Paternal imprint essential for the inheritance of telomere identity in Drosophila

Abstract

Chromatin remodeling during sperm maturation could erase epigenetic landmarks on the paternal genome, creating a challenge for its reestablishment on fertilization. Here, we show that selective retention of a chromosomal protein in mature sperm protects the identity of paternal telomeres in Drosophila. The ms(3)k81 (k81) gene is a duplication of hiphop that encodes a telomeric protein. Although HipHop protects telomeres in somatic cells, K81 is produced exclusively in males and localizes to telomeres in postmitotic cells, including mature sperm. In embryos fathered by k81 mutants, the maternal supplies fail to reestablish a protective cap on paternal telomeres, leading to their fusions. These fusions hinder the segregation of the paternal genome and result in haploid embryos with maternal chromosomes. The functional divergence between hiphop and k81 manifests not only in their expression patterns but also in the protein functions that they encode. By swapping the two coding regions, we show that K81 can replace HipHop for somatic protection; however, HipHop cannot replace K81 in the germ line to specify telomere identity, because HipHop ectopically expressed in the testis is removed from chromatin during sperm maturation. HipHop lacks a short motif in K81 that is essential for K81 to survive the remodeling process. We show that the combined functions of HipHop and K81 are likely fulfilled by the single ancestral hiphop locus in other Drosophila species, supporting the hypothesis that the evolutionary process of subfunctionalization was responsible for the preservation of the hiphop-k81 duplicate.

Fig. 1.

K81 binds and protects paternal telomeres. For A–D, samples were prepared from testes mutant for k81 but expressing GFP-K81. (A) A nucleus in meiosis II was stained with anti-GFP. Chromosomes are in white, and K81 signals in red. (A′) GFP signals only. (B–D) Merged images of GFP-K81 foci in spermatids, with DAPI signals in white and GFP signals in red. (B′, C′, and D′) GFP signals only. (E–H) Images of mitotic chromosomes from embryos. (E) From embryo with a WT father. (F–H) From embryos with k81 fathers. In F, one-half of the genome engaged in intrachromosomal telomere fusions forming circular chromosomes (marked by asterisks), and the other one-half had normal, separated telomeres. In G, one-half of the genome (marked by arrowheads) has separated telomeres, and the other one-half engaged in interchromosomal fusions forming a chain starting from the paternal Y chromosome (marked with arrow). H shows a haploid nucleus with all of the chromosomes labeled.

Fig. 2.

Functional divergence between HipHop and K81. In I, protein-coding regions are represented as rectangular boxes, with the residues defining the A, B, and C segments numbered on top of the boxes. In II, transgenes with either an mCherry or GFP tag were expressed under the control of hiphop regulation, and all lines were able to rescue hiphop lethality. 1, partial rescue with some animals dying as pharate adults (in the text). In III, transgenes were under the control of k81 regulation and were tested for their ability to rescue k81 sterility. These transgenes were also tested for their ability to form nuclear foci in spermatids of different stages under both WT and k81 mutant background. In each chimeric gene, a segment and its corresponding parental gene have the same shading. The asterisk in the last gene corresponds to the QFVH to PTV mutation in k81^PTV. 2, partial rescue (in the text).

Fig. 3.

Chromatin retention in mature sperm distinguishes K81 from HipHop. (A–I) Merged images of K81 foci in spermatids or sperm in WT males expressing various hiphop-k81 chimeric genes tagged with GFP, with DAPI signals in white and GFP signals in red. The names and schematics of the chimeric proteins are shown to the left of picture panels. For name and shading definitions for the chimera, see Fig. 2 and the text. The extent of k81 rescue for each construct is stated under the schematics for the chimeras. In J, DAPI signals from mature sperm are in white. J′ is an overexposed picture of the GFP channel showing no GFP foci on sperm chromatin shown in J.

Fig. 4.

The bimodal PTV-QFVH motif in HipHop and K81 proteins. An alignment of the Fig. 2I C segment (Fig. 2I) of HipHops from sequenced Drosophila species is shown at the top, with identical residues marked by asterisks underneath. The PTV and QFVH motifs are shaded gray. Species with K81 are labeled with a vertical line to the left. An alignment of the C segment of K81s from the sequenced melanogaster subgroup is shown in the middle, with melanogaster HipHop included at the top. At the bottom, the status of the PTV/QFVH motif is indicated for each species in reference to their evolutionary relationship and the status of the k81 duplication. –, the absence of k81.

Fig. 5.

The paternal imprint on telomeres does not contain HOAP. Samples are taken from testes with a gfp-tagged hoap locus under either live (A, A′, B, B′, D, and D′) or fixed conditions (C and C′). In the merged images A–C and image D, DAPI signals are in white. GFP signals are in red in A and B. In C, signals in red from anti-HOAP staining decorate telomeres on condensed chromosomes during meiosis II. D′ is an overexposed picture of the GFP channel showing no GFP foci in spermatids shown in D.

Fig. 6.

D. virilis HipHop foci in both somatic and sperm nuclei. A–C are merged images from a D. virilis strain transformed with a gfp-tagged hiphop transgene. DAPI signals are in white, and GFP signals in red. (A′–C′) GFP signals only. (A and A′) Cells from the mitotic compartment at the tip of the testis showing GFP-HipHop nuclear foci. (B and B′) A bundle of spermatids with HipHop foci. (C and C′) Two mature sperm with HipHop foci.