HEIP1 orchestrates pro-crossover protein activity during mammalian meiosis

Abstract

Meiotic crossovers are needed to produce genetically balanced gametes. In mammals, crossover formation is mediated by a conserved set of pro-crossover proteins via mechanisms that remain unclear. Here, we characterize a mammalian pro-crossover factor HEIP1. In mouse HEIP1 is essential for crossing over and fertility of both sexes. HEIP1 promotes crossing over by orchestrating the recruitment of other pro-crossover proteins, including the MutSγ complex (MSH4-MSH5) and E3 ligases (HEI10, RNF212, and RNF212B), that are required to mature crossover sites and recruit the crossover-specific resolution complex MutLγ. Moreover, HEIP1 directly interacts with HEI10, suggesting a direct role in controlling the recruitment of pro-crossover E3 ligases. During early stages of meiotic prophase I, HEIP1 interacts with the chromosome axes, independently of recombination, before relocalizing to the central region of the synaptonemal complex. We propose that HEIP1 is a new conserved master regulator of crossover proteins that controls different crossover maturation steps.

Figure 1.. Heip1 deletion leads to massive germline loss and infertility.

(A) Schematic representation of the Heip1 locus showing exons (blue boxes), untranslated regions (5′-UTR, purple box), and the start of the open reading frame in exon 1. View of exon 3 shows the guide RNA sequences (guide RNA rev with PAM underlined) used for CRISPR/Cas9 editing, the genomic DNA/cDNA sequence of the edited region and the corresponding wild type (WT) and Heip1^−/− protein sequences. DNA break sites are marked by red arrows; red text color marks the mutated DNA and protein sequences. (B) Representative image of wild type and Heip1^−/− testes (top). Testis to body weight ratios of 2-month-old wild type and Heip1^−/− mice. Each dot corresponds to the mean value for the two testes from one mouse. Data are the mean ± SD. ****P < 0.0001 (two-tailed Student’s t-test). (C) Representative images of seminiferous tubules stained with hematoxylin and eosin from testis sections of 60 dpp wild type and Heip1^−/− mice. In Heip1^−/− seminiferous tubules, cells progress to metaphase I (stage XII) (see arrows), normal chromosome congression is not observed, and post-meiotic spermatogenic cells (spermatids and spermatozoa) are absent. Scale bar, 50 μm (D) Left: DAPI staining of diakinesis/metaphase I chromosome spreads and magnified images from wild type and Heip1^−/− spermatocytes from 60 dpp mice. Scale bar, 10 μm. Arrow indicates a univalent in a Heip1^−/− cell. Scale bar, 1 μm. Right: number of bivalents per nucleus Each dot corresponds to one nucleus. (E) Immunostaining of MLH1 and SYCP3 on chromosome spreads from wild type and Heip1^−/− spermatocytes at 18 dpp. White squares correspond to the zoom images on the right. The number of MLH1 foci is presented on the right. Scale bar, 10 μm. For all graphs, ****P < 0.0001 (two-tailed unpaired Mann-Whitney test).

Figure 2.. Homologous recombination and synapsis in wild type and Heip1^−/− mice.

(A-D). Analysis of homologous recombination in wild type and Heip1^−/− mice. (A) SYCP3 (orange) and DMC1 (green), (B) SYCP3 (orange) and SPATA22 (green), (C) SYCP3 (orange) and MSH4 (green), and (D) SYCP3 (orange) and Bloom (green) localization in 18 dpp wild type and Heip1^−/− spermatocytes at different prophase I substages. Scale bar, 10 μm (all panels). Plots at the bottom of the panels show the quantification of foci; ns, not significant, *P < 0.1, **P < 0.01, ****P < 0.0001 (two-tailed unpaired Mann-Whitney test). (E) Chromosome synapsis is defective in Heip1^−/− mice. Representative images of chromosome spreads from 60 dpp wild type and Heip1^−/− spermatocytes immunostained for ɣH2Ax (purple), SYCP3 (red) and SYCP1 (green). Magnified images from the white boxes show representative chromosomes. Scale bars, 10 μm for full nuclei and 1 μm for the magnified images. (F) Percentages of pachytene spermatocytes with and without synapsis defects in wild type and Heip1^−/− mice. Cells were divided into three groups: with fully synapsed chromosomes (normal synapsis); with discontinued or unsynapsed chromosomes (abnormal synapsis); with synapsis between non-homologous chromosomes (non-homologous synapsis). Experiments were performed at least twice and one experiment is shown. (G) Percentages of pachytene spermatocytes with paired X-Y chromosomes in wild type and Heip1^−/− mice.

Figure 3.. HEIP1 localization dynamics in prophase I spermatocytes.

(A) HEIP1 localization in wild type spermatocyte nuclei at the different prophase I substages. Top: Representative images of chromosome spreads from prophase I spermatocyte nuclei immunostained for SYCP3 (orange), HEIP1 (green) and histone H1t (grey). Bottom: quantification of HEIP1 foci per spermatocyte nucleus at the different prophase I stages. The mean numbers of foci are shown below. Numbers of nuclei analyzed: 30, 40, 21, 77, 29, 96 and 52 for leptonema, early/mid zygonema, late zygonema, early pachynema, mid pachynema, late pachynema, respectively. (B) HEIP1 localizes to the central region of the synaptonemal complex. STED microscopy images of chromosome spreads from wild type spermatocytes at late zygonema and early pachynema immunostained for SYCP3 (red) and HEIP1 (green). Arrowheads highlight HEIP1 foci between chromosome axes. White squares correspond to the zoom images that show representative chromosomes. (C) HEIP1 colocalizes with RPA2 in prophase I spermatocytes. Left: Representative images of prophase I chromosomes from wild type mice immunostained for SYCP3 (grey), HEIP1 (red) and RPA2 (green). Right: Quantification of HEIP1 colocalization with RPA2 (top) and RPA2 colocalization with HEIP1 (bottom). (D) Localization dynamics of HEIP1 and RPA2 in wild type spermatocytes at early zygonema. Four steps of HEIP1 and RPA2 dynamics during synapsis are shown in the enlarged images 1 to 4 from the white squares. (E) Chromosome spreads from Top6bl^−/− prophase I spermatocyte nuclei immunostained for SYCP3 (grey), HEIP1 (red) and RPA2 (green). (F) Chromosome spreads from 60 dpp Sycp1^−/− prophase I spermatocyte nuclei immunostained for SYCP3 (grey), HEIP1 (red) and RPA2 (green). For each panel, magnified images show representative chromosomes. Scale bars, 10 μm for full nuclei and 1 μm for magnified images (2 μm for (B)). On graphs, bars indicate the mean ± SD and n=number of nuclei. Unless stated, 18 dpp mice were used.

Figure 4:. HEIP1 progressively localizes to crossover sites.

(A) HEIP1 colocalization with TEX11 at zygonema and pachynema. Upper panels: immunostaining of wild type spermatocytes for SYCP3 (grey), HEIP1 (red) and TEX11 (green). on the right. Bottom: quantification of HEIP1 and TEX11 foci during prophase I. (B) Quantification of TEX11 colocalization with HEIP1 and vice versa. (C) HEIP1 colocalization with MLH1 at pachytene stages. Upper panels: immunostaining for SYCP3 (grey), HEIP1 (red) and MLH1 (green) of wild type pachytene spermatocyte nuclei. Bottom: quantification of HEIP1 and MLH1 foci during prophase I. (D) Quantification of MLH1 colocalization with HEIP1 and vice versa. 18 dpp mice were used; on each panel magnified images (zoom) of representative chromosomes (white boxes) are presented, Scale bars, 10 μm for full nuclei and 1 μm for magnified images. On graphs, bars indicate the mean ± SD.

Figure 5.. Interplay between HEIP1 and the pro-CO RING finger E3 ligases HEI10, RNF212 and RNF212B.

(A) Co-immunoprecipitation of HEIP1 and HEI10 using wild type and Heip1^−/− testis protein extracts. Western blot inputs (left) and immunoprecipitation (right; IP) with rabbit antibodies against the C-terminal part of HEIP1. HEIP1 band in the wild type is detected at a size corresponding to a molecular weight of 100 KDa instead of 73 KDa expected, which could be the consequence of (i) potential posttranslational modifications (ii) or of the intrinsic biochemical property of the protein. β-tubulin was used as input control. (B) HEIP1 colocalization with HEI10 at pachytene stages. Immunostaining for SYCP3 (grey), HEIP1 (red) and HEI10 (green) in wild type spermatocytes at early, mid and late pachynema. (C) Percentage of HEIP1 foci that colocalize with HEI10 foci, and vice versa. (D) HEIP1 colocalization with RNF212. Immunostaining for SYCP3 (blue), HEIP1 (red) and RNF212 (green) on wild type spermatocytes at zygonema, early and mid-pachynema. Arrowheads show CO sites. (E) Percentage of RNF212 that colocalizes with HEIP1, and vice versa. (F) Percentage of RNF212B foci that colocalize with HEIP1 foci, and vice versa. (G) HEIP1 colocalization with RNF212B. Immunostaining for SYCP3 (blue), HEIP1 (red) and RNF212B (green) on wild type spermatocytes at zygonema, early and mid-pachynema. Arrowheads show CO sites. For all images: zoom are magnified images of representative chromosomes (white boxes); scale bars: 10 μm for full nuclei and 1 μm for magnified images. For each graph: bars indicate the mean ± SD. 18 dpp mice were used.

Figure 6.. Interdependent relationship between HEIP1 and the pro-CO RING finger E3 ligases.

(A) Chromosomal localization (left and middle) and quantification (right) of HEI10 in wild type and Heip1^−/− mice. Immunostaining for SYCP3 (orange) and HEI10 (green) on wild type and Heip1^−/− spermatocytes at mid pachynema. (B) Chromosomal localization (left) and quantification (right) of RNF212 and RNF212B in Heip1^−/− mice. Immunostaining for SYCP3 (blue), RNF212 (green) and RNF212B (red) on wild type and Heip1^−/− spermatocytes at early and mid-pachynema. (C) Left: chromosomal localization of RNF212 and HEI10 in Heip1^−/− mice. Immunostaining for SYCP3 (blue), HEI10 (green) and RNF212 (red) on wild type and Heip1^−/− spermatocytes at mid-pachynema. Right: ratio of the fluorescence intensity of RNF212 and SYCP3 on chromosomes with at least one HEI10 focus and on chromosomes without HEI10 foci. (D) Chromosomal localization (top) and quantification of HEIP1 (bottom) in wild type and Rnf212b^−/− mice. Immunostaining for SYCP3 (blue) and HEIP1 (red) on wild type and adult Rnf212b^−/− spermatocytes at late zygonema, early and midpachynema. (E) Chromosomal localization (top) and quantification (bottom) of HEIP1 in Hei10^−/− mice. Immunostaining for SYCP3 (blue) and HEIP1 (Red) on adult wild type and Hei10^−/− spermatocytes at early, mid and late pachynema. 18 dpp mice were used, otherwise stated. Zoom: higher magnification of representative chromosomes (white boxes). Scale bars, 10 μm for full nuclei and 1 μm for magnified images. Bars indicate the mean ± SD; ***P < 0.001, ****P < 0.0001 (two-tailed unpaired Mann-Whitney test) and on each graph n= numbers of analyzed nuclei.

Figure 7.. Model for HEIP1-dependent CO orchestration during prophase I of meiosis.

Proposed HEIP1 roles during homologous recombination (A) and model that illustrates HEIP1 localization on chromosomes (B). (A) After break formation, HEIP1 co-localizes with RPA2 recombination intermediates, prior to DMC1/RAD51 (a) loading and/or before second end capture (b). At these positions, HEIP1 may promote second-end capture for repair and recruit CO-promoting factors, such as the MutSγ complex and TEX11 (blue circles), to protect recombination intermediates against Bloom dissolution (NCO). Simultaneously, HEIP1 facilitates RNF212 and RNF212B relocalization, HEI10 and MutLγ recruitment. Then, at the time of crossover resolution, HEIP1 is removed. (B) (1) HEIP1 (red shapes) initially appears along chromosome axes independently of DSBs. (2) During chromosome alignment, HEIP1 relocates and colocalizes with single-stranded DNA (ssDNA) coated by RPA2 (green circles), marking recombination sites. (3) During chromosome synapsis, HEIP1 transitions from the axis to the central region of the synaptonemal complex (SC). (4) At these positions, HEIP1 may recruit CO-promoting factors, such as the MutSγ complex and TEX11 (blue circles). (–6) Then, HEIP1 presence promotes the proper deposition of HEI10, RNF212, RNF212B, and MutLγ, which will mark the future crossover sites. Once all proteins required for CO maturation are recruited, HEIP1 disappears.