Uncharted territories: Solving the mysteries of male meiosis in flies.

The segregation of homologous chromosomes during meiosis typically requires tight end-to-end chromosome pairing. However, in Drosophila spermatogenesis, male flies segregate their chromosomes without classic synaptonemal complex formation and without recombination, instead compartmentalizing homologs into subnuclear domains known as chromosome territories (CTs). How homologs find each other in the nucleus and are separated into CTs has been one of the biggest riddles in chromosome biology. Here, we discuss our current understanding of pairing and CT formation in flies and review recent data on how homologs are linked and partitioned during meiosis in male flies.

Chromosome segregation during spermatogenesis occurs through a unique center-kinetic mechanism in holocentric moth species.

Precise regulation of chromosome dynamics in the germline is essential for reproductive success across species. Yet, the mechanisms underlying meiotic chromosomal events such as homolog pairing and chromosome segregation are not fully understood in many species. Here, we employ Oligopaint DNA FISH to investigate mechanisms of meiotic homolog pairing and chromosome segregation in the holocentric pantry moth, Plodia interpunctella, and compare our findings to new and previous studies in the silkworm moth, Bombyx mori, which diverged from P. interpunctella over 100 million years ago. We find that pairing in both Bombyx and Plodia spermatogenesis is initiated at gene-rich chromosome ends. Additionally, both species form rod shaped cruciform-like bivalents at metaphase I. However, unlike the telomere-oriented chromosome segregation mechanism observed in Bombyx, Plodia can orient bivalents in multiple different ways at metaphase I. Surprisingly, in both species we find that kinetochores consistently assemble at non-telomeric loci toward the center of chromosomes regardless of where chromosome centers are located in the bivalent. Additionally, sister kinetochores do not seem to be paired in these species. Instead, four distinct kinetochores are easily observed at metaphase I. Despite this, we find clear end-on microtubule attachments and not lateral microtubule attachments co-orienting these separated kinetochores. These findings challenge the classical view of segregation where paired, poleward-facing kinetochores are required for accurate homolog separation in meiosis I. Our studies here highlight the importance of exploring fundamental processes in non-model systems, as employing novel organisms can lead to the discovery of novel biology.

Dosage compensation in Bombyx mori is achieved by partial repression of both Z chromosomes in males.

SignificanceGenes on sex chromosomes (i.e. human chX) are regulated differently in males and females to balance gene expression levels between sexes (XY vs. XX). This sex-specific regulation is called dosage compensation (DC). DC is achieved by altering the shape and compaction of sex chromosomes specifically in one sex. In this study, we use Oligopaints to examine DC in silkworms. This study visualizes this phenomenon in a species with ZW sex chromosomes, which evolved independently of XY. Our data support a long-standing model for how DC mechanisms evolved across species, and we show potential similarity between DC in silkworms and nematodes, suggesting that this type of DC may have emerged multiple independent times throughout evolution.

NURF301 contributes to gypsy chromatin insulator-mediated nuclear organization.

Chromatin insulators are DNA-protein complexes that can prevent the spread of repressive chromatin and block communication between enhancers and promoters to regulate gene expression. In Drosophila, the gypsy chromatin insulator complex consists of three core proteins: CP190, Su(Hw), and Mod(mdg4)67.2. These factors concentrate at nuclear foci termed insulator bodies, and changes in insulator body localization have been observed in mutants defective for insulator function. Here, we identified NURF301/E(bx), a nucleosome remodeling factor, as a novel regulator of gypsy insulator body localization through a high-throughput RNAi imaging screen. NURF301 promotes gypsy-dependent insulator barrier activity and physically interacts with gypsy insulator proteins. Using ChIP-seq, we found that NURF301 co-localizes with insulator proteins genome-wide, and NURF301 promotes chromatin association of Su(Hw) and CP190 at gypsy insulator binding sites. These effects correlate with NURF301-dependent nucleosome repositioning. At the same time, CP190 and Su(Hw) both facilitate recruitment of NURF301 to chromatin. Finally, Oligopaint FISH combined with immunofluorescence revealed that NURF301 promotes 3D contact between insulator bodies and gypsy insulator DNA binding sites, and NURF301 is required for proper nuclear positioning of gypsy binding sites. Our data provide new insights into how a nucleosome remodeling factor and insulator proteins cooperatively contribute to nuclear organization.

Oligopaint DNA FISH reveals telomere-based meiotic pairing dynamics in the silkworm, Bombyx mori.

Accurate chromosome segregation during meiosis is essential for reproductive success. Yet, many fundamental aspects of meiosis remain unclear, including the mechanisms regulating homolog pairing across species. This gap is partially due to our inability to visualize individual chromosomes during meiosis. Here, we employ Oligopaint FISH to investigate homolog pairing and compaction of meiotic chromosomes and resurrect a classical model system, the silkworm Bombyx mori. Our Oligopaint design combines multiplexed barcoding with secondary oligo labeling for high flexibility and low cost. These studies illustrate that Oligopaints are highly specific in whole-mount gonads and on meiotic squashes. We show that meiotic pairing is robust in both males and females and that pairing can occur through numerous partially paired intermediate structures. We also show that pairing in male meiosis occurs asynchronously and seemingly in a transcription-biased manner. Further, we reveal that meiotic bivalent formation in B. mori males is highly similar to bivalent formation in C. elegans, with both of these pathways ultimately resulting in the pairing of chromosome ends with non-paired ends facing the spindle pole. Additionally, microtubule recruitment in both C. elegans and B. mori is likely dependent on kinetochore proteins but independent of the centromere-specifying histone CENP-A. Finally, using super-resolution microscopy in the female germline, we show that homologous chromosomes remain associated at telomere domains in the absence of chiasma and after breakdown and modification to the synaptonemal complex in pachytene. These studies reveal novel insights into mechanisms of meiotic homolog pairing both with or without recombination.

Condensin II drives large-scale folding and spatial partitioning of interphase chromosomes in Drosophila nuclei.

Metazoan chromosomes are folded into discrete sub-nuclear domains, referred to as chromosome territories (CTs). The molecular mechanisms that underlie the formation and maintenance of CTs during the cell cycle remain largely unknown. Here, we have developed high-resolution chromosome paints to investigate CT organization in Drosophila cycling cells. We show that large-scale chromosome folding patterns and levels of chromosome intermixing are remarkably stable across various cell types. Our data also suggest that the nucleus scales to accommodate fluctuations in chromosome size throughout the cell cycle, which limits the degree of intermixing between neighboring CTs. Finally, we show that the cohesin and condensin complexes are required for different scales of chromosome folding, with condensin II being especially important for the size, shape, and level of intermixing between CTs in interphase. These findings suggest that large-scale chromosome folding driven by condensin II influences the extent to which chromosomes interact, which may have direct consequences for cell-type specific genome stability.

Centromeres Drive a Hard Bargain.

Centromeres are essential chromosomal structures that mediate the accurate distribution of genetic material during meiotic and mitotic cell divisions. In most organisms, centromeres are epigenetically specified and propagated by nucleosomes containing the centromere-specific H3 variant, centromere protein A (CENP-A). Although centromeres perform a critical and conserved function, CENP-A and the underlying centromeric DNA are rapidly evolving. This paradox has been explained by the centromere drive hypothesis, which proposes that CENP-A is undergoing an evolutionary tug-of-war with selfish centromeric DNA. Here, we review our current understanding of CENP-A evolution in relation to centromere drive and discuss classical and recent advances, including new evidence implicating CENP-A chaperones in this conflict.

An egg-sabotaging mechanism drives non-Mendelian transmission in mice.

Selfish genetic elements drive in meiosis to distort their transmission ratio and increase their representation in gametes, violating Mendel's law of segregation. The two established paradigms for meiotic drive, gamete killing and biased segregation, are fundamentally different. In gamete killing, typically observed with male meiosis, selfish elements sabotage gametes that do not contain them. By contrast, killing is predetermined in female meiosis, and selfish elements bias their segregation to the single surviving gamete (i.e., the egg in animal meiosis). Here, we show that a selfish element on mouse chromosome 2, Responder to drive 2 (R2d2), drives using a hybrid mechanism in female meiosis, incorporating elements of both killing and biased segregation. We propose that if R2d2 is destined for the polar body, it manipulates segregation to sabotage the egg by causing aneuploidy, which is subsequently lethal in the embryo, ensuring that surviving progeny preferentially contain R2d2. In heterozygous females, R2d2 orients randomly on the metaphase spindle but lags during anaphase and preferentially remains in the egg, regardless of its initial orientation. Thus, the egg genotype is either euploid with R2d2 or aneuploid with both homologs of chromosome 2, with only the former generating viable embryos. Consistent with this model, R2d2 heterozygous females produce eggs with increased aneuploidy for chromosome 2, increased embryonic lethality, and increased transmission of R2d2. In contrast to typical gamete killing of sisters produced as daughter cells in a single meiosis, R2d2 prevents production of any viable gametes from meiotic divisions in which it should have been excluded from the egg.

An egg sabotaging mechanism drives non-Mendelian transmission in mice.

During meiosis, homologous chromosomes segregate so that alleles are transmitted equally to haploid gametes, following Mendel's Law of Segregation. However, some selfish genetic elements drive in meiosis to distort the transmission ratio and increase their representation in gametes. The established paradigms for drive are fundamentally different for female vs male meiosis. In male meiosis, selfish elements typically kill gametes that do not contain them. In female meiosis, killing is predetermined, and selfish elements bias their segregation to the single surviving gamete (i.e., the egg in animal meiosis). Here we show that a selfish element on mouse chromosome 2, R2d2, drives using a hybrid mechanism in female meiosis, incorporating elements of both male and female drivers. If R2d2 is destined for the polar body, it manipulates segregation to sabotage the egg by causing aneuploidy that is subsequently lethal in the embryo, so that surviving progeny preferentially contain R2d2. In heterozygous females, R2d2 orients randomly on the metaphase spindle but lags during anaphase and preferentially remains in the egg, regardless of its initial orientation. Thus, the egg genotype is either euploid with R2d2 or aneuploid with both homologs of chromosome 2, with only the former generating viable embryos. Consistent with this model, R2d2 heterozygous females produce eggs with increased aneuploidy for chromosome 2, increased embryonic lethality, and increased transmission of R2d2. In contrast to a male meiotic driver, which kills its sister gametes produced as daughter cells in the same meiosis, R2d2 eliminates "cousins" produced from meioses in which it should have been excluded from the egg.

M1BP cooperates with CP190 to activate transcription at TAD borders and promote chromatin insulator activity.

Genome organization is driven by forces affecting transcriptional state, but the relationship between transcription and genome architecture remains unclear. Here, we identified the Drosophila transcription factor Motif 1 Binding Protein (M1BP) in physical association with the gypsy chromatin insulator core complex, including the universal insulator protein CP190. M1BP is required for enhancer-blocking and barrier activities of the gypsy insulator as well as its proper nuclear localization. Genome-wide, M1BP specifically colocalizes with CP190 at Motif 1-containing promoters, which are enriched at topologically associating domain (TAD) borders. M1BP facilitates CP190 chromatin binding at many shared sites and vice versa. Both factors promote Motif 1-dependent gene expression and transcription near TAD borders genome-wide. Finally, loss of M1BP reduces chromatin accessibility and increases both inter- and intra-TAD local genome compaction. Our results reveal physical and functional interaction between CP190 and M1BP to activate transcription at TAD borders and mediate chromatin insulator-dependent genome organization.

Chromosome territory formation attenuates the translocation potential of cells.

The formation and spatial arrangement of chromosome territories (CTs) in interphase has been posited to influence the outcome and frequency of genomic translocations. This is supported by correlations between the frequency of inter-chromosomal contacts and translocation events in myriad systems. However, it remains unclear if CT formation itself influences the translocation potential of cells. We address this question in Drosophila cells by modulating the level of Condensin II, which regulates CT organization. Using whole-chromosome Oligopaints to identify genomic rearrangements, we find that increased contact frequencies between chromosomes due to Condensin II knockdown leads to an increased propensity to form translocations following DNA damage. Moreover, Condensin II over-expression is sufficient to drive spatial separation of CTs and attenuate the translocation potential of cells. Together, these results provide the first causal evidence that proper CT formation can protect the genome from potentially deleterious translocations in the presence of DNA damage.

ARID1A spatially partitions interphase chromosomes.

ARID1A, a subunit of the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin-remodeling complex, localizes to both promoters and enhancers to influence transcription. However, the role of ARID1A in higher-order spatial chromosome partitioning and genome organization is unknown. Here, we show that ARID1A spatially partitions interphase chromosomes and regulates higher-order genome organization. The SWI/SNF complex interacts with condensin II, and they display significant colocalizations at enhancers. ARID1A knockout drives the redistribution of condensin II preferentially at enhancers, which positively correlates with changes in transcription. ARID1A and condensin II contribute to transcriptionally inactive B-compartment formation, while ARID1A weakens the border strength of topologically associated domains. Condensin II redistribution induced by ARID1A knockout positively correlates with chromosome sizes, which negatively correlates with interchromosomal interactions. ARID1A loss increases the trans interactions of small chromosomes, which was validated by three-dimensional interphase chromosome painting. These results demonstrate that ARID1A is important for large-scale genome folding and spatially partitions interphase chromosomes.

Co-evolving CENP-A and CAL1 Domains Mediate Centromeric CENP-A Deposition across Drosophila Species.

Centromeres mediate the conserved process of chromosome segregation, yet centromeric DNA and the centromeric histone, CENP-A, are rapidly evolving. The rapid evolution of Drosophila CENP-A loop 1 (L1) is thought to modulate the DNA-binding preferences of CENP-A to counteract centromere drive, the preferential transmission of chromosomes with expanded centromeric satellites. Consistent with this model, CENP-A from Drosophila bipectinata (bip) cannot localize to Drosophila melanogaster (mel) centromeres. We show that this result is due to the inability of the mel CENP-A chaperone, CAL1, to deposit bip CENP-A into chromatin. Co-expression of bip CENP-A and bip CAL1 in mel cells restores centromeric localization, and similar findings apply to other Drosophila species. We identify two co-evolving regions, CENP-A L1 and the CAL1 N terminus, as critical for lineage-specific CENP-A incorporation. Collectively, our data show that the rapid evolution of L1 modulates CAL1-mediated CENP-A assembly, suggesting an alternative mechanism for the suppression of centromere drive.

Establishment of Centromeric Chromatin by the CENP-A Assembly Factor CAL1 Requires FACT-Mediated Transcription.

Centromeres are essential chromosomal structures that mediate accurate chromosome segregation during cell division. Centromeres are specified epigenetically by the heritable incorporation of the centromeric histone H3 variant CENP-A. While many of the primary factors that mediate centromeric deposition of CENP-A are known, the chromatin and DNA requirements of this process have remained elusive. Here, we uncover a role for transcription in Drosophila CENP-A deposition. Using an inducible ectopic centromere system that uncouples CENP-A deposition from endogenous centromere function and cell-cycle progression, we demonstrate that CENP-A assembly by its loading factor, CAL1, requires RNAPII-mediated transcription of the underlying DNA. This transcription depends on the CAL1 binding partner FACT, but not on CENP-A incorporation. Our work establishes RNAPII passage as a key step in chaperone-mediated CENP-A chromatin establishment and propagation.

Stepwise evolution of essential centromere function in a Drosophila neogene.

Evolutionarily young genes that serve essential functions represent a paradox; they must perform a function that either was not required until after their birth or was redundant with another gene. How young genes rapidly acquire essential function is largely unknown. We traced the evolutionary steps by which the Drosophila gene Umbrea acquired an essential role in chromosome segregation in D. melanogaster since the gene's origin less than 15 million years ago. Umbrea neofunctionalization occurred via loss of an ancestral heterochromatin-localizing domain, followed by alterations that rewired its protein interaction network and led to species-specific centromere localization. Our evolutionary cell biology approach provides temporal and mechanistic detail about how young genes gain essential function. Such innovations may constantly alter the repertoire of centromeric proteins in eukaryotes.