HEIP1 is required for efficient meiotic crossover implementation and is conserved from plants to humans

Abstract

Crossovers (CO) shuffle genetic information and physically connect homologous chromosomal pairs, ensuring their balanced segregation during meiosis. COs arising from the major class I pathway require the activity of the well-conserved group of ZMM proteins, which, in conjunction with MLH1, facilitate the maturation of DNA recombination intermediates specifically into COs. The HEI10 Interacting Protein 1 (HEIP1) was identified in rice and proposed to be a new, plant-specific member of the ZMM group. Here, we establish and decipher the function of the Arabidopsis thaliana HEIP1 homolog in meiotic crossover formation and report its wide conservation in eukaryotes. We show that the loss of Arabidopsis HEIP1 elicits a marked reduction in meiotic COs and their redistribution toward chromosome ends. Epistasis analysis showed that AtHEIP1 acts specifically in the class I CO pathway. Further, we show that HEIP1 acts both prior to crossover designation, as the number of MLH1 foci is reduced in heip1, and at the maturation step of MLH1-marked sites into COs. Despite the HEIP1 protein being predicted to be primarily unstructured and very divergent at the sequence level, we identified homologs of HEIP1 in an extensive range of eukaryotes, including mammals.

Keywords: HEIP1; crossover; meiosis; recombination.

Fig. 1.

Schematic representation of the Arabidopsis HEIP1 gene, heip1 mutations, and fertility analysis of heip1 mutants. (A) The gene orientation is indicated by a horizontal arrow, and exons are indicated by solid black boxes, while introns and untranslated regions (UTR) are represented by the black line. Inverted triangles indicate T-DNA insertion points and black and blue dotted lines denote the deleted region in heip1-4 (Columbia) and heip1-5 (Landberg), respectively. The heip1-4 allele may synthesize eight amino acids shown with single letter code, whereas the heip1-5 has a complete deletion of the coding sequence. (B) Each black circle represents the average seeds per fruit for one plant, obtained by counting at least ten fruits per plant. Comparison of fertility based on the number of seeds per fruit in a series of heip1 mutants. The mean for each genotype is represented by a red bar. Each heip1 mutant allele is compared with wild-type sister plants that were cultivated together in a segregating population.

Fig. 2.

Chromosomes spreads reveal meiotic defects in heip1 mutants. Wild type (A–F): (A) Pachytene. (B) Diakinesis and (C) metaphase I with five bivalents (b). (D) telophase I, with two sets of five chromosomes. (E) anaphase II. (F) tetrad. heip1-1 (G–L): (G) Pachytene, which is indistinguishable from wild type. (H) Diakinesis with three bivalents (b) and two pairs of univalents (u). (I) Metaphase I with three bivalents and two pairs of univalents, (J) Telophase I with unbalanced chromosome distribution. (K) Anaphase II with unequal chromosome segregation, and (L) polyads. (Scale bar, 10 µm identical for all panels.) (M) Quantification of bivalents and univalents at metaphase I. Cells were categorized according to the number of pairs of univalents/bivalents. The mean bivalents number per cell and the number of cells analyzed are indicated above each bar.

Fig. 3.

Epistasis analysis of heip1-1. (A–F) Representative image of metaphase I chromosome spreads of male meiocytes in the following genotypes: (A) heip1-1 hei10, (B) heip1 msh5, (C) heip1-1 mus81, (D) heip1-1 mlh1, (E) heip1-1 fancm, (F) heip1-1 HEI10-OE. (Scale bar, 10 µm, identical for all panels.) (G) Quantification of bivalents at metaphase I. Cells were categorized according to the number of bivalents. The average number of bivalents per cell and the number of analyzed cells are indicated above each bar.

Fig. 4.

Synapsis and HEI10 dynamics are unaffected in heip1-1. The three columns on the Left panel represent wild-type meiocytes and the three columns on the Right panel show heip1-1 meiocytes in various stages of meiotic prophase I (from top to bottom) at zygotene, pachytene, late pachytene, and diplotene. Immunolocalization of ZYP1 (white, A–D and M–P), HEI10 (white, E-H and P-T; green, I-L and U–X) and REC8 (purple, I-L and U-X), on male meiocytes. ZYP1 was acquired with confocal microscopy, while HEI10 and REC8 were acquired with STED microscopy. (Scale bar, 1 µm.)

Fig. 5.

The number of HEI10-MLH1 foci is reduced in heip1. Immunolocalization of MLH1 and HEI10 on male meiocytes at diplotene/diakinesis. (A–E) Wild-type. (F–J) heip1. (K–O) heip1 mus81. (P–T) heip1 HEI10-OE. From Left to Right: HEI10 in red (A, F, K, and P), MLH1 in green (B, G, L, and Q), merge HEI10 & MLH1 (C, H, L, and Q), merge All (D, I, N, and S), and DAPI in white (E, J, O, and T). (U) Quantifications of MLH1–HEI10 cofoci. Each mutant was compared to sibling controls and separated by vertical lines. Each dot is an individual cell, and the red bar is the mean. (Scale bar, 5 µm.) P values are from Fisher’s LSD tests.

Fig. 6.

Analysis of the distribution of HEI10–MLH1 foci in heip1. (A–D) Triple immunolocalization of REC8, HEI10, and MLH1 on heip1 male meiocytes. Imaging was done with 3D-STED and the projection is shown. (Scale bar, 1 µm.) (E and F) REC8 signal was traced in 3D using the IMARIS tool. (E) All five bivalents are represented in a separate color and a similar color was used to mark HEI10/MLH1 co-foci on that bivalent. (F) Representation of one bivalent with HEI10/MLH1 co-foci. (G) The length of chromosomes (filaments) and distribution of HEI10/MLH1 foci among chromosomes analyzed in five cells are presented (The cell shown in (A–F) is cell number 5).

Fig. 7.

Analysis of CO numbers, distribution, and interference. (A) The number of COs per transmitted chromatid in female and male gametes of wild type and heip1. Fisher’s LSD test was applied. (B) The chromosomal distribution of COs in female and male wild type and heip1, with a window size of 1 Mb and step size of 50 kb. (C) CoC curves in female and male meiosis of wild type and heip1, in which chromosomes were divided into 15 intervals to estimate the mean coefficient of coincidence.

Fig. 8.

Sequence similarity between Arabidopsis HEIP1 and Human C12orf40 together with other plant and animal homologs. Homologs of HEIP1 and C12orf40 were retrieved with HHlits (Dataset S3). The N-terminal ends of proteins from representative species of diverse plant and animal clades were aligned in Jalview 2.11.2.5 using T-coffee with default parameters. Figure prepared with Biorender.