromatin (2), and reporter genes inserted into the terminal HTT array are not repressed (7).

Molecular and genetic analysis of eye color variants derived from a white (w) reporter transgene inserted between the HTT array and TAS region of the left arm of chromosome 2 (2L) revealed several components that affect TPE (22, 36, 37, 38). Higher w expression was associated with increased numbers of HeT-A elements in the HTT array in cis, indicating an activating influence of terminal retrotransposons on the repressed w reporter. These genetic studies, however, could not distinguish between indirect activation through a change in the chromatin structure of the telomeric w gene and direct activation via read-through transcription from a more distal HeT-A promoter. Altered expression of the transgene also allowed the detection of interactions between homologous telomeres, because expression of the telomeric w reporter gene is increased in response to deletion of some or all of the TAS on the homologous chromosome (22, 31, 38). Our model of HeT-A activation based on these observations postulates that telomere communication may serve to assess telomere integrity and to stimulate enhancer/promoter activity of retrotransposons. This, in turn, may result in chromosome elongation when a deleted or interrupted TAS is present (22, 37).

Here we extended the genetic studies of TPE by measuring transcription from promoters of telomeric retrotransposons. This allowed us to test several predictions of the model. First, using three different telomeric insertions, we found that transcription of telomeric transgenes can be initiated at a promoter of an upstream HeT-A element. We then used the level of this HeT-A-transgene read-through transcript as an indicator of the activity of a specific HeT-A element in a P{wvar} 11-5 strain carrying a white (w) reporter transgene between the HTT array and a truncated TAS region of the left arm of chromosome 2. The HeT-A/w read-through transcript in P{wvar} 11-5 shows a significant correlation with total w transcript, although a majority of the total w transcript originates from the w promoter. Second, a 2L TAS deficiency leads to a release in silencing of both the w and HeT-A promoters. Third, silencing of w reporter genes in nonhomologous telomeres is suppressed by 2L TAS deficiencies, suggesting a global effect of these deficiencies on TPE. 2L TAS deficiencies, however, affect the activities only of retrotransposons and transgenes located within the TAS or in the proximal end of the HTT array, close to the TAS. Finally, total HeT-A transcript does not significantly correlate with HeT-A/w read-through transcript, nor does it respond to the presence of 2L TAS deficiencies, indicating that 2L TAS deficiencies do not play a significant role in telomere maintenance in Drosophila.


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MATERIALS AND METHODS
 
Drosophila strains. Drosophila stocks were maintained and crosses performed at 25°C on cornmeal-molasses medium with dry yeast added to the surface. Except for Oregon R, which has its w gene in the normal nontelomeric position on the X chromosome, all strains carried y1 and the w67c23 deficiency for the endogenous w gene. Insertion strains P{wvar}11-5 (referred to herein as 11-5), with a copy of the genomic w gene inserted between the HTT array and the TAS (22), and P{w+}39C-5 (referred to herein as 39C-5), with a mini-w gene in the middle of the TAS array (52), have reporter genes that are read in a distal-to-proximal direction (11). P{wvar}11-10 is a variant of P{wvar} that has lost some or all of the white coding sequence (22). P{w+}39C-27 (referred to herein as 39C-27) and P{w+}39C-62 (referred to herein as 39C-62) carry mini-w insertions in 2R and 3R, respectively (52). TPE suppressor strain Df(2L)M26 (referred to herein as M26) has a complete 2L TAS deficiency and a TART element attached directly to unique sequence DNA at the 2L telomere, leaving CG11023 and l(2)gl intact (unpublished data). Df(2L)net62 is an interstitial deficiency for l(2)gl (29) that extends into the TAS but leaves the HTT array intact. l(2)glGB26 is a terminal deficiency that removes the l(2)gl gene (41), while l(2)glGB52 carries a roo insertion in l(2)gl, and l(2)glDV275 carries a roo element in place of much of the l(2)gl coding sequence (41). We also used two strains carrying insertions of EPgy2 (EY08176) or SuPorP (KG01591) elements in the HTT array (7) and four strains carrying euchromatic insertions of a mini-w gene with a 2L TAS inserted between the eye enhancer and the promoter: P{w+}836I/U1 (A6-514) and P{w+}836I/U1 (A8-2A) have <1 kb of the TAS (referred to herein as A6-514 and A8-2A), while P{w+}836I/U1 (A8-511) and P{w+}836I/U1 (B8-13Y) have 6 kb of the TAS (referred to herein as A8-511 and B8-13Y) (30).

Quantitative measurement of red-eye pigments. Five adult heads were homogenized in a 1:1 mixture of chloroform and ammonium hydroxide (0.1%); the homogenate was centrifuged, and the optical density of the upper aqueous layer was measured at 485 nm (3). Pigment analysis was performed in five samples for each strain.

RNA isolation and cDNA synthesis. RNA samples of Oregon R and 11-5 were isolated from whole wandering larvae, late-stage pupae, and 1-day-old adults using an RNeasy minikit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. RNA from ovaries was extracted using the TRIzol reagent (Gibco BRL Life Technologies, Rockville, MD). For each sample we used 30 dissected ovaries. RNA was converted to cDNA using either oligo(dT) or primers specific for the expected read-through transcripts (P-ry-4, 5'Plac-1, or white D) and Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA).

PCR primers used here were as follows: P-ry-4, 5'-CAATCATATCGCTGTCTCACTCA-3'; 3'larB (forward), 5'-CATACGTTAAGTGGATGTCTCTTGC-3'; TAHRE gag (forward), 5'-CCCCTCGATCGACAATACAG-3'; TAHRE gag (reverse), 5'-GTTAGTGTGGGTCGTTGCTG-3'; HeT-F1 (forward), 5'-CTGTCTCCGTACCTCCACCAGC-3'; HeT-F2 (forward), 5'-CCCCAAACTCACCCATGAATG-3'; HeT-F3 (forward), 5'-GCTTCCAGCGACTCGGTGCTTCCG-3'; lac1, 5'-CACCCAAGGCTCTGCTCCCACAAT-3'; 5'larB (forward), 5'-GGCTATCGACGGGACCACC-3'; 1591 upstream P, 5'-CGTCCCGCGCTTACCAATAC-3'; white D, 5'-CCACCGTTTGTAGCGTTACCTAGC-3'; wCS (forward), 5'-CCTCTTTATCGGCTCCCTAACG-3'; wCS (reverse), 5'-TCGTGTGCTGACATTTGCTGAG-3'; set 1 (forward), 5'-CACATCGTCCGTGAAAATGG-3'; set 1 (reverse), 5'-AGCACCCGTACTTCGGACAC-3'; set 2A (forward), 5'-CCGAACCCATAACACATTCCTCTC-3'; set 2A (reverse), 5'-TAAATCAGCGCTGCCATTTG-3'; set 2B (forward), 5'-CTGTTACCACATCGTCCGTG-3'; set 2B (reverse), 5'-TGTTGTCGTCCCGTTTCG-3'; set 3 (forward), 5'-GCCACCGTTTGTAGCGTTAC-3'; set 3 (reverse), 5'-AAGAAGCGAGAGGAGTTTTGG-3'; w-1 (reverse), 5'-AAGGAAGTAACTTGAACTGAGGCG-3'; HCS (forward), 5'-ATTGTCTTCTCCTCCGTCCACC-3'; HCS (reverse), 5'-TTCTCTATGCTATTGTCGCTGTGC-3'; RpL32 (forward), 5'-GGACAGTATCTGATGCCCAAC-3'; RpL32 (reverse), 5'-ATCTCGCCGCAGTAAACGC-3'; RpS17 (forward), 5'-AAGCGCATCTGCGAGGAG-3'; RpS17 (reverse), 5'-CCTCCTCCTGCAACTTGATG-3'.

Qualitative RT PCR. Qualitative RT-PCR was used to detect read-through transcripts. The PCR mixture contained 200 nM of forward and reverse primers with about 500 ng of cDNA. PCRs were denatured at 94°C for 3 min, followed by a 30-s denaturation at 94°C, annealing at 55°C for 30 s, and a 1-min extension at 72°C for 35 cycles. Products were loaded and electrophoresed in 2.5% agarose gel in TAE.

Quantitative real-time PCR (qRT-PCR). qRT-PCR was performed using an Mx3000P real-time PCR system with SYBR Green PCR Master Mix (Stratagene, La Jolla, CA). Threshold cycle was used to assess relative levels of target transcripts versus reference RpL32 transcripts. Quantification was performed in two independent experiments with five samples for each strain set up in triplicates.

Northern hybridization. RNA samples (20 µg/lane) were separated on an 0.8% agarose gel, and transferred to Hybond-N nylon membrane. Hybridization was performed with 32P-labeled RNA probes according to the method of Sambrook et al. (46).


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RESULTS
 
The M26 deficiency is a dominant suppressor of TPE. The 11-5 strain, which carries a w+ transgene inserted between the retrotransposon array and a partially deleted TAS at the tip of 2L (Fig. 1A), was characterized previously (22), but after reisolation of the chromosome carrying the transgene, we reanalyzed the sequence of the w+ insertion and found that the junction between HeT-A and the w+ transgene had moved closer to the eye enhancer (Fig. 1B and C). To determine whether silencing of the transgene was suppressed by a 2L TAS deficiency in the same way as the original isolate (22), we crossed the newly reisolated 11-5/11-5 flies with M26 flies, which carry a complete deletion of the 2L TAS (Fig. 2A). Consistent with previous results, 11-5/M26 flies exhibited red eyes, in contrast to 11-5/+, which showed yellow eyes. 11-5 homozygotes had red eyes (Fig. 2B). This confirmed that the M26 deficiency acts as a dominant suppressor of TPE on the homologue. We performed a spectrophotometric determination of red eye pigment levels in these genotypes, including wild-type Oregon R (Fig. 2C). Differences in pigment levels correlated with observed eye color phenotypes. A slightly brighter red eye color in 11-5/11-5 than Oregon R is confirmed by a slightly higher level of red pigments. To obtain a more direct measurement of TPE, we quantified w transcript levels by qRT-PCR with the primer set wCS (Fig. 1A) on RNA from late-stage pupae, because pigment deposition into eyes occurs at this stage (16). Transcript levels in pupae (Fig. 3) are in good correlation with the observed pigment levels (Fig. 2B and C). Thus, the perceived eye color in strains in which w is variegated due to TPE is a good approximation of quantitative measures of late pupal w transcription and further indicates that M26 suppresses TPE by increasing transcript levels of telomeric inserts.


Figure 1
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  		FIG. 1. P{wvar}11-5 telomeric transgene. (A) Schematic diagram of the 2L telomere in 11-5 bearing a complete white insertion between the terminal retrotransposon array and the TAS. The insert includes the upstream region of white with its eye-specific enhancer. Positions of primers used for cDNA synthesis are shown as arrows; primer pairs used to sequence the HeT-A/w junction or to identify transcripts are shown as arrowheads. Transcription of the HeT-A/w read-through product is distal to proximal. The capital "A" between HeT-A and w represents the HeT-A oligo(A) tail. (B) DNA fragment containing HeT-A/w junction was isolated by PCR using HeT-F2 and set 3 (reverse) primers, cloned into a pGEM-T-Easy vector, and sequenced with T3 and T7 primers. The positions of the HeT-F1 primer and the HeT-A/w junction with the HeT-A oligo(A) tail are shown. (C) DNA sequence of the upstream w region showing the HeT-A/w junction as it existed previously (22) and now. Enhancer and promoter regions are boxed; primer sequences are highlighted or underlined.


Figure 2
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  		FIG. 2. Activation of the P{wvar}11-5 transgene by a 2L TAS deficiency. (A) Schematic diagram showing the 2L telomere structure in homozygous 11-5 with a partially deleted TAS, in hemizygous 11-5/M26 with a complete TAS deficiency on the 2L homologue, and in hemizygous 11-5/+ with a wild-type homologue carrying a complete TAS. (B) Eyes of the wild-type Oregon R, homozygous 11-5, and hemizygous 11-5/M26 and 11-5/+. (C) Levels of red eye pigments in flies of the same genotypes.


Figure 3
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  		FIG. 3. Levels of white transcripts in Oregon R, homozygous 11-5, 11-5/M26, and 11-5/+ relative to transcripts of RpL32 at various stages of development. The RT-PCR products were derived using the wCS primers after the production of cDNA using oligo(dT).

Telomeric silencing is stage specific. Due to a lack of published information about telomeric silencing during Drosophila development, we asked if the effects of TPE on telomeric w expression may vary over time and if suppression of TPE by 2L TAS deficiencies also has a developmental component. We measured by qRT-PCR the level of w transcript in RNA extracted from wandering larvae, early,- mid-, and late-stage pupae, and 1-day-old adults (Fig. 3). During development there were significant changes in w transcript levels in both 11-5/11-5 and the Oregon R wild type, although they did not vary in concert. Transcript levels of the telomeric w transgene in 11-5/11-5 showed a tendency to increase with development, while transcript levels of the endogenous w gene showed the reverse trend (Fig. 3) (16). Levels of telomeric w transcript in larvae and early pupae are strongly repressed compared to the endogenous w of Oregon R and are not proportional to eye pigment deposition in adults (Fig. 2B and C). Considering that the level of w transcript in 11-5 is based on two copies of w, compared to one w copy in both 11-5/M26 and 11-5/+, we can see that transcript levels of w per copy in all three genotypes are comparable in larvae and early pupae. Both 11-5/11-5 and 11-5/M26 show a release in telomeric silencing during late pupal and adult stages, in contrast to 11-5/+, which shows relatively steady levels of w during development. Transcript levels from 11-5/M26 and 11-5/+ in late pupae and adults suggest that suppression of TPE by M26 leads to a roughly twofold increase in w expression. As 11-5 is associated with a partial 2L TAS deficiency (22), transcript levels of 11-5/11-5 and 11-5/M26 indicate that the effects of partial and complete TAS deficiencies on TPE are approximately equal. Thus, the developmental expression patterns of genomic w and a telomeric w are different, suggesting that telomeric silencing has a distinct developmental profile.

Read-through transcript from a HeT-A promoter into downstream sequences. It has been postulated that HeT-A transcription occurs in a distal-to-proximal direction starting at a promoter in the 3' UTR of an upstream HeT-A element (14). It also was suggested that the stimulating effect on P{wvar} expression by a TAS deficiency might be by direct read-through transcription driven from the promoters of distal retroelements through the w transgene (22). To test the possibility of HeT-A/w read-through, we examined single-copy w+ P elements inserted into an HTT array using two insertions, EY08176 and KG01591, of a P element inserted into a TAHRE element in the 2R telomere and a HeT-A element at the 3R telomere, respectively (7). In both lines, the retrotransposons with the P element insertions are flanked by upstream HeT-A elements. Maps of the insertions, which we characterized by sequencing of genomic lambda phages, are shown in Fig. 4A.


Figure 4
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  		FIG. 4. Read-through transcripts from HeT-A promoters into P elements residing within the HTT array. (A) Schematic maps of the HTT regions immediately distal to the P element insertions are shown. EY08176 has a single EPgy2 element inserted into the GAG open reading frame of a TAHRE element in the 2R telomere with a HeT-A element immediately upstream of this TAHRE. KG01591 carries a SuPor-P element inserted into the open reading frame of a HeT-A element at the 3R telomere, such that the w reporter gene is read in the opposite the direction that of the HTT array. Directly upstream of this HeT-A lies a short (168-bp) HeT-A fragment with an oligo(A) tail followed by a 3' HeT-A UTR with another oligo(A) tail. Dashed arrows indicate potential read-through transcripts from the upstream HeT-A promoters; short arrows indicate the primers used for cDNA synthesis (P-ry-4, lac1), and arrowheads indicate the primers used for RT-PCR detection of read-through transcripts. (B) An ethidium bromide-stained gel to identify read-through transcripts in both insertion lines is shown. Primer combinations are indicated below the gel. Genomic DNA served as a positive control and ovarian RNA without cDNA conversion as a negative control. The Oregon R lanes serve as a size control for the other lanes. The ribosomal gene, RpS17, served as a positive control. (C) An ethidium bromide-stained gel to determine the position of initiation of read-through transcription in EY08176 is shown. cDNA obtained with the P-ry-4 primer was subjected to PCR amplification with one primer anchored in P (3'larB) and the other in the HeT-A 3' UTR. Three primers in HeT-A (HeT-F1, -2, and -3) were used for this purpose. (D) Read-through transcript detected in 11-5. Products of RT-PCR with primer sets 1 and 2 and primers to RpS17. 11-5 and Oregon R cDNA synthesized using oligo(dT) or white D primer was used for the PCR. Samples without reverse transcriptase provided a negative control (RT–), and genomic DNA (G) provided a positive control. For positions of primers, see Fig. 1A and B. (E) Read-through transcript detected in 11-5 with primer set 3 (reverse), specific to upstream sequence of w, combined with primers HeT-F1, 2, and 3 (forward), specific to the 3' UTR of HeT-A. Specific PCR products are noted by arrows. (F) Northern hybridization of RNA extracted from larvae of Oregon R and 11-5 with a RNA probe specific to coding sequence of w.

Because strong HeT-A expression has been found in adult male and female gonads (53), we tested for transcription of the inserted elements from a retrotransposon promoter by producing cDNA from RNA samples taken from ovaries using the P-ry-4 primer anchored in EY08176 or the lac1 primer anchored in KG01591 (Fig. 4A). Transcripts were identified by PCR with primers (TAHRE-gag) specific to TAHRE and primers (5'larB and 1591 upstream P) specific to HeT-A, respectively. In parallel, RNAs were reverse transcribed with an oligo(dT) primer to test for the ribosomal RpS17 transcript as a positive control. Ethidium bromide-stained gels (Fig. 4B) clearly identified read-through transcripts in both insertion lines. To identify an initiation site for the read-through transcription, cDNA of EY08176 was subjected to PCR amplification with primer 3'larB, in the EY08176 element, combined with three primers located in the 3' UTR of HeT-A at distances of 20 bp (HeT-F1), 120 bp (HeT-F2), and 350 bp (HeT-F3) upstream from its oligo(A) tail. Although combinations of 3'larB primer with primers HeT-F2 and HeT-F3 gave no PCR product, amplification with HeT-F1 produced an ~950-bp fragment (Fig. 4C), which suggests that transcription initiated in the HeT-A element between 120 and 20 bp from its oligo(A) tail, supporting earlier hybridization experiments (14).

For all cDNA samples tested by RT PCR we also included controls for DNA contamination that had been prepared without reverse transcriptase (RT–). None of these control samples showed a PCR signal. These PCR experiments provided direct evidence of HeT-A transcriptional activity in a distal-to-proximal direction and raised the possibility of read-through transcription from a HeT-A promoter into adjacent transgenes.

To identify a read-through transcript from a distal HeT-A promoter into the adjacent w sequence in 11-5, we used PCR with three sets of primers (1, 2A, and 2B) specific to w sequence upstream of the promoter and 11-5 cDNA as a template (Fig. 1A and B). cDNA was synthesized from total adult RNA using oligo(dT) or the white D primer. No PCR product was found in Oregon R cDNA, confirming that w upstream regions are not transcribed in the normal genomic location of this gene. Neither was a product found in the RT– samples. However, a product was detected from amplifying 11-5 cDNA, indicating that the normally untranscribed, upstream region is included in a transcript of the telomeric transgene in 11-5. We infer that this transcript initiated at the promoter of an upstream telomeric retroelement. Next we used the primers from HeT-A (HeT-F1, HeT-F2, and HeT-F3) in conjunction with the primer set 3 (reverse) from the upstream w region (Fig. 1A) to identify the origin of the read-through transcript. Unlike with EY08176, amplification was obtained with all three HeT-A primers (Fig. 4E), indicating that the read-through transcript not only originates from the most proximal HeT-A promoter but also is created by a contribution of at least two retrotransposons. Further, we attempted to detect the read-through transcripts on Northern blots. We tested RNA extracted from whole larvae of 11-5 and Oregon R with an RNA probe specific to coding sequence of w. In both Oregon R and 11-5 we found only one hybridization signal, with a size corresponding to the normal, processed w transcript from a w promoter (Fig. 4F). In 11-5, the absence of a band with a size of at least 4.1 kb (2.3 kb of normal w transcript plus 1.8 kb of upstream of w to a promoter of the first HeT-A element) probably indicates a very low abundance of a HeT-A-white read-through transcript. As HeT-A elements have shown strong expression in gonads (53), we attempted to detect the read-through transcript in ovarian 11-5 RNA on a Northern blot with a probe specific to upstream w sequence but were unsuccessful. Even though HeT-A transcripts can be detected on Northern blots (20) and by whole-mount in situ hybridization (53), these transcript levels reflect the cumulative activity of all HeT-A elements in the genome, while our efforts are aimed at detecting the activity of a single HeT-A promoter or a small subset of HeT-A promoters, which might produce low levels of variably sized transcripts that are undetectable by Northern blots.

Although PCR data clearly showed the presence of HeT-A-w read-through transcript in 11-5, we conclude that this read-through transcript is only a minor component of a total w transcript and that the majority of w transcript is transcribed from the w promoter. This suggests that the stimulating effect of a TAS deficiency on telomeric w expression by read-through transcription driven from the HeT-A promoter through w, as was proposed to occur in 11-5 (22), may not be the primary component in the suppression of TPE.

A TAS deficiency stimulates promoter activity of the HeT-A element(s). The HeT-A/w read-through transcript in 11-5 we identified by PCR allowed us to test the effects of a TAS deficiency in trans on HeT-A promoters distal to the telomeric w transgene. Using qRT-PCR with the primer set 2B from the upstream region of w and the primer set HeT-F1/w-1, encompassing the 3' end of HeT-A and the upstream region of w (Fig. 1A and B), we quantified the levels of HeT-A/w read-through product in the same genotypes that were tested for w expression (Fig. 3). Quantitation was performed with larvae, late-stage pupae, and adults. Both sets of primers gave comparable data showing an increase of HeT-A/w read-through transcript in the presence of complete or partial TAS deficiencies in a manner similar to that observed with primers for the w coding sequence (Fig. 3). The data shown in Fig. 5 are for the primer set HeT-F1-w-1. The levels of read-through transcript varied in concert with the levels of total w transcript in 11-5 and increased from larvae through adults. The levels of read-through transcript also correlated with eye color and pigment levels (Fig. 2), although the measured pigment levels in 11-5/+ appear to be somewhat low compared with the quantity of transcripts. There was no amplification at any stage in Oregon R (data not shown). A comparison of w transcript levels (Fig. 3) with read-through transcript levels relative to RpL transcript (Fig. 5) suggests that a 2L TAS deficiency increases expression from both telomeric w and telomeric retrotransposon promoters but that the expression from the w promoter exceeds expression from the retrotransposon promoters.


Figure 5
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  		FIG. 5. Relative levels of HeT-A/white read-through transcripts in 11-5/11-5, 11-5/M26, and 11-5/+ at various stages of development. cDNA was generated using an oligo(dT) primer, and transcripts were identified using the primer set HeT-1F-w-1.

The TAS modulates activity of nontelomeric but TAS-adjacent w transgenes. Although a comparison of transcript levels of telomeric and nontelomeric w showed significantly different developmental profiles (Fig. 3), levels of telomeric w and HeT-A/w read-through transcripts varied in concert. To ask whether developmental changes in w transcription at the 2L telomere are induced by the TAS and whether these changes are independent of telomeric position and/or presence of adjacent HTT, we compared mini-w transcript levels in larvae and late pupae that carry insertions of TAS-mini-w fusion constructs in different euchromatic positions. The constructs are bounded by SU(HW) binding sites that act as insulators and prevent position effects at insertion sites, thus allowing us to test a direct effect of an adjacent TAS on mini-w independently of their chromosomal position. The A6-514 and A8-2A constructs have <1 kb of the TAS between the w eye enhancer and the promoter (30), and flies carrying these constructs exhibit red eyes. A6-511 and B8-13Y have 6 kb of the TAS between the eye enhancer and the promoter, and adults carrying these inserts exhibit yellow eyes. Eye color phenotypes of A6-514 and A6-511 are shown in Fig. 6A. Similar to observations on telomeric w (Fig. 2), larval transcript levels of TAS-mini-w fusions (Fig. 6B) were inconsistent with the adult eye color phenotypes and did not reflect different levels of heterochromatic silencing by the TAS. In strains with short TASs (A6-514 and A8-2A), pupal mini-w transcript was increased, whereas strains with a longer TAS array (A6-511 and B8-13Y) showed relatively steady transcript levels between larval and pupal stages. Due to the lack of a control without any TAS, we could not ask whether the relatively short TAS array in A6-514 and A8-2A might induce silenced levels of w transcript. However, the data clearly indicate that, independent of telomeric position or an adjacent HTT array, a long TAS array in cis keeps levels of w expression low during development, as we observed in both euchromatic TAS-mini-w and telomeric 11-5 insertions.


Figure 6
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  		FIG. 6. Expression of TAS-mini-w fusion transgenes. (A) Eye color phenotypes of the flies bearing nontelomeric TAS-mini-w insertions. (B) Levels of mini-white transcript in nontelomeric TAS-mini-w insertions relative to RpL 32 transcript.

Long-range effects of suppressors of TPE. To test whether 2L TAS deficiencies can suppress silencing on nonhomologous telomeres as well as on homologues, we crossed females carrying a mini-w transgene in the TAS of 2R (39C-27) or 3R (39C-62) (52) to M26/SM1 males. The resulting 39C-27/M26 and M26/+;39C-62/+ males had darker eyes than their 39C-27/SM1 and SM1/+;39C-62/+ brothers (Fig. 7A), indicating that suppression of TPE by 2L TAS deficiencies extends to other telomeres (Fig. 7B). To confirm that the observed TPE suppression is not due to another factor in the genetic background, we tested several other deficiencies of the 2L TAS: Df(2L)net62, l(2)glGB26, and P{wvar}11-10, a variant of P{wvar} that is deficient for some or all of the w reporter but has an intact l(2)gl gene (22). These deficiencies gave similar results to those observed for M26. Also, to confirm that l(2)gl, which is a locus adjacent to the TAS array, is not involved in TPE and that telomeric silencing is affected only by the TAS, we tested l(2)glDV275, which has an intact TAS array and a deficiency restricted to the l(2)gl locus, and l(2)glGB52, which has an insertion mutation in the l(2)gl locus. These mutations did not affect expression of telomeric inserts (data not shown), confirming that the 2L TAS, not the neighboring l(2)gl gene, plays a role in telomeric silencing. These data indicate that the effect of 2L TAS deficiencies on TPE are global, rather than restricted to homologues.


Figure 7
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  		FIG. 7. (A and B) Long-range regulation of telomeric silencing by a 2L TAS deficiency. (A) Females carrying a mini-w reporter gene in a TAS region were mated to M26/SM1 males, and the resulting males were examined for eye color. M26 is a 2L TAS deficiency; 39C-27 is in the 2R telomere; 39C-62 is in the 3R telomere. SM1 is a control that does not suppress telomeric silencing. (B) Diagram of the stimulating effect of 2L TAS deficiency on expression of mini-w inserted into the 2R TAS of line 39C-27 and into the 3R TAS of line 39C-62. (C) Eyes and red eye pigments in insertion lines 39C-5, bearing w in the 2L TAS as hemizygous 39C-5/M26, and 39C-5/+.

Expression of transgenes residing within the TAS. As TAS deficiencies elevate expression of w inserted into the TAS, we examined the possibility of read-through transcription from a retroelement promoter into the adjacent TAS region and a contribution of such read-through transcription on expression of w residing within the TAS. For this purpose we tested 39C-5, which carries a white insertion in the 2L TAS with its transcription occurring in a distal-to-proximal manner (11). A 2L TAS deficiency on the homologue leads to a minor but discernible increase of w expression in flies with this insertion, which we confirmed by pigment analysis (Fig. 7C). We further tested 39C-27 and 39C-62 individuals for the presence of retroelement read-through transcript, although the exact positions of these two w insertions within the TAS array are not known. For all three lines the presence of read-through transcript was tested in homozygotes and hemizygotes bearing the M26 deficiency using primer set 3 (Fig. 1A). However, in none of these cases did we observe any transcription product (data not shown). This suggests that the increase of w transcription in these TAS-deficient flies is due to direct release of TAS silencing on the w promoter with little or no contribution of direct retroelement read-through transcription.

Influence of 2L TAS deficiency on overall HeT-A transcription. Because 2L TAS deficiencies have an effect on TAS-repressed transgenes elsewhere in the genome and on HeT-A promoter activity, it was of interest to investigate the overall impact of 2L TAS deficiencies on general HeT-A transcription. For this task, we used HCS primers specific to HeT-A coding sequence and quantified overall HeT-A transcript levels in larvae, late pupae, and adults (Fig. 8A). To calculate the relative HeT-A expression per genomic HeT-A copy number, we measured relative numbers of HeT-A copies in each genotype (Fig. 8B) using the same primer set and standardized the HeT-A expression to the relative copy number in each stock (Fig. 8C). Total HeT-A transcripts did not show consistency with HeT-A/w read-through transcript levels (Fig. 5). These observations indicate that overall HeT-A transcription is independent of TAS influence and suggests that TAS influence is restricted to the proximal telomeric retroelements. Thus, the level of total HeT-A transcript is not significantly dependent on control by the TAS.


Figure 8
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  		FIG. 8. Overall HeT-A transcript activity. (A) Levels of total HeT-A transcripts in 11-5/11-5, 11-5/M26, 11-5/+ relative to RpL32 transcript at various stages of development are shown. (B) Relative HeT-A copy numbers in the genomes tested. (C) Relative levels of HeT-A transcripts standardized to relative copy number of genomic HeT-A.


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DISCUSSION
 
This study extends our earlier observations relating to a role for the TAS in telomeric silencing, telomere maintenance, and other aspects of telomere behavior in Drosophila. Based on our previous studies, we proposed the HeT-A activation model of TPE (22, 37) to explain TPE in Drosophila; this model states that the silencing effects of heterochromatin-like chromatin spread from the TAS into the HTT array, while transcriptional activity from HeT-A promoters exerts an activating influence on more proximal regions of the HTT array. TAS deficiencies lead to a stimulation of telomeric w transgenes and retrotransposons on the homologous chromosome, which suggests that homologous TAS-TAS associations may modulate interactions between telomeres. We further speculated that these interactions modulate overall HTT activity and subsequent transposition and telomere elongation. Here, we report several results that address predictions based on the model.

Telomeric gene expression. One major aspect of TPE incorporated into the HeT-A activation model is that disruption or deletion of the TAS causes suppression of telomeric silencing in trans (22, 39). Here we used the M26 deficiency, which completely deletes the 2L TAS while leaving the adjacent l(2)gl gene intact. Consistent with results on deficiencies that remove both the 2L TAS and l(2)gl, this deficiency acts as a dominant suppressor of TPE, while mutations in l(2)gl do not. Thus, the TAS, rather than l(2)gl, seems to be the relevant agent in regulating TPE. The elevated expression of the telomeric w transgene is correlated with an increase in the read-through transcript from a HeT-A promoter into w. This is the first direct evidence that the TAS can modulate HeT-A promoter activity in trans.

As shown collectively by Northern hybridization and PCR, total w expression in the 11-5 insertion between the HTT array and the TAS is produced by contributions from the telomeric w promoter and promoters of at least two telomeric retroelements. This is consistent with the observation that expression of the 11-5 reporter increases with increasing HTT array length in cis (22). Although the read-through transcript is only a minor component of total w transcript levels in 11-5, and a majority of the w transcript is produced from the w promoter, we can conclude that a 2L TAS deficiency leads to a release of silencing on both telomeric w and telomeric retroelement promoters. The relative intensities of these two promoters as seen here are consistent with the observation that the HeT-A promoter is relatively weak (53). A 2L TAS deficiency may stimulate the w promoter directly; however, transcription initiated in the HTT array may also stimulate the w promoter, even in the absence of read-through by change of chromatin structure. These two hypotheses are not mutually exclusive. Read-through transcripts from telomeric retrotransposons into other downstream sequences were detected here by RT-PCR and have also been found in expressed sequence tag libraries as cDNAs containing sequences from more than one telomeric retrotransposon (40).

Using insertion elements in TASs, we tested the possibilities that transcription from the most proximal retroelement might continue into TASs and that such transcription might be elevated in 2L TAS-deficient flies, in which TPE is suppressed. Although w transcript levels increased in 2L TAS-deficient flies, we did not see any PCR product indicating read-through transcript from the retroelement. This suggests that retroelement transcripts do not extend far into the TAS, if at all, and the elevation of w transcription in these lines may be due instead to a release of TAS silencing on w.

Telomere interactions. We demonstrate here for the first time that deficiencies of the 2L TAS can suppress TPE at nonhomologous telomeres, indicating that nonhomologues may interact. Deficiencies of the 2L TAS suppress silencing of w transgenes inserted into the 2L, 2R, and 3R telomeres. Thus, these deficiencies appear to be global suppressors of TPE, consistent with the observations that expression of a gene depends on its position within the interphase nucleus and on somatic pairing of chromosomes (12, 13, 15, 23, 25, 51). While there is no direct evidence pertaining to the question of whether physical associations between telomeres influence the genetic interactions we report here, cytological observations made with a number of organisms suggest that eukaryotic nuclei maintain reproducible long-range chromosomal organization during interphase, such as telomere interactions with other telomeres and with the nuclear lamina, and occupancy by individual chromosomes of distinct nuclear territories (10, 17, 18, 26). In addition, Polycomb group proteins bind TAS regions (2, 9) and may mediate long-range interactions. Mcp, for example, a Drosophila Polycomb response element, can mediate physical interactions between remote chromosomal sites (50). Similar long-range interactions have been described for the mouse HoxD cluster (49).

One prediction of the HeT-A activation model, that telomere-telomere interactions modulate overall HTT transcription, subsequent transposition, and chromosome elongation (22, 37), is not supported by the present data. Although 2L TAS deficiencies suppress TPE on all telomeres tested and suppress silencing of proximal telomeric retroelements, we did not see a concomitant response of overall HeT-A transcript level. Our results agree with recent evidence (7) that w genes located in the HTT array close to the TAS are under TAS control and show repression and variegation, while w genes in the HTT array far from the TAS are not repressed and behave as if they were in euchromatin independent of TAS influence. Thus, the effect of TAS deficiencies on TPE and retroelement transcription may only extend a limited distance from the TAS, and the effect of TAS deficiencies on TPE do not indicate a significant control mechanism over telomere length. More important in controlling chromosome elongation appears to be the cap structure at the telomere ends, possibly because it controls accessibility of the chromosome ends to new transpositions (42, 47). A possible role for RNAi in telomere length control and TART transposition in Drosophila was also reported (48).

Developmental changes in telomeric silencing. A novel process discovered in this study is a developmental modulation of telomeric silencing. The developmental expression profile of the read-through transcript in the upstream region of a telomeric w gene resembles the expression profile of the telomeric w itself, as well as the profile of TAS-mini-w fusion constructs in euchromatic positions, and differs from that of the w gene in its normal genomic location. It also differs from the expression profile for total HeT-A, suggesting that the TAS specifically modulates transcription in the adjacent region, i.e., telomeric w and the proximal HeT-A elements in 11-5. Overall HeT-A transcript levels reflect the sum of all HeT-A promoters, most of which are outside the range of TAS influence.

Collectively, the present results indicate that changes in expression of telomeric transgenes that occur in the presence of a shortened 2L TAS during late stages of development imply a similar modulation of the activity of telomeric retrotransposons as a result of long-range telomere-telomere interactions.


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ACKNOWLEDGMENTS
 
We thank Honza Stehlik and Sayura Ayogi for their help with RT-PCR, Miroslav Capek, Patrick Williams, Antje Michel, and Diana Le for assistance during experiments, Pratibha Hebbar and Harriet Kinyamu for valuable advice, and Daniel Menendez and Max Noureddine for critically reading the manuscript.

This research was supported, in part, by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences, and by U.S. Public Health Service grant GM-56729 to H.B.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratory of Molecular Genetics, D3-01, P.O. Box 12233, 111 T. W. Alexander Drive, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709-2233. Phone: (919) 541-4483. Fax: (919) 541-7593. E-mail: masonj{at}niehs.nih.gov Back

{triangledown} Published ahead of print on 30 April 2007. Back

{dagger} Present address: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY. Back

{ddagger} Present address: Center for Demographic Studies, Duke University, Durham, NC. Back


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Molecular and Cellular Biology, July 2007, p. 4991-5001, Vol. 27, No. 13
0270-7306/07/$08.00+0     doi:10.1128/MCB.00515-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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