HEIP1 regulates crossover formation during in rice

Yafei Lia,b,1, Baoxiang Qina,c,1, Yi Shena, Fanfan Zhanga, Changzhen Liua, Hanli Youa, Guijie Dua, Ding Tanga, and Zhukuan Chenga,b,2

aState Key Laboratory of Plant Genomics and Center for Plant Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 100101 Beijing, China; bJiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, 225009 Yangzhou, China; and cState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, Guangxi University, 530005 Nanning, China

Edited by David C. Baulcombe, University of Cambridge, Cambridge, United Kingdom, and approved September 6, 2018 (received for review May 7, 2018) During meiosis, the number of double-strand breaks (DSBs) far and MSH5 form a heterodimer complex, which binds to exceeds the final number of crossovers (COs). Therefore, to single-Holliday junctions or three-armed progenitor Holliday junc- identify proteins involved in determining which of these DSBs tions as a sliding clamp, embracing the homologous repaired into COs is critical in understanding the mechanism of CO and promoting the formation of COs (11). ZIP2 is related to WD40- control. Across species, HEI10-related proteins play important roles like repeat which appears to function in the promotion/ in CO formation. Here, through screening for HEI10-interacting stabilization of single-end invasion, while ZIP4 is a tetratricopeptide proteins via a yeast two-hybrid system, we identify a CO protein repeat protein, which may facilitate specific interactions with a HEI10 Interaction Protein 1 (HEIP1) in rice. HEIP1 colocalizes with partner protein(s) (12). Yeast ZIP3 homologs have been identified HEI10 in a dynamic fashion along the meiotic chromosomes and in many other model organisms, and the ZIP3 family members are specially localizes onto crossover sites from late pachytene to dip- divided into two subgroups: the ZIP3/RNF212 and HEI10 (13, 14), lotene. Between these two proteins, HEI10 is required for the based on their sequence similarity network. The two subgroups loading of HEIP1, but not vice versa. Moreover, mutations of the display distinct differences in enzymatic activity (15–17), although HEIP1 gene cause the severe reduction of chiasma frequency, they share similarities in protein structure and dynamic localization

whereas early processes are not dis- patterns (18), and both are required for the formation of COs. The PLANT BIOLOGY turbed and synapsis proceeds normally. HEIP1 interacts directly ZIP3/RNF212 group members appear to act solely as SUMO E3 with ZIP4 and MSH5. In addition, the loading of HEIP1 depends ligases (15), whereas the HEI10 group members appear to exhibit on ZIP4, but not on MER3, MSH4, or MSH5. Together, our results more than just ubiquitin E3 ligase activity (16, 17). suggest that HEIP1 may be a member of the ZMM group and acts Here we report the identification of a protein, HEI10 Interaction as a key element regulating CO formation. Protein 1 (HEIP1), which interacts directly with ZMM proteins HEI10, ZIP4, and MSH5, and is essential for normal meiotic CO rice | meiosis | crossover formation | HEIP1 formationinrice.LossofHEIP1 results in decreased crossover frequency during meiosis. We also show that HEIP1 displays a eiosis is a reductional type of cell division during which a dynamic localization pattern during meiosis, and only a few bright Msingle round of DNA replication is followed by two rounds foci are retained on the meiotic chromosomes from late pachytene of cell division, thus halving ploidy levels to produce haploid to diplotene, which completely colocalize with HEI10. HEIP1 may gametes. Accurate segregation of homologous chromosomes in be a type of CO protein involved in the process of CO formation. meiosis I is dependent on the formation of crossovers (COs). At least two types of COs coexist in most eukaryotes. Class I COs Results are sensitive to interference, a mechanism which ensures COs Isolation and Characterization of HEIP1. During meiosis, HEI10 are more evenly spaced along a homologous pair than would be marks the sites of crossovers in mice, rice, and Arabidopsis (13, expected by chance, and accounts for most COs. Class II COs do not exhibit interference (1). Mammals, budding yeast (Saccha- Significance romyces cerevisiae), and plants all have both types of COs (2), but some organisms have only one or the other (3, 4). According to Crossovers (COs) ensure the accurate segregation of homolo- the double-strand break (DSB) repair model in S. cerevisiae (5), gous chromosomes during meiosis. Failure to create the right the formation of class I COs is dependent on a set of meiosis- number of crossovers may lead to unequal distribution of ge- specific proteins, collectively referred to as ZMM proteins netic materials to daughter cells and sterility. CO proteins, (ZIP1, ZIP2, ZIP3, ZIP4, MER3, MSH4, and MSH5). ZMM which specially localize to crossover sites during meiosis, play proteins promote crossovers in the same recombination pathway critical roles in CO formation. Here, we identify a CO protein in as shown by the similarity in phenotypes among zmm mutants rice, named HEI10 Interaction Protein 1 (HEIP1), which is con- and colocalization of ZMM proteins (6). Several presumed ho- served in higher plants. Our results reveal its possible molec- mologs of ZMM have been identified in plants, mice, and other ular mechanism for CO control in meiosis. organisms, suggesting these proteins are conserved. Additional proteins involved in class I CO events include parting dancers Author contributions: Y.L., Y.S., D.T., and Z.C. designed research; Y.L., B.Q., Y.S., F.Z., C.L., (PTD) in Arabidopsis (7) and crossover-associated 1 (COSA-1) H.Y., G.D., and D.T. performed research; and Y.L., B.Q., and Z.C. wrote the paper. in (8). The authors declare no conflict of interest. Among ZMM proteins, ZIP1 is a central element (CE) com- This article is a PNAS Direct Submission. ponent of the synaptonemal complex (SC) (9), a meiosis-specific Published under the PNAS license. tripartite structure. MER3 is a DNA helicase protein that un- 1Y.L. and B.Q. contributed equally to this work. winds duplex DNA of various lengths in the 3′-to-5′ direction as 2To whom correspondence should be addressed. Email: [email protected]. an ATP-dependent process and extends the DNA joint molecule This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. made by RAD51 to facilitate the formation of double-Holliday 1073/pnas.1807871115/-/DCSupplemental. junctions during meiotic homologous recombination (10). MSH4

www.pnas.org/cgi/doi/10.1073/pnas.1807871115 PNAS Latest Articles | 1of6 Downloaded by guest on September 25, 2021 16, 19). To gain more insight into the CO maturation, we protein (CFP) signals were detected in cells coexpressing HEI10- screened for HEI10-interacting proteins via a two-hybrid system CFPN and HEIP1-CFPC as well as HEIP1-CFPN and HEI10- in yeast. A GAL4 activation domain (AD) fusion cDNA library CFPC (Fig. 1C). The in vivo Co-IP assays were also conducted. was prepared from young rice panicles and positive clones were As a result, HEIP1-Flag fusion proteins were immunoprecipitated selected for sequencing. Among them, one reproducible, positive with HEI10-Myc when they were transiently coexpressed in rice clone was identified, belonging to the Os01g0167700 gene. This protoplasts (Fig. 1D). gene was named HEI10 Interaction Protein 1 (HEIP1). The full- length cDNA sequence of HEIP1 was isolated by RT-PCR and HEIP1 Is the Founding Member of a Gene Family Conserved in Higher RACE. The HEIP1 cDNA was 3,476 nucleotides long and was Plants. A 2,808-bp HEIP1 ORF is predicted to encode a 935-aa composed of a 2,808-bp ORF, a 470-bp 5′-noncoding region, and protein. Searches of public databases using the Position-Specific a 198-bp 3′-noncoding region (SI Appendix, Fig. S1). Sequence Iterative Basic Local Alignment Search Tool (PSI-BLAST) from comparison between genomic DNA and cDNA revealed that the the National Center for Biotechnology Information (NCBI) HEIP1 gene was composed of 19 exons and 18 introns (Fig. 1A). website were conducted to identify HEIP1 orthologs. Proteins To verify the interaction between HEI10 and HEIP1, we cloned with sequence similarity to this gene were identified in multiple the full-length coding sequences encoding HEI10 and HEIP1 species, including LOC100835007 in Brachypodium distachyon into pGBKT7 and pGADT7 expression vectors, respectively. (64% identity and 75% similarity over a 968-aa region), The cotransformed yeast cells grew on both double dropout LOC103634850 in Zea mays (57% identity and 69% similarity medium (DDO, SD/-Leu/-Trp) and quadruple dropout medium over a 944-aa region), and AT2G30480 in Arabidopsis thaliana (QDO, SD/-Ade/-His/-Leu/-Trp) supplemented with X-α-Gal (28% identity and 45% similarity over a 475-aa region). We did and aureobasidin A, suggesting that HEI10 interacts with HEIP1 not identify candidate orthologs of HEIP1 outside the plant (Fig. 1B). Furthermore, the interaction between HEI10 and kingdom. Furthermore, additional NCBI DELTA-BLAST, Uni- HEIP1 was also validated by a bimolecular fluorescence comple- port BLAST (https://www.uniprot.org/), and EMBL-EBI HMMER mentation (BiFC) assay using rice protoplasts. Cyan fluorescent BLAST (https://www.ebi.ac.uk/Tools/hmmer/) searches were con- ducted; we could not identify the likely ortholog of HEIP1 beyond the plant kingdom either, suggesting HEIP1 likely arose in plants. We aligned the HEIP1 amino acid sequence with representative protein sequences and constructed a neighbor-joining tree for HEIP1 homologs in plants (Fig. 1E and SI Appendix,Fig.S2). We also performed a search with the full-length protein sequences of HEIP1 against motif libraries (https://www.genome.jp/tools/motif/). A potential GCK domain (E value 0.94) was detected at positions 438–488 of HEIP1 from the NCBI’s conserved domain database (SI Appendix,Fig.S2), which might be involved in intracellular signaling pathways or mediate heterodimerization according to the annotation. This domain is found only in plant proteins (https:// www.ncbi.nlm.nih.gov/cdd/).

Mutation of HEIP1 Causes a Sterile Phenotype in Rice. To investigate the function of HEIP1 in detail, we first attempted to screen our sterile mutant library, by using 60Co γ-rays as a mutagen to ir- radiate rice variety Zhongxian 3037, and isolated mutant lines with allelic disruption in Os01g0167700. Using a map-based screening approach, we identified two mutants, heip1-1 and heip1- 2.Theheip1-1 mutant contained a single C-to-T nucleotide substitution in the sixth exon, changing arginine (CGA) at position amino acid 184 to a stop codon (TGA), which led to premature termination of the protein. In heip1-2, a 1-bp deletion in the third exon led to frameshift and the formation of a premature stop codon. We generated two additional mutants designated heip1-3 and heip1-4 by CRISPR-Cas9 targeting (SI Appendix,Fig.S3). We then investigated the fertility in all heip1 mutants and found that mature pollen grains were empty and shrunken in all mutants. Moreover, the heip1 plant did not set seed when pol- linated with wild-type pollen, indicating that micro- and mega- gametogenesis in heip1 were both affected. Aside from complete sterility, the heip1 mutants exhibited normal vegetative growth (SI Appendix, Fig. S3).

Fig. 1. Isolation and characterization of HEIP1.(A) Gene structure of HEIP1 Meiosis Is Disrupted in heip1. To determine the cause of sterility in and mutation information of heip1, including mutation sites of heip1-1, heip1 mutants, we studied meiotic chromosomes of pollen mother heip1-2, heip1-3, and heip1-4. Black blocks represent exons, and un- cells (PMCs) at different meiotic stages in both wild type and the translated regions are shown as gray boxes. (B) HEIP1 interacts with HEI10 in heip1-1 mutant. In the wild type, chromosomes were condensed to yeast two-hybrid assays. AgT and murine p53 were used as positive control. form long thin threads at leptotene. During zygotene and pachy- AD, prey vector pGADT7; BD, bait vector pGBKT7. (C) BiFC assays showing tene, homologous chromosomes paired up and synapsed. From the interaction between HEI10 and HEIP1 in rice protoplasts. (D) Coimmu- noprecipitation assays of Flag-HEIP1 and Myc-Hei10. IB, immunoblot; IP, diplotene to diakinesis, SCs were disassembled, COs between immunoprecipitation. (E) Phylogenetic tree derived from the full-length homologous chromosomes matured into visible chiasmata, chro- amino acid sequences of HEIP1 and its homologs from nine plant species. mosomes condensed further, and 12 bivalents were clearly ob- (F) Meiotic behaviors in the heip1-1 mutant. (Scale bars: 5 μm.) served. The bivalents aligned on the equatorial plate in an ordered

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1807871115 Li et al. Downloaded by guest on September 25, 2021 pattern at metaphase I, and then, homologous chromosomes type deviated significantly from a Poisson distribution among separated and migrated in opposite directions at anaphase I. different PMCs (SPSS K-S test, P < 0.01). These results sug- During the second meiotic division, the sister chromatids of each gested the majority of residual chiasmata in heip1-1 are distrib- chromosome separated, resulting in the formation of four daughter uted randomly (SI Appendix, Fig. S6). cells with 12 chromosomes each (SI Appendix, Fig. S4). To further determine the role of HEIP1 in CO formation, we In the heip1-1 mutant, meiotic chromosome behavior was al- generated a double mutant between hei10 and heip1 and ana- most identical to that of wild type from leptotene to zygotene. lyzed its chiasma number. The mean chiasma number per cell During pachytene, fully aligned chromosomes were detected was 2.64 ± 1.45 (n = 50) in hei10 heip1 (SI Appendix, Fig. S5D), along the whole chromosome (Fig. 1F). However, at diakinesis, which was very close to that in the heip1 single mutant, and the bivalents were found to coexist with univalents in the same remaining chiasmata followed a Poisson distribution (SPSS K-S mutant PMC. At metaphase I, the bivalents aligned at the test, P > 0.05) (SI Appendix, Fig. S6). equator of the spindle, whereas the univalents were distributed randomly. During anaphase I, the bivalents separated normally γH2AX, COM1, and DMC1 Localize Normally in heip1. To examine but the univalents segregated randomly, resulting in an unequal whether CO deficiency is due to an early recombination defect, distribution of chromosomes in the two daughter cells. The we detected γH2AX, COM1, and DMC1, three marker proteins second meiotic division subsequently occurred. A tetrad with present in early meiotic progress, in heip1-1 mutants by immu- uneven chromosomes was detected. Similar defects were ob- nostaining assays. γH2AX, the histone H2AX variant, is con- served during male meiosis in heip1-2, heip1-3, and heip1-4 mu- sidered a reliable marker for DSBs. COM1, the homolog of – tants (SI Appendix, Fig. S5 A C), further indicating that the budding yeast COM1/SAE2, participates in DSB end resection, mutant phenotype was caused by the disruption of HEIP1. whereas DMC1, the meiotic recombinase, mediates single-strand invasion. In wild type, both of these proteins were observed as CO Levels Are Deficient in heip1. We further quantified the chiasma punctuate foci on meiotic chromosomes. Similar levels were frequency at metaphase I in both wild type and the heip1-1 γ mutant using previously described criteria (20). Rod- and ring- observed in the heip1-1 mutant. The number of H2AX foci was not significantly different (unpaired t test, P = 0.848) between shaped bivalents were treated as having one and two chiasmata, ± = ± respectively. In wild type, the mean chiasma frequency per cell meiocytes of heip1-1 (213 57, n 6) and the wild type (218 = ± was 20.85 ± 1.25 (n = 46), while in the heip1-1 mutant, the 27, n 9) (Fig. 2A). The number of COM1 foci in heip1-1 (257 ± 32, n = 8) did not significantly differ (unpaired t test, P = 0.307) chiasma frequency was 2.89 1.50 per cell which corresponds to PLANT BIOLOGY ± = 2.56 ± 1.21 bivalents per PMC (n = 54). Thus, the mutation of from that in wild type (278 51, n 10) (Fig. 2B). In wild type, heip1-1 led to a significant reduction in both chiasma frequency the mean number of DMC1 foci at zygotene was 227 ± 25 (n = (unpaired t test, P < 0.01) and the number of bivalents (unpaired 26). Similarly, the mean number of DMC1 foci in heip1-1 was t test, P < 0.01). The distribution of the remaining chiasmata was 219 ± 29 (n = 24) at the same stage, which did not differ from the also analyzed in the heip1-1 mutant and compared with that in wild type (unpaired t test, P = 0.338) (Fig. 2C). Two additional the wild type. Statistical analysis showed that the residual chi- members of the ZMM family, MER3 and ZIP4, are required for asmata distribution per cell in heip1-1 was consistent with a CO formation, and determining their localization may also in- predicted Poisson distribution [SPSS Kolmogorov–Smirnov (K- dicate potential defects at early steps in crossover formation. In S) test, P > 0.05], while the distribution of chiasmata in wild heip1-1, both MER3 and ZIP4 appeared normal (SI Appendix,

Fig. 2. HEIP1 is not required for the localization of γH2AX, COM1, DMC1, PAIR2, PAIR3, and ZEP1 onto chromosomes. (A) Dual immunolocalization of REC8 (green) and γH2AX (red) in the wild type and heip1.(B) Dual immunolocalization of REC8 (red) and COM1 (green) in the wild type and heip1.(C) Dual immunolocalization of REC8 (red) and DMC1 (green) in the wild type and heip1.(D) Triple immunolocalization of PAIR2 (red), PAIR3 (green), and ZEP1 (blue) in the heip1 meiocytes. (Left) Merge results of PAIR2 and PAIR3. (Right) Merge results of PAIR2, PAIR3, and ZEP1. (Scale bars: 5 μm.)

Li et al. PNAS Latest Articles | 3of6 Downloaded by guest on September 25, 2021 Fig. S7). These results suggest that the early homologous re- combination could be implemented in heip1.

Full Synapsis of Homologous Chromosomes Is Achieved in heip1. Synaptonemal complexes connect homologs along their lengths during the pachytene stage. An SC is a tripartite protein struc- ture consisting of two parallel axial elements (AEs), a structure that forms between sister chromatids before synapsis, also called lateral elements (LEs) after synapsis, and a CE. To explore the role of HEIP1 in synapsis, dual immunodetection was carried out in the heip1-1 mutant with antibodies related to both AEs/LEs and CEs. PAIR2 is only associated with unsynapsed AEs at leptotene and zygotene, and PAIR3 is associated with both unsynapsed AEs and LEs of SC during prophase I, whereas the distribution of ZEP1 signals indicates the extent of synapsis. Detailed analysis of PAIR2, PAIR3, and ZEP1 progression in wild-type meiotic prophase I was described by Wang et al. (21). In heip1-1, the localization of LEs and CEs from leptotene to early pachytene was indistinguishable from that of wild type (Fig. 2D). Therefore, we determined that HEIP1 may not be required for synapsis to proceed. Furthermore, the synapsis initiation sites were quantified by counting the number of ZEP1 stretches in early to midzygotene according to criteria previously described (22). We found that the mean number of synapsis sites per cell was not significantly different between heip1-1 and the wild type (unpaired t test, P > 0.05). In heip1-1, the mean number of ZEP1 stretches was nearly 19 (n = 16, range 8–27), whereas it was about 18 (n = 19, range 9–25) for the wild type.

HEIP1 Proteins Are Present as Punctuate Foci and Colocalize with HEI10. To monitor the spatial and temporal location of HEIP1 during rice meiosis, coimmunolocalization was performed with antibodies against HEIP1 and REC8 (a component of the mei- otic cohesin complex). HEIP1 was first visible as numerous punctuated foci at early leptotene and the mean number of HEIP1 foci increased rapidly, reaching its peak at late leptotene/ early zygotene (324 ± 46, n = 12) (Fig. 3 A and B). With the proceeding of homologous pairing in zygotene, the foci of HEIP1 decreased quickly and only 90 ± 29 foci (n = 12) remained at pachytene (Fig. 3C). However, with the further progression of Fig. 3. The localization pattern of HEIP1 in wild type. HEIP1 (green) presents meiosis, only a few bright foci were retained from late pachytene as punctuate foci on chromosomes (REC8 labeled red). (A)Leptotene.(B)Zy- to diplotene (Fig. 3 D and E). The mean number of HEIP1 foci gotene. (C)Pachytene.(D)Latepachytene.(E) Diplotene. (Scale bars: 5 μm.) per cell at this stage was 25 ± 3(n = 20). They disappeared at diakinesis and could be detected thereafter. HEI10 Is Required for Loading of HEIP1, but Not Vice Versa. Considering A previous study showed that punctuate foci of HEI10 is also the close localization pattern between HEIP1 and HEI10, dual retained at a limited number of sites during late prophase I (19). immunolocalization experiments were performed to investigate To establish the relationship between HEI10 and HEIP1, we the mutual dependency in the loading of HEI10 and HEIP1 onto performed dual immunostaining experiments using antibodies chromosomes. In hei10 meiocytes, no obvious HEIP1 signals were against HEIP1 and HEI10. In the leptotene stage, discrete foci detected (SI Appendix,Fig.S10), suggesting that the proper lo- of HEIP1 almost completely colocalized with foci of HEI10 calization of HEIP1 relies on the presence of HEI10. In heip1-1, (97.80% of HEI10 foci contained HEIP1, 98.23% of HEIP1 foci the loading of HEI10 was indistinguishable from that of wild type contained HEI10; n = 5) (SI Appendix, Fig. S8 A–D). From zy- when small dot signals gradually developed into linear signal from gotene to pachytene, HEI10 began to elongate along chromo- leptotene to early pachytene. However, at late pachytene, the di- somes and still showed high colocalization with HEIP1 foci. vergence was apparent since the prominent foci did not develop Almost all HEI10 short stretches had at least one HEIP1 focus, normally (Fig. 4A). As a result, heip1-1 meiocytes had only 9 ± 2 and most HEIP1 foci localized on the linear HEI10 signals (SI prominent foci, compared to 25 ± 3inwildtype(n = 21) (Fig. 4B). Appendix, Fig. S8 E–H). Interestingly, during diplotene, HEI10 It seems that HEI10 loaded normally onto chromosomes until foci and HEIP1 foci completely colocalized in all wild-type early pachytene in heip1; however, the formation of prominent foci PMCs observed (n = 20) (SI Appendix, Fig. S8 I–K). At diaki- at late pachytene was severely disrupted. Western blotting was also nesis, prominent HEI10 foci appeared and persisted to later performed to test the HEIP1 levels in both wild type and hei10, stages, whereas HEIP1 foci were no longer visible. and vice versa. Our results showed that HEI10 levels in the wild Furthermore, the colocalization pattern between HEIP1 and type and heip1-1 were comparable; nevertheless, almost no HEIP1 HEI10 was investigated in zep1. Although linear signal formation was detected in hei10 (SI Appendix,Fig.S10). of HEI10 was severely disrupted in zep1, prominent foci appeared normal on chromosome axes and also showed high The Interplay of HEIP1 and ZMM Proteins. MER3, ZIP4, MSH4, and colocalization with HEIP1 foci (SI Appendix, Fig. S9). MSH5 are members of the ZMM gene family, whose deletion or

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1807871115 Li et al. Downloaded by guest on September 25, 2021 Fig. 4. The loading pattern of HEI10 in heip1 and interactions between HEIP1 and ZMM proteins. (A) The immunodetection of HEI10 (green) in wild type and heip1 during prophase I. (Scale bars: 5 μm.) (B) Number of the HEI10 foci on chromosomes at late pachytene in wild type and heip1. Each symbol rep- resents a single nucleus. ***P < 0.001. (C) Schematic diagram of full-length and four truncated proteins, A–D, of HEIP1 used in yeast two-hybrid assays. (D) HEIP1 interacts with ZIP4 and MSH5. AD, prey vector pGADT7; BD, bait vector pGBKT7. (E) Coimmuno- precipitation assays of Myc-HEIP1 and Flag-MSH5. IB, immunoblot; IP, immunoprecipitation.

mutation results in a severe reduction in crossover number. In heip1 mutants showed a reduced number of chiasmata with rice, ZIP4, MER3, MSH4, and MSH5 have been well character- remaining chiasmata distributed randomly among cells. Similarly,

ized (23–26). To further characterize the effect of ZMM proteins residual chiasmata in hei10 heip1 mutants also displayed a random PLANT BIOLOGY on HEIP1 localization, fluorescence immunolocalization studies distribution. Second, the HEI10 prominent foci correspond to the were performed using antibodies against REC8 and HEIP1 on class I CO sites in rice. In heip1, the formation of prominent meiocytes of rice zmm mutants. In zip4, HEIP1 was not loaded HEI10 foci was severely disrupted at late pachytene, and none of onto meiotic chromosomes. Almost no immunostaining of HEIP1 them was located on the remaining bivalents at diakinesis. Third, was detected in zip4 meiotic chromosomes, suggesting that normal we revealed that HEIP1 interacted directly with HEI10, ZIP4, and loading of HEIP1 is dependent on ZIP4. However, the localiza- MSH5. Thus, we suspect HEIP1 and ZMM may act in the same tion of HEIP1 in mer3, ,andmsh5 differed from that in zip4. CO formation pathway, which is likely interference sensitive. In mer3, msh4,andmsh5, HEIP1 foci appeared normally at zy- gotene (SI Appendix,Fig.S11). In addition, we performed dual HEIP1 Marks Putative CO Sites from Late Pachytene to Diplotene. We showed that the mean number of HEIP1 foci per cell from late immunostaining experiments using antibodies against HEIP1 and ∼ MER3, raised in mice and rabbits, respectively. As shown in SI pachytene to diplotene was 24 foci, similar to that of HEI10. Appendix,Fig.S12, discrete foci of HEIP1 almost completely Previous studies revealed that HEI10 prominent foci appear during colocalized with MER3 at zygotene. late prophase I, where they marked the class I CO site (19). From The similar function of these proteins in CO formation also late pachytene to diplotene, HEI10 and HEIP1 foci almost com- prompted us to further investigate possible interactions between pletely colocalized in all wild-type nuclei observed. Furthermore, previous studies showed that in zep1 mutants, HEI10 foci (class I HEIP1 and ZMM proteins. We cloned ORFs of HEIP1 and COs) increased ∼1.5-fold compared with that in wild type, which was ZMM proteins into the binding domain (BD) vector pGBKT7 further confirmed by genetic analysis (27). We found that HEI10 and the AD vector pGADT7, which cotransformed into the Y2H prominent foci always colocalized with HEIP1 in zep1 mutants. Gold yeast strain, triggering their coexpression. We confirmed a Across species, there appears to be four groups of proteins direct interaction between the full-length HEIP1 and ZIP4 localized at putative CO sites at late prophase I: DNA mismatch proteins, whereas the BD vector containing full-length HEIP1 or repair protein MutL (MLH1–MLH3) (28–30), ZIP3/ZHP-3/ MSH5 may autonomously activate the reporter in the Vilya/RNF212 SUMO E3 ligases (14, 18), HEI10 ubiquitin li- absence of interacting partners (25). The interaction between gases (13, 31), and the cyclic domain-containing proteins COSA- HEIP1 and MSH5 was validated by the yeast Y2H assay using 1/CNTD1, which are conserved only in metazoans (8, 32). Given truncated HEIP1 protein with no self-activation activity and Co- the localization of HEIP1 from late pachytene to diplotene, it – IP assays in rice protoplasts (Fig. 4 C E). However, no in- might also belong to a group of CO proteins. teraction was detected between HEIP1 and the other ZMM proteins (MER3 and MSH4) (Fig. 4D). We propose that HEIP1 How Might HEIP1 Act in Meiosis? Recombination is initiated at a may interact with ZIP4 and MSH5 to promote CO formation. large number of DSBs generated by Spo11 transesterase (33). These DSBs are then processed to generate free 3′ single- Discussion stranded overhangs which are rapidly coated with recombi- HEIP1 Is Required for Class I CO Formation. By screening for HEI10- nases (34). In most species, the number of recombination sites interacting proteins via a yeast two-hybrid system, we identified far exceeds the number of CO events (34, 35). Recombination the HEIP1 protein. In heip1, the chiasma frequency was severely nodules were originally identified as small electron-dense ovoid reduced compared with that in the wild type. Roughly 14% of structures appearing on the SCs (36). Recombination nodules chiasmata remained in heip1, which is similar to that in (26). have been mainly classified into two classes: early nodule (EN) As previously shown in budding yeast, Arabidopsis, and rice, there and late nodule (LN) (37). ENs present during zygotene–early are two classes of CO occurring: one appears to be sensitive to pachytene and have been postulated to correspond to initial interference, while the other is not. First, our investigations into recombination sites, whereas LNs are believed to indicate COs

Li et al. PNAS Latest Articles | 5of6 Downloaded by guest on September 25, 2021 that are assumed to mature into chiasmata during mid-to-late suggesting that HEI10 may play a role in the stabilization of pachytene (37, 38). Previous studies in mice suggest that most HEIP1. In Arabidopsis, the exquisite dosage sensitivity of HEI10 early RAD51–DMC1 complexes (300 ENs) acquire recombina- for crossover formation was well shown (44, 45). Considering the tion factors (39), including the meiosis-specific MutSγ complex close functional relationship between HEI10 and HEIP1, the ef- MSH4–MSH5, whereas the RAD51–DMC1 component is lost fect of increasing HEI10 and/or HEIP1 dosage on crossovers also from such “transition nodules” (100–200 TNs) (40). Only about deserves further study in rice. In addition, the lack of conservation 25 TNs acquire the MLH1 protein that marks the sites of chi- suggests HEIP1 may possibly play a structural role in a similar asmata (LNs) (18, 41). The dynamic localization pattern of manner to the axis-associated proteins and the central element of HEIP1 during meiosis resembles LNs identified in other or- the synaptonemal complex (36). These proteins are also essential ganisms. Thus, we propose that HEIP1 plays an important role in for crossover formation yet exhibit considerable sequence di- meiotic crossover maturation. vergence between species. In our investigation of HEIP1 and ZMM protein interactions, we found that HEIP1 interacted directly with HEI10, ZIP4, and Materials and Methods MSH5, which further supports the hypothesis that HEIP1 acts as The heip1-1 and heip1-2 were isolated from an indica rice variety Zhongxian the backbone component in crossover formation. In hei10 and zip4, 3037, and the heip1-3 and heip1-4 were generated in a japonica variety Yandao no obvious HEIP1 signals were detected, suggesting that HEIP1 8 by CRISPR-Cas9 targeting. Details on the following are available in SI Ap- may act downstream of HEI10 and ZIP4. However, in mer3, msh4, pendix, SI Materials and Methods: plant materials, full-length cDNA cloning of and msh5, the HEIP1 foci appeared normal on chromosomes at HEIP1, multiple alignments and phylogenetic tree construction, library screen- zygotene, indicating that the localization of HEIP1 is independent ing and Y2H assay, BiFC assay, coimmunoprecipitation assay, meiotic chromo- of MER3, MSH4, and MSH5. Furthermore, prominent axis some preparation, antibody production, immunofluorescence, and Western blot assay. The primers used in this study are listed in SI Appendix, Table S1. staining of ubiquitin was detected on rice meiotic chromosomes (SI Appendix,Fig.S13), indicating that ubiquitination is also in- ACKNOWLEDGMENTS. This work was supported by grants from the National volved in meiosis of plants, which has been studied in yeast and Key Research and Development Program of China (2016YFD0100901) and mouse (42, 43). Almost no HEIP1 was detected in hei10 mutant, the National Natural Science Foundation of China (31460278 and 31771363).

1. Youds JL, Boulton SJ (2011) The choice in meiosis–Defining the factors that influence 23. Wang K, et al. (2009) MER3 is required for normal meiotic crossover formation, but crossover or non-crossover formation. J Cell Sci 124:501–513. not for presynaptic alignment in rice. J Cell Sci 122:2055–2063. 2. Osman K, Higgins JD, Sanchez-Moran E, Armstrong SJ, Franklin FC (2011) Pathways to 24. Shen Y, et al. (2012) ZIP4 in synapsis and crossover for- meiotic recombination in Arabidopsis thaliana. New Phytol 190:523–544. mation in rice meiosis. J Cell Sci 125:2581–2591. 3. Youds JL, et al. (2010) RTEL-1 enforces meiotic crossover interference and homeo- 25. Zhang L, et al. (2014) Crossover formation during rice meiosis relies on interaction of stasis. Science 327:1254–1258. OsMSH4 and OsMSH5. Genetics 198:1447–1456. 4. Davis L, Smith GR (2001) Meiotic recombination and in 26. Luo Q, et al. (2013) The role of OsMSH5 in crossover formation during rice meiosis. Schizosaccharomyces pombe. Proc Natl Acad Sci USA 98:8395–8402. Mol Plant 6:729–742. 5. Börner GV, Kleckner N, Hunter N (2004) Crossover/noncrossover differentiation, syn- 27. Wang K, Wang C, Liu Q, Liu W, Fu Y (2015) Increasing the genetic recombination aptonemal complex formation, and regulatory surveillance at the leptotene/zygotene frequency by partial loss of function of the synaptonemal complex in rice. Mol Plant 8: transition of meiosis. Cell 117:29–45. 1295–1298. 6. Lynn A, Soucek R, Börner GV (2007) ZMM proteins during meiosis: Crossover artists at 28. de Boer E, Stam P, Dietrich AJ, Pastink A, Heyting C (2006) Two levels of interference – work. Chromosome Res 15:591–605. in mouse meiotic recombination. Proc Natl Acad Sci USA 103:9607 9612. 7. Wijeratne AJ, Chen C, Zhang W, Timofejeva L, Ma H (2006) The Arabidopsis thaliana 29. Lhuissier FG, Offenberg HH, Wittich PE, Vischer NO, Heyting C (2007) The mismatch PARTING DANCERS gene encoding a novel protein is required for normal meiotic repair protein MLH1 marks a subset of strongly interfering crossovers in tomato. Plant – homologous recombination. Mol Biol Cell 17:1331–1343. Cell 19:862 876. 8. Yokoo R, et al. (2012) COSA-1 reveals robust homeostasis and separable licensing and 30. Chelysheva L, et al. (2010) An easy protocol for studying chromatin and recombination reinforcement steps governing meiotic crossovers. Cell 149:75–87. protein dynamics during Arabidopsis thaliana meiosis: Immunodetection of cohesins, – 9. Sym M, Engebrecht JA, Roeder GS (1993) ZIP1 is a synaptonemal complex protein histones and MLH1. Cytogenet Genome Res 129:143 153. required for meiotic chromosome synapsis. Cell 72:365–378. 31. De Muyt A, et al. (2014) E3 ligase Hei10: A multifaceted structure-based signaling – 10. Nakagawa T, Flores-Rozas H, Kolodner RD (2001) The MER3 helicase involved in molecule with roles within and beyond meiosis. Genes Dev 28:1111 1123. 32. Holloway JK, Sun X, Yokoo R, Villeneuve AM, Cohen PE (2014) Mammalian CNTD1 is meiotic crossing over is stimulated by single-stranded DNA-binding proteins and critical for meiotic crossover maturation and deselection of excess precrossover sites. unwinds DNA in the 3′ to 5′ direction. J Biol Chem 276:31487–31493. J Cell Biol 205:633–641. 11. Snowden T, Acharya S, Butz C, Berardini M, Fishel R (2004) hMSH4-hMSH5 recognizes 33. Keeney S, Giroux CN, Kleckner N (1997) Meiosis-specific DNA double-strand breaks are Holliday junctions and forms a meiosis-specific sliding clamp that embraces homolo- catalyzed by Spo11, a member of a widely conserved protein family. Cell 88:375–384. gous chromosomes. Mol Cell 15:437–451. 34. Lambing C, Franklin FC, Wang CR (2017) Understanding and manipulating meiotic 12. Perry J, Kleckner N, Börner GV (2005) Bioinformatic analyses implicate the collabo- recombination in plants. Plant Physiol 173:1530–1542. rating meiotic crossover/chiasma proteins Zip2, Zip3, and Spo22/Zip4 in ubiquitin la- 35. Baudat F, de Massy B (2007) Regulating double-stranded DNA break repair towards beling. Proc Natl Acad Sci USA 102:17594–17599. crossover or non-crossover during mammalian meiosis. Chromosome Res 15:565–577. 13. Chelysheva L, et al. (2012) The Arabidopsis HEI10 is a new ZMM protein related to 36. Page SL, Hawley RS (2004) The genetics and molecular biology of the synaptonemal Zip3. PLoS Genet 8:e1002799. complex. Annu Rev Cell Dev Biol 20:525–558. 14. Lake CM, et al. (2015) Vilya, a component of the recombination nodule, is required 37. Zickler D, Kleckner N (1999) Meiotic chromosomes: Integrating structure and func- for meiotic double-strand break formation in Drosophila. eLife 4:e08287. tion. Annu Rev Genet 33:603–754. 15. Cheng CH, et al. (2006) SUMO modifications control assembly of synaptonemal complex 38. Hamant O, Ma H, Cande WZ (2006) Genetics of meiotic prophase I in plants. Annu Rev – and polycomplex in meiosis of . Genes Dev 20:2067 2081. Plant Biol 57:267–302. 16. Toby GG, Gherraby W, Coleman TR, Golemis EA (2003) A novel RING finger protein, 39. Moens PB, et al. (1997) Rad51 immunocytology in rat and mouse and human enhancer of invasion 10, alters mitotic progression through regulation of oocytes. Chromosoma 106:207–215. – cyclin B levels. Mol Cell Biol 23:2109 2122. 40. Moens PB, Marcon E, Shore JS, Kochakpour N, Spyropoulos B (2007) Initiation and 17. Strong ER, Schimenti JC (2010) Evidence implicating CCNB1IP1, a RING domain- resolution of interhomolog connections: Crossover and non-crossover sites along containing protein required for meiotic crossing over in mice, as an E3 SUMO ligase. mouse synaptonemal complexes. J Cell Sci 120:1017–1027. Genes (Basel) 1:440–451. 41. Moens PB, et al. (2002) The time course and chromosomal localization of 18. Reynolds A, et al. (2013) RNF212 is a dosage-sensitive regulator of crossing-over recombination-related proteins at meiosis in the mouse are compatible with models during mammalian meiosis. Nat Genet 45:269–278. that can resolve the early DNA-DNA interactions without reciprocal recombination. 19. Wang K, et al. (2012) The role of rice HEI10 in the formation of meiotic crossovers. J Cell Sci 115:1611–1622. PLoS Genet 8:e1002809. 42. Rao HB, et al. (2017) A SUMO-ubiquitin relay recruits proteasomes to chromosome 20. Sanchez Moran E, Armstrong SJ, Santos JL, Franklin FC, Jones GH (2001) Chiasma axes to regulate meiotic recombination. Science 355:403–407. formation in Arabidopsis thaliana accession Wassileskija and in two meiotic mutants. 43. Ahuja JS, et al. (2017) Control of meiotic pairing and recombination by chromoso- Chromosome Res 9:121–128. mally tethered 26S proteasome. Science 355:408–411. 21. Wang M, et al. (2010) The central element protein ZEP1 of the synaptonemal complex 44. Ziolkowski PA, et al. (2017) Natural variation and dosage of the HEI10 meiotic E3 li- regulates the number of crossovers during meiosis in rice. Plant Cell 22:417–430. gase control Arabidopsis crossover recombination. Genes Dev 31:306–317. 22. Chelysheva L, et al. (2007) Zip4/Spo22 is required for class I CO formation but not for 45. Serra H, et al. (2018) Massive crossover elevation via combination of HEI10 and recq4a synapsis completion in Arabidopsis thaliana. PLoS Genet 3:e83. recq4b during Arabidopsis meiosis. Proc Natl Acad Sci USA 115:2437–2442.

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