Hydra Meiosis Reveals Unexpected Conservation of Structural Synaptonemal Complex Proteins Across Metazoans

Hydra meiosis reveals unexpected conservation of structural synaptonemal complex proteins across metazoans

Johanna Fraunea,1, Manfred Alsheimera,1,2, Jean-Nicolas Volffb, Karoline Buscha, Sebastian Fraunec, Thomas C. G. Boschc, and Ricardo Benaventea,2

aDepartment of Cell and Developmental Biology, Biocenter, University of Würzburg, D-97074 Würzburg, Germany; bInstitut de Génomique Fonctionnelle de Lyon, Université de Lyon, Ecole Normale Supérieure de Lyon, CNRS, Université Lyon 1, 69364 Lyon Cedex 07, France; and cInstitute of Zoology, Christian- Albrechts-University of Kiel, D-24098 Kiel, Germany

Edited by Joseph G. Gall, Carnegie Institution of Washington, Baltimore, MD, and approved August 27, 2012 (received for review April 24, 2012)

The synaptonemal complex (SC) is a key structure of meiosis, medi- joined by several crosswise arranged transverse filaments (TFs). ating the stable pairing (synapsis) of homologous chromosomes Chromatin of homologous chromosomes is associated with one during prophase I. Its remarkable tripartite structure is evolutionarily LE each. The central region of the SC where the TFs of opposing well conserved and can be found in almost all sexually reproducing LEs overlap is referred to as central element (CE). organisms. However, comparison of the different SC protein compo- In mammals, protein SYCP1 is the main constituent of TFs (9) nents in the common meiosis model organisms Saccharomyces cer- whereas LEs are largely composed of the proteins SYCP2 and evisiae, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila SYCP3 (10, 11). In addition to the N-terminal head domain of SYCP1 molecules, the CE is composed of the CE-specific pro- melanogaster, and Mus musculus revealed no sequence homology. teins SYCE1, SYCE2, SYCE3, and Tex12 [(12–14); for review This discrepancy challenged the hypothesis that the SC arose only see (15)]. SYCP1 and SYCP3 are particularly interesting as they once in evolution. To pursue this matter we focused on the evolution are the only bona fide structural SC proteins so far characterized of SYCP1 and SYCP3, the two major structural SC proteins of mam- in mammals (16). SYCP3 can self-assemble in the absence of mals. Remarkably, our comparative bioinformatic and expression other SC proteins, thereby forming thin fibrils that can associate studies revealed that SYCP1 and SYCP3 are also components of the laterally to generate higher-order structures resembling LEs (16, SC in the basal metazoan Hydra. In contrast to previous assumptions, 17). SYCP1 forms so-called polycomplexes on its own that rep- we therefore conclude that SYCP1 and SYCP3 form monophyletic resent stacks of normally appearing SC central regions (17). groups of orthologous proteins across metazoans. As the fine structure of the SC is well conserved in evolution, it is reasonable to assume that this would be also the case for the ost eukaryotic organisms reproduce sexually, a process that structural protein components. However, according to the litera- Minvolves the fusion of gametes of the two sexes to form ture, this is apparently not the case. Comparison of LE or TF a new organism. A prerequisite is that the chromosome com- protein components in the most commonly used meiosis model organisms—S. cerevisiae, Arabidopsis thaliana, C. elegans, D. mel- plement becomes reduced from diploid to haploid during germ — cell differentiation. This is achieved during a special type of cell anogaster, and Mus musculus revealed very little sequence simi- division, the meiosis. The original diploid state is then recon- larity between these proteins. stituted during fertilization. The emerging picture is rather puzzling. On the one hand, the The reduction of the chromosome number is achieved by the SC shows a conserved ladderlike organization from yeast to human and plays a key role in meiotic recombination. On the other hand, succession of two rounds of chromosome segregation after a sin- ’ gle round of DNA replication. During the first and most crucial SC s protein composition appears to be not conserved, which round, homologous chromosomes need to pair and go through suggests that the SC might have evolved independently in different cross-over recombination to ensure their correct segregation into taxonomic groups (5, 6, 18). To address this apparent discrepancy two new daughter cells. The association of sister chromatids is we performed comparative bioinformatic and expression studies maintained and will not be abrogated until the onset of the second focused on SC structural proteins in the basal metazoan Hydra.We round of cell division, which resembles a mitotic one. provide clear evidence that, in contrast to previous assumptions, As the essential features of meiosis, i.e., pairing and cross-over SYCP1 and SYCP3 form monophyletic groups of orthologous SC recombination of homologs, can be found in a wide range of dif- proteins across metazoans down to organisms as basal as Hydra. ferent taxonomic groups, it is assumed that meiosis evolved only Results and Discussion once early in eukaryotic life (1). The meiotic recombination ma- chinery, which is responsible for the synthesis of crossing over, SYCP1 and SYCP3 Orthologs in a Basal Metazoan. Mammalian fi α shows strong evolutionary conservation and can be found in the SYCP1 is a long brillar molecule composed of a central -he- meiotic model organisms from the yeast Saccharomyces cerevisiae lical domain, most likely involved in the formation of coiled coil fl across Caenorhabditis elegans and Drosophila melanogaster to structures, and the anking globular N and C termini (9, 17). mammals with only little variation (2, 3). The mechanism of homology search is still not well understood, but the assembly of a proteinaceous structure known as synaptonemal complex (SC), Author contributions: M.A. and R.B. designed research; J.F., M.A., J.-N.V., K.B., and S.F. which mediates synapsis of homologs, seems likely to be an im- performed research; J.F., M.A., J.-N.V., S.F., T.C.G.B., and R.B. analyzed data; and J.F., M.A., portant facilitator or stabilizer of homologous pairing (4, 5). Syn- and R.B. wrote the paper. aptic defects consequently impair meiosis and lead to failure of The authors declare no conflict of interest. cross-over recombination and chromosome segregation (chro- This article is a PNAS direct submission. mosome nondisjunction) in diverse organisms [for reviews see (6, Data deposition: The sequences reported in this paper have been deposited in the Gen- 7)] that can cause aneuploidy as well as infertility (5, 8). Therefore, Bank database (accession nos. JQ906932–JQ906936). the functions of the SC, which is implemented by its remarkable 1J.F. and M.A. contributed equally to this work. tripartite structure, seem to be evolutionarily conserved (5). 2To whom correspondence may be addressed. E-mail: [email protected] Electron microscopic data revealed that the fine structure of burg.de or [email protected]. the SC is also well conserved in evolution. It is similar in shape to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. a ladder in which two parallel rodlike lateral elements (LEs) are 1073/pnas.1206875109/-/DCSupplemental.

16588–16593 | PNAS | October 9, 2012 | vol. 109 | no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1206875109 Downloaded by guest on October 1, 2021 Recently, Iwai and colleagues (19) reported the presence of an SYCP1 ortholog also in a nonmammalian vertebrate, the medaka fish. Comparing the sequence of mammalian (rat) and fish (medaka) SYCP1 proteins we identified, as in the case of SYCP3 (20), conserved features within taxonomically distant vertebrate species (Fig. S1). Sequence analysis of medaka SYCP1 (Gen- Bank accession no. JQ906936) revealed that the protein has the same general organization as the rat ortholog, with a sequence identity of about 20% (sequence similarity of about 45%) in the nonhelical terminal domains and the α-helix. It is notable that we identified two previously unnoticed domains that display a much higher homology [conserved motif 1 (CM1) and 2 (CM2)]. CM1 is 83 amino acids long and comprises part of the nonhelical N terminus and the head of the coiled coil (amino acids 106–188 in the rat). It is this domain that shows one of the highest conser- Fig. 1. Schematic comparison of SYCP1 proteins of rat (GenBank accession vation among vertebrates (65% identity and 80% similarity be- no. NM_012810) and Hydra (GenBank accession no. JQ906934). Sequence tween rat and medaka; Fig. S1). CM2 consists of 31 amino acids identity and sequence similarity (in parentheses) at the amino acid level are and is located in the last third of the protein (amino acids 724– given (%). The gray boxes represent putative coiled coil domains. Position of conserved motifs CM1 (amino acids 106–188 in the rat; amino acids 56–138 in 754 in the rat). It reveals a sequence identity of 74% (97% se- – – quence similarity) between rat and medaka (Fig. S1). Hydra) and CM2 (amino acids 724 754 in the rat; amino acids 674 704 in Hydra) is denoted by red boxes. NLS, nuclear localization signal. We next asked whether SYCP1 is actually restricted to vertebrate species. To address this issue, we performed extensive TBlastN searches (i.e. searches of a translated nucleotide database more revealed sequence conservation across metazoans (Fig. S3; using a protein query) with the sequence corresponding to the for further sequences see Table S2). highly conserved domain of rat SYCP1 (i.e., CM1). By this means fi we could nd a great number of apparently homologous sequences Hydra across a broad range of metazoans including the cnidarians as one SYCP1 and SYCP3 Are Bona Fide SC Proteins. The obtained of the earliest phylogenetic stages. The fact that TBlastN search results of a given similarity of the proteins in sequence and returned significant hits even to cnidarian Hydra magnipapillata structure provided compelling evidence that the Hydra genome EST and genomic sequences was rather astounding because it was contains genes that appear to be the orthologs of mammalian previously suggested that SC proteins are not well conserved over SYCP1 and SYCP3. However, it remained to be proven whether metazoans and might have evolved independently in distant taxa they also exhibit a similar functionality by being expressed in the (5, 6, 18). Therefore, we intended to test whether these sequences same cellular context and playing a similar role in Hydra meiosis. actually relate to a real Hydra SYCP1 ortholog, which would share Therefore, we started a detailed expression analysis of the Hydra a common origin with the mammalian SYCP1 from the same Sycp1 and Sycp3 genes. ancestral gene. A good indicator of orthology of proteins is the To investigate the expression pattern of these genes in Hydra similarity of sequence, structure, and function. Thus, we checked we performed RT-PCR on the H. vulgaris strain AEP. To this end, we isolated mRNA from head, midpiece, and foot. With the for sequence similarity and structural similarity of the SYCP1 fi proteins in the first instance. We cloned and sequenced the re- aid of sequence-speci c primers, we were able to selectively spective full-length cDNA of H. vulgaris (GenBank accession no. amplify Hydra Sycp1 and Sycp3 cDNAs in the midpiece fraction JQ906934) and performed a detailed comparison of the deduced where testes are associated with the body column (Fig. 3 A and Hydra protein, which is 1,016 amino acids long, and the rat SYCP1 B). Using the same assumption that SYCP1 and SYCP3 are protein. It revealed that the general organization of the putative meiotic proteins, we could amplify the message from isolated SYCP1 protein was conserved between cnidarians and mammals testis samples as well. Coanalysis of Hydra actin expression (Fig. 1; see also below). The highly conserved SYCP1 domains we served as internal control for RT-PCR (Fig. 3 A and B). defined above were also contained in the Hydra ortholog (41% In a next step, we applied Western blot analysis to monitor the expression of SYCP1 and SYCP3 proteins in the different identity and 67% similarity of CM1 and 32% identity and 65% fi similarity of CM2 between rat and Hydra). Alignment of CM1 tissue fractions. As expected, speci c protein bands with the sequences of a representative range of organisms further con- calculated molecular mass of Hydra SYCP1 and SYCP3 ap- firmed its sequence conservation during metazoan evolution (Fig. peared in the midpiece and the testis tissue. We could not detect S2; for further sequences see Table S1). We next extended our study to SYCP3, the other major structural SC protein in mammals. In previous studies it has been shown that SYCP3 is composed of a central coiled coil region and the flanking N- and C-terminal domains (10). We have reported that the coiled coil domain and two short highly conserved flanking sequence motifs of the N and C termini (CM1 and CM2) are not only essential but sufficient for SYCP3 polymerization (20, 21). It is interesting to note that the calculated length of the coiled coil domain according to the Lupas algo- rithm may differ among vertebrates. However, the distance between CM1 and CM2 was remarkably constant from fish to mammals, namely 146 amino acids (20). TBlastN searches using BIOLOGY

the described SYCP3 domains involved in polymerization also DEVELOPMENTAL revealed homologous sequences across metazoans including the cnidarians. As in the case of SYCP1, we cloned the full-length cDNA of the putative H. vulgaris SYCP3 (GenBank accession Fig. 2. Schematic comparison of SYCP3 proteins of rat (GenBank accession no. JQ906932). Analysis of the deduced Hydra SYCP3 peptide no. NM_013041) and Hydra (GenBank accession no. JQ906932). Sequence again disclosed an evolutionarily conserved domain structure of identity and sequence similarity (in parentheses) at the amino acid level are the protein, including the constant distance of 146 amino acids given (%). The gray boxes represent putative coiled coil domains. Position of between the ﬁrst and second CM (Fig. 2; see also below). In conserved motifs CM1 (amino acids 86–105 in the rat; amino acids 64–82 in addition, sequence alignment of these domains (CM1, CM2, and Hydra) and CM2 (amino acids 252–257 in the rat; amino acids 230–235 in the 146 amino acids in between) from different species once Hydra) is denoted by red boxes.

Fraune et al. PNAS | October 9, 2012 | vol. 109 | no. 41 | 16589 Downloaded by guest on October 1, 2021 Fig. 3. Identiﬁcation and characterization of Hydra SYCP1 and SYCP3. Expression pattern of SYCP1 (A) and SYCP3 (B) mRNAs as shown by RT-PCR. Expres- sion pattern of SYCP1 (C) and SYCP3 (D)atthe protein level as shown by immunoblotting. Testis- speciﬁc expression of SYCP1 (E and F, arrow) and SYCP3 (G and H, arrow) mRNA as shown by in situ hybridization on whole-mount Hydra.

any protein signal either in the head or in the foot fraction. magnification, SYCP3 localizes to the two parallel running axes Again, visualization of Hydra actin was used as loading control of a bivalent and SYCP1 localizes in between (Fig. 5). (Fig. 3 C and D). In summary, our results provide compelling evidence that Hydra Once we were able to demonstrate the tissue-specificsynthesis SYCP1 and Hydra SYCP3 are bona fide components of the cni- of SYCP1 and SYCP3, we now aimed to narrow down the spatial darian SC. expression within the Hydra testis. Whole-mount in situ hybridization with digoxigenin (DIG)-labeled RNA probes against mRNA of Hydra Sycp1 and Sycp3 showed that both mRNAs are selectively synthesized in the basal located cells of the testis (Fig. 3 E–H). This finding fits well with the general organization of meiotic cells in Hydra testis where primary spermatocytes locate in pe- ripheral chambers, forming a basal layer close to the mesoglea (22). As expected, electron microscopic analysis of Hydra spermatocytes revealed the presence of SCs showing the typical fine structure (Fig. 4A). Hydra SYCP1 and SYCP3 proteins specifically localized to the spermatocytes as shown by immunofluorescence microscopy on cryosections of Hydra testes (Fig. 4B). No signals were detected in spermatogonia or spermatids (Fig. 4B). In pachytene spermatocytes, both antibodies selectively stained typical threadlike structures corresponding to the SCs (Figs. 4 and 5, Insets). As expected, the number of 15 threads in spread pachytene spermatocytes (Fig. 5) correlates with the number of synapsed homologous chromosomes in H. vulgaris (23). From previous studies in mice it is known that the assembly of the SC begins with the formation of the axial elements (AEs) during leptotene by the recruitment of SYCP3 to the chromosome axis. During zygotene, the TFs consisting of SYCP1 start to assemble between the aligned AEs (now called lateral elements) thereby mediating synapsis of the homologous chromosomes. From several initiation sites synapsis finally spreads along the axis in a zipperlike manner until it is completed during pachytene. In diplotene stage, the SC disassembles and SYCP1 dis- appears gradually from the chromosome axis [for a review see (15)]. Consistent with these studies in mammals, in Hydra, zygotene meiocyte chromosome axes are formed by SYCP3; but synapsis is just initiated at several sites as denoted by SYCP1 labeling that apparently colocalizes with SYCP3. In pachytene, all chromosomes are fully synapsed and SYCP3 and SYCP1 Fig. 4. Ultrastructure of a Hydra SC showing the typical components: LE, CE, show the same localization pattern. Finally during diplotene and NE, nuclear envelope (A). Immunolocalization of SYCP1 and SYCP3 on when the homologous chromosomes split, SYCP1 starts to dis- cryosections of Hydra testis (B). Sg, Spermatogonia; Sp, Spermatocytes; St, sociate from the axes at several sites (Fig. 5). As shown at higher Spermatids. Bar, 20 μm.

16590 | www.pnas.org/cgi/doi/10.1073/pnas.1206875109 Fraune et al. Downloaded by guest on October 1, 2021 Fig. 5. Immunolocalization of SYCP1 and SYCP3 on spread meiotic chromosomes as shown by confocal microscopy. During zygotene and pachytene, SYCP1 selectively localizes to the synapsed areas of homologous chromosomes (arrows) and SYCP3 labels the whole length of lateral elements. During diplotene, SYCP1 dissociates from the lateral elements (arrows). Image enlargements of diplotene SCs show the localization of SYCP3 in the two aligned LEs (arrows); whereas, SYCP1 is restricted to the synapsed regions in between. Bar, 10 μm.

Structural Synaptonemal Complex Proteins SYCP1 and SYCP3 Form Evolutionary Conservation of the SC: More than Just the Ladderlike Monophyletic Groups of Orthologous Proteins in Metazoans. Organization. So far it was assumed that only the organization of the According to the results presented above, Hydra SYCP1 and SC, but not the protein components, would be evolutionarily con- SYCP3 are SC proteins representing orthologs to the mammalian served. However, according to our bioinformatic and expression SYCP1 and SYCP3. This notion received support from the cal- analysis we concluded that the major structural SC protein compo- culation of evolutionary trees for metazoan SYCP1 and SYCP3 nents SYCP1 and SYCP3 form monophyletic groups of orthologous (Tables S1 and S2). As shown in Figs. 6 and 7, sequences from proteins across metazoans down to organisms as basal as Hydra.In most multicellular clades (cnidaria, annelida, mollusca, echino- other terms, evolutionary conservation of metazoan SCs would in- dermata, and chordata) cluster together as would be expected for volve more than just the ladderlike organization. SYCP1 and SYCP3 monophyletic groups of proteins. Beyond that, we also found sequences are consistent between quite distant metazoan clades and homologous SYCP1 and SYCP3 sequences in the early branching share a common origin more than 500 million years ago. Therefore, (and much divergent) metazoan taxa of demosponges, placo- we can assume that there is selection pressure operating not only on zoans, and ctenophores, which suggests that the origin of these the ladderlike structure of the SC but also on certain sequence SC proteins might even be as old as metazoan themselves [for motifs of single SC protein components. This casts another light not metazoan phylogeny, see (24)]. only on the topic of chromosome synapsis but also provides further Only in the clade of ecdysozoans (i.e., molting animals) hardly support to the hypothesis of a single origin of the SC. any sequence homologs could be identiﬁed. In the case of SYCP1 Finally, an interesting outcome of our work is the under- (Fig. 6) sequences of the crab appear in the cluster as sole repre- standing that Hydra fulﬁls the major criteria to become an impor- sentatives of this clade. This is an interesting point as the two current tant model organism also in meiosis research: The genome of H. multicellular invertebrate meiosis model organisms C. elegans and magnipapillata and the transcriptome of H. vulgaris AEP are fully D. melanogaster belong to the clade of ecdysozoans. An explanation sequenced, genetic manipulation methods are available, and for the strong differences in SC protein sequences between these clones of genetically manipulated animals can be easily expanded BIOLOGY

two species and the other metazoans described here may be the via asexual reproduction (30–32). Thus, studies comparing Hydra DEVELOPMENTAL evolutionary drift of ecdysozoans. This view is not without pre- and mammals promise to provide deep insight into the essential cedent in the literature as it has been already proposed for other features of meiosis over the last 500 million years of metazoan structural proteins as is the case of lamins, the major components of evolution. the nuclear lamina (25, 26), as well as for ribosomal DNA (27). In support of this, our bioinformatic analysis with C(3)G [the TF Materials and Methods protein of D. melanogaster (28)] and SYP1-4 [the TF proteins of C. Experiment details are described in SI Material and Methods. elegans (29)] in each case revealed only protein sequences from the respective genus, underscoring the proposal of a great sequence Sequence Analysis. Multiple amino acid sequence alignments were generated diversity and a rapid evolution within this clade (27). using ClustalX (33). Phylogenetic trees were constructed with the distance-

Fraune et al. PNAS | October 9, 2012 | vol. 109 | no. 41 | 16591 Downloaded by guest on October 1, 2021 Fig. 6. Molecular phylogeny of SYCP1 proteins. The tree was calculated from an 83 amino acid alignment using Bayesian inference [MrBayes (36); posterior probability values are indicated]. CCDC39 sequences were used as an outgroup. Phylogenetic grouping of SYCP1 proteins was also supported using the neighbor-joining method (34).

based neighbor-joining method [(34); 1,000 bootstrap replicates] with Poisson (36). Four Markov chains were run from random starting trees for 106 gen- correction for multiple substitutions, as implemented in Seaview (35). Bayesian erations. The mixed model option was used to choose the appropriate ﬁxed- inference of phylogeny was done with MrBayes v3.0b4, a program using rate model for amino acid substitutions. Trees were sampled every 100 gen- Markov Chain Monte Carlo to approximate the posterior probability of trees erations and the ﬁrst 25% of samples were discarded as burn-in. BLAST analysis

Fig. 7. Molecular phylogeny of SYCP3 proteins. The tree was calculated from a 171 amino acid alignment corresponding to the Cor1/Xlr/Xmr conserved region (CM1 to CM2) using the neighbor-joining method [(34); 1,000 pseudosamples; bootstrap values are indicated]. SYCP2 sequences were used as an outgroup. Phylogenetic grouping of SYCP3 proteins was also supported using Bayesian inference MrBayes (36).

16592 | www.pnas.org/cgi/doi/10.1073/pnas.1206875109 Fraune et al. Downloaded by guest on October 1, 2021 was performed using sequence databases accessible from the National Center In Situ Hybridization. Whole mount in situ hybridization on Hydra was for Biotechnology Information (NCBI) server (www.ncbi.nlm.nih.gov). performed as described in (42) with minor modifications. Digoxigenin- Annotated sequence alignments were designed using CHROMA Version labeled RNA probes targeted on Hydra Sycp3 and Sycp1 were synthe- 1.0 (www.llew.org.uk/chroma/index.html) (37). Identity threshold for sized according to the manufacturer’s instructions (DIG RNA labeling Kit grouping of the residues was set to 80%. Depending on the different fea- SP6/T7; Roche). tures of residues, the following seven groups were created: identical, charged, Ser/Thr, aliphatic, aromatic, polar, and hydrophobic. Immunocytochemistry. The 6-μm cryosections of Hydra midpieces with associated testis were cut and transferred to Superfrost Plus slides (Thermo Animal Strains and Culture Conditions. Experiments were performed using the Scientific). animal strain AEP from the H. vulgaris group (38; for sequence database see ref. 39). Animals were cultured following the standard procedures (32). Preparation of Hydra meiotic chromosome spreads was performed as described by De Boer et al. (43) with minor modifications. Immunostaining of Isolation of RNA, Reverse Transcription, PCR, and Cloning of cDNA. Total RNAs cryosections was performed following the protocol described in (44), whereas from whole animals or tissues were reversely transcribed into cDNAs. These immunostaining of the chromosome spreads was carried out in accordance to were used for either cloning of full-length cDNA sequences or tissue-specific De Boer et al. (43). The following affinity-purified primary antibodies were expression analysis. used: guinea pig anti-HySYCP1 (1:50), rabbit anti- HySYCP1 (1:400), and guinea- pig anti-HySYCP3 (1:200). Secondary antibodies (Dianova) were applied fol- Antibodies. Antibodies recognizing Hydra SYCP1 were generated against lowing instructions provided by the manufacturer. a peptide (ENRTDANVLKKNSDYK), as well as the C-terminal region of Hydra SYCP1. Anti-Hydra SYCP3 antibody was generated against the peptide SDEA- Microscopy and Imaging. Fluorescent images were taken on a confocal laser PALLSKSSLKRK. All antibodies were applied as affinity-purified antibodies (40). scanning microscope and processed using Adobe Photoshop (Adobe Systems). For electron microscopy, fixation and Epon embedding of Hydra pieces were SDS/PAGE and Immunoblot Analysis. SDS/PAGE and immunoblot analysis (41) done according to standard procedures (14). were carried out on protein fractions of different tissues of 50–100 animals. fi fi For protein detection the following af nity-puri ed antibodies were used: ACKNOWLEDGMENTS. We thank Elisabeth Meyer-Natus for excellent guinea pig anti-HySYCP1 (1:1,500), guinea-pig anti-HySYCP3, mouse anti- technical assistance and Georg Krohne for generous support. This study actin (1:10,000). Peroxidase-conjugated secondary antibodies (Dianova) were was supported by Priority Program SPP1384 “Mechanisms of Genome Hap- used as recommended by the manufacturer. loidization” (Deutsche Forschungsgemeinschaft).

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