A map of human PRDM9 binding provides evidence for novel behaviors of PRDM9 and other zinc-finger proteins in meiosis

Altemose, Nicolas; Noor, Nudrat; Bitoun, Emmanuelle; Tumian, Afidalina; Imbeault, Michaël; Chapman, J. Ross; Aricescu, A. Radu; Myers, Simon

doi:10.7554/elife.28383

Cited by 93 publications

(158 citation statements)

References 94 publications

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“…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 Supplementary Figure 12). This perturbation experiment provides strong evidence that PRDM9 causes recruitment of ZCWPW1 (as opposed to for example independent recruitment of both proteins).…”

Section: Zcwpw1 Is Recruited To Prdm9 Binding Sites In An Allele-specmentioning

confidence: 98%

“…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 hypothesized that it would be recruited to PRDM9-bound genomic sites, where these marks are deposited upon binding in HEK293T cells (Altemose et al, 2017).…”

Section: Zcwpw1 Is Recruited To Prdm9 Binding Sites In An Allele-specmentioning

confidence: 99%

“…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 ZF-array, as studied previously (Altemose et al, 2017), and then performed ChIP-seq against the ZCWPW1 tag. 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 (Figure 5A, B).…”

Section: Zcwpw1 Is Recruited To Prdm9 Binding Sites In An Allele-specmentioning

confidence: 99%

“…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), mouse anti-SYCP3 (Santa Cruz Biotechnology sc-74569, D-1) or biotinylated rabbit anti-SYCP3 (Novus NB300-232), rabbit anti- (Altemose et al, 2017), using mouse anti-human ZCWPW1 (SIGMA SAB1409478) and mouse anti-polyhistidine (SIGMA H1029). The purified protein was used to immunize 2 rabbits (Eurogentec, Belgium), and the resulting immune antisera were tested against the recombinant antigen by ELISA (Eurogentec), and the endogenous protein in mouse testes from WT and KO mice (data not shown), alongside the preimmune sera.…”

Section: Immunostaining Of Spermatocytesmentioning

confidence: 99%

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

Wells

Bitoun

Moralli

et al. 2019

Preprint

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During 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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Section: Zcwpw1 Is Recruited To Prdm9 Binding Sites In An Allele-specmentioning

confidence: 98%

Section: Zcwpw1 Is Recruited To Prdm9 Binding Sites In An Allele-specmentioning

confidence: 99%

Section: Zcwpw1 Is Recruited To Prdm9 Binding Sites In An Allele-specmentioning

confidence: 99%

Section: Immunostaining Of Spermatocytesmentioning

confidence: 99%

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

Wells

Bitoun

Moralli

et al. 2019

Preprint

Self Cite

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“…Expression of PRDM9 ex vivo recapitulates aspects of hotspot activation including allelespecific H3K4me3 deposition (Altemose et al, 2017;Baker et al, 2015b;Thibault-Sennett et al, 2018). To develop a model for temporal molecular characterization of hotspot activation, we choose a human cell line that expresses HELLS and created a stably-integrated FLAG-PRDM9 C allele under inducible control of the tetracycline promoter (HEK293-P9 C , Fig.…”

Section: Hells Forms a Complex With Prdm9mentioning

confidence: 99%

HELLS and PRDM9 form a Pioneer Complex to Open Chromatin at Meiotic Recombination Hotspots

Spruce

Dlamini

Ananda

et al. 2019

Preprint

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Chromatin barriers prevent spurious interactions between regulatory elements and DNAbinding proteins. One such barrier, whose mechanism for overcoming is poorly understood, is access to recombination hotspots during meiosis. Here we show that the chromatin remodeler HELLS and DNA-binding protein PRDM9 function together to open chromatin at hotspots and provide access for the DNA double-strand break (DSB) machinery. Recombination hotspots are decorated by a unique combination of histone modifications, not found at other regulatory elements. HELLS is recruited to hotspots by PRDM9, and is necessary for both histone modifications and DNA accessibility at hotspots. In male mice lacking HELLS, DSBs are retargeted to other sites of open chromatin, leading to germ cell death and sterility. Together, these data provide a model for hotspot activation where HELLS and PRDM9 function as a pioneer complex to create a unique epigenomic environment of open chromatin, permitting correct placement and repair of DSBs.

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The RNA-binding protein FUS/TLS interacts with SPO11 and PRDM9 and localize at meiotic recombination hotspots

Giannattasio

Testa

Palombo

et al. 2023

Cell. Mol. Life Sci.

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In mammals, meiotic recombination is initiated by the introduction of DNA double strand breaks (DSBs) into narrow segments of the genome, defined as hotspots, which is carried out by the SPO11/TOPOVIBL complex. A major player in the specification of hotspots is PRDM9, a histone methyltransferase that, following sequence-specific DNA binding, generates trimethylation on lysine 4 (H3K4me3) and lysine 36 (H3K36me3) of histone H3, thus defining the hotspots. PRDM9 activity is key to successful meiosis, since in its absence DSBs are redirected to functional sites and synapsis between homologous chromosomes fails. One protein factor recently implicated in guiding PRDM9 activity at hotspots is EWS, a member of the FET family of proteins that also includes TAF15 and FUS/TLS. Here, we demonstrate that FUS/TLS partially colocalizes with PRDM9 on the meiotic chromosome axes, marked by the synaptonemal complex component SYCP3, and physically interacts with PRDM9. Furthermore, we show that FUS/TLS also interacts with REC114, one of the axis-bound SPO11-auxiliary factors essential for DSB formation. This finding suggests that FUS/TLS is a component of the protein complex that promotes the initiation of meiotic recombination. Accordingly, we document that FUS/TLS coimmunoprecipitates with SPO11 in vitro and in vivo. The interaction occurs with both SPO11β and SPO11α splice isoforms, which are believed to play distinct functions in the formation of DSBs in autosomes and male sex chromosomes, respectively. Finally, using chromatin immunoprecipitation experiments, we show that FUS/TLS is localized at H3K4me3-marked hotspots in autosomes and in the pseudo-autosomal region, the site of genetic exchange between the XY chromosomes.

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A map of human PRDM9 binding provides evidence for novel behaviors of PRDM9 and other zinc-finger proteins in meiosis

Cited by 93 publications

References 94 publications

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

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

HELLS and PRDM9 form a Pioneer Complex to Open Chromatin at Meiotic Recombination Hotspots

The RNA-binding protein FUS/TLS interacts with SPO11 and PRDM9 and localize at meiotic recombination hotspots

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