Decrease in Testis-Specific Transcripts Reflects Under-Representation of cav⁻ Males

In an effort to identify candidate HOAP-regulated genes, the Affymetrix Drosophila Genome Array 1 was used to compare the expression profile of wild type larvae to those that were mutant for the HOAP-encoding gene (caravaggio or cav). The original recessive lethal cav¹ allele was used in the study. This allele contains a 5 bp insertion that causes truncation of the HOAP protein after two of three copies of a C-terminal HP1-binding repeat [27], [30]. The y¹w^67c23 stock, which provides the genetic background for all genetic manipulations in the lab, was used as the wild type control. Larvae of each genotype were collected at the first and second instar stage, prior to the lethal phase of the cav¹ mutant. Out of 13,500 transcription units represented on the array, 183 genes were found to have significantly altered expression (log₂R+2.0, p<0.01) in cav¹ mutant larvae. Within this set, 142 genes had reduced transcript levels and 41 had increased levels.

Two strategies were then used to catalogue the normal expression profiles of genes in each data set. A gene's relative representation in publically available tissue-specific cDNA libraries [35]–[37] provided the first method for assessing its normal tissue distribution. This analysis was later complemented by data from two published microarray profiling studies of Drosophila sex- or tissue-specific gene expression [38], [39]. The results of these analyses are summarized in Tables S1 and S2. Genes categorized as “multiple” were represented in cDNA libraries of at least three different developmental specificities and enriched in at least three different tissue types in the Chintapalli et al. study [38]. Those categorized as “rare” were represented by a single or few cDNA clones and detected at low levels in multiple tissues by Chintapalli et al. [38]. Those categorized as tissue-specific (e.g., testis or midgut) were represented only, or predominantly, in cDNA libraries of that tissue-specificity and also specifically enriched in that tissue in Chintapalli et al. [38]. The most striking pattern to emerge from these analyses was the relative enrichment of testis-enriched genes (67%) in gene set with reduced transcript levels in the cav¹ mutant. The Parisi et al. [39] study of sex-specific gonad expression provided corroboration for the testis-specificity of 67% of these genes, as they were both enriched in testes relative to whole animals and in testes relative to ovaries. This is in contrast to a complete absence of testis-specific genes in the gene set with elevated transcript levels, and also far exceeds the ∼12% of Drosophila genes reported to have testis-specific expression [40].

The enrichment of testis-specific genes in the reduced transcript level data set could indicate a requirement for HOAP in testis-specific gene expression. Alternatively, it could reflect an early lethal phase for cav¹ mutant males and, thus, under-representation of male-specific transcripts in the cav¹ RNA sample. RNA interference (RNAi) against the HP1-encoding gene (Su(var)205) in Drosophila has revealed enhanced vulnerability of males to partial HP1 knockdown [41]. To determine if males were under-represented in our cav¹ larval sample, we used the X-linked yellow (y⁺) marker to sex individual cav¹ mutant larvae, allowing us to sex the animals through a phenotype other than gonad size. By this criterion, 2.83-fold fewer male cav¹ larvae were observed than female cav¹ larvae. Using PCR with Y-linked primers to sex individual cav¹ homozygous embryos, we also observed approximately two-fold more male embryos fail to progress to the larval stage.

We then used RNAi to partially knockdown HOAP and determine its effect on adult male viability. The GAL4/UAS binary expression system was used to drive expression of a cav RNAi transgene (Figure 1). Ubiquitous expression of cav RNAi from two transgenic lines (F8 and 543) through a maternally introduced Actin5C GAL4 driver resulted in a 2.5- and 1.5-fold reduction in adult male viability relative to females, respectively (Figure 1A). A third cav RNAi transgenic line (662) resulted in lethality of both males and females. The severity in effect of cav RNAi expression in different transgenic lines correlated with the degree to which endogenous cav mRNA was reduced as monitored through semi-quantitative RT-PCR, with ∼80% cav knockdown resulting in a 2.5-fold reduction in male viability (Figure 1B). The dose-dependent effects of cav RNAi expression on male viability are similar to those observed with Su(var)205 RNAi expression (2.54-fold reduction with 60–90% Su(var)205 knockdown and lethality of both sexes with >90% knockdown) [41]. A less severe, but significant, reduction in viability of male progeny was also observed in the reciprocal cross in which the Actin5C GAL4 transgene was introduced through the fathers. Although the results in both sets of crosses indicate a zygotic requirement for HOAP in male viability, the more pronounced effect was with maternally contributed Actin5C GAL4. This suggests maternally expressed GAL4 is needed to drive earlier expression of the cav RNAi for maximum effect.

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Figure 1. cav RNAi reduces adult male viability.

(A) The GAL4/UAS binary system using maternally contributed Actin-5C GAL4 to drive expression of cav RNAi shows reduced viability of males carrying the GAL4 driver and cav RNAi transgene #8; both sexes carrying the GAL4 driver and cav RNAi transgene #662 had reduced viability, in comparison to comparison to their control siblings containing either cav RNAi or Act5C-GAL4 transgene alone. (p<0.01^**) Male viability was also significantly reduced (albeit less dramatically) in the reciprocal cross in which the Actin-5C GAL4 transgene was paternally derived. (p<0.01^**) (B) RT-PCR assays of endogenous cav and RpA70 (normalizing standard) in larvae expressing cav RNAi transgenes from lines #8 and #662. The triangles represent a 5-fold dilution series of RNA from animals carrying either the cav RNAi transgene only (wt) or cav RNAi and Act5C-GAL4 (cav RNAi). (C) Effect of the compound X-Y chromosome, C(1;Y), in animals expressing cav RNAi. The presence of C(1;Y) did not significantly affect viability of adult females or males expressing cav RNAi, in comparison to their control siblings containing either cav RNAi or Act5C-GAL4 transgene alone (p>0.10). By contrast, the animal's sex significantly affected viability, only in the class of animals expressing cav RNAi, regardless of whether they did or did not carry C(1;Y). (p<0.01^**).

https://doi.org/10.1371/journal.pgen.1002122.g001

A dominant effect on male viability was also observed for a newly recovered cav allele (cav²²⁴⁸). Heterozygosity for this allele reduces male viability 1.84-fold (n≥312, p<0.01). The cav²²⁴⁸ carries a nonsense mutation at nucleotide 111 of the cav-PB coding sequence within the region of similarity to the SRY HMG box. This allele appears to exert dominant negative activity on the wild type protein, as its effect on male viability is similar to that caused by RNAi-induced HOAP knockdown but is more pronounced than a deficiency for the locus (Df(3R)F89-4). The prematurely truncated protein in this mutant, or a reinitiation product from the next Met within the HMG box, is apparently responsible for this dominant negative effect. Smaller, but significant, reductions in male viability were also observed in progeny from wild type crossed to either parent carrying the cav²²⁴⁸ allele (data not shown). The lethal phase for cav²²⁴⁸ heterozygous males was determined to be late in embryogenesis after denticle belt formation; cav²²⁴⁸ homozygous embryos had an earlier lethal phase which is apparently due to telomeric defects (Figure S1).

Presence of Y Chromosome Is Not Responsible for Reduced Male Viability

Although the Y chromosome is not required for viability or sexual differentiation in Drosophila males, its heterochromatic composition enables it to act as a sink for heterochromatin proteins in suppressing position effect variegation of euchromatic genes artificially juxtaposed to heterochromatin [42], [43]. A limited pool of heterochromatin proteins might then render males more vulnerable to reductions in HOAP (and other heterochromatin proteins). To test whether the Y chromosome has a role in reducing viability of males that are deficient for HOAP, a compound X-Y chromosome [C(1;Y)] was introduced into animals expressing a cav RNAi transgene (Figure 1C). While the C(1;Y) chromosome modestly reduced viability of both male and female progeny by 30% relative to siblings lacking it, the sex of the animal had a pronounced effect on viability. Males expressing cav RNAi showed a 2.3-fold decrease in viability, regardless of whether they also carried C(1;Y).

Defects in Sex Determination Pathway Are Responsible for Reduced Male Viability

Defects in the sex determination pathway, causing inappropriate dosage compensation in Drosophila, result in sex-specific lethality. Sex-lethal (SXL) protein acts as the master switch regulator of this pathway at the level of splicing and translation [44]. As described earlier, the critical decision is made early in embryogenesis by the activation of the Sxl establishment promoter (Sxl_Pe) in only females, to generate functional SXL protein [32]. Failure to activate Sxl_Pe, thus leads to improper male splicing of Sxl_Pm transcripts and female lethality. Conversely, inappropriate activation of Sxl_Pe in males results in the female mode of splicing of Sxl_Pm transcripts and male lethality.

To determine if the enhanced male lethality of cav mutants is associated with inappropriate SXL activity in males, RT (reverse transcriptase)-PCR assays were used to monitor sex-specific splicing of Sxl_Pm transcripts in heterozygous cav²²⁴⁸ and homozygous cav¹ male embryos that failed to progress to the larval stage. Individual embryos of the appropriate genotype were identified through a GFP-marked wild type balancer chromosome (described more fully in Materials and Methods) and sexed by PCR with Y-linked primers (Figure S2). RNA was then purified from pools of male or female embryos and used in RT-PCR assays with Sxl primer pairs (P1/P3 and P2/P3) designed to discriminate between Sxl_Pm transcripts spliced in the male vs. female mode (Figure 2A). Assays of RNA from wild type (y¹w^67c23) embryos showed correct sex-specific splicing of Sxl_Pm transcripts in both males and females (Figure 2B). By contrast, a minor fraction of transcripts had undergone the female-mode of splicing in heterozygous cav²²⁴⁸ and homozygous cav¹ male embryos (arrowhead in Figure 2B).

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Figure 2. Aberrant sex-specific Sxl_Pm-derived transcripts are observed in mutants for HOAP and in mutants for HP1.

(A) RT-PCR was used to specifically amplify male- or female-specific Sxl_Pm transcripts. The P1/P3 primer pair amplifies a 482 bp product from the female-specific transcript and a 672 bp product from the male-specific transcript in wild type animals (labeled wt in all three panels). The P2/P3 pair amplifies a 284 bp product from a male-specific transcript in males only. (B) Pools of RNA from individually sexed male or female cav¹ homozygous or cav²²⁴⁸ heterozygous embryos failing to progress to the larval stage were used in RT-PCR assays with both P1/P3 (top panel) and P2/P3 (bottom panel) primer pairs, revealing the presence of aberrant female-specific Sxl_Pm transcripts in male embryos (arrowhead). RT-PCR assays of RNA from four different individually sexed Su(var)205⁴/Su(var)205⁵ female (♀) or male (♂) larvae, each with both P1/P3 (top panel) and P2/P3 (bottom panel) primer pairs revealed the presence of aberrant sex-specific Sxl_Pm transcripts in both sexes. The methods used to genotype and sex individual embryos and larvae are described in Materials and Methods.

https://doi.org/10.1371/journal.pgen.1002122.g002

If inappropriate SXL expression is the cause of the enhanced male lethality in the cav²²⁴⁸ heterozygous males, we would expect a loss of function Sxl allele to rescue them. To this end, the Sxl^f1 point null allele was introduced into heterozygous male and female cav²²⁴⁸ progeny through their mothers. Closely linked recessive markers, cut and carmine, which flank Sxl were used to follow the Sxl^f1 allele in males, while in females white⁺, which should segregate with Sxl^f1 82.3% of the time, was used as a close approximation. As shown in Table 1, the viability of cav²²⁴⁸ males carrying a defective Sxl allele (Sxl^f1) was not significantly different from their female siblings, unlike their Sxl⁺ brothers (yw). It should be noted that in this cross, the effect of the cav²²⁴⁸ mutation on male viability was somewhat reduced; we ascribe this difference to the temperature at which the cross was done. For reasons that are not clear at present, all progeny from this cross were nonviable at 25°C, so the cross was performed at room temperature. An overall rescue in male viability was also observed by the introduction of the Sxl^fP7BO allele, although for this cross the markers on the deletion allele chromosome did not allow identification of the different progeny classes.

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Table 1. Mutations in HOAP reduce male viability, and loss of Sxl partially rescues the lethality.

https://doi.org/10.1371/journal.pgen.1002122.t001

As HP1 and HOAP interact, and HP1 reduction also preferentially affects male viability, we wondered whether male larvae mutant for the HP1-coding gene [Su(var)205⁵/Su(var)205⁴] would also show altered splicing of Sxl_Pm transcripts. GFP-marked balancer chromosomes were used to identify individual larvae of the correct genotype, and both an X-linked genetic marker (y⁺) and PCR assays of a Y-linked gene were used to sex them. Surprisingly, we found aberrant Sxl_Pm transcripts in multiple individual Su(var)205⁵/Su(var)205⁴ larvae of both sexes (arrowheads in Figure 2C), suggesting HP1 has a dual role, of opposite consequence in each sex.

We therefore explored whether a maternal mutation in either cav or Su(var)205 would affect the viability of female progeny that also have a reduction in Sxl dose (Table 2). Female viability is not compromised in a cross between either wild type females and Sxl⁻ males (fP7B0 or f1), or between cav mutant females and Sxl⁻/Y fathers (Note: the modest reduction in male viability in the progeny from cav²²⁴⁸ females and Sxl⁻ males is essentially the same as that observed in progeny from cav²²⁴⁸ females and wild type males, as described earlier). However, in progeny from Su(var)205 heterozygous females crossed to Sxl⁻/Y males, female viability was dramatically reduced, particularly with the strong loss of function Su(var)205⁵ allele. The effect of Su(var)205 mutations is strictly maternal; no significant reduction in female viability is observed in the reciprocal cross (Table 2). Interestingly, this effect is allele-specific. Whereas mothers carrying the Su(var)205⁵ null allele or the Su(var)205⁴ carboxyl-terminally deleted allele had greatly reduced viability in their female progeny, mothers carrying a point mutation in the MeK9-H3-binding chromodomain allele [Su(var)205²] had a modest, but significant, effect. Although neither cav allele affected sex-specific viability of progeny from Sxl mutant fathers, the introduction of either allele into Su(var)205 mothers rescued the Su(var)205 maternal effect on female viability (Table 3). Both the dominant negative cav²²⁴⁸ allele and the C-terminally truncated cav¹ allele, with compromised HP1-binding activity [27], [30], were capable of rescuing the Su(var)205 maternal effect, and this rescue was also strictly maternal.

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Table 2. Mutations in HP1 interact with loss of function Sxl alleles to reduce female viability.

https://doi.org/10.1371/journal.pgen.1002122.t002

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Table 3. Maternal mutations in HOAP and HP1 have antagonistic effects on female viability.

https://doi.org/10.1371/journal.pgen.1002122.t003

Transcription of Sxl_Pe Is Affected by Mutations in Either cav or Su(var)205

The rescue of cav²²⁴⁸ male viability by the loss of Sxl and the antagonistic maternal effects of cav and Su(var)205 on the viability of their Sxl⁻-bearing female progeny, suggested a role for maternal HOAP and HP1 in regulating Sxl_Pe. To directly assess the effect of reduced HOAP or HP1 on Sxl_Pe, in situ hybridizations were performed with probes that distinguish the early from maintenance Sxl mRNAs on 0–4 hr embryos from cav²²⁴⁸ and Su(var)205⁵ heterozygous parents. For Sxl_Pe, wild type embryos show two dots on the chromosomes, one for each female X, beginning in cycle 12 through to early cycle 14. Cycle 14 is also when the maintenance promoter, Sxl_Pm, is activated and the autoregulatory splicing loop set in motion in females. In embryos from cav²²⁴⁸ heterozygous parents, two key changes were observed (Figure 3A). First, expression of Sxl_Pe in females began two cycles earlier than normal – cycle 10, and overall expression appeared more robust than in wild type embryos. Second, male embryos also showed Sxl_Pe activation (single in situ dot), although the expression was not as strong or as early as in females. The level of expression in male embryos was more variable both between embryos and at the level of individual nuclei. The majority had sporadic or scattered positive nuclei, while others had much greater numbers, as shown in Figure 3A. A count of early cycle 14 male embryos suggests >95% of the male embryos (n = 33) had at least some positive nuclei. In embryos from Su(var)205⁵ heterozygous parents, Sxl_Pe expression was not observed in male embryos (single in situ signal) and was weaker and more variable than normal in females (Figure 3B). Analysis of the female embryos indicates that ∼85% express the promoter weakly during cycles 12 and 13 (n = 14–20 for each cycle) and less than half the embryos have normal levels of expression at cycle 14 (n>25). These results are consistent with the early expression of Sxl_Pe being heavily reliant on the maternal deposit of HP1 protein and/or mRNA, with the zygotic contribution becoming more apparent at cycle 14. Expression of the maintenance Sxl_Pm transcripts did not show significant changes in embryos from either genotype (Figure S3). Although HP1 binds to Sxl_Pm in 4–18 hr embryos, at cellular blastoderm the promoter does not appear to be sensitive to a reduction in maternal HP1.

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Figure 3. In situs for Sxl_Pe transcripts in embryos from wild type, cav²²⁴⁸/TM3, Sb, or Su(var)205⁵/CyO parents.

Comparisons of the same-sized area of images taken at 40×. (A) Sxl_Pe transcripts are present only in Ore R wild type females (containing 2 dots per nucleus) during cycle 12 to 14. Sxl_Pe transcripts are present in female embryos from cav²²⁴⁸/TM3 parents as early as cycle 10. Sxl_Pe transcripts are also frequently present in male embryos from cav²²⁴⁸/TM3 parents (single dot per nucleus, usually near nuclear periphery where the dosage compensated X chromosome resides [62]. Not much signal is detected before cycle 13 in males. (B) Poor Sxl_Pe expression is observed in embryos from Su(var)205⁵/CyO parents in comparison to simultaneously stained embryos from Ore R wild type parents.

https://doi.org/10.1371/journal.pgen.1002122.g003

Both HOAP and HP1 Are Associated with the Sxl_Pe Regulatory Region

Chromatin immunoprecipitation assays were then used to determine if the effects of cav²²⁴⁸ and Su(var)205⁵ on Sxl_Pe activity are mediated through direct physical association of the proteins with the Sxl locus (Figure 4). Cross-linked chromatin was prepared from developmentally staged collections of wild type (y¹w^67c23) embryos and immunoprecipitated with antibodies against HP1, HOAP, and non-immune IgG. Embryos were staged to monitor both Sxl promoters: before and during Sxl_Pe activation (1–3 hr), during Sxl_Pe activation (2–3 hr), and during Sxl_Pm expression (4–18 hr). Quantitative real time PCR was then used to measure enrichment of Sxl sequences in each ChIP fraction. As summarized in Figure 4A, significant enrichment of sequences in the immediate vicinity of Sxl_Pe (+138 and −101 fragments) was observed in the HP1 and HOAP ChIP fractions but not in the IgG ChIP fraction from 1–3 hr embryos. No enrichment of Sxl_Pm sequences was observed at this stage. Significant enrichment of sequences upstream of Sxl_Pe (−1132,−1800, and −2224) was also observed in the HOAP ChIP fraction. Using an anti-Myc antibody to probe chromatin from a stock expressing Myc-tagged HOAP from the endogenous cav locus [29], similar results were obtained (data not shown).

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Figure 4. Chromatin immunoprecipitation (ChIP) assays show association of both HOAP and HP1 with the Sxl locus.

(A) Relative enrichment (ChIP/input-Sxl sequence/ChIP/input-RpA-70) of specific Sxl locus sequences in HOAP- and HP1-ChIP fractions from 1–3 hr embryos plotted against the molecular map of the Sxl locus (HOAP in red; HP1 in blue). The positions of Sxl fragments monitored for enrichment are shown relative to the Sxl_Pe initiation site, below the map. The positions of E-box binding sites for positive (blue diamonds) and negative (red squares) bHLH factors [63] and Sxl transcripts are also indicated below the map. (p_{(no enrichment)}<0.05*) (B) Bar graphs show enrichment of Sxl sequences in HOAP- (red bars) and HP1- (blue bars) ChIP fractions during development (1–3 hr, 2–3 hr, and 4–18 hr staged embryos). (p_{(no enrichment)}<0.05*; change between 1–3 hr and 2–3 hr embryos, p_{(no effect)} p_{(no change)}<0.10^(#), <0.05^#).

https://doi.org/10.1371/journal.pgen.1002122.g004

A comparison of the ChIP data from 1–3 hr versus 2–3 hr embryos shows a significant decrease (p<0.5) in the associations of both HOAP and HP1 with the −101 region of Sxl_Pe. Both proteins retained significant associations with the +138 region of Sxl_Pe in 2–3 hr embryos at a time that is coincident with Sxl_Pe expression. HOAP enrichment in both the +138 region and the −1132 region appeared reduced at this time, although only at the 90% confidence level. No enrichment of either protein was found more 3′ in Sxl gene coding sequences or in the Sxl_Pm region in either the 1–3 hr or 2–3 hr embryos. Also, Sxl_Pe sequences were not enriched in either the HP1 or HOAP ChIP fractions from 4–18 hr embryos. However, HP1 enrichment in the vicinity of Sxl_Pm (−5094 and −5331) and a slight, but significant, enrichment of HOAP in the −1132 region was observed in later staged embryos.

HOAP and HP1 Affect Sxl_Pe Chromatin

The ChIP assays described above confirmed physical association of HOAP and HP1 with the Sxl_Pe promoter, but do not distinguish between the sexes. We, therefore, took advantage of the feminizing and masculinizing effects of maternal mutations in cav and Su(var)205 to gain insight into the requirement for each protein in regulating Sxl_Pe activity.

The in situs, as well as the lethal effect of a maternal mutation in Su(var)205 on female progeny lacking one functional copy of Sxl, indicated an activation function for HP1 at Sxl_Pe. To further test this hypothesis and examine its nature, we determined the effect of reducing maternal HP1 on RNA polymerase II (RNAP II) association with Sxl_Pe (Figure 5A). Chromatin was prepared from 2–3 hr. embryos from wild type or heterozygous Su(var)205⁵ mothers mated to Sxl^f1 fathers, as Sxl_Pe is active at this stage in wild type embryos. Antibodies that recognize three different phosphoisoforms of RNAP II, which mark distinct stages of transcription initiation and elongation, were used to gain insight into the level of transcriptional activation at Sxl_Pe at which HP1 is functioning. In embryos from wild type mothers, both unphosphorylated RNAP II (blue bar) and Ser 5-phorphorylated carboxyterminal domain RNAP II (green bar), which marks early steps of transcription elongation, were enriched in the proximal regions of Sxl_Pe and the hunchback (hb) promoter. Both genes are active at this stage. Ser 2-phosphorylated RNAP II (red bar) which identifies polymerase in its elongation phase was mostly absent from both promoters. All RNAP II isoforms, particularly those associated with transcription initiation (unphosphorylated RNAP II, blue bars) and early events in transcription elongation (Ser 5-phosphorylated RNAP II, green bars), were drastically reduced from Sxl_Pe proximal sequences in embryos from Su(var)205 mutant mothers and Sxl^f1/Y fathers (Figure 5A). By contrast, the Su(var)205 mutation did not affect RNA polymerase II association with the hb promoter (Figure 5A).

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Figure 5. Chromatin immunoprecipitation (ChIP) assays show perturbations in Sxl_Pe chromatin in embryos from HOAP and HP1 mutant mothers.

(A) Bar graphs show Sxl_Pe (Sxl +138 and −101) and hunchback (hb −140) promoter sequences in RNA polymerase II ChIP fractions from embryos produced by either wild type (wt) or Su(var)205⁵/+ mothers crossed to Sxl^f1/Y fathers. ChIP assays for unphosphorylated (blue bar), Ser2-phosphorylated (red bar), or Ser5-phosphorylated (green bar) RNA polymerase II are shown. (p_{(no enrichment)}<0.05*; change between wild type and mutant embryos, p_{(no change)}<0.05^#, <0.01^##) (B) Bar graphs show changes in the enrichment of Sxl sequences in HOAP- (red bars) and HP1- (blue bars) ChIP fractions in embryos produced by Su(var)205⁵/CyO parents (left) and by cav²²⁴⁸/TM3, Sb parents (right). (p_{(no enrichment)}<0.05*; change between wild type and mutant embryos, p_{(no change)}<0.05^#, <0.01^##) (C) Bar graphs show changes in the enrichment of Sxl sequences in di-MeK9H3- (blue bars) and tri-MeK9H3- (red bars) ChIP fractions in embryos produced by wt, Su(var)205⁵/CyO and cav²²⁴⁸/TM3, Sb parents. (p_{(no enrichment)}<0.05*; change between wild type and mutant embryos, p_{(no change)}<0.05^#, <0.01^##).

https://doi.org/10.1371/journal.pgen.1002122.g005

The feminizing effects of reducing maternal HOAP, most notably the effect of the cav²²⁴⁸ mutation on transcription from Sxl_Pe, support a repressive role for HOAP at this promoter. The partial rescue of the maternal Su(var)205 masculinizing effect by the HP1-binding-defective cav¹ allele implicates HP1 in this repression as well. To examine the interdependency of HOAP and HP1 in their association with Sxl_Pe sequences, we performed ChIP assays of each protein in 1–3 hr embryos with a reduced maternal dose of the other protein. As shown in Figure 5B, a maternal mutation in one copy of the gene for either protein significantly reduced association of its encoded protein with Sxl_Pe sequences. These data also indicate a strong reliance of HP1 on HOAP for its association with Sxl_Pe proximal sequences in embryos of this stage. HP1 association with these sequences was significantly reduced in embryos from cav²²⁴⁸ heterozygous parents. HOAP association with Sxl_Pe sequences, also appeared educed in embryos from Su(var)205⁵ heterozygous parents, but this reduction was not statistically significant. These data support a role for HOAP in recruiting HP1 to Sxl_Pe prior to the time of Sxl_Pe activation; failure to form this repressive chromatin in embryos from heterozygous cav²²⁴⁸ mothers apparently negates the need for HP1 in Sxl_Pe activation slightly later in embryonic development.

K9-methylated histone H3 has a well known conserved role in HP1 association with constitutive pericentric heterochromatin [7]–[9] and some euchromatic genes [5]. The activating function of HP1 at Sxl_Pe does not appear to rely on this histone modification, as a maternal mutation for the Su(var)205² allele, which encodes a protein that is defective for MeK9H3-binding, does not strongly affect the viability of female progeny from Sxl mutant fathers. It is still possible, however, that HP1-binding to MeK9H3 has a role in HP1 repression prior to the time of Sxl_Pe activation. We, therefore, used ChIP assays to monitor the presence of both di- and tri-methylated histone H3 at the major site of HP1enrichment in the −101 region of Sxl_Pe. The hb promoter, which is active at the same time as Sxl_Pe but does not appear to require HP1 for activation, was used as the normalizing standard in these experiments.

Di-MeK9H3 was detected at above background levels (p<0.05) at Sxl_Pe, but not at the simultaneously expressed hb promoter, in embryos from wild type mothers (Figure 5C). Although no significant enrichment was observed at Sxl_Pe relative to hb in wild type embryos, a maternal cav²²⁴⁸ or Su(var)205⁵ mutation resulted in significant enrichment of both di- and tri-MeK9H3 at Sxl_Pe (Figure 5C). Assuming, as in pericentric heterochromatin, a repressive activity for these histone modifications at Sxl_Pe, their increased enrichment in this region in mutants for HP1 and HOAP may reflect a feedback mechanism to compensate the loss of HP1.

Heterochromatin Proteins in Sex-Specific Viability

We find that reductions in HOAP preferentially compromise male viability. This was observed for two different cav mutant alleles and by reducing HOAP through RNAi. The presence of Sxl_Pm-derived transcripts that have been spliced in the female mode in cav mutant males suggested inappropriate Sxl activation to be responsible for this reduced viability. In situ data indicating inappropriate firing of Sxl_Pe in male embryos from cav²²⁴⁸ heterozygous parents support this view, as does the rescue of the cav²²⁴⁸ male viability defect by Sxl loss of function mutations. The more pronounced male lethality observed from reducing HOAP by RNAi expression driven by maternal, versus paternal, contribution of Actin5C GAL4 is consistent with such an early requirement for HOAP for male viability (Figure 1).

Previous reports have shown that reducing HP1 by RNAi similarly reduces male viability preferentially [41]. RT-PCR assays of Sxl_Pm transcripts in HP1 mutants, however, suggested a more complex scenario as incorrect sex specific transcripts were observed in both sexes (Figure 2C). This pointed to an activation, as well as repressor, role for HP1. Consistent with an activation role, reduction of maternal HP1 severely compromised female viability when the dose of Sxl was also reduced in the progeny (Table 2), and ChIP assays of embryos from this cross showed recruitment of RNAP II to Sxl_Pe to be impaired (Figure 5A). This effect of reducing HP1 on female viability was strictly maternal, as was the antagonizing effect of simultaneously reducing maternal HOAP (Table 3). Moreover, the partial rescue of the Su(var)205 maternal effect by the C-terminally truncated cav¹ allele, which produces a protein that is compromised for HP1-binding [27], [30], points to an involvement of HP1 in the antagonizing activity of HOAP. Finally, ChIP assays show a dependence of HP1 on HOAP for its association with Sxl_Pe. Combined, these data indicate both antagonistic and cooperative roles for these heterochromatin proteins in regulating Sxl_Pe, whereby HOAP acts as a repressor and HP1 acts as both an activator and repressor. The reliance of HP1 on HOAP for recruitment to the promoter would suggest HOAP may also have a role in the activation function of HP1 at the promoter, although this was not readily apparent in our assays.

Although our data clearly show maternal roles for HOAP and HP1 in regulating the activity of Sxl_Pe, for both HOAP and HP1 [41] the RNAi knockdown data indicate a substantial zygotic component in their effects on male viability. These zygotic effects, observed only in progeny carrying both an interference RNA transgene and a GAL4 driver transgene (Figure 1A), suggest additional later sex-specific roles for both proteins. Such roles could be related to those observed for HP1 and SU(VAR)3-7 in male dosage compensation [47]. Because the effect of reducing these proteins on the chromosomal distribution of DCC proteins [47] is the opposite of those observed for males that are deficient for DCC proteins [48], as predicted to occur with inappropriate Sxl_Pe expression, the activities of heterochromatin proteins in dosage compensation appear to be distinct from the early roles of HP1 and HOAP at Sxl_Pe. In addition, there may be zygotic roles for heterochromatin proteins in sex-specific gene expression, as proposed for HP1 by Liu et al. [41].

Dynamic Roles of HOAP and HP1 at Sxl_Pe

Previous analysis of Sxl_Pe indicated that 400 bp immediately upstream of the promoter are sufficient for sex-specific regulation, but distal sequences, extending to −1700 bp, are required for wild type levels of expression [33]. As shown in Figure 4A, E-box binding sites for antagonistically acting bHLH proteins, which are encoded by zygotically expressed X-linked and autosomal signal elements (XSE and ASE) and direct an X counting mechanism, are distributed throughout both regions [49].

Both HP1 and HOAP are enriched in the region proximal to Sxl_Pe which contains binding sites for both positive and negative E-box proteins. Within the Sxl_Pe promoter distal region, HOAP alone is enriched in two peaks where there is a striking relationship with E-box binding sites for positive factors, but those for negative factors appear essentially devoid of HOAP. HOAP may antagonize positive factors but permit negative factors to bind in the Sxl_Pe distal region, in an HP1-independent repressing role. Whereas loss of HOAP de-represses Sxl_Pe in males, the strength and uniformity of expression does not approach that in wild type females. This indicates continued influence from the X counting mechanism in cav mutant males. Sxl_Pe is also expressed prematurely in female embryos. This de-repression by reduced levels of maternal HOAP in both sexes indicates that HOAP is present at Sxl_Pe in both sexes of wild type embryos. However, whether the proximal and distal Sxl_Pe regions have the same or different compositions of HOAP and HP1 in the two sexes cannot be determined from our ChIP assays, as the embryos are of mixed sexual identity.

The interdependency of HOAP and HP1 for their binding to the Sxl_Pe proximal region, most notably the dependence of HP1 on HOAP, also indicates both proteins are in this region in, at least, wild type female embryos. In spite of this interdependency, the genetic data show HOAP repression antagonizes HP1 activation. HOAP repression appears to also be partly HP1-dependent; the mutant HOAP protein from the cav¹ allele which lacks HP1-binding also antagonizes HP1 activation. This combination of antagonistic and cooperative interactions suggests a model in which maternal HOAP and HP1 first cooperate to repress Sxl_Pe prior to its activation (Figure 6A). The repressive structure formed by maternal HOAP and HP1 likely serves to reduce the sensitivity of Sxl_Pe to spurious fluctuations in zygotic XSE levels, ensuring it is only activated in females where an effective ratio of activating to repressing transcription factors exists (Figure 6B). HP1 is retained at Sxl_Pe during its activation in females, where it presumably switches into an activation role. In early embryos constitutive heterochromatin proteins may be more appropriate for such regulation than the Polycomb Group of facultative heterochromatin proteins, as they would not be subject to cross regulatory signals from body plan specification pathways.

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Figure 6. Model of the interactions between HOAP and HP1 at Sxl_Pe.

(A) Maternal HOAP and HP1 cooperate to form a repressive complex which serves to reduce the sensitivity of Sxl_Pe to spurious fluctuations in zygotic expression of positive (e.g. Sis-a, Sis-b) and negative (e.g. Dpn) regulatory proteins. Pre-loading of RNA pol II requires HP1. (B) Binding of bHLH proteins to Sxl_Pe sequences is dependent on their zygotic dose and binding sites relative to those for HOAP. At 2–3 hr of development, the two X chromosome dose of positive factors in females, is able to result in activation of Sxl_Pe, and allow the pre-loaded RNA pol II to extend. In males, the single X chromosome presumably does not produce enough positive factors to displace (all of) the negative components. For simplicity not all known X∶A factors are shown. The triangle depicts low levels of H3K9 methylation at the promoter.

https://doi.org/10.1371/journal.pgen.1002122.g006

How HP1 switches over to transcriptional activation mode in the Sxl_Pe proximal region is unclear. Changes in HP1 phosphorylation and/or association with other factors could alter its activity [50], [51]. Several XSE binding sites are nearby, making them strong candidates. Presumably, this would only occur in females where the XSE dose surpasses a threshold and Sxl_Pe is activated.

Mechanism of HP1 in Transcriptional Activation at Sxl_Pe

This report provides the most clearly defined role for HP1 in developmental control of a euchromatic gene in a metazoan species, and the first evidence of a bifunctional regulatory role for it in such a context. Prior reports describing HP1 in transcriptional activation have focused on it in the context of transcription elongation [11]–[13], [52], [53]. Our ChIP data at Sxl_Pe, however, show a requirement of it for association of RNAP II with the promoter, more consistent with a role in transcription initiation. A role in initiation is also in keeping with the position of HP1 on the gene; we find very little HP1 elsewhere on the Sxl gene, even during the time of Sxl_Pe activity. This dependence of RNAP II association on HP1 is similar to what is observed in the accumulation of noncoding RNAs at S. pombe centromeric repeats and mating type locus [54]. Nonetheless, it is possible that the loss of RNAP II at Sxl_Pe reflects reduced stability of all RNAP II isoforms as a consequence of an early defect in transcription elongation, rather than a defect in RNAP II recruitment to the promoter.

Pausing of RNAP II in promoter proximal regions prior to activation has been observed in a high proportion of genes under developmental control in Drosophila embryos [55], and such pauses have also been implicated in regulation of alternative splicing [56]. While Sxl_Pm appears to have the features of a promoter with paused RNAP II in a genome wide RNAP II ChIP study of 0–4 hr embryos (Flybase MODENCODE), RNAP II was absent from Sxl_Pe. It is likely that the collection window for this study did not precisely coincide with the time of Sxl_Pe activity. Our more narrowly timed collection indicates paused RNAP II at Sxl_Pe, suggesting that, like Sxl_Pm, it is a pre-loaded promoter. A preloaded Sxl_Pe also readily explains how generalized up-regulation of phosphorylation of the RNAP II CTD by the loss of Nanos, causes Sxl_Pe activation in males with an unchanged X∶A ratio [57].

Evolutionary Implications of HOAP, Similarity to SRY?

Finally, the dominant negative activity of the cav²²⁴⁸ allele suggests a role for the partially deleted SRY-like HMG box in HOAP association with Sxl_Pe. Our ChIP data show HOAP association with the Sxl_Pe proximal region is required for HP1 association. This proposed role for the HMG box of HOAP in Sxl_Pe regulation is of particular interest with regards to a recent report linking HP1 and KAP-1 (TIF1β) to SRY-dependent repression of testis-specific genes in the ovary [26]. Because mammalian sex determination is inextricably linked to gonad sex determination, SRY and HOAP each appear to constitute early decision points in their respective sex determination pathways. There are, perhaps, unexpected parallels between these divergent pathways.

Genetic Analyses

The cav RNAi lines were obtained by germline transformation of the pUAST vector containing an inverted repeat of a near full length cDNA sequence for the cav-RB transcript (See Table S3 for primer sequences). The cav²²⁴⁸ allele was recovered in a screen of progeny from ethane methyl sulfonate (EMS)-mutagenized males which failed to complement the original cav¹ allele, and the sequence of the allele was determined from PCR products obtained from homozygous cav²²⁴⁸ embryos identified through their lack of the homologous twi-GAL4, UAS-GFP marked balancer chromosome.

Flies in all crosses were reared under uncrowded conditions on standard cornmeal medium enriched with active dry yeast. Unless otherwise noted, all crosses were done at 25°C; Ore R or y¹w^67c23 were used as the wild type control. Progeny were counted for 8 days beginning on the first day of eclosion. The Z test was used in statistical analyses of distributions in two populations [58]. Description of genes can be found in Flybase (http://www.flybase.org/).

Microarray Expression Profiling Study

RNA was TRIzol-extracted from y¹w^67c23; cav¹ homozygous (those lacking the Tm6B,Tb balancer chromosome used to maintain the cav¹ mutation in heterozygous condition) and wild type first and second instar larvae and purified through Qiagen RNeasy columns. The RNA was then used by the University of Kentucky Microarray Facility to prepare biotin-labeled cRNA that was hybridized to separate Affymetrix Drosophila Genome microarrays (version 1). The y¹w^67c23 stock to which all mutant stocks are out-crossed during genetic manipulations was used as the wild type control in these experiments. The data were obtained from two biological replicate samples from each stock. The statistical analyses of the arrays were carried out by A.J. Stromberg, PhD (UK Dept. of Statistics) associated with this facility according to standard Affymetrix specifications. Of 13,982 affytags to Drosophila genes on the array, an average of 46% and 53% were present in the cav¹ and wild type y¹w^67c23 samples, respectively. Data from publically available tissue-specific cDNA libraries [35]–[37] and from two published microarray profiling studies of Drosophila sex- or tissue-specific gene expression [38], [39] were used to catalogue normal gene expression profiles of genes with reduced or elevated transcript levels (log₂R>2.0, p<0.01).

RT-PCR Analysis

RNA was similarly prepared from pools of male and female cav²²⁴⁸ heterozygous and cav¹ homozygous embryos and from individual Su(var)205⁴/Su(var)205⁵ larvae. The cav²²⁴⁸ heterozygous embryos were identified from a pool of tightly staged embryos that failed to progress to the larval stage and contained intermediate levels of GFP expression from the P{GAL4-twi.C}2.3, P{UAS-2xEGFP}AH2.3-marked TM3Sb balancer chromosome. The cav¹ homozygous embryos were similarly identified through their lack of GFP expression from this balancer chromosome. Su(var)205⁴/Su(var)205⁵ larvae were identified through their lack of GFP expression from the P{Act5C-GAL4}25F01-marked CyO balancer chromosome. The sex of individual cav²²⁴⁸ heterozygous and cav¹ homozygous embryos was first determined through PCR of the Y-linked kl-2 gene from a total nucleic acid extraction from individual embryos (See Table S3 for primer sequences). RNA was then purified from pools of nucleic acids from individual male or female embryos. The sex of individual Su(var)205⁴/Su(var)205⁵ larvae was first determined through the presence or absence of the y⁺-marker from the paternally derived X chromosome and then substantiated through PCR assays of the Y-linked kl-2 gene. RT-PCR assays were carried out with RNA purified from these pools using the Qiagen One-Step RT-PCR kit (Qiagen 210212). The sequences of all primers used are shown in Table S3. Primers for the Drosophila RpA-70 were used in PCR reactions to determine that all RNA samples were DNA-free before they were used in RT-PCR as a positive control and in normalization of all RT-PCR data (Table S3).

In Situ Hybridization

These were done as described in [59]. Digoxygenin labeled RNA probes complementary to Sxl exon E1 or L1 region were prepared using T7 RNA polymerase in vitro transcription of plasmid- or PCR-derived templates. The establishment (407 nt) and maintenance (1039 nt) transcript specific probes were generated by the primers shown in Table S3. All in situs were repeated at least once. Each batch was done simultaneously with an Ore R control and had sufficient embryos so that several representatives of each cycle could be examined.

Chromatin Preparation

A modification of the protocol described in [60] was used to prepare cross-linked chromatin from embryonic progeny from parents of the following genotypes: wild type (y¹w^67c23 or yw), yw; cav²²⁴⁸/TM3Sb, yw; Su(var)205⁵/CyO, yw females×Sxl^f1/Y males, and yw; Su(var)205⁵/CyO females×Sxl^f1/Y males. Embryos from yw parents were collected at the following developmental stages: 0.75 to 2.75 hr (labeled 1–3 hr), 2–3 hr, and 4–18 hr. Embryos were collected from yw; Su(var)205⁵/CyO females crossed to Sxl^f1/Y males in parallel to those from yw females crossed to Sxl^f1/Y males (at 2–3 hr stage). Embryos from yw; cav²²⁴⁸/TM3Sb and from yw; Su(var)205⁵/CyO parents were also collected in parallel (at 0.75 to 2.75 stage). All embryo collections and staging were done at 22°C. Chromatin was prepared from 6.0 g yw embryos in 6.0 ml homogenization buffer (50 mM Hepes at pH 7.6, 60 mM potassium chloride, 0.25 M sucrose, protease inhibitor cocktail [15]). The homogenate was first clarified by centrifugation at 500× g for 10 minutes before the addition of formaldehyde to 2%. Cross-linked chromatin was then washed 3 times in phosphate buffered saline (150 mM sodium chloride (NaCl), 10 mM sodium phosphate, pH 7.6) (with centrifugation at 3,000× g for 10 minutes after each wash) and re-suspended in 6.0 ml RIPA buffer (50 mM Tris-HCl, pH 7.6, 1 mM ethylene diamine tetraacetic acid (EDTA), 0.5 mM ethylene glycol tetraacetic acid (EGTA), 140 mM NaCl, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS) and sonnicated to an average length of 500 bp. A scaled down version of the protocol was carried out with 0.5 g mutant embryos in 1.5 ml homogenization buffer.

Chromatin Immunoprecipitations

Chromatin immunoprecipitations were performed with 0.2 ml clarified chromatin, 20–40 µg antibody [anti-HOAP [61], anti-HP1 [15], anti-RNA polymerase II antibody (Covance MMS-126R, MMS-134, or MMS-129R), anti-di and tri MeK9 histone H3 (Millipore 05-1249 and 05-1242) or non-immune IgG (Santa Cruz Biotech. SC-2027 or SC-2025)] and 100 µl anti-rabbit (Sigma A914) or anti-mouse (Sigma A6531) IgG agarose in 1.5 ml RIPA buffer. Washes were performed as described in Alekseyenko et al., [60]. The immunoprecipitated material was eluted from the beads by incubation at 37°C for 1 hr in 500 µl TE (1 mM EDTA, 50 mM Tris, pH 8.0) containing 0.5% sodium dodecyl sulfate (SDS) and proteinase K (0.1 mg/ml), followed by 12 hr at 65°C after the addition of NaCl to 0.3 M and SDS to 1%. The samples were then extracted once with phenol/chloroform, once with chloroform before ethanol precipitation in the presence of glycogen.

Protocol for Quantitative Real-Time PCR

The iCycler iQ real-time PCR detection system (Bio Rad) was used to quantitate Sxl sequences in the precipitated DNA from each ChIP fraction. The primer pairs shown in Table S3 were used to amplify fragments spanning the Sxl locus as shown in Figure 4 and RpA-70 normalizing standard (average length 288 bp). Similar enrichment values were obtained for all data when calculated as % of total in ChIP vs. input fraction. PCR amplification was performed in duplicate in 50 µl SYBR Green qPCR SuperMix (Bio-Rad 170–8880) on two biological replicates of each ChIP fraction. Dissociation curve analysis was performed at the end of 40 cycles, and quantification was carried out by Bio-Rad comparative C_T methodology with standard curves constructed for each primer pair with a serial dilution of input DNA having PCR efficiencies of 80–120%. A one sample t-test was performed to identify sequences that were enriched in ChIP fractions above background (i.e., >1); a student's t test was used to determine significance of differences between two samples of equal variance.