Dual Histone Methyl Reader ZCWPW1 Facilitates Repair of Meiotic Double Strand Breaks

Mahgoub, Mohamed; Paiano, Jacob; Bruno, Melania; Wu, Wei; Pathuri, Sarath; Zhang, Xing; Ralls, Sherry; Cheng, Xiaodong; Nussenzweig, André; Macfarlan, Todd S.

doi:10.1101/821603

Cited by 5 publications

(5 citation statements)

References 69 publications

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“…Immunofluorescence images in this study revealed that Zcwpw1 functioned in DNA repair and synapsis. Very recently, Zcwpw1 was identified as a histone H3K4me3 reader required for the repair of PR domain zinc finger protein 9-dependent DNA DSBs and synapsis by three independent research groups ( 49 , 50 , 51 , 52 ), which was consistent with our observations. Tesmin and 1700102P08Rik have also recently been identified as essential genes in spermatogenesis ( 53 , 54 ), yet their mechanisms of involvement in meiosis-essential biological events still require further exploration.…”

Section: Discussionsupporting

confidence: 92%

Prediction and Validation of Mouse Meiosis-Essential Genes Based on Spermatogenesis Proteome Dynamics

Fang

et al. 2021

Molecular & Cellular Proteomics

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The molecular mechanism associated with mammalian meiosis has yet to be fully explored, and one of the main reasons for this lack of exploration is that some meiosis-essential genes are still unknown. The profiling of gene expression during spermatogenesis has been performed in previous studies, yet few studies have aimed to find new functional genes. Since there is a huge gap between the number of genes that are able to be quantified and the number of genes that can be characterized by phenotype screening in one assay, an efficient method to rank quantified genes according to phenotypic relevance is of great importance. We proposed to rank genes by the probability of their function in mammalian meiosis based on global protein abundance using machine learning. Here, nine types of germ cells focusing on continual substages of meiosis prophase I were isolated, and the corresponding proteomes were quantified by high-resolution mass spectrometry. By combining meiotic labels annotated from the MGI mouse knockout database and the spermatogenesis proteomics dataset, a supervised machine learning package, FuncProFinder, was developed to rank meiosis-essential candidates. Of the candidates whose functions were unannotated, four of ten genes with the top prediction scores, Zcwpw1, Tesmin, 1700102P08Rik and Kctd19, were validated as meiosis-essential genes by knockout mouse models. Therefore, mammalian meiosis-essential genes could be efficiently predicted based on the protein abundance dataset, which provides a paradigm for other functional gene mining from a related abundance dataset.

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Section: Discussionsupporting

confidence: 92%

Prediction and Validation of Mouse Meiosis-Essential Genes Based on Spermatogenesis Proteome Dynamics

Fang

et al. 2021

Molecular & Cellular Proteomics

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“…Immunofluorescence images in this study divulged that Zcwpw1 played a role of DNA repair and synapsis. Recently, Zcwpw1 was identified as a histone H3K4me3 reader required for synapsis and repair of PRDM9-dependent DSBs by other three research groups [37][38][39][40] , which was consistent with our observations. Whereas, knockout of the other two genes, Tesmin and 1700102P08Rik, were found have no effect on DNA repair and synapsis, indicating they could be involved in presently unknown molecular events inspected by midPachytene checkpoint.…”

Section: Dissucssionsupporting

confidence: 92%

Prediction of meiosis-essential genes based on dynamic proteomes responsive to spermatogenesis

Fang

Wei

et al. 2020

Preprint

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Mammalian meiosis is a cell division process specific to sexual reproduction, whereas a comprehensive proteome related to different meiotic stages has not been systematically investigated. Here, we isolated different types of germ cells from the testes of spermatogenesis-synchronized mice and quantified the corresponding proteomes with high-resolution mass spectrometry. A total of 8,002 proteins were identified in nine types of germ cells, while the protein signatures of spermatogenesis were characterized by the dynamic proteomes. A supervised machine learning package, FuncProFinder, was developed to predict meiosis-essential candidates based on the proteomic dataset. Of the candidates with unannotated functions, four of the ten genes at the top prediction scores, Zcwpw1, Tesmin, 1700102P08Rik and Kctd19, were validated as meiosis-essential genes by knockout mouse models. The proteomic analysis towards spermatogenic cells indeed setups a solid evidence to study the mechanism of mammalian meiosis. The proteome data are available via ProteomeXchange with identifier PXD017284.Up to now, transcriptional gene expression in thousands of germ cells covering various developmental stages of spermatogenesis were quantified at single cell level 9-15 , resulting in very detail transcriptome landscape throughout spermatogenesis, yet few studies explored the data for further functional excavating. As gene expression at protein level are downstream of transcription, proteomic abundance change of genes could be more directly associated with phenotype or functional change. Importantly, multiple studies clarified a poor correlation between mRNA and protein abundance in testes 16,17 , therefore, a global proteomic profiling of gene expression in spermatogenesis is of great meaningful to unravel functional molecules of meiosis. However, the report regarding systematic profiling of proteomics during meiosis was limited. Only one type of meiotic cells-pachytene spermatocytes was quantified in previous proteomics studies 7, 17 , leaving protein expression remained unknown in most of the stages in meiosis. Therefore, in contrast to meiotic dependence of transcriptomes in details, quantified profiling of meiotic proteome has remained a large room to be improved, as well as digging for functional molecules from a big omics dataset.In this work, to understand the molecular basis of mouse meiosis and predict meiosis-essential proteins, 7 consecutive types of meiotic cells plus pre-meiotic spermatogonia and post-meiotic round spermatids were isolated and the proteins in each cell-type were identified and quantified by high-resolution mass spectrometry with a label-free mode. The meiosisdependent signatures were characterized by protein abundance changes. Furthermore, a supervised ensemble machine learning package, FuncProFinder, was developed to predict the meiosis-essential proteins. The meiosis-related phenotypes for the five proteins at the top scores of the prediction, Pdha2, Zcwpw1, Tesmin, Kctd19 and 1700102P08Rik were verified by knoc...

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“…However, an increased lifespan of resected DSBs is not always associated with the removal of SPO11-RI. For example, loss of ZCWPW1, a dual histone methylation reader of PRDM9, does not impact DSB placement or SPO11-RI formation 48 . Zcwpw1 -/spermatocytes display decreased DSB repair and asynapsis phenocopying loss of PRDM9 48 ; however, SPO11-RI is present at WT levels in Zcwpw1 -/spermatocytes.…”

Section: Discussionmentioning

confidence: 99%

“…For example, loss of ZCWPW1, a dual histone methylation reader of PRDM9, does not impact DSB placement or SPO11-RI formation 48 . Zcwpw1 -/spermatocytes display decreased DSB repair and asynapsis phenocopying loss of PRDM9 48 ; however, SPO11-RI is present at WT levels in Zcwpw1 -/spermatocytes. Thus, failure in synapsis and DSB repair does not necessarily lead to an increased fraction of breaks that are fully and symmetrically processed by MRE11.…”

Section: Discussionmentioning

confidence: 99%

ATM and PRDM9 Regulate SPO11-bound Recombination Intermediates During Meiosis

Paiano

Yamada

et al. 2019

Preprint

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Meiotic recombination is initiated by genome-wide SPO11-induced double-strand breaks (DSBs) that are processed by MRE11-mediated release of SPO11. The DSB is then resected and loaded with DMC1/RAD51 filaments that invade homologous chromosome templates. In most mammals, DSB locations ("hotspots") are determined by the DNA sequence specificity of PRDM9. Here, we demonstrate the first direct detection of meiotic DSBs and resection in vertebrates by performing END-seq on mouse spermatocytes using low sample input. We find that DMC1 limits both the minimum and maximum lengths of resected DNA, whereas 53BP1, BRCA1 and EXO1 play surprisingly minimal roles in meiotic resection. Through enzymatic modifications to the END-seq protocol that mimic the in vivo processing of SPO11, we identify a novel meiotic recombination intermediate ("SPO11-RI") present at all hotspots. The SPO11-bound intermediate is dependent on PRDM9 and caps the 3' resected end during engagement with the homologous template. We propose that SPO11-RI is generated because chromatin-bound PRDM9 asymmetrically blocks MRE11 from releasing SPO11. In Atm -/spermatocytes, SPO11-RI is reduced while unresected DNA-bound SPO11 accumulate because of defective MRE11 initiation. Thus in addition to their global roles in governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing. MainRecombination between homologous chromosomes during meiosis requires DNA double-strand break (DSB) formation by the topoisomerase-like protein SPO11 1 . After cutting, SPO11 remains covalently bound to a two-nucleotide, 5' overhang at both ends of the DNA via phosphotyrosyl linkage. Recombination then begins with the processing of SPO11-bound DSBs into resected 3' single-stranded DNA (ssDNA) tails that preferentially invade the homologous chromosome by the recombinases DMC1 and RAD51. Studies in budding yeast Saccharomyces cerevisiae determined that the MRE11/RAD50/NBS1 (MRN) complex detects SPO11 and cooperates with Sae2 to produce a nick on the SPO11-bound strand via MRE11 endonuclease activity 2 . The nick serves as an entry point for both short-range MRE11 3'-5' exonuclease activity to degrade back to the DSB, thereby removing covalently bound SPO11 attached to a ssDNA oligonucleotide, as well as for more extensive long-range processing of 5' strands (Extended Data Fig. 1a) 2 . In budding yeast, Exo1 nuclease is uniquely responsible for this long-range 5'-3' resection 3 .Moreover, short-and long-range resection are tightly coupled in a single processive reaction (Extended Data Fig. 1a). As a result, meiotic DSBs are maximally resected as soon as they appear and unresected SPO11-bound DSBs are extremely rare 4-6 . While ATM has been shown to regulate DSB numbers and locations 7,8 , its remains unclear whether it also functions downstream in regulating SPO11 processing and resection.Distinct from yeast, DSB hotspots in mice and humans are determined by the DNA binding specificity of the PRDM9 methyltransferase 9 . Besides positioning DSBs, PRDM9...

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Dual Histone Methyl Reader ZCWPW1 Facilitates Repair of Meiotic Double Strand Breaks

Cited by 5 publications

References 69 publications

Prediction and Validation of Mouse Meiosis-Essential Genes Based on Spermatogenesis Proteome Dynamics

Prediction and Validation of Mouse Meiosis-Essential Genes Based on Spermatogenesis Proteome Dynamics

Prediction of meiosis-essential genes based on dynamic proteomes responsive to spermatogenesis

ATM and PRDM9 Regulate SPO11-bound Recombination Intermediates During Meiosis

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