Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Mar 26;80(4):107.
doi: 10.1007/s00018-023-04744-5.

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

Teresa Giannattasio  1 , Erika Testa #  1 , Ramona Palombo #  2 , Lidia Chellini #  2 , Flavia Franceschini  1 , Álvaro Crevenna  3 , Petko M Petkov  4 , Maria Paola Paronetto  5   6 , Marco Barchi  7
Affiliations

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

Teresa Giannattasio et al. Cell Mol Life Sci. .

Abstract

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.

Keywords: EWSR1; H3K4me3; Meiosis; REC114; SPO11α; SPO11β; XY.

PubMed Disclaimer

Conflict of interest statement

The authors have no relevant financial or non-financial interests to disclose.

Figures

Fig. 1
Fig. 1
Analysis of FUS expression and localization. A Western Blot (WB) of FUS expression in total testes extracts from mice at the indicated age. At least two testes were used for each time point. The expression of clathrin proteins served as a normalizer. B WB analysis of FUS protein level in enriched cell populations of the testis. Somatic cells and pre-leptotene (pLe)/leptotene (Le)/zygotene (Zyg) stage cells were isolated from five wild-type 10 dpp old mice; pachytene/diplotene (Pach/Dip) stage cells and round spermatids (rSpe) were isolated from four wild-type adult mice. Tubulin served as a normalizer. C Immunolocalization of FUS (red) and cell nuclei (blue) in seminiferous tubules at the indicated stages of the epithelial cell cycle. The white arrows point to somatic Sertoli cells (Se) and germ cells at different stages of development: spermatogonia (Sg), primary spermatocytes (SpI), secondary spermatocytes (SpII), elongated spermatids (eSp), and spermatozoa (Sp). Magnification bar represents 50 μm. D Immunolocalization of FUS (red) and SYCP3 (green) on surface chromosome spreads of germ cells, at the indicated stages of development. Images were captured using an inverted fluorescence microscope. Magnification bar represents 10 μm. E Confocal images of FUS (gray) and SYCP3 (red) on surface chromosome spreads at the indicated stages. The white arrows in magnifications (a–c) point at sites of colocalization of FUS with the unsynapsed chromosome axes identified by SYCP3. The yellow arrow in C points to FUS on synapsed chromosome axes. Magnification bar represents 10 μm
Fig. 2
Fig. 2
FUS colocalizes/interacts with PRDM9 and coimmunoprecipitates with REC114. A Confocal imaging of FUS, SYCP3, and PRDM9 in surface chromosome spreads at leptonema. The white arrows indicate SYCP3 colocalization with FUS (left panel), SYCP3 colocalization with PRDM9 (mid panel) or FUS colocalization with PRDM9 (right panel). Magnification bar represents 10 μm. B Pulldown by maltose-binding protein (pMAL-MBP) and PRDM9-maltose binding protein (PRDM9-MBP) of FUS from 11–12 dpp total testis extracts (four testes per lane). C Pulldown by the PRDM9-MBP protein of endogenously expressed FUS in HEK293 cells. The Coomassie stained gel shows the purified PRDM9-MBP protein. D IP/WB analysis of FUS and WB of PRDM9 in total extracts from juvenile mice testes, isolated populations of germ cells and somatic cells obtained from 10 dpp old mice. IP from somatic cells of the testes and 10 dpp testes with anti-FUS and nonspecific IgG, respectively, served as negative controls. pLe/Le/Zyg is a mix population of pre-leptotene, leptotene and zygotene stage cells. E IP/WB analysis of FUS and WB of PRDM9 from total testis extracts from mice of the indicated genotypes. Wild types (wt) are six testes from 14 dpp old C57BL6/J mice, Prdm9−/−; Prdm9Tg/Tg are two testes from adult mice, Prdm9−/−; Prdm9YF/YF are four testes from adult mice, Prdm9−/− are two testes from adult mice. F Top panel, identification of the REC114 specific band in inputs used for FUS immunoprecipitation. The Rec114−/− adult testes served as a control. pLe/Le/Zyg is a mix population of pre-leptotene, leptotene and zygotene stage cells obtained from 10 dpp old mice. “Somatic” indicates a mix population of testicular somatic cells obtained from 10 dpp old mice. The total testis extract was prepared from 14 dpp old mice. Lower panel, IP/WB analysis of the FUS-REC114 interaction in the extracts indicated above. The IP with a non-specific IgG served as negative control. Seven testes were used for each point. The asterisk indicates a non-specific band
Fig. 3
Fig. 3
FUS interacts with SPO11 in vivo and in vitro. A IP/WB analysis of SPO11 and WB of FUS, PRDM9, and REC114 coimmunoprecipitating from juveniles and adult mice total testes extracts and from isolated populations of germ cells. Pach/Dip and rSpe are pachytene/diplotene stages enriched fractions and round spermatids, respectively, obtained from adult mice. Total extracts of juvenile mice testes were prepared from five 12 dpp mice. The total extracts of the testes of adult mice were from a 6-month-old mouse. The IP with a non-specific mouse IgG served as negative control. The immunoprecipitation of SPO11 from a purified fraction of r-Spe served as a control for PRDM9 and REC114 signal specificity. The asterisks indicate nonspecific bands (see Fig. 2F, top panel). B FUS and REC114 expression in total extracts of the input. SYCP3 served as a measure of spermatocyte cell content, while tubulin was used as an independent loading control. C Pulldown by the SPO11β-GST and SPO11α-GST recombinant proteins, of FUS from 12 dpp testis extracts, or D from extracts of HEK293 cells. Coomassie-stained gels in B and C show SPO11-GST fusion proteins. E IP/WB analysis of SPO11β/SPO11α and WB of FUS coimmunoprecipitated from total testis extracts of adult knock-in mice expressing only either SPO11β or SPO11α cDNA. The higher exposure of the film of SPO11 IP is shown to allow a better visualization of the SPO11α splice isoform. IgG indicates immunoglobulin. The asterisk in the input indicates a nonspecific band
Fig. 4
Fig. 4
FUS localizes at H3K4me3-marked hotspots. A Chromatin Immunoprecipitation (ChIP) of H3K4me3 and quantitative Real-Time PCR (qPCR) of the indicated PRDM9Dom2 hotspots. The intragenic region of Pol II (Pol II-intra) is an H3K4me3 coldspot. B ChIP-qPCR of FUS at the indicated hotspots and the coldspot. C ChIP-qPCR of H3K4me3 and qPCR at two distinct pseudoautosomal regions named 1 and 2 (PAR1 and PAR2). X-Hs is a PRDM9Dom2 hotspot of a non-PAR genomic region of the X chromosome. X-Cs is a PRDM9RJ2 hotspot in a non-PAR genomic region of the X chromosome, expected to be a coldspot in the C57 background. D ChIP-qPCR of FUS at the indicated hotspots. In all cases, the analyses were performed from at least three 12dpp old mice testes, and the data were normalized against the input. Nonspecific IgG-based qPCR served as a control. Each ChIP-qPCR experiment was performed at least in triplicate. Error bars are mean ± standard error of the mean. Statistical significances (*p < 0.05; **p < 0.005; ***p < 0.001; n.s. = not significant) indicates enrichment to IgGs (two tails t-test). E Putative model of the action of FUS in early meiosis, according to the axis loop tethering model. FUS by interacting concomitantly (directly or indirectly) with PRDM9 and SPO11 provides a link between the H3K4me3-marked chromatin loops and the SPO11/TOPOVIBL cleavage complex. The interaction of FUS with REC114 may promote the association of the PRDM9-marked hotspot with the axis. EWS by interacting (directly) with PRDM9 and (directly or indirectly) with SPO11, may support the function of FUS
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Svetoni F, Frisone P, Paronetto MP. Role of FET proteins in neurodegenerative disorders. RNA Biol. 2016;13(11):1089–1102. doi: 10.1080/15476286.2016.1211225. - DOI - PMC - PubMed
    1. Andersson MK, Stahlberg A, Arvidsson Y, Olofsson A, Semb H, Stenman G, et al. The multifunctional FUS, EWS and TAF15 proto-oncoproteins show cell type-specific expression patterns and involvement in cell spreading and stress response. BMC Cell Biol. 2008;9:37. doi: 10.1186/1471-2121-9-37. - DOI - PMC - PubMed
    1. Tan AY, Manley JL. The TET family of proteins: functions and roles in disease. J Mol Cell Biol. 2009;1(2):82–92. doi: 10.1093/jmcb/mjp025. - DOI - PMC - PubMed
    1. Paronetto MP. Ewing sarcoma protein: a key player in human cancer. Int J Cell Biol. 2013;2013:642853. doi: 10.1155/2013/642853. - DOI - PMC - PubMed
    1. Bertolotti A, Lutz Y, Heard DJ, Chambon P, Tora L. hTAF(II)68, a novel RNA/ssDNA-binding protein with homology to the pro-oncoproteins TLS/FUS and EWS is associated with both TFIID and RNA polymerase II. EMBO J. 1996;15(18):5022–5031. doi: 10.1002/j.1460-2075.1996.tb00882.x. - DOI - PMC - PubMed

LinkOut - more resources

-