HEIP1 orchestrates pro-crossover protein activity during mammalian meiosis

Abstract

Meiotic crossovers (COs) are needed to produce genetically balanced gametes. In mammals, CO formation is mediated by a conserved set of pro-CO proteins via mechanisms that remain unclear. Here, we characterize a mammalian pro-CO factor HEIP1. In mouse HEIP1 is essential for crossover and fertility of both sexes. HEIP1 promotes crossover by orchestrating the recruitment of other pro-CO proteins, including the MutSγ complex (MSH4-MSH5) and E3 ligases (HEI10, RNF212, and RNF212B), that are required to mature CO sites and recruit the CO-specific resolution complex MutLγ. Moreover, HEIP1 directly interacts with HEI10, suggesting a direct role in controlling the recruitment of pro-CO 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 conserved master regulator of CO proteins that controls different CO maturation steps.

Keywords: E3-ligases; HEIP1; crossovers; homologous recombination; meiosis.

Fig. 1.

Heip1 is essential for chiasma and crossover formation. (A, 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.) Arrows indicate univalents in a Heip1^−/− cell. (Scale bar, 1 μm.) (Right) Number of bivalents per nucleus. Each dot corresponds to one nucleus. (B, Left) Immunostaining of MLH1 and SYCP3 on chromosome spreads from wild type and Heip1^−/− spermatocytes at 18 dpp. White squares correspond to the zoom images. (Right) number of MLH1 foci per nucleus. (Scale bar, 10 µm.) For all graphs, ****P < 0.0001 (two-tailed unpaired Mann–Whitney test).

Fig. 2.

HR and synapsis in wild type and Heip1^−/− mice. (A and B) Analysis of HR in wild type and Heip1^−/− mice. (A) SYCP3 (orange) and MSH4 (green), and (B) 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). (C) Chromosome synapsis is defective in Heip1^−/− mice. Representative images of chromosome spreads from 60 dpp wild type and Heip1^−/− spermatocytes immunostained for γH2Ax (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). (D) 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 nonhomologous chromosomes (nonhomologous synapsis). Experiments were performed at least twice and one experiment is shown. (E) Percentages of pachytene spermatocytes with paired X-Y chromosomes in wild type and Heip1^−/− mice.

Fig. 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 (gray, stained nucleus presented in a dashed box). (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 SC. STED microscopy images of chromosome spreads from wild type spermatocytes at late zygonema and early pachynema immunostained for SYCP3 (red) and HEIP1 (green). Arrowheads: 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 (gray), 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 and F) Chromosome spreads from Top6bl^−/− (E) and Sycp1^−/− (F) prophase I spermatocytes from 17 and 60 dpp mice respectively, immunostained for SYCP3 (gray), HEIP1 (red), and RPA2 (green). 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.

Fig. 4.

HEIP1 progressively localizes to CO sites. (A) HEIP1 colocalization with TEX11 at zygonema and pachynema. (Upper) Immunostaining of wild type spermatocytes for SYCP3 (gray), HEIP1 (red), and TEX11 (green). (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) Immunostaining for SYCP3 (gray), 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.

Fig. 5.

Interplay between HEIP1 and the pro-CO RING finger E3 ligases HEI10, RNF212, and RNF212B. (A) Coimmunoprecipitation 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 (gray), 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.

Fig. 6.

Interdependent relationship between HEIP1 and the pro-CO RING finger E3 ligases. (A) Chromosomal localization (Left) 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 (Up) and quantification (Bottom) 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 and D) Chromosomal localization (Top) and quantification of HEIP1 (Bottom) in wild type and (C) Rnf212b^−/− and (D) Hei10^−/− mice. Immunostaining for SYCP3 (blue) and HEIP1 (red) on wild type and adult Rnf212b^−/− and Hei10^−/− spermatocytes at different prophase I stages. 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.

Fig. 7.

Model for HEIP1-dependent CO orchestration during prophase I of meiosis. (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 transits from the axis to the central region of the synaptonemal complex (SC). (4) At these positions, HEIP1 may recruit CO-promoting factors (MutSγ and TEX11 - blue circles). (5–6) Then, HEIP1 promotes the proper deposition of HEI10, RNF212, RNF212B, and MutLγ. Once all proteins required for CO maturation are recruited, HEIP1 disappears.