Chromatin structural changes around satellite repeats on the female sex chromosome in Schistosoma mansoni and their possible role in sex chromosome emergence
© Lepesant et al.; licensee BioMed Central Ltd. 2012
Received: 23 November 2011
Accepted: 29 February 2012
Published: 29 February 2012
In the leuphotrochozoan parasitic platyhelminth Schistosoma mansoni, male individuals are homogametic (ZZ) whereas females are heterogametic (ZW). To elucidate the mechanisms that led to the emergence of sex chromosomes, we compared the genomic sequence and the chromatin structure of male and female individuals. As for many eukaryotes, the lower estimate for the repeat content is 40%, with an unknown proportion of domesticated repeats. We used massive sequencing to de novo assemble all repeats, and identify unambiguously Z-specific, W-specific and pseudoautosomal regions of the S. mansoni sex chromosomes.
We show that 70 to 90% of S. mansoni W and Z are pseudoautosomal. No female-specific gene could be identified. Instead, the W-specific region is composed almost entirely of 36 satellite repeat families, of which 33 were previously unknown. Transcription and chromatin status of female-specific repeats are stage-specific: for those repeats that are transcribed, transcription is restricted to the larval stages lacking sexual dimorphism. In contrast, in the sexually dimorphic adult stage of the life cycle, no transcription occurs. In addition, the euchromatic character of histone modifications around the W-specific repeats decreases during the life cycle. Recombination repression occurs in this region even if homologous sequences are present on both the Z and W chromosomes.
Our study provides for the first time evidence for the hypothesis that, at least in organisms with a ZW type of sex chromosomes, repeat-induced chromatin structure changes could indeed be the initial event in sex chromosome emergence.
The origin and evolution of sexuality is one of the most fascinating topics in evolutionary biology. Sex can be determined by several mechanisms, such as environmental stimuli (environmental sex determination) or genetic differences between males and females (genetic sex determination). Genetic sex determination is mainly based on the acquisition of sex chromosomes, a more stable strategy than environmental determinism, especially when the environment becomes variable. The principle steps leading to the emergence and evolution of sex chromosomes have been proposed by Charlesworth et al.  and Rice . In this model, the emergence of a locus with female fertility and male sterility and another locus with male fertility and female sterility led to the establishment of a small sex-determining region on ordinary autosomes in hermaphrodite ancestors. These so-called proto-sex chromosomes are hardly distinguishable. To prevent the production of infertile individuals, recombination of these loci becomes restricted [3, 4]. This crucial step is intensively debated and two mechanisms of action have been proposed: (i) structural changes by translocation or inversion (reviewed in ); or (ii) chromatin status changes involving heterochromatization of the heterosexual chromosome [4, 6–9]. Heterochromatization of the sex-determining region has been shown in species with primitive or nascent sex chromosomes, such as in papaya or tilapia (reviewed in ). The suppression of recombination between the heterochromosome and its homologue would trigger gradual degradation of the heterochromosome (Y in XY systems, or W in WZ systems) because genes that are not essential for males (in XY systems) or females (in WZ systems) show accelerated rates of mutation and deletion. Consequently, the heterochromosome becomes progressively gene-poor (for example, ) and in the extreme case the degradation process can lead to the complete loss of the heterochromosome (for example, ).
We decided to investigate the role of chromatin structural changes in sex chromosome emergence by using a basal metazoan species harboring a ZW system, the acoelomate Schistosoma mansoni. Schistosomes are parasitic plathyhelminthes that are responsible for schistosomiasis (bilharziosis), an important parasitic human disease ranking second only to malaria in terms of parasite-induced human morbidity and mortality . S. mansoni's life cycle is characterized by passage through two obligatory hosts: the fresh-water snail Biomphalaria glabrata (or other Biomphalaria species, dependent on the geographical location), for the asexual stage; and human or rodents for the sexual adult stage. The sex of the parasite is determined in the eggs (syngamic determination). Eggs are excreted with the host feces and free-swimming larvae (miracidia) are released when the eggs come into contact with water. These miracidiae infect the freshwater mollusk host and transform into primary and secondary sporocysts. Finally, a third larval stage, the cercariae, capable of infecting the vertebrate host, is released into the water. Once in the human or rodent host, morphological differences between female and male adults develop, and these then mate and produce eggs. In the larval stages, schistosome males and females are genetically different but morphologically identical; the sexual dimorphism (that is, the phenotypic expression of sex differentiation) is restricted to the adult stage. All stages are experimentally accessible, which allows the study of chromatin structural modifications for all stages of the life cycle.
Analysis of metaphase spreads indicates that sex is determined in schistosomes by sex chromosomes, with female being the heterogametic sex (ZW) and male the homogametic sex (ZZ) . In some schistosoma species, there is a clear size difference between W and Z, while in other species, such as S. mansoni, discrimination is solely based on chromatin structure . This makes S. mansoni a model of choice to study the involvement of chromatin structural changes in sex determination of a model harboring a ZW system. In addition, and in contrast with most other plathyhelminth species, schistosomes are gonochoric . This suggests that, in general, being hermaphrodite is an advantage in this phylum, probably through minimizing the risk that is associated with finding a mate inside the host . In Schistosomatidae, the acquisition of separated sexes was concomitant with the invasion of warm-blooded animals . This could be explained by the benefit that genetic diversity provides against the sophisticated immune system of warm-blooded vertebrate hosts and/or by the specialization of each gender for a limited set of 'domestic tasks' [16, 18, 19]. This particular feature of schistosomes in the plathyhelminth phylum provides the opportunity to study sex chromosome emergence.
The genome of S. mansoni was sequenced and initially only partially assembled (version 3.1 with 19,022 scaffolds) . During the preparation of this manuscript, an improved version with assembly at the chromosome level became available (version 5.2 with 882 scaffolds) , and Criscione et al.  constructed a linkage map for 210 version 3.1 scaffolds using microsatellite markers. They identified eight linkage groups corresponding to the seven autosomes and one sex chromosome , indicating that the sex chromosomes recombine. Nevertheless, Criscione et al. discovered a small region of roughly 18 Mb on the sex chromosome that shows recombination repression. Several open questions remain to be answered. First, it is not clear what are the genetic differences between W and Z chromosomes of S. mansoni, or in other words, what are the W- and what are the Z-specific sequences. Second, the mechanism of recombination repression between S. mansoni sex chromosomes is not clear. As outlined above, either inversion events or heterochromatization [7, 9, 23] have been proposed for other species. The specific objectives of the present study were to determine what the sex-specific DNA sequences of S. mansoni are, and how heterochromatization of the W chromosome might be initiated. We present here evidence that S. mansoni sex chromosomes contain large pseudoautosomal regions. Outside these regions, Z-specific sequences are composed of unique sequences and interspersed repeats. W-specific sequences are almost entirely composed of satellite-type repeats located in the heterochromatic region of the W chromosome. While no female-specific gene could be identified, many of the female repeats are transcribed in the larval stages of the parasite but never in the adults. This loss of transcriptional activity and the development into adults is accompanied by chromatin structural changes around the W-specific repeats. We develop a model in which female-specific repeats are expressed to induce a change in chromatin structure of the W chromosome specifically in the sexual part of the life cycle, leading to functional heterogametism.
The S. mansoni sex chromosomes Z and W share large pseudoautosomal regions
Comparison of the ratio of relative amounts of genomic DNA in male and female adults of S. mansoni
Male (ZZ)/female (WZ)
NGS hit-count ratio
2.00 ± 0.15
1.76 ± 0.11 (region 1)
1.83 ± 0.11 (region 2)
1.61 ± 0.03
0.87 ± 0.04 (region 1)
0.87 ± 0.12 (region 2)
0.87 ± 0.06 (region 1)
0.99 ± 0.13 (region 2)
1.76 ± 0.07
1.10 ± 0.08
1.04 ± 0.09
1.07 ± 0.13
0.99 ± 0.21
0.95 ± 0.09
0.93 ± 0.03
0.93 ± 0.02
The Z-specific region of the Z chromosome is composed of unique sequences and interspersed repeats
The region that is covered by the 15 Z-specific scaffolds contains 205 putative genes (according to the gene predictions in SchistoDB). For 118 genes, a function could be predicted based on sequence similarities (Additional file 1). Among those there are at least four genes that code for proteins that are predicted to be involved in spermatogenesis or for which paralogous genes show testis-specific expression. Nevertheless, for the moment it cannot be concluded that these genes are involved in sex differentiation and further analysis is necessary to clarify the role of these genes. Interspersed repeats were also observed in this genomic region but none of them are Z-specific. The Z-specific region in assembly 3.1 is 6.5 Mb in size. In assembly version 5.2 it spans about 18 Mb and, according to , contains 782 genes.
A region on the sex chromosomes with repressed recombination contains Z-specific sequences but also pseudoautosomal sequences
The female-specific region of the W chromosome is composed of repetitive sequences
W-chromosome-specific repeats of S. mansoni
GenBank accession number
Percentage female hits
Copy number estimateb
60,000 - 70,000
Middle of q-arm at frontier between heterochromatin and euchromatin as satellite, middle of q-arm
LTR, highly similar to W2, highly similar to R = 407
No transcription (RT-PCR)
As satellite in the middle of q-arm at frontier between heterochromatin and euchromatin or also in the pericentromeric region
Highly similar to R = 879
Same location as W1
LTR, similar to Perere-2, identical to R = 564
EST and RT-PCR
Either at the frontier of heterochromatin and euchromatin of the q-arm or in the pericentromeric region, or at both locations
In the pericentromeric region
DNA transposon, 97% identical to GenBank accession number XP_002570219 (hypothetical protein Smp_186230)
In the pericentromeric region
Tandem repeat (previously described as TR266), DNA transposon
Either at the frontier of heterochromatin and euchromatin of the q-arm or in the pericentromeric region
LINE2, similar to SjR2 retrotransposon
LINE, similar to R = 170
Retro, 97 to 100% identical to several hypothetical S.m. proteins
In the middle of the heterochromatic part of the q-arm as satellite
DNA transposon, similar to R = 170
Similar to R = 116
Retro, 100% identical to GenBank accession number XP_002569391 (hypothetical protein Smp_181820)
DNA transposon, similar to R = 116
Retro, similar to Sh122 repeat and R = 31
DNA transposon, similar to R = 133 and Sh microsatellite C2
Similar to Sh microsatellite C140
Similar to Sb Sbov20 repeat
We used SchistoDB to identify genes that could be located within the region that is spanned by the repeats. Eight putative genes were identified in the vicinity of the repeats (not more than 5 kb away). Manual inspection of all loci showed that female next generation sequencing hits can be found for four putative genes, and male hits are absent (Smp_186230, Smp_190410, Smp_117150, Smp_117160). However, three genes (Smp_190410, Smp_117150, and Smp_117160) are identical and the predicted coding regions are small (243 bp for Smp_190410, 327 bp for Smp_186230). No significant similarity to known proteins could be found with blastx. Blast against the genome shows that these putative genes are not unique and it remains to be answered whether these sequences are actually transcribed and code proteins.
Female-specific repeats are arranged as large satellite type blocks in the heterochromatic region of chromosome W
Several of the female-specific repeats are transcribed in larvae but not in adults
The chromatin structure around the female-specific repeats changes during the life cycle
Histone deacetylase inhibition does not induce transcription of W-specific repeats in adults
We tested whether the observed changes in chromatin structure are a result or the cause of the changes in transcription. If hypoacetylation of histones were the cause of transcriptional inactivation, then inactivation of histone deacetylase would relieve repression. On the other hand, if transcription of repeats is the origin of chromatin structural changes, inhibition treatment should not lead to detectable changes in transcription because each transcriptional increase would reinforce deacetylation and counteract the inhibition. We treated adult parasites with trichostatin A (TSA), an inhibitor of histone deacetylases at increasing concentrations in vitro. After 2 hours of treatment with ≥20 µM TSA, mobility changes were observed (worms first straightened up and ceased moving, and convulsive movements were observed at higher concentrations and longer incubation times (Additional file 4)). We then measured the transcription levels for repeats W4, W5 and Sm-alpha-female at 20 µM TSA and for 4 hours. In none of the cases was transcription activated. In contrast, an increase of transcription of retrotransposons Perere3 and Saci7, used as control, was observed (by 45 and 23%, respectively). The lactate dehydrogenase test shows no difference in cytotoxicity between TSA-treated and mock-treated worms.
Despite tremendous advancements in the past, the elements that are responsible for the establishment of sex chromosomes remain still enigmatic. According to Müller's ratchet model, sexual reproduction evolved because deleterious mutations could be eliminated by recombination between the parental autosomes . To maintain isolation of two different sexes, recombination must, however, be repressed (at least partially) between the sex chromosomes. Zones in which recombination is repressed between sex chromosomes were meanwhile identified in many species. Accumulation of repeats on the heterogametic sex chromosome was also found in many examples, although their role is unknown and many authors still consider them as junk DNA. The view that repetitive DNA is non-functional was challenged by the discovery of transcription from repeats on autosomes and the production of small RNA that could be related to heterochromatization events . The presence of large heterochromatic blocks is also a common feature of sex chromosomes. So far, these observations were made in isolation from each other, and generally in different species, which makes the construction of a hypothetical model difficult. Here we present for the first time a comprehensive analysis of sequence composition, gene and repeat content, chromatin structure and repeat transcription of the sex-specific chromosome regions of the Z and W chromosomes of our biological model S. mansoni. Recombination repression has been described before in this region of interest . Our data, in relation to previous reports, allows the current models for the suite of events that led to sex chromosome differentiation in S. mansoni to be refined and could represent a general model for this process in species with genetic sex determination of the Z/W type.
Z- and W-specific sequences
Criscione et al.  identified a region of 20 scaffolds in which recombination repression was observed and suggested that these are Z-specific sequences. We indeed found a male/female sequence reads hit and/or qPCR ratio of ≥1.5 for 13 of these scaffolds, indicating an overrepresentation in the male genome. However, seven scaffolds in this region showed no disequilibrium of hit counts and/or qPCR between males and females (male/female hit ratio ≤1.4), that is, the sequences are not specific to the Z chromosome (Figure 1). In other words, recombination is repressed but the homologous sequences on the sister chromosomes are still present. We find at least two blocks of sequences that are shared between the Z and W chromosome located in the large region with recombination repression. This result was confirmed with the most recent version of the genome assembly. We see three possible conclusions that can be drawn from our results. Either the Z/W sequence blocks are inverted, and additionally or alternatively the sequences are heterochromatic, thus preventing recombination. It is also possible that the scaffolds in the original assembly of the S. mansoni genome were chimeric. Indeed, of the 48 scaffolds originally found in linkage group Z/W , 4 are on other chromosomes in the 5.2 assembly. It will be difficult to formally exclude the possibility that our results are due to misassembly.
We did not find any paralogues to sex determination genes among the predicted genes on the Z-specific scaffolds. The specific region of the W chromosome is largely composed of large satellite blocks of at least 36 different W-specific repeats. These repeats are abundant on the W chromosome but our PCR analysis on different male individuals indicates that these sequences can also sometimes be found on other chromosomes. The strength of the PCR signal suggests, however, that they are present in very low copy number there. Analysis of the genomic sequence shows that they can occur intermingled with other repeats on autosomal scaffolds as individual sequences or as small blocks of up to five repeats in tandem. Our understanding of these results is that these repeats exist as large satellite blocks on the W chromosome but can occasionally be transferred to autosomes by a so far unknown mechanism. Such a behavior was described for W1  and could depend on the chromatin structure around the repeats and/or flanking regions. Several of these W-specific repeats are transcribed in the miracidia and cercariae stages but never in the adults.
Role of W-specific repeats
One could argue that the function of repeat-induced silencing is purely defensive and down-regulates retrotransposon expression in general. Such a mechanism was described as the repeat-associated small interfering RNA (rasiRNA)-mediated pathway  in Drosophila ovary cells and is believed to protect the (female) germ line from transposable elements. If this were the case for S. mansoni, transcription should be observed in the ovary. Our data do not support this view.
Most authors agree that suppression of recombination is an initial event in sex chromosome emergence, although it is not clear by what mechanism it is caused. Chromosome rearrangements (for example, inversions) or the action of modifier genes have been proposed (reviewed, for example, in ). Other authors see conformation differences (chromatin structural changes, differences in replication timing) as the origin for recombination inhibition [3, 5]. Accumulation of repeats is a general feature of Y/W-type chromosomes. Some consider it an important feature with unknown function , while others see repeat accumulation as the result of recombination suppression  or solely as a genome defense mechanism , placing it late in the suite of events that characterize evolution of sex chromosomes.
With the present work we contribute two new elements that allow us to exclude some of the current hypotheses and to refine others. First, we show that the presence of satellite repeats on the W chromosome does not lead in all life cycle stages to heterochromatization. Consequently, it is not their presence itself that induces the heterochromatin formation. We show that all W-specific repeats are euchromatic in the miracida stage. Our ChIP-Seq data tell us that this is not a general feature of autosomal and pseudoautosomal repeats, but specific for the W-specific satellites. Second, we demonstrate that the euchromatization occurs concomitantly with transcription and that transcription always precedes heterochromatization.
Based on these findings, we propose two not necessarily exclusive scenarios for the emergence of sex chromosomes. In the first model, transcription of non-coding RNA from repetitive DNA elements was the initial event in sex chromosome evolution of schistosomes. Non-coding RNA would have induced heterochromatization and suppression of recombination. Both favored expansion of repeats and organization in large blocks (satellites). Satellite expansion would have reinforced the system and led finally to the beginning of genetic changes in the W chromosome. The very basal phylogenetic position of leuphotrochozoans such as S. mansoni permits a general model for the main stages of sex chromosome evolution to be proposed: the establishment of a sex-determining region, recruitment of repeats for production of non-coding RNA, RNA-directed heterochromatization and repeat expansion, local suppression of recombination, and shrinkage of the chromosome by deletion.
In the second model, a small mutation and/or local heterochromatization could have been the initial event, leading to recombination repression in the first place. Repetitive DNA accumulated subsequently. During germ cell formation or during early embryogenesis euchromatization occurs. Cytogenetic evidence in other species in which the female is the heterogametic sex shows that the W chromosome is often condensed in somatic cells, and becomes euchromatic in early oocytes (reviewed in ). This releases transcription repression and repeats are transcribed, leading subsequently to heterochromatization. Our preliminary data suggest that chromatin structural changes do not occur in trans - that is, not on the Z chromosome but on the adjacent regions of the W chromosome (not shown).
We cannot formally exclude that sex determination is based on a specific protein-coding gene that is absent or present on the W chromosome. But we show that the most pronounced difference in transcription between ZZ and ZW individuals is at the level of 'non-coding' RNA. We therefore favor the hypothesis that sex differentiation in S. mansoni is based on developmental stage-dependent tagging of the W chromosome by non-coding RNA and a chromatin marking system. Our model predicts that chromatin structural changes influence transcription of one or several genes in the close vicinity of the core heterochromatic region and that transcriptional activation or inactivation of these leads to morphological and/or physiological changes that are the bases for development of the male and female phenotypes in the adult stage.
Materials and methods
Parasite culture and drug treatment
Eggs were axenically recovered from 60-day infected hamster livers and miracidia were hatched from eggs in 5 ml of spring water over 2 to 3 hours under light. Miracidia were concentrated by sedimentation on ice for 15 minutes. Cercariae were recovered from infected snails (4 weeks post-infection) and collected by pipetting. They were then concentrated by cold centrifugation (4°C) at 1,200 rpm for 5 minutes and the supernatant was removed. Eight-week-old adult worms were recovered by portal perfusion of hamsters with 0.8% (w/v) NaCl and 0.8% (w/v) trisodium citrate . If necessary, miracidia, cercariae and adults were kept at -80°C.
For infection with a single sex, B. glabrata snails 4 to 5 mm in diameter were individually exposed to a single miracidium in 5 ml of springwater. The snails were then each isolated and maintained in round, clear plastic containers for 24 hours and kept all together for 5 weeks. Snails were fed fresh lettuce ad libitum and the water was maintained at 25°C and changed weekly. The photoperiod during the entire experiment was equilibrated to 12 hours light:12 hours dark .
Adults were recovered by portal perfusion of hamsters. Ten individuals were kept in 250 µl RPMI medium (Invitrogen-Gibco, Carlsbad, USA) and treated with an ethanol solution of the histone deacetylase inhibitor TSA (Invitrogen) at different final concentrations (2 µM, 20 µM, 50 µM, 100 µM and 200 µM). To the untreated control, a corresponding volume of ethanol was added. The cytotoxic effect of the drug was measured using the Roche Cytotoxicity Detection Kit (Roche no. 04744926001), which is based on the measurement of lactate dehydrogenase activity released from dead and lysed cells into the supernatant . Behavior was observed every hour until 6.5 hours and after 21 hours of treatment. Individuals were filmed with a conventional numerical camera adapted to a stereomicroscope after 5, 6.5 and 21 hours of treatment.
Sequencing of genomic DNA, alignment, and assembly of repeats
Solexa sequencing was performed at the sequencing facilities of GenomiX Montpellier (France) on a Genome Analyzer II (Illumina) by single end sequencing (36 bp) according to the manufacturer's protocol. The software SOAP is usually employed to map unique sequences and reject repetitive sequences. We took advantage of this algorithm and used SOAP 2.17 , evoking the -u and -r 0 options to split the sequence reads into those corresponding to unique or repetitive sequences. The resulting fasta files of unmapped reads (-u) was assembled with velvet using a coverage cutoff of 4 and a minimum contig length of 80 bp. For a second assembly round Sequencher v4.5 was used with minimum match 93%, minimum overlap 60 bp.
In silico analysis
Velvet-assembled repeats were then used for the whole-genome in silico subtractive hybridization (WISH) procedure . This method compares different massive sequencing datasets with a reference genome and identifies sequences that are under-represented in one data set. Censor , Teclass  and blast  were used for repeat annotation.
For identification of genes in the vicinity of W-specific repeats, all repeat sequences were compared to the genome using blast searches of the SchistoDB database  and genes 5 kb upstream and downstream of regions containing these repeats were manually analyzed.
Confirmation of sex-specific sequences by PCR
PCRs were carried out in a final volume of 25 µl containing 0.2 µmol of each oligonucleotide primer (Additional file 5), 0.2 mmol of each dNTP (Promega), 0.625 U of GoTaq polymerase (Promega) used with the recommended buffer and completed to the final volume with DNase-free water. The PCR program consisted of an initial denaturation phase at 95°C for 5 minutes followed by 20 cycles at 95°C for 30 s, 60°C for 90 s, 72°C for 30 s and a final extension at 72°C for 5 minutes. The PCR products were separated by electrophoresis through a 2% TBE agarose gel.
FISH on S. mansoni metaphases
Metaphase spreads were prepared essentially as described by Hirai and LoVerde . Sporocysts were obtained by dissection of two to three snails, each infected with five miracidia, at 28 to 29 days post-infection. Probes for repetitive DNA were prepared by cloning PCR products (for primers see Additional file 5) on genomic DNA as template into pCR2.1-TOPO (Invitrogen #K4510-20). Clones were sequenced to confirm the repeat assembly, labeled with the BioPrime DNA labeling system (Invitrogen #18094-011) and hybridized as described before . Chromosomes were counterstained with propidium iodide and observed under an epifluorescence microscope (AKIOSKOP 2, Zeiss) equipped with a Leica DC 300 FX digital camera. Between 7 and 34 female metaphases were studied for each repeat.
RNA extraction, cDNA synthesis and qPCR
Total RNA was purified from three independent preparations of larvae and adults. For the larval stages, RNA was extracted from 10,000 miracidia and 10,000 cercariae using 500 µl Trizol (Invitrogen). Fifty adult couples were solubilized in 500 μl Trizol with a MagNA Lyser and Green beads (Roche). RNA was treated with DNase I (Invitrogen) for 15 minutes at 37°C, followed by inhibition of the enzyme for 10 minutes at 65°C. PCR of 28s rDNA was used to test for genomic DNA contaminations. The DNase I treatment was repeated as many times as necessary to eliminate contaminations with genomic DNA. RNA was purified with the QIAGEN RNeasy kit. First strand cDNA was synthesized using 10 μl of the total RNA preparation, in a final volume of 20 μl (10 mM dNTPs, 0.1 M DTT, 40 U RNase out, 0.15 μM random primers) with 200 U of SuperScript II RT (Invitrogen). After reverse transcription, the cDNAs were purified with the PCR clean-up system (Promega) and eluted into 40 μl 10 mM Tris/Cl (ph 7.5). Real-time PCR analyses were performed using the LightCycler 2.0 system (Roche Applied Science) and LightCycler Fast-start DNA Master SYBR Green I kit (Roche Applied Science).
qPCR amplification was done with 2.5 μl of cDNA in a final volume of 10 μl (3 mM MgCl2, 0.5 μM of each primer, 1 μl of master mix). Primers were designed with the LightCycler Probe design software or the primer3plus web based interface . The following protocol was used: denaturation, 95°C 10 minutes; amplification and quantification (40 cycles), 95°C for 10 s, 60°C for 5 s, 72°C for 16 s; melting curve, 60 to 95°C with a heating rate of 0.1 C/s and continuous fluorescence measurement, and a cooling step to 40°C. For each reaction, the crossing point (Ct) was determined using the 'fit point method' of the LightCycler Software 3.3. PCR reactions were done in duplicates and the mean value of Ct was calculated. 28s rRNA was used as an internal control and the amplification of a unique band was verified by electrophoresis through 2% TBE agarose gels for each qPCR product. Primer sequences and expected PCR product size are listed in Additional file 5. For all qPCR, efficiency was at least 1.89.
Chromatin status analysis by ChIP and qPCR
Antibodies used for native ChIP (N-ChIP)
Saturating quantity used for N-ChIPa
chromatin immunoprecipitation followed by quantitative PCR
chromatin immunoprecipitation followed by massively parallel sequencing
expressed sequence tag
fluorescence in situ hybridization
histone H3 tri-methylated on lysine 27
histone H3 tri-methylated on lysine 4
histone H3 lysine 9
histone H3 acetylated on lysine 9
histone H3 tri-methylated on lysine 9
- NCBI SRA:
Sequence Read Archive at the National Center for Biotechnology Information
polymerase chain reaction
The authors are grateful to the Plant Genome and Development Laboratory (UMR5096) of the University of Perpignan for access to their fluorescence microscope. Anne Rognon, Bernard Dejean and Kristina Smith provided important support. The work received financial support from the CNRS (PostDoc fellowship to CC) and the programs 'Schistophepigen' and 'Monogamix' from the French National Agency for Research (ANR).
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