Yukiko M. Yamashita

Germline-restricted chromosomes (GRCs) constitute a unique class of chromosomes confined to germ cells. Arising in multiple evolutionarily distant lineages, GRCs have been identified in 3 insect families (nonbiting midges, gall midges, and fungus gnats), each within the order Diptera. Genomic characterization in fungus gnats has revealed GRCs to be large, gene-rich chromosomes, which, together with their persistence over millennia, implies they play important biological roles within this clade. However, transcription from these chromosomes has yet to be demonstrated, leaving key questions about their function and activity unresolved. Here, we present the first direct analysis of GRC transcription in Diptera, combining immunofluorescence staining with bulk RNA-seq data in the fungus gnat Bradysia coprophila (Sciaridae). We find that transcription from GRCs is highly limited during key developmental stages. In larval and adult gonads, elongating polymerase II appears to be active at all chromosomes except the GRCs in most cells. Furthermore, using stringent filtering criteria, we identify only 10 confidently expressed GRC-linked genes, mostly of unknown function. In addition, we report the surprising presence of a ∼290 kb region of bacterial-derived sequence on GRC2, likely acquired through horizontal transfer from germline-associated endosymbionts. Overall, these findings identify GRCs in fungus gnats as unique chromosomes, largely transcriptionally quiescent during key stages of B. coprophila development, yet capable of maintaining both endogenous and potentially horizontally acquired genes over evolutionary timescales. This raises fundamental questions about the mechanisms that maintain gene content in these chromosomes and their contribution, if any, to germline biology.

Genome integrity is fundamental to cellular and organismal health, with the germline genome bearing the most profound impact. During spermatogenesis, post-meiotic germ cells, called spermatids, undergo a series of morphological changes over an extended period (a week in fruit flies, two weeks in humans) to yield mature sperm. Having undergone consecutive meiotic divisions, these spermatids lack homologous chromosomes or sister chromatids, critical templates for accurate DNA repair. How genome integrity of spermatids is ensured during this vulnerable yet prolonged period remains poorly understood. In this study, we show that the process of spermatogenesis is specifically compromised to eliminate damaged nuclei upon γ-irradiation. We further find a non-canonical role of histone variant H2Av in the process of spermatid elimination. Our findings demonstrate the presence of a haploid-specific quality control program that ensures the integrity of the sperm genome transmitted to the next generation.

The nucleolus is widely regarded as a specialized compartment for RNA polymerase I (Pol I)-driven ribosomal RNA transcription and ribosome biogenesis. Yet the presence of “atypical nucleoli”, or nucleolus-like bodies (NLBs), which lack rRNA transcription despite containing canonical nucleolar components, has long been recognized, most notably during mammalian oogenesis and spermatogenesis. NLBs have been shown to have an essential function independent of rRNA transcription, but the nature of that function remained unclear. Here, we demonstrate that the nucleolus becomes an NLB during spermatocyte development in Drosophila melanogaster and, surprisingly, that this NLB serves as a platform for RNA polymerase II (Pol II)-mediated transcription. We find that the Y chromosome-linked fertility genes, which are heterochromatic in most cell types but highly expressed in spermatocytes, are transcribed at the spermatocyte NLB. We further show that the recruitment of active Pol II to the NLB requires known spermatocyte-specific transcriptional regulators. In their absence, the Y-linked fertility genes embedded within heterochromatin are not properly transcribed. Our findings reveal an active role for an NLB as a Pol II platform, and we propose that other NLBs may have similar functionality.

Male germ cells have complex transcriptomes, with a large fraction of the genome being transcribed. This includes protein-coding genes (often not translated), non-coding DNA, and repetitive DNA, such as transposons and satellite DNA, which are normally silenced as heterochromatin. The significance of such widespread transcription remains unknown. Here, we show that a heterochromatin protein, HP2, is required for the transcription of AAGAG satellite DNA in Drosophila spermatocytes. HP2 depletion leads to abnormal retention of heterochromatin histone marks (H3K9me3) and spermatid death during sperm DNA packaging, leading to a model that transcription of AAGAG satellite DNA facilitates the remodeling of its heterochromatic nature in preparation for sperm DNA packaging. Strikingly, the severity of the spermatid death correlates with the amount of AAGAG satellite DNA carried by the spermatids, leading to preferential death of Y-chromosome-containing spermatids over X-containing spermatids, and hence sex-ratio meiotic drive phenotype. We propose that widespread spermatocyte transcription may reflect the process of chromatin remodeling to allow sperm DNA packaging. We further propose that differential composition and amount of satellite DNA on chromosomes may underlie naturally occurring male meiotic drive.

Adult stem cells maintain tissue homeostasis, yet are themselves vulnerable to loss. One common mechanism to replace lost stem cells is dedifferentiation, in which progeny revert to stem cell identity. It is a paradox how stem cells and progeny retain the same stem cell potential while exhibiting distinct current identities of self-renewal, differentiation, and dedifferentiation. Here, we show that the Drosophila male germline lineage solves this paradox via two parallel and complementary mechanisms to separate potential and identity. First, differentiating progeny maintain stem cell potency by inheriting perdurant stem cell mRNAs without actively transcribing them. Second, two known niche signals (Bmp and Jak-Stat) activate distinct sets of targets, defining three identities (self-renewal, differentiation, and dedifferentiation) based on the combination of their on/off states. Together, this study reveals how a pool of dedifferentiation-competent progeny is maintained to regenerate stem cells as needed without resulting in stem cell overproduction and resolves the puzzle of why most stem cell systems require multiple independent niche signals.

The Y chromosome evolves rapidly, often differing dramatically even between closely related species. While such divergence has long been suspected to contribute to hybrid male sterility, leading to reproductive isolation and thus speciation, the underlying mechanisms remain elusive. Here, we identify a molecular basis linking Y chromosome divergence to reproductive isolation in Drosophila. We show that male hybrids between D. simulans and D. mauritiana fail to properly express key Y-linked fertility genes. These genes contain unusually large introns, exceeding megabases and show substantial sequence divergence between species. In the hybrids, these gigantic introns are misprocessed, resulting in widespread splicing defects, including aberrant “back-splicing” events that join later exons to earlier ones. Our findings suggest that sequence divergence within introns can disrupt essential gene expression through defective splicing, providing a mechanistic link between rapid Y chromosome evolution and hybrid sterility. This work highlights the underappreciated role of intronic divergence in speciation.

Meiotic drive is a phenomenon that violates Mendel's Law of Equal Segregation, leading to biased transmission of the meiotic driver to the offspring. Drosophila melanogaster Stellate (Ste) is an X-linked meiotic driver that preferentially harms Y-chromosome-bearing spermatids, thereby favoring the transmission of the X chromosome to the next generation. We have recently shown that Ste protein segregates asymmetrically during meiosis I with a strong bias toward the Y-chromosome-inheriting side, leading to the eventual demise of the Y-chromosome-containing spermatids. However, the cellular mechanisms by which Ste protein interferes with spermatid development remain unknown. Here, we show that Ste-containing spermatids are delayed in the process of nuclear envelope remodeling, an essential process during sperm DNA compaction. We show that components of the nuclear lamina (such as Lamin Dm0, and the LEM domain proteins Otefin and Bocks) are rapidly removed during nuclear envelope remodeling during the early stages of normal spermatid development. However, Ste-containing spermatids retained these nuclear lamina proteins for a prolonged time. Their delayed removal is associated with defective formation of the dense complex, which is composed of a bundle of microtubules and serves as a structural support for sperm nuclear morphogenesis. Defective dense complex formation in Ste-containing spermatids led to defective sperm DNA compaction. Together, the present study reveals an unexpected cellular mechanism by which a meiotic driver, Ste, sabotages sperm development.

Meiotic drive is a phenomenon that violates Mendel’s Law of Equal Segregation, leading to biased transmission of the meiotic driver to the offspring. D. melanogaster Stellate (Ste) is an X-linked meiotic driver that preferentially harms Y-chromosome-bearing spermatids, thereby favoring the transmission of the X chromosome to the next generation. We have recently shown that Ste protein segregates asymmetrically during meiosis I with a strong bias toward the Y-chromosome-inheriting side, leading to the eventual demise of the Y-chromosome-containing spermatids. However, the cellular mechanisms by which Ste protein interferes with spermatid development remain unknown. Here, we show that Ste-containing spermatids are delayed in the process of nuclear envelope remodeling, an essential process during sperm DNA compaction. We show that components of the nuclear lamina (such as Lamin Dm0, and the LEM domain proteins Otefin and Bocks) are rapidly removed during nuclear envelope remodeling during the early stages of normal spermatid development. However, Ste-containing spermatids retained these nuclear lamina proteins for a prolonged time. Their delayed removal is associated with defective formation of the dense complex, which is composed of a bundle of microtubules and serves as a structural support for sperm nuclear morphogenesis. Defective dense complex formation in Ste-containing spermatids led to defective sperm DNA compaction. Together, the present study reveals an unexpected cellular mechanism by which a meiotic driver, Ste, sabotages sperm development. Article summary: Stellate is an X-chromosome-linked meiotic driver in D. melanogaster , which interferes with the process of spermatogenesis and causes preferential death of the Y-chromosome-containing spermatids. However, the cellular mechanisms by which Stellate interferes with spermatogenesis remain unknown. This study shows that Stellate-containing spermatids are defective in the process of nuclear envelope remodeling, an essential process during sperm DNA compaction. Defective nuclear envelope remodeling was associated with a failure to assemble the dense complex, a microtubule-rich structure that serves as structural support for sperm nuclear morphogenesis. Together, the study provides insights into a cellular strategy employed by a meiotic driver.

Programmed DNA elimination, a phenomenon wherein cells eliminate a subset of genetic material during certain stages of development, is observed in a broad range of organisms. The fungus gnat Bradysia (formerly Sciara) coprophila undergoes a series of programmed DNA elimination events during their development, including elimination of germline-restricted chromosomes (called L chromosomes) in the soma and elimination of paternal chromosomes during male meiosis. However, a lack of understanding surrounding the nature of eliminated chromosomes poses a barrier to studying programmed DNA elimination in this system. Highly repetitive satellite DNA, which often shows chromosome-specific distribution, is a possible candidate for sequences involved in programmed DNA elimination. In this study, we utilized recent genomic data and genome assemblies to identify new satellite DNA sequences of B. coprophila, and characterized their distribution on chromosomes. The results imply that the X and autosomes do not share centromeric satellite DNA sequence (BcopSat-155) with the L chromosomes. We further provide cytological evidence that confirms a recent finding based on the genome assembly that there are 2 distinct L chromosomes that were not previously distinguished cytologically. Together, our work lays a foundation for future studies to explore the possible connection between satellite DNA and the mechanism of programmed DNA elimination in B. coprophila.