Heterochromatin protein 1 (HP1) is intrinsically required for post-transcriptional regulation of Drosophila Germline Stem Cell (GSC) maintenance

A very important open question in stem cells regulation is how the fine balance between GSCs self-renewal and differentiation is orchestrated at the molecular level. In the past several years much progress has been made in understanding the molecular mechanisms underlying intrinsic and extrinsic controls of GSC regulation but the complex gene regulatory networks that regulate stem cell behavior are only partially understood. HP1 is a dynamic epigenetic determinant mainly involved in heterochromatin formation, epigenetic gene silencing and telomere maintenance. Furthermore, recent studies have revealed the importance of HP1 in DNA repair, sister chromatid cohesion and, surprisingly, in positive regulation of gene expression. Here, we show that HP1 plays a crucial role in the control of GSC homeostasis in Drosophila. Our findings demonstrate that HP1 is required intrinsically to promote GSC self-renewal and progeny differentiation by directly stabilizing the transcripts of key genes involved in GSCs maintenance.

Decapentaplegic (Dpp) and Glass bottom boat (Gbb) are produced from the somatic niche and activate bone morphogenetic protein (BMP) signaling in the GSC to directly repress the Bam-dependent differentiation pathway and to maintain GSC identity [17][18][19][20] . Besides extrinsic mechanisms, stem cell intrinsic programs are crucial to control the binary germ line cell fate in Drosophila. Nanos and Pumilio are intrinsic factors essential to maintain stem cell identity [21][22][23] . They are key components of an evolutionarily conserved translational repressor complex 24-28 that bind to specific recognition sequences in the 3′untranslated regions (3′UTRs) of differentiating mRNAs to repress their translation 24,29,30 .
A fast-growing body of experimental data provides strong evidences that also epigenetic mechanisms involving chromatin architecture and histone modification are equally important for the regulation of GSC maintenance and differentiation in Drosophila [39][40][41][42][43] . For example, the chromatin remodeling factor Iswi and the putative transcription factor Stonewall are intrinsically required for GSC maintenance [39][40][41][42][43] . The H3K4 demethylase Lsd1 controls non-autonomously the germ cell differentiation presumably through repressing dpp expression 41 . Moreover, other interesting studies show that the histone H2B ubiquitin protease Scrawny (Scny) 40 and the histone H3K9 trimethylase Eggless (Egg) are required for maintaining self-renewal of GSC 42 .
Although different experimental evidence confirms the relevance of epigenetic regulatory programs in the GSC regulation, a complete picture of such mechanisms is still far to be resolved.
Heterochromatin protein 1 (HP1) is an evolutionarily conserved multifunctional epigenetic adaptor that is involved in heterochromatin formation and epigenetic gene silencing in different species including humans [44][45][46] . In addition to its role in heterochromatin structural organization, emerging evidence in Drosophila and mammals has highlighted the importance of HP1 in telomere capping, telomere length homeostasis 47,48 and, more surprisingly, in positive regulation of gene expression [49][50][51][52][53][54] .
A recent study showed that HP1 and Su(var)3-9 are both necessary for GSC maintenance and that HP1 is sufficient for GSC self-renewal in Drosophila testis 55 . It has also been demonstrated that planarian HP1, induced upon injury, is able to promote regenerative proliferation of adult stem cells 56 . In mice, loss of HP1 gamma significantly reduces the number of primordial germ cells (PGCs) by regulating their cell cycle progression 57 . Moreover, HP1 gamma is essential for male germ cell survival and spermatogenesis 58 . Recently, a large-scale RNAi screen in Drosophila female germline stem cells identified HP1 as potentially involved in oogenesis 59 even though the precise molecular mechanisms by which it exerts its function still remain elusive and need to be defined.
Here, we report our experiments showing an important function for Drosophila HP1 in female gametogenesis. In this study, we establish that HP1 is necessary for Drosophila oogenesis and is required cell autonomously to control the fine balance between stem cell self-renewal and differentiation. Finally, we show that HP1 exerts its functions positively regulating the stability of key mRNAs involved in the control of female germ line stem cells development.

Results and Discussion
Functional inactivation of HP1 by in vivo RNA interference (RNAi) causes severe germ line defects that result in agametic ovarioles and female sterility. HP1 is a protein constitutively expressed in almost all larval and adult tissues with highest enrichment in adult ovaries (flybase.org). Immunostaining experiments performed by a specific anti-HP1 antibody on wild type ovaries, showed that HP1 localizes in the nucleus of both somatic and germline cells, from the anterior tip of the germarium (GSCs and CBs) until late stages of oogenesis (Fig. 1a). Specifically, HP1 immunosignals were mainly detected in dense pericentric heterochromatic foci in all germarium and developing egg chamber cells; HP1 also accumulated in the germinal vesicle and on the karyosome of the oocyte (Fig. 1a). HP1 was particularly enriched within and next to heterochromatic regions also in larval and pupal gonads ( Supplementary Fig. S1).
Since homozygous HP1 mutants die at third instar larvae, to investigate in vivo the function of HP1 in adult female germline, we took advantage of the Gal4-UAS binary system 60 . We performed tissue-specific HP1 knockdown by independently crossing two different transgenic lines carrying HP1 short small hairpin RNAs (shRNA) 61 under the control of Gal4-responsive UAS promoter, with nanos-Gal4-NGT (hereafter referred as nos-Gal4) that provides a robust and uniform Gal4 expression in the germarium 62 . We found that the functional inactivation of HP1 in the F1 female progeny resulted in complete sterility thus suggesting an essential role for HP1 in female gametogenesis.
In order to further investigate the molecular basis underlying this female sterility, ovaries from nos-Gal4 > HP1 RNAi females ranging from 1-to 15-day-old, were dissected and immunostained with a specific antibody against Vasa, a DEAD-box RNA helicase which is a well-characterized marker of germ cells lineage in insects and vertebrates 63,64 . We found that knocking down HP1 upon nos-Gal4 driver expression ( Supplementary  Fig. S2), resulted in ovaries that were completely agametic (Fig. 1b,c) as compared to control ovaries (Fig. 1d); 86% of HP1 depleted germaria from 0-to 1-day-old females were completely devoid of germ cells (Fig. 1b) whereas 14% contained only a few germ cells at the tip of the ovariole (less than 10 per germarium) and one or two abnormal egg chambers (Fig. 1c) (n = 250 ovaries). From 5-to 15-day-old females, all the HP1 depleted ovaries exhibited a typical germline less morphology confirmed by the total absence of Vasa-positive cells (data not shown).
These findings strongly suggest for HP1 a specific and crucial role in germ line stem cell maintenance and differentiation; we could not, however, completely exclude a general role for HP1 in cell viability.
To discriminate between these possibilities, we knocked down HP1 with a maternal tubulin (Mat) Gal4 that induces transgenic expression of short hairpin RNAs against HP1 outside the germarium, starting in stage 2 65 www.nature.com/scientificreports www.nature.com/scientificreports/ ( Supplementary Fig. S3a,b). We found that HP1 knockdown females were fertile and showed no obvious oogenesis defects ( Supplementary Fig. S3c) thus suggesting for HP1 an essential and cell autonomous function in early oogenesis and not a general requirement for cell survival.
HP1 is required during multiple processes in early oogenesis. Germ cell-specific knockdown of HP1 causes almost complete loss of germ cells before adulthood. In order to determine the phenocritical period for HP1 requirement during normal oogenesis, we cytologically examined larval and pupal HP1 depleted ovaries following the germ cell fate, starting from early stages of germ cell development to adulthood ( Fig. 2 and Supplementary Fig. S4) Vasa staining analysis showed that larval ovaries from nos-Gal4 > HP1 RNAi females displayed a normal cellular organization as compared to control; consistent with this the total number of PGCs resulted unaffected (nos-G4/+, 107.5 ± 8.5; nos-G4 > HP1 RNAi , 106.6 ± 7.0) (Fig. 2a).
On the contrary, HP1 depleted pupal ovaries were almost completely devoid of differentiated egg chambers when compared to the control pupal gonads (Fig. 2b).
Taken together, these findings suggest that HP1 is required during the earliest stages of oogenesis at the larval/ pupal transition when GSCs are established 66 .
In order to gain a more complete understanding of the altered phenotypes observed in pupal ovaries and to better investigate how HP1 regulates the behavior of germ cells, we performed an accurate cytological analysis on nos-Gal4 > HP1 RNAi pupal ovaries. We performed double-immunostaining experiments with antibodies against Vasa and α-Spectrin; α-Spectrin is a cytoskeletal protein that specifically labels spectrosomes and fusomes and can be used to trace the germline differentiation. Spectrosomes are spherical and mark GSCs and cystoblasts, whereas fusomes are branched and mark 2, 4, 8, and 16-cell cysts 67 .
These complex phenotypic defects suggest for HP1 a functional role in regulating the germline stem cell (GSC) maintenance.
We asked whether the low number of germ cells in HP1 depleted ovarioles could be related to defects in the division rate of ovarian stem cells and their progeny. These defects might contribute to germ line cells loss over time. In order to verify the capacity of germ cells to undergo mitotic divisions, we immunostained wild type and HP1 knockdown ovaries with a specific antibody to phosphorylated H3S10 (phospho-H3, PH3) to detect  www.nature.com/scientificreports www.nature.com/scientificreports/ germline cells undergoing mitosis at a given time (Fig. 3c). In HP1 depleted ovaries we observed an almost complete loss of PH3 positive nuclei (5%, n = 56 ovarioles) respect to the control ovaries (35%, n = 46 ovarioles) (Fig. 3d); this result establishes that the functional inactivation of HP1 severely impairs the correct germ cells division.
We also assessed apoptosis by using anti-cleaved Caspase-3 antibody that is a proven marker for cells that are dying. The results clearly indicated that the few remaining germline cells detected in HP1 depleted ovaries are strongly stained with cleaved Caspase-3 suggesting that the germ cells that fail to properly divide die prematurely (Fig. 3e).
HP1 promotes germ cell differentiation by post-transcriptionally regulating bam expression and function. Germline division defects are often associated to an altered differentiation program.
Previous studies demonstrated that Bag of Marbles (Bam) protein is necessary and sufficient for promoting GSC and cystoblasts differentiation, since bam mutations completely block germ cell differentiation (causing GSC hyperplasia), whereas bam ectopic expression in GSCs results in their complete and precocious differentiation 18,68,69 .
To determine whether the phenotypic defects observed in HP1 depleted pupal ovaries could be related to bam repression, we firstly evaluated, by quantitative real-time PCR (qRT-PCR), the expression of bam gene in HP1 knockdown pupal ovaries. We found that ovaries lacking HP1 exhibited a significant reduction of bam transcript levels (close to about 80%) as compared to control ovaries (Fig. 4a).
Consistent with the down regulation of bam mRNAs, we also observed a drastic diminution of Bam protein by immunostaining with a specific monoclonal antibody against Bam (Fig. 4b). In wild type ovaries, Bam protein was detected, as expected, in cystoblasts and early developing cysts (2-, 4-, and 8-cell cysts) whereas in HP1 mutant ovaries Bam protein was almost undetectable (Fig. 4b).
Altogether, these data strongly suggest that HP1 blocks Bam driving germ cell differentiation.
Previously we have demonstrated that in Drosophila HP1 takes part in positive regulation of gene expression by stabilizing RNA transcripts and protecting them against premature and rapid degradation 53 ; in particular, we found that HP1 is able to directly bind the transcripts of more than one hundred euchromatic genes in Drosophila and physically interacts with DDP1 70 , HRB87F 71 and PEP 72 , which belong to different classes of heterogeneous nuclear ribonucleoproteins (hnRNPs) involved in RNA packaging, stability and processing. Moreover, in our previous work we also demonstrated that HP1 is cotranscriptionally recruited on nascent transcripts through its chromodomain 49,53 .
In order to verify if HP1 was directly involved in post-transcriptional regulation of bam gene by binding in vivo its mRNA, we performed HP1 CLIP (UV cross-linking and immunoprecipitation) experiments 73,74 on whole adult ovaries dissected from 0-to 1-day-old-wild type females.
The results of RT-PCR from HP1 CLIP experiments clearly showed that bam transcripts were significantly enriched in the CLIP sample when compared to the mock control sample (Fig. 4c) and demonstrated that HP1 is able to specifically bind bam transcripts in vivo.
In order to further investigate whether HP1 is cotranscriptionally recruited on bam nascent transcripts, we performed ChIP experiments on cross-linked chromatin purified from 0-to 1-day-old wild-type ovaries. To evaluate the presence of bam sequences among the immunoprecipitated DNA, a PCR analysis was performed with specific primer pairs covering both the promoter and the coding regions of bam gene.
The results of ChIP assays demonstrated that HP1 is clearly associated to bam gene (Fig. 4d). To completely exclude any direct role for HP1 on bam transcriptional control and to confirm that HP1 binding on bam gene was exclusively mediated by the presence of bam nascent transcripts, ChIP experiments were repeated in presence of RNaseA/T1 mix that specifically degrades single stranded RNA (ssRNA). The RNase-ChIP results demonstrated that chromatin RNase treatment prior to immunoprecipitation completely remove HP1 from bam gene thus confirming that the recruitment of HP1 on bam gene is clearly RNA-dependent (Fig. 4d). RNase treatment did not affect, as expected, the HP1 occupancy over Het-A telomeric retrotransposon (Fig. 4d) since, at the telomeres, HP1 is capable to directly bind HeT-A sequences through its hinge domain 48 .
To determine the stability of bam transcripts, we analyzed, by qRT-PCR, RNA samples purified from wild type and HP1 knockdown ovaries treated with Actinomycin D to inhibit de novo RNA synthesis. Previous work showed that a 30 min treatment was sufficient to inhibit transcription in the ovaries 75 . As shown in Fig. 4e, in HP1 lacking ovaries we observed a strong and rapid decay rate of bam transcript when compared to the control (Fig. 4e).
These observations strongly suggest that HP1 may regulate bam mRNAs in a post-transcriptional manner.
To confirm our findings and to verify if HP1 can effectively control germ cells differentiation in a bam-dependent manner, we overexpressed bam from a heat shock inducible transgene carrying the full-length bam cDNA 68 in the HP1 knockdown germ cells. To assess the effectiveness of hs-bam transgene expression we analyzed bam mRNA and protein in HP1 depleted ovaries with or without heat-shock ( Supplementary Fig. S5).
Nos-Gal4/UAS-HP1 RNAi ; P[hs-bam]/+ and nos-Gal4/HP1 RNAi ; +/+females were heat-shocked at pupal stage (96 hours) at 37 °C for 1 hour and, 24 hours after heat shock (HS) treatment, adult ovaries were dissected and stained with anti-Vasa antibody (Fig. 5a,b). As showed in Fig. 5b, heat-shock induced bam can only partially rescue the phenotypic defects induced by HP1 knockdown since its forced expression under control of the heat shock promoter generates only few normally developed egg chambers (see Fig. 5c for quantification of ovarioles containing developing egg chambers in heat shocked HP1 depleted females carrying the P[hs-bam] transgene). This finding suggests that oogenesis defects observed in HP1 depleted ovaries may be only partially imputable to defective differentiation mechanisms.
It is well known that loss of bam blocks germ cell differentiation resulting in GSC hyperplasia 68 , a characteristic phenotype that we never observed in HP1 depleted ovaries by nos-Gal4.
www.nature.com/scientificreports www.nature.com/scientificreports/ Altogether, these findings strongly suggest that the complex phenotypic defects arising from HP1 knockdown in the female germline are only partially dependent on bam repression and are probably due to a duplex coordinated control operated by HP1 in both GSCs self-renewal and differentiation.
In order to verify this hypothesis we inactivated HP1 only in Bam-expressing germline cells by using P{bam promoter-Gal4:VP16} 17 that drives the expression of shHP1 only in the dividing cystoblast and cystocytes but not in GSCs where the function of HP1 protein remains completely wild-type. In this case, we observed the classical ovarian tumor phenotype (Fig. 5d) albeit at very low frequency (less than 1%) due to the low effectiveness of bam-Gal4 driver in knocking down HP1 protein (Supplementary Fig. S6). www.nature.com/scientificreports www.nature.com/scientificreports/ HP1 controls GSCs self-renewal by post-transcriptional regulation of stemness genes. Consistent with the conclusion stated above, we wondered if HP1 was able to post-transcriptionally regulate also key stemness genes. First, we analyze by qRT-PCR the expression profiles of some important genes that are intrinsically involved in GSCs self-renewal by repressing Bam differentiation pathways [76][77][78] .
We found that some of them as nos, cup, piwi and vasa were significantly down regulated in HP1 knockdown pupal ovaries respect to the control (Fig. 6a). These results allowed us to hypothesize that also nos, cup, piwi and vasa genes might be post-transcriptionally regulated by HP1. So we dissected ovaries from 0-to 1-day-old wild type females to repeat both CLIP and ChIP experiments. CLIP-PCR analysis, clearly showed that nos, cup and piwi RNAs were significantly enriched in the IP sample respect to the mock control sample whereas vasa mRNA did not (Fig. 6b). These genes resulted strongly enriched also in ChIP IP sample but not in RNAse-ChIP IP sample (Fig. 6c) indicating that their RNAs are co-transcriptionally bound by HP1. To determine the mRNA decay of these genes, we repeated the Actinomycin D treatment that allowed us to conclude that HP1 is able to stabilize nos, cup and piwi mRNAs (Fig. 6d).
Altogether, these data strongly indicate that HP1 is intrinsically required for post-transcriptional regulation of Drosophila GSC maintenance. Our findings suggest that HP1 exerts its function through the formation of an HP1-containing hnRNP nuclear complex that protects and stabilizes key mRNAs involved in the control of GSC homeostasis and behavior. Intriguingly there are different experimental evidences demonstrating that also mutations in genes coding for the HP1-interacting hnRNPs DDP1, HRB87F and PEP induce female-sterile phenotypes 79-81 . For example, the hypomorphic insertional allele of DDP1 (Dp1 15.1 ) causes complete sterility in females but not in males. Homozygous Dp1 15.1 females show abnormal ovaries with ovarioles undeveloped, egg chambers often fused and containing an irregular number of cells 79 ; finally, also PEP and HRB87F are essential for normal gonadal development and female fecundity 80,81 . www.nature.com/scientificreports www.nature.com/scientificreports/ In conclusion, the above results demonstrate, for the first time, an essential role for HP1 in post-transcriptional regulation of GSC maintenance and certainly add a new dimension to our understanding of HP1 targeting and functions in epigenetic regulation of GSC behavior.

Methods
Drosophila strains. All flies were raised at 24 °C on standard cornmeal-sucrose-yeast-agar medium.
For a detailed list of all stocks used in this study, see Supplementary Methods. Immunofluorescent staining of larval, pupal and adult whole-mount ovaries. Pupal and adult ovaries were stained according to Grieder 82 .
Larval ovaries were dissected, fixed, and immunostained as described previously by Pisano 83 . Further details can be found in Supplementary Methods.

Western blot analysis. Protein extracts fractionated by 10% SDS-PAGE and electroblotted onto
Immobilion-P polyvinyldifluoride membranes (Bio-rad) were probed with antibodies against HP1 (1:500, 9A9 monoclonal mouse), α-Tubulin (mouse 1:2000, Sigma). Proteins of interest were detected with HRP-conjugated goat anti-mouse or anti-rabbit IgG antibody (1:10000, Santa Cruz) and visualized with the ECL Western blotting substrate (GE Healthcare), according to the provided protocol. The chemiluminescence detection was performed on the ChemiDoc XRS+ System (Bio-rad) and analyzed using the included ImageLab software.
Cross-linking immunoprecipitation (CLIP) assay. CLIP assay was performed as previously reported 84 with some modifications. Approximately 20 mg ovaries from 0-to 1-day-old wild-type females were UV crosslinked (3 × 2000 µJ/cm 2 ), homogenized on ice in 1 mL RCB buffer (50 mM HEPES pH 7.4, 200 mM NaCl, www.nature.com/scientificreports www.nature.com/scientificreports/ 2.5 mM MgCl 2 , 0.1% Triton X-100, 250 mM sucrose, 1 mM DTT, 1× EDTA-free Complete Protease Inhibitors, 1 mM PMSF) supplemented with 300 U RNAseOUT (Invitrogen) and placed on ice for 30 min. The homogenate was sonicated on ice, at 80% power, five times in 20 s bursts with a 60 s rest in between using the Hielscher Ultrasonic Processor UP100H (100 W, 30 kHz) and centrifuged (16000 × g for 5 min at 4 °C). Soluble extract was precleared with 20 µl Protein-G dynabeads (Invitrogen) for 20 min at 4 °C. After removal of samples for immunoblotting and quantitation of RNA input (1%), HP1 was immunoprecipitated with anti-HP1 9A9 antibody from 450 µl precleared extract by incubation for 4 h with 50 µl Protein-G dynabeads. Immunoprecipitates were washed 4 times with RCB. To elute the immunoprecipitated RNAs, the pelleted beads were boiled in 100 µL of UltraPure DEPC-treated Water for 5 min. 900 µL Qiazol Reagent was added to the supernatant recovered for RNA preparation. The RNA purified was used as a template to synthesize cDNA using oligo dT, random hexamers and SuperScript reverse transcriptase III (Invitrogen) according to the manufacturer's protocol.
Chromatin immunoprecipitation assay. Chromatin immunoprecipitation was performed according to the method described by Menet 85 with minor modifications. Approximately 20 mg ovaries from 0-to 1-day-old wild-type females were homogenized in 1 mL of NEB buffer (10 mM HEPES-Na at pH 8.0, 10 mM NaCl, 0.1 mM EGTA-Na at pH 8, 0.5 mM EDTA-Na at pH 8, 1 mM DTT, 0.5% NP-40, 0.5 mM Spermidine, 0.15 mM Spermine, 1× EDTA-free Complete Protease Inhibitors) with a Polytron homogenizer (Kinematica Swizerland) with a PT300 tip for 1 min (at 3000 rpm). The homogenate was transferred to a pre-chilled glass dounce (Wheaton) and 15 full strokes were applied with a tight pestle. Free nuclei were then centrifuged at 6000xg for 10 min at 4 °C. The nuclei-containing pellets were resuspended in 1 mL of NEB and centrifuged at 20000 × g for 20 min on sucrose gradient (0.65 mL of 1.6 M sucrose in NEB, 0.35 mL of 0.8 M sucrose in NEB). The pellet was resuspended in 1 mL of NEB and formaldehyde to a final concentration of 1%. Nuclei were cross-linked for 10 min at room temperature and quenched by adding 1/10 vol of 1.375 M glycine. The nuclei were collected by centrifugation at 6000 × g for 5 min. Nuclei were washed twice in 1 mL of NEB and resuspended in 1 mL of Lysis Buffer (15 mM HEPES-Na at pH 7.6, 140 mM NaCl, 0.5 mM EGTA, 1 mM EDTA at pH 8, 1% Triton X-100, 0.5 mM DTT, 0.1% Na Deoxycholate, 0.1% SDS, 0.5% N-lauroylsarcosine and 1× EDTA-free Complete Protease Inhibitors). Nuclei were sonicated using a Hielscher Ultrasonic Processor UP100H (100 W, 30 kHz) six times for 20 s on and 1 min on ice. Sonicated nuclei were centrifuged at 13000 × g for 4 min at 4 °C. The majority of sonicated chromatin was 500 to 1000 base pairs (bp) in length. For each immunoprecipitation, 15 µg of chromatin was incubated in the presence of 10 µg of HP1 9A9 monoclonal antibody (3 h at 4 °C in a rotating wheel). Then, 50 µl of dynabeads protein G (Invitrogen) was added and incubation was continued overnight at 4 °C. The supernatants were discarded and samples were washed twice in Lysis Buffer (each wash 15 min at 4 °C) and twice in TE Buffer (1 mM EDTA, 10 mM TrisHCl at pH 8). Chromatin was eluted from beads in two steps; first in 100 µl of Eluition Buffer 1 (10 mM EDTA, 1% SDS, 50 mM TrisHCl at pH 8) at 65 °C for 15 min, followed by centrifugation and recovery of the supernatant. Beads material was re-extracted in 100 µl of TE + 0.67% SDS. The combined eluate (200 µl) was incubated overnight at 65 °C to reverse cross-links and treated by 50 µg/ml RNaseA for 15 min at 65 °C and by 500 µg/ml Proteinase K (Invitrogen) for 3 h at 65 °C. Samples were phenol-chloroform extracted and ethanol precipitated. DNA was resuspended in 25 µl of water. For maximising the molecular analyses with DNA immunoprecipitated, candidate genes were amplified in pairs through an optimized duplex-PCR protocol by using two different sets of primers having similar melting temperatures in a single reaction.
RNAse-Chromatin immunoprecipitation was performed essentially as described for ChIP but with an important modification: sheared chromatin was treated with RNAse mix (Roche) for 1 h at 37 °C before immunoprecipitation.
Primers design and PCR amplification. All PCR specific primers (18-25 mers with a minimum GC content of 50% and average Tm of 60 °C) (listed in Supplementary Table S1) were designed using the Invitrogen OligoPerfect ™ designer web tool and oligonucleotide sequences were screened using a BLAST search to confirm the specificity. PCR amplifications were performed with Platinum ® Taq DNA Polymerase Kit (Invitrogen) according to the manufacturer's instructions.
The thermal profile for PCR amplification of CLIP samples was as follows: initial denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, and ending with a final extension at 72 °C for 7 min.
The thermal profile for duplex-PCR amplification of ChIP samples was as follows: initial denaturation at 94 °C for 5 min, followed by 28 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, and ending with a final extension at 72 °C for 7 min. The PCR products were analyzed by 2% agarose gel electrophoresis.
Total RNA extraction and qRT-PCR. RNA samples from ovaries were isolated by Qiazol reagent (Qiagen) according to the manufacturer's instructions. The concentration and purity of RNAs were determined using NanoDrop 1000 Spectrophotometer (Thermo Scientific). 5 µg of total RNA was reverse transcribed using oligo dT and SuperScript Reverse Transcriptase III (Invitrogen) according to the manufacturer's protocol. The qPCR reactions were carried out with QuantiFast SYBR Green PCR Kit (Qiagen) according to manufacturer's protocol. Relative abundance of the different transcripts was determined using the 2 −ΔΔCt method 86 using rp49 transcript as control. qRT-PCR experiments were performed in three independent biological replicates; all reactions were run in triplicates in 96-well plates over 40 cycles of 95 °C for 15 s and 60 °C for 60 s in a two-step thermal cycle preceded by an initiation step of 95 °C for 10 min. Melting-curve analysis was performed on each sample to control for nonspecific amplification and primer dimer formation. Primer sequences were listed in Supplementary  Table S1. Statistical significance was determined by Mann-Whitney tests using GraphPad Prism Software. A p value ≤ 0.05 was considered statistically significant.