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Nuclear localization of PRDM9 and its role in meiotic chromatin modifications and homologous synapsis

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Abstract

Developmental progress of germ cells through meiotic phases is closely tied to ongoing meiotic recombination. In mammals, recombination preferentially occurs in genomic regions known as hotspots; the protein that activates these hotspots is PRDM9, containing a genetically variable zinc finger (ZNF) domain and a PR-SET domain with histone H3K4 trimethyltransferase activity. PRDM9 is required for fertility in mice, but little is known about its localization and developmental dynamics. Application of spermatogenic stage-specific markers demonstrates that PRDM9 accumulates in male germ cell nuclei at pre-leptonema to early leptonema but is no longer detectable in nuclei by late zygonema. By the pachytene stage, PRDM9-dependent histone H3K4 trimethyl marks on hotspots also disappear. PRDM9 localizes to nuclei concurrently with the deposition of meiotic cohesin complexes, but is not required for incorporation of cohesin complex proteins into chromosomal axial elements, or accumulation of normal numbers of RAD51 foci on meiotic chromatin by late zygonema. Germ cells lacking PRDM9 exhibit inefficient homology recognition and synapsis, with aberrant repair of meiotic DNA double-strand breaks and transcriptional abnormalities characteristic of meiotic silencing of unsynapsed chromatin. Together, these results on the developmental time course for nuclear localization of PRDM9 establish its direct window of function and demonstrate the independence of chromosome axial element formation from the concurrent PRDM9-mediated activation of recombination hotspots.

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Acknowledgments

We are indebted to members of the Handel and Paigen laboratories for discussion of this work in progress, to Kristina Palmer and Sabrina Petri for animal care and technical assistance, to Dr. G. Carter for statistical consultation, and to Drs. G. Carter, J. Eppig and S. Handel for critical comments on the manuscript. We are grateful to Y. Watanabe and S. Namekawa for providing antibodies. This work was supported by P01 grants from the NIH (HD42137 and GM99640) and by fellowships to YF from the Japan Society for the Promotion of Science (JSPS) and the Strategic Young Researcher Oversea Visits Program for Accelerating Brain Research. Research reported in this publication was also partially supported by the National Cancer Institute under award number P30 CA034196; the content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

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  1. The Jackson Laboratory, 600 Main Street, Bar Harbor, ME, 04609, USA

    Fengyun Sun, Yasuhiro Fujiwara, Laura G. Reinholdt, Jianjun Hu, Ruth L. Saxl, Christopher L. Baker, Petko M. Petkov, Kenneth Paigen & Mary Ann Handel

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  1. Fengyun Sun
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  2. Yasuhiro Fujiwara
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Correspondence to Mary Ann Handel.

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Supplementary Fig. 1

Expression of Prdm9 and nuclear localization of PRDM9 protein. a qRT-PCR showing the developmental pattern of Prdm9 transcript expression during the juvenile onset of spermatogenesis. b Western blot showing developmental pattern of expression of PRDM9 protein in testis during the juvenile onset of spermatogenesis. TUBA1A is the loading control. c Nuclear localization of PRDM9 was visualized in HEK293 cells transfected with FLAG-tagged Prdm9 after immunolabeling with pre-immune serum (left panel), with anti-PRDM9 antibody in green (middle panel), and with anti-FLAG antibody in green (right panel). DNA was counterstained with DAPI (blue). The co-localization of PRDM9 with nuclear DAPI signal suggests inherent nuclear localization of PRDM9 protein, even in a heterologous system. Scale bar = 20 μm (GIF 112 kb)

High resolution image (TIFF 1738 kb)

Supplementary Fig. 2

PRDM9 in the nuclei of germ cells during early meiotic prophase I. a, b PRDM9 in germ cells at early meiosis I prophase is revealed by immunolabeling of fibrin clot-embedded germ cells with a combination of anti-SYCP3 (red) to mark the SC and anti-PRDM9 (green). PRDM9 was not detected in pachytene spermatocytes (arrows in a) but was detected in germ cells with the patchy SYCP3 labeling typical of pre-leptotene/leptotene spermatocytes (arrows in b). c–f PRDM9 is detected in the nuclei of spermatocytes co-labeled with EdU during the pre-meiotic S-phase. Juvenile males (8 dpp) were injected with EdU for a 6-h pulse, followed by removal of testes for immunolabeling. Whole-mounted seminiferous tubules were immunolabeled with PRDM9 antibody (green); the incorporation of EdU was detected with Alexa Fluor®594 (red); and nuclei were counterstained with DAPI (blue in c and f). Localization of PRDM9 in EdU-positive germ cells provides further evidence that PRDM9 is initially expressed in S-phase/post-S-phase pre-leptotene/leptotene spermatocytes (arrow in e–f). Scale bars = 20 μm (GIF 113 kb)

High resolution image (TIFF 1535 kb)

Supplementary Fig. 3

Abnormal silencing of unsynapsed chromatin in Prdm9-deficient pachytene-like spermatocytes. Spread chromatin preparations from wild-type (a) and Prdm9 M1045Lja-mutant (b) spermatocytes were labeled with antibodies recognizing SUMO1 (green), a marker of transcriptional silencing, SYCP3 (blue) to mark the SC, P-H2AFX (red). The co-localization of SUMO1 and P-H2AFX (arrows in b) provides evidence for transcriptional silencing. Scale bar = 20 μm (GIF 29 kb)

High resolution image (TIFF 274 kb)

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Sun, F., Fujiwara, Y., Reinholdt, L.G. et al. Nuclear localization of PRDM9 and its role in meiotic chromatin modifications and homologous synapsis. Chromosoma 124, 397–415 (2015). https://doi.org/10.1007/s00412-015-0511-3

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