ZCWPW1 is recruited to recombination hotspots by PRDM9 and is essential for meiotic double strand break repair
Meiosis is a specialised cell division , producing haploid gametes essential for reproduction . Uniquely , during this process homologous maternal and paternal chromosomes pair and exchange DNA ( recombine ) before undergoing balanced independent segregation . Alongside de novo mutations , this generates all genetic diversity – providing a substrate on which natural selection can act . In humans , mice , and likely many other vertebrates ( Baker et al . , 2017 ) , the locations at which recombination occur are determined by the binding locations of PRDM9 , which recognises a specific sequence motif encoded by its zinc finger array ( Baudat et al . , 2010; Myers et al . , 2010; Parvanov et al . , 2010 ) . At these sites , SPO11 catalyses recombination-initiating double strand breaks ( DSBs ) , a subset of which are repaired by recombination with the homologous chromosome . The sister chromatid can also provide a repair substrate , for example for DSBs on the X chromosome in males , and there is evidence that this also occurs at some autosomal hotspots ( Allen and Latt , 1976; Li et al . , 2019b; Lu and Yu , 2015 ) . In Prdm9-null mice , DSBs form at promoter regions rich in H3K4me3 ( Brick et al . , 2012 ) , and fail to repair resulting in severe asynapsis , meiotic arrest at pachytene , and sterility in both males and females on the B6 background ( Hayashi et al . , 2005 ) . We previously showed ( Davies et al . , 2016 ) that across DSB sites , increased binding of PRDM9 to the homologous chromosome aids synapsis and fertility in hybrid mice , implying an impact of binding downstream of DSB positioning . Mice for which most DSBs occur at sites where PRDM9 does not bind the homologue show widespread asynapsis ( Davies et al . , 2016; Gregorova et al . , 2018 ) . Following their formation , DSB sites are processed by resection , resulting in single-stranded DNA ( ssDNA ) that becomes decorated with DMC1 ( Hong et al . , 2001; Neale and Keeney , 2006; Sehorn et al . , 2004 ) . In wild-type ( WT ) mice , DMC1 foci start to appear in early zygotene cells . From mid-zygotene to early pachytene , as part of the recombinational repair process , DMC1 dissociates from the ssDNA and counts decrease until all breaks ( except those on the XY chromosomes ) are repaired at late pachytene ( Moens et al . , 2002 ) . At DSB sites where the homologous chromosome is not bound by PRDM9 , the DMC1 signal is strongly elevated ( Davies et al . , 2016 ) , suggesting persistent DMC1 foci and consistent with delayed DSB repair at these sites . In addition , fewer homologous recombination events occur at these sites ( Hinch et al . , 2019; Li et al . , 2019b ) implying that ( eventual ) DSB repair may sometimes use a sister chromosome pathway . However , the underlying mechanism ( s ) by which PRDM9 effectively contributes to DSB repair are not yet known . PRDM9 deposits both H3K4me3 and H3K36me3 histone methylation marks at the sites it binds , and this methyltransferase activity is essential for its role in DSB positioning ( Diagouraga et al . , 2018; Powers et al . , 2016 ) . What factors ‘read’ this unique combination of marks at recombination sites , however , is currently unknown . Notably , outside of hotspots and the pseudoautosomal region ( PAR ) on sex chromosomes , H3K4me3 and H3K36me3 occur at largely non-overlapping locations ( Powers et al . , 2016 ) , suggesting potentially highly specialised reader ( s ) . Indeed , in somatic cells H3K4me3 is deposited mainly at promoters , in particular by the SET1 complex targeted by CXXC1/CFP1 and Wdr82 which binds Ser5 phosphorylated polymerase II ( Barski et al . , 2007; Lee and Skalnik , 2008; Lee and Skalnik , 2005 ) . H3K36me3 is deposited by different methyltransferases , including SETD2 bound to Ser2 phosphorylated ( elongating ) polymerase II , and is enriched at exon-bound nucleosomes , particularly for 3’ exons ( reviewed in McDaniel and Strahl , 2017; Wagner and Carpenter , 2012 ) . H3K36me3 has multiple important roles , including in directing DNA methylation by recruiting DNMT3B , somatic DSB repair by homologous recombination ( Aymard et al . , 2014; Carvalho et al . , 2014; Pfister et al . , 2014 ) , mismatch repair by recruiting MSH6 ( Huang et al . , 2018; Li et al . , 2013 ) , and V ( D ) J recombination during lymphopoiesis ( Ji et al . , 2019 ) . Using single-cell RNA-sequencing ( RNA-seq ) of mouse testis , we identified a set of genes co-expressed in ( pre ) leptotene cells which are highly enriched for genes involved in meiotic recombination ( Jung et al . , 2019 ) . Zcwpw1 , which ranks 3rd in this set after Prdm9 ( 2nd ) , is of unknown function but contains two recognised protein domains: CW and PWWP , shown to individually bind H3K4me3 and H3K36me3 respectively ( He et al . , 2010a; Rona et al . , 2016 ) . This raises the attractive possibility that ZCWPW1 might recognise and physically associate with the same marks deposited by PRDM9 ( Jung et al . , 2019 ) . In humans , ZCWPW1 is expressed in testis ( Carithers et al . , 2015; Uhlén et al . , 2015; Figure 1—figure supplement 1 ) . It is also one of 104 genes specifically expressed in meiotic prophase within murine fetal ovaries ( Soh et al . , 2015 ) , further suggesting a conserved meiotic function . This was confirmed by a recent study showing that ZCWPW1 is required for male fertility in mice , with ZCWPW1 hypothesised to recruit the DSB machinery to hotspot sites ( Li et al . , 2019a ) . Here , we show that ZCWPW1 co-evolves with PRDM9 and is recruited to recombination hotspots by the combination of histone marks deposited by PRDM9 . However ZCWPW1 is not in fact required for the positioning of DSBs at PRDM9-bound sites , which occurs normally in Zcwpw1-null mice . Instead , ZCWPW1 is required for proper inter-homologue interactions: synapsis and the repair of DSBs . In Zcwpw1-null mice , DMC1 dynamics are strongly perturbed , with signals at autosomal hotspots resembling those on the X-chromosome , which does not have a homologue . Thus , ZCWPW1 represents the first protein directly positioned by PRDM9’s dual histone marks , but impacting homologous DSB repair , not positioning .
Based on publicly available databases , we identified likely ZCWPW1 orthologues in 167 species ( Materials and methods , Figure 1—source data 1 ) , aligned each to the human reference ZCWPW1 protein sequence , and compared against a previous analysis of PRDM9 ( Baker et al . , 2017 ) . The regions containing the CW and PWWP domains are well-conserved ( Figure 1A , B ) , while other parts of the protein appear absent in some species . Additionally , there is a region of moderate conservation downstream of the PWWP domain , not overlapping any known domain . Further , an SYCP1 ( SCP1 ) domain is annotated in the mouse protein only which , although only suggestive , is notable given that SYCP1 physically connects homologous chromosomes in meiosis . The C-terminal end of ZCWPW1 may include a methyl-CpG-binding domain ( Materials and methods , Lobley et al . , 2009 ) , at least in some species . Incomplete currently available DNA and protein sequences mean we are almost certain to miss some species where either ZCWPW1 and/or PRDM9 ( Baker et al . , 2017 ) are present . Despite this , we see extremely high overlap between PRDM9 and ZCWPW1 occurrence ( Figure 1C , D ) . ZCWPW1 appears to mainly occur in species possessing PRDM9 ( and even more clearly in groups of species possessing PRDM9 ) . Furthermore , with the exception of a small number of amphibians , ZCWPW1 orthologues are only identified in clades previously identified to possess PRDM9 ( Baker et al . , 2017 ) , and the majority of species ( 78% of 167 ) possessing ZCWPW1 also possess a close relative ( species or family ) where PRDM9 is identified ( Figure 1C ) . We leveraged a previous analysis of PRDM9 evolution ( Baker et al . , 2017 ) to extend this comparison ( Figure 1D , Figure 1—source datas 1 and 2 ) . Among seven clades previously shown to possess PRDM9 copies with all N-terminal domains ( KRAB , SSXRD and PR/SET domains ) intact , ZCWPW1 is found in each case , typically in the same species ( Figure 1D ) . In some species with several PRDM9 copies , including many teleost fish and two other confirmed independent events , PRDM9 has lost N-terminal domain sequences in some but not all copies , while in at least three further species clades , particular N-terminal domains have been lost in all PRDM9 copies , but the PR/SET domain remains intact . ZCWPW1 is also found across these cases ( Figure 1D ) , so it is widely conserved across species possessing one or more copies of PRDM9 with an intact PR/SET domain . Previously ( Baker et al . , 2017 ) , three independent groups of species have been identified possessing mutations in the PR/SET domain of PRDM9 , at key catalytic residues for the deposition of the H3K4me3/H3K36me3 mark ( s ) . Complete loss of PRDM9 is also identified in three groups: bird/crocodiles , canids , and ( tentatively ) amphibians . Given that ZCWPW1 is predicted to recognise the H3K4me3/H3K36me3 marks catalysed by the PR/SET domain of PRDM9 , we would expect each of these six events to disrupt any potential interaction between the two proteins . Strikingly , we failed to identify ZCWPW1 orthologues in any of the previously examined species impacted by these six events ( Figure 1D ) , except in canids ( and one amphibian species ) . Thus , at least four of the six may be completely mirrored by ZCWPW1 loss . Even if PRDM9 is retained , species with PR/SET domain mutations are much more likely not to possess an identified ZCWPW1 orthologue within their class/phylum compared to species without such mutations ( odds ratio = 285; indicative p<10−15 by FET , although we note that these observations are not all independent ) . In conclusion , among vertebrates at least , it appears the set of species with ZCWPW1 is extremely similar to those species possessing PRDM9 with an intact PR/SET domain , with considerable evidence of co-evolution of gain/loss events for each protein . In some species with apparently intact PR/SET domains ( for example Tasmanian Devil , Mexican Tetra , Platypus ) , it is hypothesised that PRDM9 does not position DSBs due to loss of other N-terminal domains and/or the PRDM9 zinc-finger array ( Baker et al . , 2017 ) . These example species still possess identified ZCWPW1 orthologues ( Figure 1—source data 1 ) , so it does not seem that PRDM9’s ability to position DSBs is essential for retention of ZCWPW1 . Instead , our results are consistent with ZCWPW1’s main function involving recognition of the histone modifications catalysed by PRDM9-SET during early meiosis , in agreement with the functional evidence from ZCWPW1’s CW and PWWP domains . We previously detected Zcwpw1 transcript expression in pre-leptotene to leptotene spermatocytes of adult WT mice ( Jung et al . , 2019 ) . To investigate the role of ZCWPW1 during meiosis in vivo , we produced and validated the specificity of an antibody against the full-length recombinant mouse protein ( Figure 2—figure supplement 1 ) , and studied the phenotype of a newly generated knockout ( KO ) mouse line for Zcwpw1 , with a particular focus on fertility and meiotic recombination . In testes from WT mice , we observe a dynamic localisation of ZCWPW1 protein ( Figure 2 ) , similar but non-identical to that reported in a recent study ( Li et al . , 2019a ) . ZCWPW1 shows a strong , punctate nuclear staining excluding the pericentromeric regions ( clustered into chromocenters brightly stained with DAPI ) in zygotene and early pachytene cells . In mid-pachytene cells , ZCWPW1 levels drop , with the protein now mainly localised in the XY body and as bright foci at the ends of the synaptonemal complex labeled by SYCP3 , not previously observed using an antibody raised against a 174 amino acid C-terminal region of the protein ( Li et al . , 2019a ) . By diplotene , little expression is visible . Using fluorescent in-situ hybridisation ( FISH ) to label centromeric and telomeric ( distal and proximal to centromeres ) regions of chromosomes , we established that these discrete foci of ZCWPW1 are consistently positioned at the ends of the synaptonemal complex , with the majority at both ends simultaneously , lying close to telomeres and centromeres themselves ( Figure 2—figure supplement 2 ) . We next studied mice from a constitutive Zcwpw1 KO line ( Materials and methods ) , carrying a ~ 1 . 5 kb frameshift deletion encompassing exons 5 to 7 upstream of the CW and PWWP domains . This creates a premature stop codon that would produce a predicted heavily truncated 492bp ( vs 1893bp for WT ) transcript lacking either of these domains ( Figure 3A ) , and a candidate for nonsense-mediated mRNA decay ( Kurosaki et al . , 2019 ) . Indeed , we observed a complete lack of detectable ZCWPW1 protein expression in testis protein extracts ( Figure 3—figure supplement 1A ) and chromosome spreads from Zcwpw1−/− mice , in zygotene cells ( Figure 3B ) and all other meiotic stages of prophase I where expression is detected in WT mice ( Figure 3—figure supplement 1B ) . Confirming recent findings in a different Zcwpw1−/− mouse ( Li et al . , 2019a ) , we observed no overt fertility phenotype in either sex in the heterozygous Zcwpw1+/− mice ( data not shown ) . However , Zcwpw1−/− male mice were sterile with complete azoospermia and reduced testis size and weight ( Figure 3C , D , Figure 3—source data 1 ) , while female mice retained fertility until around 7–8 months of age ( Figure 3—source data 2 ) , and otherwise both sexes develop normally . As in Li et al . , 2019a , in male mice we observe no meiotic progression beyond ( pseudo ) pachytene with widespread asynapsed chromosomes marked by γ-H2AX and HORMAD2 , failure to form the sex body ( Figure 3B , Figure 3—figure supplement 2A ) , a complete absence of MLH1 foci marking recombination crossover sites ( Figure 3—figure supplement 2B ) , and persistent DMC1 foci marking unrepaired DSBs ( Figure 4 ) . Each of these properties resembles observations in the Prdm9−/− mutant ( Hayashi et al . , 2005 ) , and so we compared to this mutant . In our Zcwpw1−/− male mice , >98% of pachytene cells failed to properly synapse at least one pair of chromosomes ( Figure 3B , E ) , similar to Prdm9−/− males . However , the nature of the synaptic defects observed differed ( Figure 3—figure supplement 2—source data 1 ) . In the Prdm9−/− mutant , 63 . 2% of the pseudo-pachytene cells showed mispairing of non-homologous chromosomes in a typical branched structure ( referred to as ‘tangled’ , Figure 3—figure supplement 2—source data 1 ) . In contrast , we only observed 24 . 8% of Zcwpw1−/− pseudo-pachytene cells with this type of error , while the majority ( 74 . 5% ) of cells contained multiple bundles of HORMAD2-positive asynapsed chromosomes , which resemble the XY body and may merge with the sex chromosomes ( thus referred to as ‘multibodies’ , Figure 3—figure supplement 2 ) . These results imply that the Prdm9−/− mutant often mispairs chromosomes , while Zcwpw1−/− spermatocytes mainly fail to pair a subset of chromosomes at all . The expression levels and staining pattern of ZCWPW1 were not visibly altered in testis nuclear spreads ( data not shown ) and protein extracts ( Figure 3—figure supplement 1A ) from Prdm9−/− mice . However , since the Zcwpw1 KO and Prdm9 KO lines are on a different genetic background ( C57/BL6N and B6×129 , respectively ) , we cannot rule out the possibility that this explains partly or fully the differences in meiotic defects observed between the two mutants . Comparing levels of DMC1 foci as a proxy for DSB repair , or a potential cause for the asynapsis in Zcwpw1−/− males , in both Zcwpw1−/− and Prdm9−/− mice foci count was significantly elevated from zygotene onwards , indicating delayed repair of DSBs ( Figure 4 ) . However , we observed a wider spread for Zcwpw1−/− males . A similar increase was observed at pachytene stage in the levels of RAD51 ( Figure 4—figure supplement 1 ) , a strand exchange protein which functions in concert with DMC1 , with the large majority of RAD51 and DMC1 foci co-localising ( Brown et al . , 2015; Tarsounas et al . , 1999 ) . Like DMC1 , RPA2 levels were also significantly elevated in the Zcwpw1−/− mouse from zygotene onwards ( Figure 4—figure supplement 2 ) . In Zcwpw1−/− males , like in the Prdm9−/− mutant , DSBs form and recruit RPA2 , RAD51 and DMC1 in similar numbers ( Huang et al . , 2020; Li et al . , 2019a; Mahgoub et al . , 2020 ) , but fail to repair efficiently , accompanied by asynapsis and meiotic arrest at pseudo-pachytene . Indeed , we observe late unrepaired DMC1 foci mainly on HORMAD2-positive asynapsed chromosomes ( Figure 4—figure supplement 3 ) . We previously studied the binding properties of human PRDM9 and established a genome-wide map in transfected human mitotic ( HEK293T ) cells by ChIP-seq ( Altemose et al . , 2017 ) , observing binding to the majority of human meiotic recombination hotspots . Based on the presence of H3K4me3 and H3K36me3 recognition domains in ZCWPW1 , we hypothesised that it would be recruited to PRDM9-bound genomic sites , where these marks are deposited upon binding in HEK293T cells ( Altemose et al . , 2017 ) . To test this , we co-transfected HEK293T cells with full-length human HA-tagged ZCWPW1 and either no other protein , or full-length PRDM9 alleles carrying the human or chimpanzee zinc finger array , as studied previously ( Altemose et al . , 2017 ) , and then performed ChIP-seq against the ZCWPW1 tag ( the endogenous levels of these proteins in HEK293T cells , and of the closely related family member ZCWPW2 , are extremely low so unlikely to confound the analysis - see Figure 5—source data 1 ) . Confirming the recruitment hypothesis , in the presence of human PRDM9 and compared to cells lacking human PRDM9 , ZCWPW1 shows a strong enrichment at human PRDM9-binding sites with higher enrichment at sites with higher PRDM9 enrichment ( Figure 5A , B , Pearson’s correlation = 0 . 43 - Figure 5—figure supplement 1 ) . Notably , even without PRDM9 we observed many ZCWPW1 binding peaks across the genome , some coinciding with PRDM9-binding sites ( Figure 5C and Figure 5—figure supplement 2 ) . However , upon co-transfection with PRDM9 , 92% of the strongest 10 , 000 ZCWPW1 peaks are at PRDM9-bound sites ( 98% of the strongest 3 , 000 vs 7% expected overlap with randomised peaks , and only 47% overlap for the strongest 3 , 000 ZCWPW1 peaks in cells without PRDM9 transfection ) . Hence , PRDM9 is able to strongly reprogram ZCWPW1 binding ( Figure 5C , see also Figure 5—figure supplement 3 ) , suggesting that loci with peaks in cells without PRDM9 transfection are bound more weakly . Because our transfection has <100% efficiency ( Figure 5—figure supplement 4 ) , some cells containing ZCWPW1 will not possess PRDM9 , and so it is possible that an even greater fraction of ZCWPW1 is redirected to PRDM9 binding sites in cells where both proteins are present . Importantly , when co-transfecting with a modified version of PRDM9 in which the zinc finger array is replaced with that from chimp ( which binds different locations in the genome [Altemose et al . , 2017] ) , we find that the enrichment at human binding sites disappears , but instead ZCWPW1 is enriched at chimp PRDM9-binding sites ( Figure 5A and Figure 5—figure supplement 5 ) . This perturbation experiment provides strong evidence that PRDM9 causes recruitment of ZCWPW1 ( as opposed to for example independent recruitment of both proteins ) . Notably , the strength of ZCWPW1 binding in HEK293T cells at human PRDM9-binding sites provides a better predictor of human meiotic DMC1 binding status ( a proxy for DSB formation ) than does human PRDM9 binding strength itself ( Figure 5—figure supplement 6 ) . We show ( see below ) that ZCWPW1 is not directly involved in DSB positioning , this result might therefore instead suggest that similar features to those ZCWPW1 recognises are involved in recruiting DSB formation machinery . We tested whether the recruitment of ZCWPW1 to sites bound by PRDM9 might be mediated by the dual histone modifications H3K4me3 and H3K36me3 . Consistent with this idea , ZCWPW1 binding is positively associated with levels of both H3K4me3 and H3K36me3 marks ( Figure 6—figure supplements 1 and 2 , tested separately , not necessarily ‘dual’ marks coincident within an individual cell , although locations of these marks in meiotic cells are rarely coincident except at hotspots [Powers et al . , 2016] ) . While we cannot rule out a direct interaction between ZCWPW1 and PRDM9 , others have shown that dual modified peptides have a greater affinity to ZCWPW1 than peptides carrying the single modifications , supporting that the marks themselves are responsible for ZCWPW1’s recruitment ( Mahgoub et al . , 2020 ) . We examined transcription start sites , which possess H3K4me3 at high levels , but lack H3K36me3 , observing some ZCWPW1 signal at these sites , but with a uniformly lower mean ZCWPW1 enrichment compared to those with evidence of PRDM9 binding ( and hence both histone marks ) ( Figure 6 ) . Although it is not possible to measure H3K4me3 presence/absence in individual cells in our system , even the most weakly bound PRDM9 sites – which must therefore possess H3K4me3 in only a fraction of cells – show stronger ZCWPW1 enrichment than the strongest promoters , which are likely to have near 100% H3K4me3 marking , and possess >2 fold more H3K4me3 than even the strongest PRDM9-binding sites . We conclude that H3K4me3 alone endows only relatively weak binding , while PRDM9 is therefore able to recruit ZCWPW1 with a much greater efficiency than sites marked by H3K4me3 alone , suggesting that both histone modifications might aid efficient binding . While the increase in ZCWPW1 enrichment at sites with pre-existing H3K4me3 is consistent with both marks contributing to recruitment , we have no evidence that these weaker binding sites serve a functional role , and indeed concurrent investigations did not find strong evidence of ZCWPW1-driven transcriptional changes ( Huang et al . , 2020 ) . Previous work has shown that Prdm9−/− mice use a new set of DSB hotspots , localising at CpG islands and/or promoter regions ( Brick et al . , 2012 ) . Given that PRDM9 recruits ZCWPW1 , one possible function of ZCWPW1 may be that , in turn , it recruits the DSB machinery and hence forms part of the causal chain in normal positioning of DSBs at PRDM9-specified hotspots . Alternatively , the Zcwpw1−/− mutant phenotypes we observe might reflect a more downstream role . To distinguish these hypotheses , we gathered data on DSB positioning and repair dynamics genome-wide , by carrying out single ssDNA sequencing ( SSDS ) by ChIP-seq against DMC1 ( Khil et al . , 2012 ) . In the Zcwpw1−/− mutant males , we observed normal localisation of DMC1 at the expected B6 WT hotspots for this background , with no signal at Prdm9−/− hotspots ( Figure 7A ) . We only fail to see evidence of DMC1 signal in a subset of the weakest hotspots , where we are likely to lack statistical power ( Figure 7—figure supplement 1 ) . Thus , DSBs occur in unchanged hotspot regions in Zcwpw1−/− males , relative to WT . To check if they occur at the same locations within hotspot regions , we leveraged data for SPO11-mapped DSB sites ( Lange et al . , 2016 ) . Specifically , we identified three sets of hotspots whose respective mapped breaks occur mainly upstream , central , or downstream of the PRDM9-binding site ( Materials and methods ) , and compared their DMC1 ChIP-seq signal profiles , as well as their SPO11 signals ( Figure 7—figure supplement 2 ) . This revealed that in the Zcwpw1−/− males , as in the WT , DMC1 signals mirror this break positioning . Therefore , ZCWPW1 is not required to specify hotspot locations , and neither does it strongly influence where DSBs occur within hotspots . One perhaps important difference between the Zcwpw1−/− and WT DMC1 profiles is that the latter signal is slightly wider ( up to ~200bp ) in hotspots with increasing PRDM9-induced H3K4me3 ( Figure 7B ) , while this effect is absent in the Zcwpw1−/− case . This might be explained by small differences in chromatin accessibility subtly impacting SPO11 locations , DNA end resection which generates the 3’ ssDNA tail to which DMC1 binds , or downstream processing differences altering DMC1 span in the mutant mice . Despite very similar DSB locations , we saw much greater , and systematic , differences in the strength of the DMC1 signal at individual hotspots . As previously ( Davies et al . , 2016; Khil et al . , 2012 ) , we note that observed average DMC1 signal strength at a hotspot reflects the product of the frequency at which DSBs occur there , and the average length of time DMC1 remains bound to the ssDNA repair intermediate , with the latter reflecting DSB processing/repair time . In contrast , available SPO11 oligo-seq data ( Lange et al . , 2016 ) reflect mainly the frequency of DSBs . We therefore compared DMC1 and SPO11 signal strength at each autosomal and X-chromosome hotspot , in WT and Zcwpw1−/− male mice . In WT mice , the non-PAR X-chromosome shows a very strong elevation of DMC1 signal strength ( as seen previously in other mice [Davies et al . , 2016] including , albeit somewhat more weakly , even sterile hybrids ) . This elevation reflects the persistent DMC1 foci on this chromosome also visible using microscopy , at DSB sites that eventually repair using the sister chromatid . Moreover , in WT mice there is a sub-linear ( curved ) relationship between SPO11 and DMC1 at hotspots ( Figure 7C ) . This is thought to reflect a wider phenomenon of faster DSB repair occurring within those hotspots whose homologue is more strongly bound by PRDM9 , that is those hotspots with a stronger H3K4me3 signal in the WT mouse ( Davies et al . , 2016; Hinch et al . , 2019; Li et al . , 2019b ) . The X-chromosome DMC1 elevation is in a sense an extreme case of slower repair of DSBs whose homologue is not PRDM9-bound , because no homologue exists in this case . However , we see a striking departure in the Zcwpw1−/− mouse , where the X-chromosome behaves more similarly to the autosomes with respect to DMC1 vs WT SPO11 signal strength ( Figure 7C ) . Moreover , we see a simple linear relationship between DMC1 and WT SPO11 binding in this mouse ( Figure 7C ) . This implies that the effect of PRDM9 binding on DMC1 removal is eliminated in this mouse ( assuming SPO11 levels are not greatly affected by Zcwpw1 KO ) ; so PRDM9 appears unable to ‘assist’ in homologue pairing and synapsis in this mouse ( whether because ZCWPW1 directly aids the process , or indirectly ) . Indeed , the data imply a widespread perturbation of DSB repair in this mouse , with autosomal DMC1 foci persisting as long as those on the X-chromosome . In the previously studied Hop2−/− mouse ( Khil et al . , 2012; Petukhova et al . , 2003; Smagulova et al . , 2011 ) , we identified a very similar pattern ( Figure 7—figure supplement 3 ) : in this mouse , DMC1 is loaded onto ssDNA , but DSBs are not repaired at all , indicating that DSB repair pathways in Zcwpw1−/− mice are profoundly altered . These results are consistent with , and extend , our microscopy observations ( Figure 4 ) that many DSBs persist in the Zcwpw1−/− mouse , and some may never repair . We are unable to say whether DSB repair involves the homologue or not in this mouse , but the partial synapsis we observe suggests some repair does likely occur . To further understand DMC1 persistence at individual hotspots , we estimated the relative DMC1 heat of autosomal hotspots in the Zcwpw1−/− mouse , compared to the WT mouse . The best individual predictor of this ratio among H3K4me3 , DMC1 , and SPO11 in WT was the level of H3K4me3 ( r = 0 . 60 Generalised Additive Model; Figure 7—figure supplement 4 , while r = 0 . 63 when using all three predictors together ) . The average KO:WT DMC1 ratio increases around ninefold from the most weakly to the most strongly H3K4me3-marked hotspots . Therefore , if DMC1 signal changes are indeed explained by slower DSB processing in the Zcwpw1−/− mouse at hotspots bound strongly by PRDM9 , then this implies a very strong effect of PRDM9’s ‘assistance’ in aiding repair . Whatever the cause , the overall strong correlation implies perturbation of DMC1 behaviour is widespread , rather than impacting any small subset of hotspots . Moreover , it appears to be mainly controlled by local levels of PRDM9 histone modification , in keeping with the evidence that ZCWPW1 recognises this mark at PRDM9-bound sites , on either the broken or ( identical ) homologous chromosome . In addition to the strong PRDM9-dependent ZCWPW1 peaks described earlier , there are many locations in HEK293T cells at which ZCWPW1 binds , typically more weakly , and independently of PRDM9 . Indeed we identified over 800 , 000 ZCWPW1 peaks . Surprisingly , a large proportion of these binding sites overlap Alu repeats ( Figure 8A , Figure 5C , and Figure 5—figure supplement 2 ) ( of which there are 1 . 1 million in the human genome [Deininger , 2011] ) . The weakest ZCWPW1 peaks overlap Alus most frequently , whilst the strongest peaks are depleted of Alus relative to chance overlap ( Figure 8A ) . Because Alus are rich in CpGs ( containing >23% of human CpG dinucleotides , with deamination depleting CpGs in older Alus [Luo et al . , 2014] ) , we tested if binding correlates with CpG presence/absence . Indeed , the binding of ZCWPW1 to Alus depends on the presence of CpGs , which are mainly methylated ( Xie et al . , 2011 ) . Alus with no CpGs were bound at a rate lower than by chance , but almost all Alus containing 10–20 CpGs are bound ( Figure 8B ) . This dependence is not Alu-specific: even outside genomic repeats , 300bp regions with zero CpGs have negligible probability of overlapping a ZCWPW1 binding site , while the overlap probability rises to >50% for regions containing 10 or more CpGs , suggesting CpGs are essential for PRDM9-independent ZCWPW1 binding ( Figure 8C , D and Figure 8—figure supplement 1 ) . ZCWPW1 appears to have greater affinity for methylated CpG pairs , but retains some affinity even for non-methylated regions . This binding mode was unexpected a priori , and does not appear to be easily explained by patterns of H3K4me3/H3K36me3 in the genome , which do not concentrate as strongly in these regions .
Here , we exploited previous work on single-cell transcript profiling of mouse testis , which has established spatio-temporal ( and likely functional ) components of co-expressed genes ( Jung et al . , 2019 ) , to identify co-factors relevant to PRDM9 function in specification of recombination hotspots and DSB repair during meiosis . We show that the histone methylation reader ZCWPW1 , which is both highly co-expressed and co-evolving with PRDM9 , is recruited by PRDM9 to its binding sites , likely mediated through the recognition of the dual H3K4me3-H3K36me3 histone mark that PRDM9 deposits . Like Mahgoub and colleagues in a parallel analysis ( Mahgoub et al . , 2020 ) , we find co-evolution of ZCWPW1 with PRDM9 , consistent with proteins functioning in the same pathway . We find that this association appears to depend on particular domains . ZCWPW1 co-occurs with PRDM9 in species where PRDM9 possesses a SET domain predicted to retain catalytic activity for H3K4me3/H3K36me3 , which the domains of ZCWPW1 are predicted to recognise , strongly suggesting a partnership totally dependent on this recognition . This co-occurrence can persist even in species where PRDM9 is not believed to position DSBs ( Baker et al . , 2017 ) , suggesting the co-operation between these proteins does not completely depend on this aspect of PRDM9’s function . Interestingly , a predicted functional SET domain of PRDM9 mainly occurs when PRDM9 possesses a SSXRD domain , which in other proteins ( Banito et al . , 2018 ) recruits CXXC2 which binds to CpG islands – whether this connects to our observation of a potential CpG-binding domain in ZCWPW1 , and CpG-binding behaviour in a human cell line , remains to be explored . A recent study reported phenotyping of an independent Zcwpw1−/− mouse ( Li et al . , 2019a ) , and we observe similar complete loss of fertility in males , but not in young females . Why females deficient for ZCWPW1 are initially fertile ( but lose fertility by ~8 months; Li et al . , 2019a ) is unknown , but resembles sexual dimorphism in loss-of-function mutants for other meiotic genes involved in homologous recombination , where females exhibit a milder fertility phenotype ( Cahoon and Libuda , 2019; Morelli and Cohen , 2005; Zhang et al . , 2019 ) . However , we observe a distinct intracellular localisation of ZCWPW1 from that reported ( Li et al . , 2019a ) : while we also detect ZCWPW1 as a diffuse nuclear signal across the nucleus ( although excluding the chromocenters ) and localisation to the XY body in pachytene , we see an additional signal in mid-pachytene to diplotene cells at the ends of the synaptonemal complex . This might reflect the specificity of our polyclonal antibody , which is raised against the full-length mouse protein vs a monoclonal antibody raised against a C-terminal region , hence recognising a wider spectrum of ZCWPW1 . Given that the Zcwpw1−/− mouse arrests in early pachytene , it is not possible to test later stages , so a caveat is that we cannot completely exclude off-target antibody recognition . If accurate , the late-stage localisation of ZCWPW1 at subtelomeric regions resembles other proteins ( Speedy A , CDK2 , TFR1 , SUN1 , TERB1/2 , MAJIN , and KASH ) , which form a complex tethering the telomeres to the nuclear envelope to facilitate chromosome movement and pairing — essential for synapsis and fertility ( Ashley et al . , 2001; Ding et al . , 2007; Horn et al . , 2013; Shibuya et al . , 2014; Tu et al . , 2017 ) . However , no evidence of impaired telomeric attachment was reported in the other Zcwpw1−/− mutant , based on normal localisation of TRF1 ( Li et al . , 2019a ) . In parallel with this work , two other studies of ZCWPW1 Huang et al . , 2020; Mahgoub et al . , 2020 have performed complementary analyses , and together with our own results allow some clear conclusions to be drawn . We mapped positions of ZCWPW1 recruitment ( of the human protein ) in mitotic HEK293T cells , in the presence of human or chimpanzee PRDM9 and compared to assayed peaks for PRDM9 , H3K4me3 and H3K36me3 ( Altemose et al . , 2017 ) . We find that in co-transfected HEK293T cells ZCWPW1 is strongly recruited to PRDM9-bound sites , in an allele-specific manner . Mahgoub et al . , 2020 also report PRDM9 allele-specific recruitment of ZCWPW1 , but in hybrid mice carrying Prdm9Dom2 and Prdm9Cast alleles which encode proteins with distinct zinc finger array DNA binding specificities . Together with ZCWPW1 binding measurements from Huang et al . , 2020 , these results imply that across PRDM9 alleles and species - humans , mice , and likely chimpanzees - PRDM9 binding alone is sufficient to strongly recruit ZCWPW1 without requiring DSB formation ( which does not occur in our system ) or meiosis-specific co-factors ( which would not be expected to be present in our cellular system ) . A likely explanation of this recruitment is recognition of H3K4me3 and H3K36me3 that PRDM9 deposits by the CW and PWWP domains of ZCWPW1 respectively . Previously , Li and colleagues ( Li et al . , 2019a ) showed co-immunoprecipitation of H3K4me3 with ZCWPW1 . In HEK293T cells we observed that ZCWPW1 recruitment correlates with the levels of H3K4me3 and H3K36me3 that PRDM9 deposits , while Huang et al . , 2020 reported that in mice with a non-functional CW domain no ZCWPW1 binding peaks were observed , suggesting that H3K4me3 recognition at least might be necessary for the recruitment of ZCWPW1 at hotspots in vivo . However , H3K4me3 cannot be the only factor because we observed much stronger recruitment of ZCWPW1 to PRDM9 binding sites than to other sites possessing similar H3K4me3 levels , which might be explained by the additional presence of H3K36me3 at PRDM9 binding sites increasing ZCWPW1’s recognition and/or its stable association at these sites . Indeed in cells with PRDM9 , the strongest ZCWPW1-binding sites are almost all PRDM9-bound sites . Similarly in mice , >85% of ZCWPW1 peaks overlap both marks concomitantly ( Huang et al . , 2020; Mahgoub et al . , 2020 ) . Mahgoub et al . , 2020 additionally reported that in vitro binding affinity of recombinant mouse ZCWPW1 is at least five fold greater for dual-modified H3K4me3/K36me3 peptides compared to peptides carrying the single modifications . Together , this paints a clear picture that ZCWPW1 likely directly recognises sites possessing the dual histone marks deposited by the PRDM9-HELLS complex ( Spruce et al . , 2020 ) , the first such protein identified . Our evolutionary analyses suggest that this function might occur across almost all species where PRDM9 deposits these marks , even species where PRDM9 does not specify DSB locations ( Baker et al . , 2017 ) . Although it was suggested that ZCWPW1 might recruit the DSB machinery to PRDM9 binding sites ( Li et al . , 2019a; Spruce et al . , 2020 ) , we and Mahgoub et al . , 2020 ( using distinct assays ) find that DSB positioning is completely unaffected by loss of ZCWPW1 . However , the additional role PRDM9 plays besides DSB positioning , in enabling homologous chromosome pairing ( Davies et al . , 2016; Hinch et al . , 2019; Li et al . , 2019b ) , apparently malfunctions in Zcwpw1−/− mice . Our DMC1 data , together with microscopy data , are most easily explained if , unlike in WT , DSBs at sites bound more strongly by PRDM9 do not repair more quickly . Instead , in Zcwpw1−/− males , all DSBs behave similarly to those on the X-chromosome — which repairs DSBs very slowly , even in WT animals , and does not synapse . This implies an unexpected role for ZCWPW1: in aiding PRDM9 in its second function , synapsis . ZCWPW1 therefore offers a ( thus far ) unique protein , directly recruited by PRDM9 binding and so linking PRDM9 binding with its downstream functions in aiding rapid DSB processing/repair at hotspots whose homologue is bound by PRDM9 ( Davies et al . , 2016; Hinch et al . , 2019; Li et al . , 2019b ) . PRDM9 is thought to aid homology search/chromosome pairing by such binding to the homologous chromosome , and hybrids where hotspots are highly ‘asymmetric’ ( i . e . the homologues are not bound at the sites where DSBs occur , due to evolutionary hotspot erosion ) show multiple features identical to Zcwpw1−/− mice . These include asynapsis , persistent DMC1 foci ( at the asymmetric hotspots ) , and complete sterility in males only ( Davies et al . , 2016 ) . In Zcwpw1−/− mice , unlike in WT mice , there are many persistent DMC1 foci and we observe that even strong PRDM9 binding to the homologue causes no reduction in DMC1 persistence . These mice therefore behave , in many ways , as if all hotspots are completely asymmetric . Thus , a parsimonious explanation of our findings is that ZCWPW1 functions by attaching to PRDM9-bound sites on the matching homologous chromosome , thus marking possible repair partner locations for DSB sites , to help pair homologues . This might for example lead to recruitment to the chromosomal axis ( where DMC1 foci are found ) , for potential use as the homologous repair template of DSBs , perhaps mediated by the SCP-1-like domain identified in some ( although not all ) orthologous copies . Given we observe ZCWPW1 throughout the nucleus in early meiosis , such recruitment would likely be transient in nature . Alternatively , ZCWPW1’s part in mediating PRDM9’s downstream roles might also depend ( or even , depend only ) on it binding the chromosome on which DSBs occur . Given that we still observe some synapsis , similar to Prdm9−/− mice , we suggest that a ‘back-up’ mechanism , which is independent of PRDM9 binding level , is acting to attempt homology search/chromosome pairing in Zcwpw1−/− mice . In fact , some such mechanism is likely necessary , to repair DSBs happening within a subset of hotspots that are asymmetric due to naturally occurring variation ( as seen in for example humans and heterozygous mice ) . If our observed >9 fold changes in relative DMC1 levels for strongly vs weakly bound hotspots purely reflect increased DMC1 persistence , it might be that this mechanism is greatly slower than the PRDM9-dependent pathway for WT mice . In turn , this could explain many – or all – of the downstream phenotypes in these mice , as consequences of delayed homology search . The simplest explanation of our observation that ZCWPW1 binds weakly to many sites in a CpG-dependent manner in HEK293T cells is that these sites might be directly bound by a methyl-CpG binding domain ( MBD ) putatively observed in the human , mouse , and coelacanth Zcwpw1 , among others . Alternatively , recruitment might be indirect , with ZCWPW1 forming a complex with an MBD containing protein , for example SETDB1 which is expressed in HEK293T cells , co-expressed with ZCWPW1 and PRDM9 ( Jung et al . , 2019; Schultz et al . , 2002 ) , and interacts with several other identified PRDM9 partner proteins ( Mulligan et al . , 2008; Parvanov et al . , 2017 ) . Given the precisely regulated developmental changes in methylation status , ZCWPW1/PRDM9 protein abundance , and chromatin during meiosis ( Gaysinskaya et al . , 2018; Seisenberger et al . , 2013 ) it is unclear whether ZCWPW1 binding will also be affected by CpGs in vivo . Although more modest , Huang et al . , 2020 did also report enrichment of ZCWPW1 at B1 elements in mice , the closest equivalent of human Alus . Unlike Mahgoub et al . , 2020 , Huang et al . , 2020 also observed overlap of ZCWPW1 peaks with CpG islands ( CGI ) more frequently than expected . Given ( i ) they and Mahgoub et al . , 2020 detect a much lower number of peaks ( 14 , 688 and 4 , 487 respectively ) than we do in our HEK293T system ( 800 , 000 ) , and ( ii ) we only find Alu enrichment in weaker ZCWPW1 peaks ( of our top 10 , 000 peaks , >90% would be PRDM9-bound sites ) , these results are not conflicting , and instead might depend on detection/behaviour of weaker peaks , and/or presence of PRDM9 in ZCWPW1-containing cells . If CpG dinucleotides do ( although weakly , relative to PRDM9 ) affect in vivo ZCWPW1 binding affinity and given that CpG methylation is associated with transposons , it is interesting to speculate whether ZCWPW1 might play a role in germline transposon recognition , silencing , or facilitate repair of DSBs occurring within transposable elements . Zcwpw1 possesses a paralogue , Zcwpw2 , which is also co-expressed in testis with Prdm9 ( although at a lower level ) , contains both CW and PWWP domains , and is able to recognise H3K4me3 at least ( Liu et al . , 2016 ) . Further studies are required to investigate what function if any Zcwpw2 has in meiosis . One interesting possibility is that ZCWPW2 , rather than ZCWPW1 , might act to help position DSBs – if so , its low abundance relative to ZCWPW1 would be analogous to the low number of DSBs per meiosis ( ~300 ) relative to the number of PRDM9 binding sites . In this study , we have shown that ZCWPW1 co-evolves with PRDM9 , binds to sites marked by PRDM9 , and is required for the proper processing , removal of DMC1 , and repair of DSBs . How exactly it mediates repair , if for example by recruitment of other proteins , remains to be determined . This study also further demonstrates that genes co-expressed with Prdm9 represent a rich source of undiscovered meiotic genes , with important functional implications for recombination , meiosis progression and ultimately fertility . ZCWPW1 impacts synapsis , downstream of DSB formation . Therefore the protein ( s ) responsible for recognising PRDM9-marked sites for DSB formation , and for aiding synapsis , are at least partly different , and we anticipate that future studies will likely uncover additional PRDM9-recruited proteins .
We identified ZCWPW1 orthologues across species using four data sources: first , we used BlastP ( blast . ncbi . nlm . nih . gov ) , against the full-length human reference ZCWPW1 sequence ( identifier NP_060454 . 3 , against nr_v5 database ) storing the top 1000 hits using the default parameters ( set 1 ) . Secondly , we downloaded the two sets of Ensembl identified ZCWPW1 orthologues ( against ENSMUSG00000037108 . 13; ensembl . org , 105 orthologues ) , and identified NCBI ZCWPW1 protein orthologues ( 146 species , www . ncbi . nlm . nih . gov ) . Initial examination of identified orthologues revealed conservation among orthologues mainly of the CW and PWWP domains; therefore , to find additional orthologous sequences we performed tBLASTn ( against the nr/nt nucleotide collection 17th July 2019; top 1000 hits ) to identify orthologues in the NCBI nucleotide database , to the partial sequence ( amino acids 256–339 of the reference sequence NP_060454 . 3 ) , corresponding to the CW and PWWP domains of ZCWPW1 . Protein sequences were then aligned against full-length human ZCWPW1 ( NP_060454 . 3 ) using BLASTP2 . 9 ( Altschul et al . , 2005; Altschul et al . , 1997 ) . We obtained taxonomy information from the NIH classification https://ftp . ncbi . nlm . nih . gov/pub/taxonomy/new_taxdump/ , for comparisons among species and between ZCWPW1 and PRDM9 . Previous work has identified highly conserved ‘KWR’ and ‘PWWP’ patterns among CW and PWWP domains , respectively ( He et al . , 2010b; Qin and Min , 2014 ) . We thus identified an initial set of ‘clear’ ZCWPW1 orthologues , and then used these to identify further less precise matches . ‘Clear’ orthologues are defined as proteins within set one containing perfect matches to both these sequences , and such that in the NCBI alignment , at least 39% of each sequence aligns to the human protein , and conversely . This second step is required to avoid spurious matches to , for example , ZCWPW2 , which overlaps 19% of ZCWPW1 , by requiring >2 fold this match length; although several ZCWPW2 copies are in our initial list , no gene annotated as most similar to ZCWPW2 attains the 39% overlap . For proteins within the ‘clear’ set , we identified the longest alignment for each given species , resulting in 136 species with a likely ZCWPW1 orthologue . This initial screen identified three groups of fish , placental mammals , marsupials , monotremes and reptiles as likely possessing ZCWPW1 , similar to our final conclusions . Moreover , it defined amino acids 247–428 in the human reference sequence as being conserved in the alignment ( at most one sequence not aligning ) , including the annotated CW and PWWP domains . Using this initial set , we identified additional orthologous sequences and refined our results , by identifying conserved bases within ZCWPW1 . Specifically , we divided the 136 species into major clades , as in Baker et al . , 2017 , and gave each sequence a weighting so that the overall weight for each clade was the same ( so , for example , each Placental mammal sequence was downweighted as this clade was over-represented ) . Within the consistently aligned region 247–428 , we calculated the overall weighted probability of each of the 20 amino acids , or a gap ( adding 10−5 to exclude zero weights ) . This identified 31 completely conserved amino acids , and 78 amino acids whose entropy was below 1 ( equivalent to two amino acids having equal probability , so implying one amino acid present in >50% of species ) . Finally , we defined ZCWPW1 orthologues as those sequences matching at least 90% of those 31 perfectly conserved bases which were aligned , and aligning to at least 50% of these bases ( this last condition allows for inclusion of incomplete sequencing or protein assembly ) . We note that while this 90% condition is arbitrary , by definition all annotated orthologues exceed this threshold , while thresholds below ~70–80% are exceeded by orthologues of ZCWPW2 among other genes , offering some justification . Nonetheless , it is worth pointing out that our analysis does not rule out the existence of more poorly conserved ZCWPW1 copies in more distantly related species . This approach identified a final set of 167 genes , which we annotated as likely ZCWPW1 orthologues and used for the majority of results . For each ZCWPW1 orthologue , we also identified the taxonomic relationship of the closest species possessing such a PRDM9 copy ( Figure 1—source data 1 ) . We also reciprocally annotated each PRDM9 copy previously identified ( Baker et al . , 2017 ) according to the closest species also possessing ZCWPW1 ( Figure 1—source data 1 ) . Our analyses revealed a relationship between ZCWPW1 predicted functional domains and PRDM9 histone modifications . Therefore , we identified additional potential conserved sites by identifying perfectly conserved bases among those species possessing both ZCWPW1 and PRDM9 orthologues , and where three key SET domain catalytic amino acids within PRDM9 are intact , meaning PRDM9 is predicted to have normal histone modification activity ( Baker et al . , 2017 ) . This identified a slightly larger number of conserved amino acids ( 37 ) . Only eight of these varied in any of the 167 species with a potential ZCWPW1 orthologue: 260 , 404 and 411 ( in two Xenopus frogs , with 411 also in white-headed capuchin ) , 19 and 22 ( in three canines ) , 25 ( in Ocelot gecko ) , 257 ( in Anolis lizard and in Wombat ) , 325 ( in elephantfish ) . While these changes might alter ZCWPW1 function , the true behaviour of ZCWPW1 is uncertain in these cases , although it is interesting that canids and frogs represent clades that all appear to have lost PRDM9 , and possess multiple , clustered , amino acid changes . pDomThreader ( Lobley et al . , 2009 ) was used via the PSIPRED server ( http://bioinf . cs . ucl . ac . uk/psipred/ ) on the following uniProt amino acid sequences ( Q9H0M4|ZCPW1_HUMAN , Q6IR42|ZCPW1_MOUSE , E2RFJ2|E2RFJ2_CANLF , M3XJ39_LATCH , A0A3S5ZP38_BOVIN , M3WDY6_FELCA , and G3ULT5|G3ULT5_LOXAF ) all of which identified a match to 1ub1A00 in the C terminal section after the PWWP domain , except in LOXAF in which this match was slightly below the p-value threshold of 0 . 001 . SCP1 prediction used NCBI conserved domain search server ( https://www . ncbi . nlm . nih . gov/Structure/cdd/wrpsb . cgi ? ) with the uniProt amino acid sequences Q9H0M4|ZCPW1_HUMAN and Q6IR42|ZCPW1_MOUSE . KO mice for Zcwpw1 ( C57BL/6N-Zcwpw1em1 ( IMPC ) Tcp ) were generated by the Toronto Centre for Phenogenomics ( Canada ) . A 1485bp deletion on chromosome 5 spanning exons 5 to 7 of Zcwpw1 ( chr5:137799545–13780101029 ) was engineered by CRISPR/Cas9 using guide RNAs 5’-GACTGCACTCACGGCCATCT-3’ and 5’-GCCCGGTTCTTCATCCAATT-3’ . The frameshift deletion introduces a stop codon in Exon 8 , leading to a predicted unstable short truncated 492bp transcript . Mice were genotyped at the Zcwpw1 locus using the following primers , and standard cycling conditions: KO allele-Forward , 5’-CACAGGCTCATGTATGTTTGTCTC-3’; KO allele-Reverse , 5’-CTGCTTCGTCCTCTTTCCTTATCTC-3’; WT allele-Forward , 5’-TGCCACCACACTTCATTTGT-3’; WT allele-Reverse CCTGTTTCCTTCCCAACTCA-3’ . The deletion was verified by direct Sanger sequencing of the KO genotyping PCR product ( Source Bioscience , UK ) , following purification with the QIAquick PCR Purification Kit ( Qiagen ) . Sequence analysis was carried out using Chromas LITE ( version 2 . 1 . 1 ) . KO mice for Prdm9 were described previously ( Hayashi et al . , 2005; Mihola et al . , 2019 ) and obtained from the RIKEN BioResource Research Center in Japan ( strain name B6 . 129P2-Prdm9 < tm1Ymat> , strain number RBRC05145 ) . Mice were genotyped at the Prdm9 locus using the following primers , and standard cycling conditions: WT allele-Forward 5’-AGGAATCTTCCTTCCTTGCTGTCG-3’; WT allele-Reverse 5’-ATTTCCCTGTATCTTCTTCAGGACT-3’; KO allele-Reverse 5’-CGCCATTCAGGCTGCGCAACTGTT-3’ . All animal experiments received local ethical review approval from the University of Oxford Animal Welfare and Ethical Review Body ( Clinical Medicine board ) and were carried out in accordance with the UK Home Office Animals ( Scientific Procedures ) Act 1986 . Fertility was assessed in mice ranging from 9 to 12 weeks of age , either by mating with WT littermates and recording the average litter size and frequency , or by measuring paired testes weight ( normalized to lean body weight ) , sperm count ( per paired epididymides ) and chromosome synapsis rate ( by immunostaining of pachytene spermatocytes ) in males . Lean body weight was measured using the EchoMRI-100 Small Animal Body Composition Analyzer . Mouse testis chromosome spreads were prepared using surface spreading ( Barchi et al . , 2008; Peters et al . , 1997 ) and immunostained as previously described ( Davies et al . , 2016 ) . The following primary antibodies were used: custom ZCWPW1 rabbit antiserum ( 1:100 dilution ) , mouse anti-SYCP3 ( Santa Cruz Biotechnology sc-74569 , RRID:AB_2197353 ) or biotinylated rabbit anti-SYCP3 ( Novus NB300-232 , RRID:AB_2087193 ) , rabbit anti-DMC1 ( Santa Cruz Biotechnology sc-22768 , RRID:AB_2277191 , discontinued; 1:100 dilution ) , rabbit anti-HORMAD2 ( Santa Cruz Biotechnology sc-82192 , RRID:AB_2121124; 1:300 dilution ) , mouse anti-RAD51 ( Abcam ab88572 , RRID:AB_2042762; 1:50 dilution ) , rabbit anti-RPA2 ( Abcam ab10359 , RRID:AB_297095; 1:1000 dilution ) , mouse ( Sigma-Aldrich 05–636 , RRID:AB_309864; 1:250 dilution ) or chicken ( Biorbyt orb195374 , discontinued; 1:1000 dilution ) anti-phospho γ-H2AX and Alexa Fluor 488- , 647- or 594-conjugated secondary antibodies against rabbit , mouse or chicken IgG/Y ( A-11008 , RRID:AB_143165; A-11001 , RRID:AB_2534069; A11012 , RRID:AB_141359; A-1105 , RRID:AB_141372; A-21235 , RRID:AB_2535804; A-21449 , RRID:AB_2535866; Thermo Fisher Scientific; 1:250 dilution ) , as well as streptavidin Alexa Fluor 647 ( Thermo Fisher Scientific ) were used to detect the primary antibodies . Images were acquired using either a BX-51 upright wide-field microscope equipped with a JAI CVM4 B and W fluorescence CCD camera and operated by the Leica Cytovision Genus software , or a Leica DM6B microscope for epifluorescence , equipped with a DFC 9000Gt B and W fluorescence CCD camera , and operated via the Leica LASX software . Image analysis was carried out using Fiji ( ImageJ-win64 ) . The specificity of the ZCWPW1 signal was verified using the pre-immune serum from the same rabbit: no staining was visible at any stage of prophase I ( Figure 2—figure supplement 1B shows representative images of mid-zygotene and early pachytene cells ) . Staging and sub- ( early , mid and late ) staging of spermatocytes during prophase I was carried out according to defined staining patterns against SYCP1 , SYCP3 , and DAPI-stained DNA appearance , and morphological criteria previously published ( Gaysinskaya et al . , 2014 ) . Briefly , cells showing large chromocentres ( brightly stained by DAPI and corresponding to pericentromeric regions ) with short tracts of SYCP3 were assigned as ( mid- ) leptotene . Cells with larger pooled chromocentres , associated with longer tracts of SYCP3 were identified as early zygotene . Cells where some synapsis was initiated ( with more than two paired SYCP3 tracts , resulting in a thicker synaptonemal complex region ) , were assigned as ( mid- ) zygotene . Cells where the majority of chromosomes were synapsed , with three or less completely or partially asynapsed , were identified as late zygotene . Following immunostaining of spermatocytes , centromeres and telomeres were labelled using a biotin labelled minor satellite probe ( pIRESMinor , Chan et al . , 2017 ) together with the Telomere PNA FISH Kit/Cy3 ( Agilent ) , following the manufacturer’s instructions , but without protease treatment . The hybridisation and signal detection were carried out using standard techniques . Constructs encoding full-length human PRDM9 ( B allele ) in frame with a C-terminal V5 tag and a self-cleaving YFP tag ( hPRDM9-V5-YFP ) , or the chimp construct ( cPRDM9-V5-YFP ) where the sequence that encodes the human zinc finger array in hPRDM9-V5-YFP was replaced with the equivalent sequence from the chimp PRDM9 w11a allele , for expression in mammalian cells ( pLENTI CMV/TO Puro DEST backbone vector ) were described previously ( Altemose et al . , 2017 ) . Constructs encoding full-length human ( h ) and mouse ( m ) ZCWPW1 or ZCWPW2 for expression in mammalian cells were purchased from GenScript ( hZCWPW1 and hZCWPW2 cDNA with a C-terminal HA tag in pCDNA3 . 1 clones ID OHu16813 and OHu31001C , respectively ) and OriGene ( mZCWPW1 cDNA with a FLAG-Myc dual tag in pCMV6-Entry; clone ID MR209594 ) , respectively . mZCWPW2 cDNA was synthesised by GenScript and subcloned into pCMV6-Entry in-frame with a C-terminal FLAG tag . For expression in E . coli , full-length mZCWPW1 cDNA was subcloned into the pET22b ( + ) vector ( Novagen ) with a C-terminal poly-His tag . pET22b-mZCWPW1-His construct was transformed into BL21 ( DE3 ) E . coli cells ( Thermo Fisher Scientific ) . Bacterial cultures were grown to a density with O . D600 ~0 . 7 , and expression of recombinant His-tagged ZCWPW1 protein was induced overnight at 20°C by addition of IPTG to 0 . 5 mM . The protein was purified using TALON metal affinity resin , according to the manufacturer’s instructions ( Takara 635502 ) . Further purification was carried out by size exclusion chromatography ( Superdex HiLoad 200 16/60 , GE Life Sciences ) . WB validation was carried out as described previously ( Altemose et al . , 2017 ) , using mouse anti-human ZCWPW1 ( Sigma-Aldrich SAB1409478; 1:2000 dilution ) and mouse anti-polyhistidine ( Sigma-Aldrich H1029 , RRID:AB_260015; 1:2000 dilution ) antibodies . The purified protein was used to immunise two rabbits ( Eurogentec , Belgium ) , and the resulting immune antisera ( and pre-immmune sera from the same rabbits ) were tested against the recombinant antigen by ELISA ( Eurogentec ) , the overexpressed FLAG-tagged mouse ZCWPW1 protein ( and the closely related ZCWPW2 protein to address specificity ) in transfected HEK293T cells , and the endogenous protein in mouse testes ( Figure 2—figure supplement 1 ) . Both antisera showed high titers and similar reactivity by IP , WB and IF staining . The antiserum with the lowest level of background ( corresponding to the signal detected by the pre-immune serum ) was chosen to carry out the experiments . Human embryonic kidney ( HEK ) 293 T cells were purchased from the ATCC ( ATCC CRL-3216 ) , with a certificate of analysis confirming cell line identity by Short Tandem Repeat profiling and lack of mycoplasma contamination . All experiments were carried out on cells cultured for less than five passages from the purchased stock reference strain . Cells were cultured and transfected using Fugene HD as previously described ( Altemose et al . , 2017 ) . High and comparable expression of the target proteins across all samples was verified by IF staining of duplicate transfected cultures before proceeding to ChIP ( Figure 5—figure supplement 4 ) . PRDM9 expression was visualised either directly from live cells through YFP fluorescence , or by IF staining of fixed cells using rabbit anti-V5 ( Abcam ab9116 , RRID:AB_307024; 1:500 dilution ) as described previously ( Altemose et al . , 2017 ) . ZCWPW1-HA was detected by immunostaining as above , using mouse anti-HA ( Sigma-Aldrich H3663 , RRID:AB_262051 , 1:500 dilution ) . The fraction of cells co-expressing ZCWPW1-HA and PRDM9-V5 was determined by co-staining with both antibodies , as above ( Figure 5—figure supplement 4 ) . To assess the specificity of the ZCWPW1 antiserum , cells transfected with FLAG-tagged mZCWPW1 or mZCWPW2 were co-immunostained with the antiserum ( 1:100 dilution ) and a mouse anti-FLAG ( Sigma-Aldrich F3165 , RRID:AB_259529; 1:500 dilution ) . Images were acquired using a ZOE fluorescent cell imager ( Bio-Rad ) . RT-qPCR analysis of human PRDM9 , ZCWPW1 and ZCWPW2 transcript expression in HEK293T cells was performed as previously described ( Altemose et al . , 2017 ) , with minor modifications listed here . RNA was extracted from one biological replicate per sample using TRI Reagent ( Sigma-Aldrich T9424 ) , following the manufacturer’s guidelines . For qPCR , each sample was analysed in triplicate ( technical replicates ) using primer sets from Origene: GAPDH ( NM_002046 ) forward 5’-GCTCCTCTGACTTCAACAGCG-3’ and reverse 5’-ACCACCCTGTTGCTGTAGCCAA-3’; PRDM9 ( NM_020227 ) forward 5’-ACGAAGAGGCAGCCAACAATGG-3’ and reverse 5’-GCCACCAGGTTCTGCTCTTCAT-3’; ZCWPW1 ( NM_017984 ) forward 5’-GATGGCTCAAGAGGCAGAACAG-3’ and reverse 5’-TGGGCTGTTCAAACCAGAGAGC-3’; ZCWPW2 ( NM_001040432 ) forward 5’- AAGAGCTGGAGCAAATGCTGCAG-3’ and reverse 5’-CAGGAGCTTCTGGGCTGCATTT-3’ . PCR reactions were carried out in triplicate ( three technical replicates per sample ) , using Fast SYBR Green Master Mix according to the manufacturer’s instructions ( Applied Biosystems 4385610 ) . Relative gene expression was calculated using the ΔΔCt method after averaging the technical replicates for each sample and normalising to the GAPDH gene . Raw Ct values and detailed calculations are given in Figure 5—source data 1 . Cell and testes protein extracts were prepared in lysis buffer containing 50 mM Tris-HCl pH8 . 0 , 150 mM NaCl , 1% Triton X-100 and a cocktail of protease inhibitors ( Sigma-Aldrich P8340 ) , followed by gentle rotation at 4°C for 30 to 45 min , respectively . Cell debris were pelleted by centrifugation at 4°C for 20 min at 20 , 000 g . Protein extracts were quantified using the Pierce BCA protein assay kit ( Thermo Fisher Scientific 23227 ) and equal quantities were incubated overnight at 4°C with 5 or 10 µl ( on transfected cells and mouse testis , respectively ) of rabbit ZCWPW1 immune antiserum or pre-immune serum , or 3 µg of mouse anti-FLAG antibody ( Sigma-Aldrich F3165 , RRID:AB_259529 ) , setting aside 5–100 µg of protein extract for direct western blot detection . Immunocomplexes were pulled down with 25–75 µl of Dynabeads M-280 sheep anti-rabbit ( 11203D , RRID:AB_2783009 ) or anti-mouse ( 11202D , RRID:AB_2783009 ) IgG ( Thermo Fisher Scientific ) for 2 hr at 4°C with gentle rotation . After five washes in lysis buffer , they were eluted from the beads by boiling in Laemmli sample buffer for 5 min . Immunocomplexes and total protein extracts were resolved on either 8% or 4–20% Novex Tris-Glycine precast gels ( Thermo Fisher Scientific XP00080BOX , XP04200BOX ) . Proteins were transferred onto PVDF membranes and the proteins of interest were detected by western blotting following standard procedures . Blots were blocked for 1 hr at room temperature ( RT ) in PBS containing 0 . 2% Tween-20 ( PBS-T ) and 5% milk , and incubated for 1 hr at RT with rabbit ZCWPW1 immune antiserum or pre-immune serum ( 1:1000 dilution ) , mouse anti-β-actin ( Sigma-Aldrich A1978 , RRID:AB_476692 , 1:2000 dilution ) , or mouse anti-FLAG antibody ( Sigma-Aldrich F3165 , RRID:AB_259529; 1 , 2000 dilution ) , washed three times in PBS-Tween buffer and incubated for 1 hr at RT with HRP-conjugated donkey anti-rabbit ( NA934 , RRID:AB_772206 ) or sheep anti-mouse ( NA931 , RRID:AB_772210 ) IgG antibody ( 1:10 , 000 dilution; GE Healthcare ) . Protein signals were revealed using the ECL Prime western blotting detection reagent according to the manufacturer’s recommendations ( GE Healthcare 10308449 ) . The specificity of the ZCWPW1 signal observed in B6 testis extract was verified by immunoprecipitation using the pre-immune serum from the same rabbit: no signal was detected ( Figure 2—figure supplement 1C ) . No cross-reactivity was observed with ZCWPW2 in either B6 testis extract or transfected HEK293T cells overexpressing FLAG-tagged ZCWPW2 ( Figure 2—figure supplement 1C , D ) . For the HEK293T experiments , reads were mapped to either hg38 ( NCBI’s GCA_000001405 . 15_GRCh38_no_alt_plus_hs38d1_analysis_set . fna . gz ) using bwa mem ( version 0 . 7 . 17-r1188 ) ( Li , 2013 ) . Duplicates were removed using picard’s markDuplicates ( version 2 . 20 . 4-SNAPSHOT ) . Unmapped , mate unmapped , non primary alignment , failing platform , and low MAPQ reads were removed using samtools with parameters ‘-q 30 F 3852 f 2 u’ ( version 1 . 9 ( using htslib 1 . 9 ) ) . Other unmapped reads and secondary alignments were removed using samtools fixmate . Fragment position bed files were created using bedtools bamtobed ( v2 . 28 . 0 ) . For the DMC1 mouse experimental data , we processed the data following the algorithm provided by Khil et al . , 2012 to map the reads to the mouse mm10 reference genome ( Lunter and Goodson , 2011 ) , and obtain type I reads . We called DMC1 peaks , as described previously ( Davies et al . , 2016 ) . For the HEK293T experiments , peaks for ZCWPW1 , PRDM9 , H3K4me3 , and H3K36me3 were called using a peak caller previously described ( Davies et al . , 2016 ) and available at https://github . com/MyersGroup/PeakCaller ( archived at https://doi . org/10 . 5281/zenodo . 3783600 ) . Single base peaks were called with parameters pthresh 10−6 and peakminsep 250 on 22 autosomes . For each experiment , we used available IP replicates , and sequenced input DNA to estimate background , in calling peaks . Most of our analyses refer to these peaks , except in Figure 5A , B which plot the change in ZCWPW1 occupancy following PRDM9 transfection , for either the chimp or human PRDM9 alleles . To estimate ZCWPW1 levels with vs without PRDM9 , we called ZCWPW1 peaks in HEK293T cells also transfected with PRDM9 as before , but now replacing the ‘input’ lane with the IP data for ZCWPW1 in cells not transfected with PRDM9 . Figure 5—figure supplement 6 also uses this measure , of ZCWPW1 recruitment attributable to PRDM9 binding , to predict DMC1 levels , while Figure 5—figure supplement 2 ( right panel ) checks that changes in ZCWPW1 occupancy occur largely at PRDM9-bound sites , and Figure 5—figure supplement 1 shows the correlation of PRDM9 binding strength with the change in ZCWPW1 enrichment . The peak calling algorithm allows for calculation of enrichment and p-values at arbitrary pre-specified locations or windows in the genome , we refer to these as ‘force called’ . Note that the enrichment values from peak calling represent coverage due to signal , after subtracting coverage due to background , relative to background coverage . Hence a value less than one does not imply depletion — any value greater than 0 is enriched . For more details see Altemose et al . , 2017 . Peaks were filtered if: center within 2 . 5kb of PRDM9-independent H3K4me3 ( promoters ) , input coverage ≤5 , in top five by likelihood , greater than 99 . 9 percentile input coverage . For human PRDM9 peaks the PRDM9 motif position ( Altemose et al . , 2017 ) was inferred using the getmotifs function in MotifFinder within the 300bp region around the peak ( with parameters alpha = 0 . 2 , maxits = 10 , seed = 42 , stranded_prior = T ) , and peaks were recentered and stranded at these locations . Peaks within 4kb of one another are removed to avoid double counting . Mean coverage was calculated with bwtool 1 . 0 using the aggregate command with parameters ‘-fill = 0 -firstbase’ at a width of +/− 2kb . Each profile was normalised by the total coverage of all of the fragments and the normalised sample profile was divided by the relevant normalised comparison/control dataset to calculate enrichment . Random profiles were created using bedtools random with 10 , 000 locations , with seed 72346 , and were also filtered such that none were within 4kb of one another . Mapped type one reads were filtered by removing non-canonical chromosomes ( with underscores in names ) , sex chromosomes , and mitochondrial reads . Bedgraphs were converted to bigWig format using UCSC bedGraphToBigWig v4 . Profiles were created using bwtool 1 . 0 using the aggregate command with parameters 5000:2000 and -fill = 0 . B6 WT hotspot locations were filtered to remove non-autosomal chromosomes , retain only B6 allele hotspots , and remove hotspots with PRDM9-independent H3K4me3 . Prdm9−/− hotspots were filtered to remove the X chromosome . Motif centered coordinates were used for WT hotspots where the motif positions were inferred using the MotifFinder software and PRDM9 position weight matrices from Altemose et al . , 2017 . To normalise background signal , the mean signal between −5000 and −3000 was subtracted from each strand-mouse combination . Mean coverage was normalised by the sum of coverage over each strand-mouse combination across both WT and KO locations . Alu locations were downloaded from UCSC tables and filtered for Alu repeats with a width between 250 and 350bp . DNA sequence was extracted from the genome ( Ensembl 95 h38 primary assembly ) at these locations using the bedtools getfasta command and CpG dinucleotides were counted using ‘stringr::str_locate_all’ command in R . Data is from Libertini et al . , 2015 ( GSE51867 ) , non CpG methylated sites were removed , and the two strands were summed . High copy number ( >1 . 5 ) CpGs were removed . PRDM9-dependent ZCWPW1 enrichment was force-called at the human PRDM9 peaks . Human testis DMC1 sites are from Pratto et al . , 2014 ( GSE59836 ) and were subsetted to ‘A or B’ autosomal peaks with a width less than 3kb . These peaks were then trimmed to a maximum of 800bp before overlapping with PRDM9 peaks to create a binary target variable . Regions with input coverage ≤5 in either predictor were removed in addition to outliers with input >200 and/or ZCWPW1 enrichment >10 . A binomial generalised linear model with a logit link ( logistic regression ) was fitted using the ‘glm’ function from the ‘stats’ package in R ( 3 . 6 . 0 ) . Chromosomes 1 , 3 , and 5 were used as the test data with the remaining autosomes being the training data . Regions were extracted from bigWig using bwtool matrix-fill = 0 -decimals = 1 -tiled-averages=5 , and width parameters 2000 . Coverage was normalised by total coverage ( scaled by 1010 ) . A pseudocount of 1 was added to both input and sample , and the sample was then normalised by the input for each region . Values outside the quantile range 0 . 01–0 . 99 were thresholded . The profile plots are created by taking the ratio of the mean coverage of the sample and input separately . Ordering of the regions was determined by the mean coverage of a 200bp window centered on the peak center . Computational analysis was performed using R ( R Development Core Team , 2018 ) and snakemake ( Köster and Rahmann , 2012 ) . BigWig aggregations by bwtool ( Pohl and Beato , 2014 ) , BED manipulation with BEDtools ( Quinlan and Hall , 2010 ) , and FASTA processing with seqkit ( Shen et al . , 2016 ) . We also used BigWig tools ( Kent et al . , 2010 ) , IGV ( Thorvaldsdóttir et al . , 2013 ) , and GNU parallel ( Tange , 2018 ) . Plots were created using the ggplot2 package ( Wickham , 2016 ) and extensions ggforce ( Pedersen , 2016 ) , RColorBrewer ( Neuwirth , 2014 ) , viridis ( Garnier , 2018 ) , drawProteins ( Brennan , 2018 ) , and cowplot ( Wilke , 2018 ) . In addition , the following R packages were used: data . table ( Dowle and Srinivasan , 2019 ) , mgcv ( Wood , 2011 ) , PRROC ( Grau et al . , 2015 ) , sangerseqR ( Hill et al . , 2014 ) , and ComplexHeatmap ( Gu et al . , 2016 ) . DMC1 SSDS ChIP-seq in human testis is from Pratto et al . , 2014 ( GSE59836 ) . DMC1 SSDS ChIP-seq ( Brick et al . , 2012 ) ( GSE35498 ) , H3K4me3 ChIP-seq ( Davies et al . , 2016 ) ( GSE73833 ) , and SPO11 oligo-seq ( Lange et al . , 2016 ) ( GSE84689 ) are in WT B6 mouse testis . PRDM9 ( h/c-V5 tagged ) , H3K4me3 , and H3K36me3 ChIP-seq in HEK293T cells is from Altemose et al . , 2017 ( GSE99407 ) . Whole-genome bisulfite sequencing ( BS-seq ) of HEK293 cells is from Libertini et al . , 2015 ( GSE51867 ) .
During meiosis , homologous chromosomes pair and recombine , enabling balanced segregation and generating genetic diversity . In many vertebrates , double-strand breaks ( DSBs ) initiate recombination within hotspots where PRDM9 binds , and deposits H3K4me3 and H3K36me3 . However , no protein ( s ) recognising this unique combination of histone marks have been identified . We identified Zcwpw1 , containing H3K4me3 and H3K36me3 recognition domains , as having highly correlated expression with Prdm9 . Here , we show that ZCWPW1 has co-evolved with PRDM9 and , in human cells , is strongly and specifically recruited to PRDM9 binding sites , with higher affinity than sites possessing H3K4me3 alone . Surprisingly , ZCWPW1 also recognises CpG dinucleotides . Male Zcwpw1 knockout mice show completely normal DSB positioning , but persistent DMC1 foci , severe DSB repair and synapsis defects , and downstream sterility . Our findings suggest ZCWPW1 recognition of PRDM9-bound sites at DSB hotspots is critical for synapsis , and hence fertility .
Sexual reproduction – that is , the combination of sex cells from two different individuals to produce an embryo – is one of the many mechanisms that have evolved to maintain genetic diversity . Most human cells contain 23 pairs of chromosomes , with each chromosome in a pair carrying either a paternal or maternal copy of the same gene . To form an embryo with the right number of chromosomes , each sex cell ( the egg or sperm cell ) must only contain one chromosome from each pair . Sex cells are produced from parent cells containing two sets of paternal and maternal chromosomes: these cells then divide twice to form four sex cells which contain only one chromosome from each pair . Before the parent cell divides , a process known as ‘recombination’ takes place , which allows chromosomes in a pair to exchange bits of genetic information . This reshuffling ensures that each chromosome in a sex cell is unique . A protein called PRDM9 helps control which sections of genetic information are recombined by modifying proteins attached to the chromosomes , marking them as locations for exchange . The DNA at each of these sites is then broken and repaired using the genetic sequence of the chromosome it is paired with as a template , thus causing the two chromosomes to swap genes . In 2019 , a group of researchers found a set of genes in the testis of mice that are expressed at the same time as the gene for PRDM9 . This suggested that another protein called ZCWPW1 is likely involved in recombination , but the precise role of this protein was unclear . To answer this question , Wells , Bitoun et al . – including many of the researchers involved in the 2019 study – examined human cells grown in the laboratory to determine where ZCWPW1 binds to in the chromosome . This revealed that ZCWPW1 can be found at the same sites as PRDM9 , which is responsible for bringing it there . Furthermore , cells from male mice lacking the gene for ZCWPW1 cannot complete the exchange of genetic information between chromosomes , meaning that the mice are infertile . As such , ZCWPW1 seems to connect location selection by PRDM9 to the DNA repair mechanisms needed for gene exchange between chromosomes . Infertility is a significant issue for humans affecting as many as one in every six couples . Fertility is complex and many of the biological mechanisms involved are not fully understood . This work suggests that both PRDM9 and ZCWPW1 are key to the production of sex cells and may be worth investigating as factors that affect fertility in humans .