ZCWPW1 is recruited to recombination hotspots by PRDM9, and is essential for meiotic double strand break repair

AbstractDuring meiosis, homologous chromosomes pair (synapse) and recombine, enabling balanced segregation and generating genetic diversity. In many vertebrates, recombination initiates with double-strand breaks (DSBs) within hotspots where PRDM9 binds, and deposits H3K4me3 and H3K36me3. However, no protein(s) recognising this unique combination of histone marks have yet been identified.We identified Zcwpw1, which possesses H3K4me3 and H3K36me3 recognition domains, as highly co-expressed 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 recognizes CpG dinucleotides, including within many Alu transposons.Male Zcwpw1 homozygous knockout mice show completely normal DSB positioning, but persistent DMC1 foci at many hotspots, particularly those more strongly bound by PRDM9, severe DSB repair and synapsis defects, and downstream sterility. Our findings suggest a model where ZCWPW1 recognition of PRDM9-bound sites on either the homologous, or broken, chromosome is critical for synapsis, and hence fertility.Graphical Abstract LegendIn humans and other species, recombination is initiated by double strand breaks at sites bound by PRDM9. Upon binding, PRDM9 deposits the histone marks H3K4me3 and H3K36me, but the functional importance of these marks has remained unknown. Here, we show that PRDM9 recruits ZCWPW1, a reader of both these marks, to its binding sites genome-wide. ZCWPW1 does not help position the breaks themselves, but is essential for their downstream repair and chromosome pairing, and ultimately meiotic success and fertility in mice.

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ZCWPW1 is recruited to recombination hotspots by PRDM9 and is essential for meiotic double strand break repair

eLife ◽

10.7554/elife.53392 ◽

2020 ◽

Vol 9 ◽

Author(s):

Daniel Wells ◽

Emmanuelle Bitoun ◽

Daniela Moralli ◽

Gang Zhang ◽

Anjali Hinch ◽

...

Keyword(s):

Binding Sites ◽

Protein S ◽

Strand Break ◽

Double Strand Breaks ◽

Dsb Repair ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Cpg Dinucleotides ◽

Recombination Hotspots ◽

Highly Correlated

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.

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De novo deletion mutations at recombination hotspots in mouse germlines

10.1101/2020.06.23.168138 ◽

2020 ◽

Author(s):

Agnieszka Lukaszewicz ◽

Julian Lange ◽

Scott Keeney ◽

Maria Jasin

Keyword(s):

De Novo ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Atm Kinase ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Recombination Hotspots ◽

Genome Wide ◽

Meiotic Dsbs ◽

Meiotic Recombination Hotspots

AbstractNumerous DNA double-strand breaks (DSBs) arise genome-wide during meiosis to ensure recombination between homologous chromosomes, which is required for gamete formation1,2. The ATM kinase plays a central role in controlling both the number and position of DSBs3-5, but the consequences of deregulated DSB formation have not been explored. Here we discovered that an unanticipated type of DNA deletion arises at meiotic recombination hotspots in the absence of ATM. Deletions form via joining of ends from two closely-spaced DSBs at adjacent hotspots or within a single hotspot. Deletions are also detected in normal cells, albeit at much lower frequency, revealing that the meiotic genome has a hidden potential for deletion events. Remarkably, a subset of deletions contain insertions that likely originated from DNA fragments released from hotspots on other chromosomes. Moreover, although deletions form primarily within one chromosome, joining between homologous chromosomes is also observed. This predicts in turn gross chromosome rearrangements, with evidence of damage to multiple chromatids and aborted gap repair. Thus, multiple nearby meiotic DSBs are normally suppressed by ATM to protect genomic integrity. We expect the de novo germline mutations we observe to affect human health and genome evolution.

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End-resection at DNA double-strand breaks in the three domains of life

Biochemical Society Transactions ◽

10.1042/bst20120307 ◽

2013 ◽

Vol 41 (1) ◽

pp. 314-320 ◽

Cited By ~ 30

Author(s):

John K. Blackwood ◽

Neil J. Rzechorzek ◽

Sian M. Bray ◽

Joseph D. Maman ◽

Luca Pellegrini ◽

...

Keyword(s):

Dna Repair ◽

Homologous Recombination ◽

Strand Break ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Domains Of Life ◽

Strand Invasion ◽

End Resection

During DNA repair by HR (homologous recombination), the ends of a DNA DSB (double-strand break) must be resected to generate single-stranded tails, which are required for strand invasion and exchange with homologous chromosomes. This 5′–3′ end-resection of the DNA duplex is an essential process, conserved across all three domains of life: the bacteria, eukaryota and archaea. In the present review, we examine the numerous and redundant helicase and nuclease systems that function as the enzymatic analogues for this crucial process in the three major phylogenetic divisions.

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i-BLESS is an ultra-sensitive method for detection of DNA double-strand breaks

Communications Biology ◽

10.1038/s42003-018-0165-9 ◽

2018 ◽

Vol 1 (1) ◽

Cited By ~ 22

Author(s):

Anna Biernacka ◽

Yingjie Zhu ◽

Magdalena Skrzypczak ◽

Romain Forey ◽

Benjamin Pardo ◽

...

Keyword(s):

Cell Fate ◽

Genome Stability ◽

Strand Break ◽

Double Strand Breaks ◽

Universal Method ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Agarose Beads ◽

Genome Wide ◽

Dna Double Strand Break

AbstractMaintenance of genome stability is a key issue for cell fate that could be compromised by chromosome deletions and translocations caused by DNA double-strand breaks (DSBs). Thus development of precise and sensitive tools for DSBs labeling is of great importance for understanding mechanisms of DSB formation, their sensing and repair. Until now there has been no high resolution and specific DSB detection technique that would be applicable to any cells regardless of their size. Here, we present i-BLESS, a universal method for direct genome-wide DNA double-strand break labeling in cells immobilized in agarose beads. i-BLESS has three key advantages: it is the only unbiased method applicable to yeast, achieves a sensitivity of one break at a given position in 100,000 cells, and eliminates background noise while still allowing for fixation of samples. The method allows detection of ultra-rare breaks such as those forming spontaneously at G-quadruplexes.

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A global view of meiotic double-strand break end resection

Science ◽

10.1126/science.aak9704 ◽

2017 ◽

Vol 355 (6320) ◽

pp. 40-45 ◽

Cited By ~ 79

Author(s):

Eleni P. Mimitou ◽

Shintaro Yamada ◽

Scott Keeney

Keyword(s):

Meiotic Recombination ◽

Strand Break ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Naked Dna ◽

Global View ◽

Genome Wide ◽

End Resection

DNA double-strand breaks that initiate meiotic recombination are exonucleolytically processed. This 5′→3′ resection is a central, conserved feature of recombination but remains poorly understood. To address this lack, we mapped resection endpoints genome-wide at high resolution inSaccharomyces cerevisiae. Full-length resection requires Exo1 exonuclease and the DSB-responsive kinase Tel1, but not Sgs1 helicase. Tel1 also promotes efficient and timely resection initiation. Resection endpoints display pronounced heterogeneity between genomic loci that reflects a tendency for nucleosomes to block Exo1, yet Exo1 also appears to digest chromatin with high processivity and at rates similar to naked DNA in vitro. This paradox points to nucleosome destabilization or eviction as a defining feature of the meiotic resection landscape.

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Identification of regulators of poly-ADP-ribose polymerase (PARP) inhibitor response through complementary CRISPR knockout and activation screens

10.1101/871970 ◽

2019 ◽

Cited By ~ 1

Author(s):

Kristen E. Clements ◽

Anastasia Hale ◽

Nathanial J. Tolman ◽

Claudia M. Nicolae ◽

Anchal Sharma ◽

...

Keyword(s):

Histone Acetyltransferase ◽

Positive Response ◽

Parp Inhibitor ◽

Strand Break ◽

Double Strand Breaks ◽

Repair Pathway ◽

Genetic Determinants ◽

Strand Breaks ◽

Genome Wide ◽

End Resection

AbstractInhibitors of poly-ADP-ribose polymerase 1 (PARPi) are highly effective in killing cells deficient in the homologous recombination (HR) DNA repair pathway, such as those lacking BRCA2. In light of this, PARPi have been utilized in recent years to treat BRCA2-mutant tumors, with many patients deriving impressive clinical benefit. However, positive response to PARPi is not universal, even among patients with HR-deficient tumors. Here, we present the results of three genome-wide CRISPR knockout and activation screens which provide an unbiased look at genetic determinants of PARPi response in wildtype or BRCA2-knockout cells. Strikingly, we reveal that depletion of the histone acetyltransferase TIP60, a top hit from our screens, robustly reverses the PARPi sensitivity caused by BRCA2 deficiency. Mechanistically, we show that TIP60 depletion rewires double strand break repair in BRCA2-deficient cells by promoting 53BP1 binding to double strand breaks to suppress end resection. Our work provides a comprehensive set of putative biomarkers that serve to better understand and predict PARPi response, and identifies a novel pathway of PARPi resistance in BRCA2-deficient cells.

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Genome-Wide Redistribution of Meiotic Double-Strand Breaks in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.02063-06 ◽

2006 ◽

Vol 27 (5) ◽

pp. 1868-1880 ◽

Cited By ~ 63

Author(s):

Nicolas Robine ◽

Norio Uematsu ◽

Franck Amiot ◽

Xavier Gidrol ◽

Emmanuel Barillot ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Meiotic Recombination ◽

Binding Sites ◽

Chromosomal Region ◽

Double Strand Breaks ◽

Dna Binding Domain ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Genome Wide ◽

Local Stimulation

ABSTRACT Meiotic recombination is initiated by the formation of programmed DNA double-strand breaks (DSBs) catalyzed by the Spo11 protein. DSBs are not randomly distributed along chromosomes. To better understand factors that control the distribution of DSBs in budding yeast, we have examined the genome-wide binding and cleavage properties of the Gal4 DNA binding domain (Gal4BD)-Spo11 fusion protein. We found that Gal4BD-Spo11 cleaves only a subset of its binding sites, indicating that the association of Spo11 with chromatin is not sufficient for DSB formation. In centromere-associated regions, the centromere itself prevents DSB cleavage by tethered Gal4BD-Spo11 since its displacement restores targeted DSB formation. In addition, we observed that new DSBs introduced by Gal4BD-Spo11 inhibit surrounding DSB formation over long distances (up to 60 kb), keeping constant the number of DSBs per chromosomal region. Together, these results demonstrate that the targeting of Spo11 to new chromosomal locations leads to both local stimulation and genome-wide redistribution of recombination initiation and that some chromosomal regions are inherently cold regardless of the presence of Spo11.

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Identification of regulators of poly-ADP-ribose polymerase inhibitor response through complementary CRISPR knockout and activation screens

Nature Communications ◽

10.1038/s41467-020-19961-w ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Kristen E. Clements ◽

Emily M. Schleicher ◽

Tanay Thakar ◽

Anastasia Hale ◽

Ashna Dhoonmoon ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Histone Acetyltransferase ◽

Positive Response ◽

Strand Break ◽

Double Strand Breaks ◽

Genetic Determinants ◽

Mechanisms Of Resistance ◽

Strand Breaks ◽

Genome Wide ◽

Polymerase Inhibitor

AbstractInhibitors of poly-ADP-ribose polymerase 1 (PARPi) are highly effective in killing cells deficient in homologous recombination (HR); thus, PARPi have been clinically utilized to successfully treat BRCA2-mutant tumors. However, positive response to PARPi is not universal, even among patients with HR-deficiency. Here, we present the results of genome-wide CRISPR knockout and activation screens which reveal genetic determinants of PARPi response in wildtype or BRCA2-knockout cells. Strikingly, we report that depletion of the ubiquitin ligase HUWE1, or the histone acetyltransferase KAT5, top hits from our screens, robustly reverses the PARPi sensitivity caused by BRCA2-deficiency. We identify distinct mechanisms of resistance, in which HUWE1 loss increases RAD51 levels to partially restore HR, whereas KAT5 depletion rewires double strand break repair by promoting 53BP1 binding to double-strand breaks. Our work provides a comprehensive set of putative biomarkers that advance understanding of PARPi response, and identifies novel pathways of PARPi resistance in BRCA2-deficient cells.

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Mouse TEX15 is essential for DNA double-strand break repair and chromosomal synapsis during male meiosis

The Journal of Cell Biology ◽

10.1083/jcb.200709057 ◽

2008 ◽

Vol 180 (4) ◽

pp. 673-679 ◽

Cited By ~ 75

Author(s):

Fang Yang ◽

Sigrid Eckardt ◽

N. Adrian Leu ◽

K. John McLaughlin ◽

Peijing Jeremy Wang

Keyword(s):

Meiotic Recombination ◽

Strand Break ◽

Male Meiosis ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Recombination Proteins ◽

Meiotic Chromosomes ◽

Dna Double Strand Break

During meiosis, homologous chromosomes undergo synapsis and recombination. We identify TEX15 as a novel protein that is required for chromosomal synapsis and meiotic recombination. Loss of TEX15 function in mice causes early meiotic arrest in males but not in females. Specifically, TEX15-deficient spermatocytes exhibit a failure in chromosomal synapsis. In mutant spermatocytes, DNA double-strand breaks (DSBs) are formed, but localization of the recombination proteins RAD51 and DMC1 to meiotic chromosomes is severely impaired. Based on these data, we propose that TEX15 regulates the loading of DNA repair proteins onto sites of DSBs and, thus, its absence causes a failure in meiotic recombination.

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The bakers’s yeast Msh4-Msh5 associates with double-strand break hotspots and chromosome axis during meiosis to promote crossovers

10.1101/2020.07.24.219295 ◽

2020 ◽

Author(s):

Krishnaprasad G. Nandanan ◽

Ajith V. Pankajam ◽

Sagar Salim ◽

Miki Shinohara ◽

Gen Lin ◽

...

Keyword(s):

Mismatch Repair ◽

Baker’S Yeast ◽

Double Strand Breaks ◽

Holliday Junction ◽

Baker's Yeast ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Genome Wide ◽

Chromosome Axis

ABSTRACTSegregation of homologous chromosomes during the first meiotic division requires at least one obligate crossover/exchange event between the homolog pairs. In the baker’s yeast Saccharomyces cerevisiae and mammals, the mismatch repair-related factors, Msh4-Msh5 and Mlh1-Mlh3 generate the majority of the meiotic crossovers from programmed double-strand breaks (DSBs). To understand the mechanistic role of Msh4-Msh5 in meiotic crossing over, we performed genome-wide ChIP-sequencing and cytological analysis of the Msh5 protein in cells synchronized for meiosis. We observe that Msh5 associates with DSB hotspots, chromosome axis, and centromeres. We found that the initial recruitment of Msh4-Msh5 occurs following DSB resection. A two-step Msh5 binding pattern was observed: an early weak binding at DSB hotspots followed by enhanced late binding upon the formation of double Holliday junction structures. Msh5 association with the chromosome axis is Red1 dependent, while Msh5 association with the DSB hotspots and axis is dependent on DSB formation by Spo11. Msh5 binding was enhanced at strong DSB hotspots consistent with a role for DSB frequency in promoting Msh5 binding. These data on the in vivo localization of Msh5 during meiosis have implications for how Msh4-Msh5 may work with other crossover and synapsis promoting factors to ensure Holliday junction resolution at the chromosome axis.AUTHOR SUMMARYDuring meiosis, crossovers facilitate physical linkages between homologous chromosomes that ensure their accurate segregation. Meiotic crossovers are initiated from programmed DNA double-strand breaks (DSBs). In the baker’s yeast and mammals, DSBs are repaired into crossovers primarily through a pathway involving the highly conserved mismatch repair related Msh4-Msh5 complex along with other crossover promoting factors. In vitro and physical studies suggest that the Msh4-Msh5 heterodimer facilitates meiotic crossover formation by stabilizing Holliday junctions. We investigated the genome-wide in vivo binding sites of Msh5 during meiotic progression. Msh5 was enriched at DSB hotspots, chromosome axis, and centromere sites. Our results suggest Msh5 associates with both DSB sites on the chromosomal loops and with the chromosome axis to promote crossover formation. These results on the in vivo dynamic localization of the Msh5 protein provide novel insights into how the Msh4-Msh5 complex may work with other crossover and synapsis promoting factors to facilitate crossover formation.

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