Biology subjects

Simmons, J. R.

Publications and source records attributed to Simmons, J. R..

9 recordsLinked to original sources

bioRxiv2026

A sequence motif for DNA double-strand break and telomere healing during programmed DNA elimination

Programmed DNA Elimination (PDE) is an exception to the paradigm of genome integrity, removing selected DNA sequences during development. PDE is observed in dozens of metazoan species from diverse phyla, but its molecular mechanisms and biological significance in most metazoa remain largely unknown. During PDE in the nematode Oscheius tipulae, DNA double-strand breaks (DSBs) are generated at subtelomeric regions, followed by the loss of DNA at chromosome ends and the healing of the DSBs by de novo telomere synthesis. DSBs occur at a 29-bp degenerate palindromic Sequence For Elimination (SFE) motif. We determined the sequence requirement for DSB generation and demonstrated that the conserved GGC/GCC sites are used for neotelomere formation. Introducing the SFE into a retained DNA region adjacent to a native SFE induces DNA cleavage, telomere healing, and loss of additional DNA between the two SFEs. Moreover, insertion of the SFE in the middle of the sex chromosome splits it into two functional somatic chromosomes, demonstrating that the function of SFE is not necessarily constrained by its genomic location. Overall, our data show that the SFE motif is both necessary and sufficient for the generation of DSBs and healing of DSB ends via telomere addition, providing molecular insights into the mechanisms of metazoan PDE.

genetics↗

bioRxiv2025

Spatial genome reorganization is associated with chromosomal breakage regions and karyotype changes induced by programmed DNA elimination

Programmed DNA elimination (PDE) is a notable exception to genome integrity, characterized by significant DNA loss during development. In many nematodes, PDE is initiated by DNA double-strand breaks (DSBs), which lead to chromosome fragmentation and subsequent DNA loss. However, the mechanism of nematode programmed DNA breakage remains largely unclear. Interestingly, in the human and pig parasitic nematode Ascaris, no conserved motif or sequence structures are present at chromosomal breakage regions (CBRs), suggesting the recognition of CBRs may be sequence-independent. Using Hi-C, we revealed that Ascaris CBRs engage in three-dimensional (3D) interactions before PDE, indicating that physical contacts between break regions may contribute to the PDE process. The 3D interactions are established in both Ascaris male and female germlines, demonstrating inherent genome organization associated with the CBRs and to-be-eliminated sequences. In contrast, in the unichromosomal horse parasite Parascaris univalens, transient pairwise interactions between neighboring CBRs that will form the ends of future somatic chromosomes were observed only during PDE. Intriguingly, we found that Ascaris PDE, which converts 24 germline chromosomes into 36 somatic ones, induces specific compartmentalization changes. Remarkably, Parascaris PDE generates the same set of 36 somatic chromosomes, and the 3D compartment changes following PDE are consistent between the two species. Overall, our findings suggest that CBRs spatially demarcate the retained and eliminated DNA and may contribute to their spatial organization during Ascaris PDE. We also demonstrated that the 3D genome reorganization of the somatic chromosomes in these nematodes following PDE is evolutionary and developmentally conserved.

genomics↗

bioRxiv2023

Chromosome fusion and programmed DNA elimination shape karyotypes of parasitic nematodes

A growing list of metazoans undergo programmed DNA elimination (PDE), where a significant amount of DNA is selectively lost from the somatic genome during development. In some nematodes, PDE leads to the removal and remodeling of the ends of all germline chromosomes. In several species, PDE also generates internal breaks that lead to sequence loss and an increased number of somatic chromosomes. The biological significance of these karyotype changes associated with PDE and the origin and evolution of nematode PDE remain largely unknown. Here, we assembled the single germline chromosome of the horse parasite Parascaris univalens and compared the karyotypes, chromosomal gene organization, and PDE features among ascarid nematodes. We show that PDE in Parascaris converts an XX/XY sex-determination system in the germline into an XX/XO system in the somatic cells. Comparisons of Ascaris, Parascaris, and Baylisascaris ascarid chromosomes suggest that PDE existed in the ancestor of these parasites, and their current distinct germline karyotypes were derived from fusion events of smaller ancestral chromosomes. The DNA breaks involved in PDE resolve these fused germline chromosomes into their pre-fusion karyotypes, leading to alterations in genome architecture and gene expression in the somatic cells. Cytological and genomic analyses further suggest that satellite DNA and the heterochromatic chromosome arms play a dynamic role in the Parascaris germline chromosome during meiosis. Overall, our results show that chromosome fusion and PDE have been harnessed in these ascarids to sculpt their karyotypes, altering the genome organization and serving specific functions in the germline and somatic cells.

genomics↗

bioRxiv2023

Interactions Between the Histone Variant H2Av and the Suppressor of Hairy wing Insulator Influence the DNA Damage Response and Lymph Gland Development in Drosophila

Genome architecture is regulated by chromatin insulator proteins, and misregulation of insulator function is associated with genome instability and transcriptional regulatory defects in both vertebrates and Drosophila. Indeed, mutations of the sole insulator protein in humans, CTCF, are carcinogenic and mutations in the Drosophila insulator protein Suppressor of Hairy wing [Su(Hw)] lead to chromosomal rearrangements. However, the mechanism that links the DNA damage response and the regulation of transcription with insulator function is not yet understood. Here we show that enrichment of Su(Hw) insulator proteins at insulator sites increases after DNA damage. Additionally, Su(Hw) is necessary for phosphorylation of the histone variant H2Av in response to both UV treatment and X-ray irradiation. The requirement of Su(Hw) for H2Av phosphorylation appears to be tissue-specific, since H2Av is phosphorylated in response to DNA damage also in neurons, where Su(Hw) is not normally expressed. Similarly, we provide evidence that Su(Hw) and H2Av work together to ensure proper development of the lymph gland in Drosophila larvae. We show that H2Av regulates formation of the larval lymph gland, and mutation of H2Av causes formation of large melanotic tumors that are rescued by mutation of su(Hw) in the His2Av810 mutant background. Double mutants of su(Hw)- and His2Av810 also form supernumerary lymph gland lobes that are larger than in wild type. Our findings support a role for the Su(Hw) in the DNA damage response through the regulation of H2Av phosphorylation and suggest that Su(Hw) and H2Av also work together to ensure proper development of the lymph gland.

molecular biology↗

bioRxiv2022

The Nematode Oscheius tipulae as A Genetic Model for Programmed DNA Elimination

SUMMARYProgrammed DNA elimination (PDE) is a notable exception to the paradigm of genome integrity. In metazoa, PDE often occurs during early embryogenesis, coincident with germline to somatic cell differentiation. During PDE, portions of genomic DNA are lost, resulting in reduced somatic genomes. Recent studies have described the sequences lost and chromosome behavior in metazoa. However, a system for studying the mechanisms and consequences of PDE in metazoa is lacking. Here, we established a functional and genetic model for PDE in the free-living nematode Oscheius tipulae, a member of the Rhabditidae family, which includes Caenorhabditis elegans. O. tipulae was recently suggested to eliminate DNA. Using staged embryos, we show that O. tipulae PDE occurs during embryogenesis at the 2-16 cell stages, similar to the human/pig parasitic nematode Ascaris. We identified a conserved motif, named Sequence For Elimination (SFE), for all 12 break sites on the six chromosomes at the junction of retained and eliminated DNA. SFE mutants exhibit a "fail-to-eliminate" phenotype only at the modified sites. END-seq revealed that breaks can occur at multiple positions within the SFE, with extensive end resection followed by telomere addition to both retained and eliminated ends. We identified through END-seq in the wild-type embryos, genome sequencing of SFE mutants, and comparative genomics of 23 wild isolates a large number of functional SFEs at the chromosome ends. We suggest these alternative SFEs provide flexibility in the sequences eliminated and a fail-safe mechanism for PDE. These studies establish O. tipulae as a new, attractive model to study the mechanisms and consequences of PDE in a metazoan.

genetics↗

bioRxiv2021

A Phosphorylated Histone H2A Variant Displays Properties of Chromatin Insulator Proteins in Drosophila

Chromatin insulators are responsible for mediating long-range interactions between enhancers and promoters throughout the genome and align with the boundaries of topologically associating domains (TADs). Here, we demonstrate an interaction between proteins that associate with the gypsy insulator and the phosphorylated histone variant H2Av ({gamma}H2Av), a marker of DNA double strand breaks. Gypsy insulator components colocalize with {gamma}H2Av throughout the genome. Mutation of insulator components prevents stable H2Av phosphorylation in polytene chromatin. Phosphatase inhibition strengthens the association between insulator components and {gamma}H2Av and rescues {gamma}H2Av localization in insulator mutants. We also show that {gamma}H2Av is a component of insulator bodies, and that phosphatase activity is required for insulator body dissolution after recovery from osmotic stress. We further demonstrate a tight association between {gamma}H2Av and TAD boundaries. Together, our results indicate a novel mechanism linking insulator function with a histone H2A variant and with genome stability.

molecular biology↗

bioRxiv2019

A Drosophila Insulator Interacting Protein Suppresses Enhancer-Blocking Function and Modulates Replication Timing

Insulators play important roles in genome structure and function in Drosophila and mammals. More than six different insulator proteins are required in Drosophila for normal genome function, whereas CTCF is the only identified protein contributing to insulator function in mammals. Interactions between a DNA binding insulator protein and its interacting partner proteins define the properties of each insulator site. The different roles of insulator protein partners in the Drosophila genome and how they confer functional specificity remain poorly understood. Functional analysis of insulator partner proteins in Drosophila is necessary to understand how genomes are compartmentalized and the roles that different insulators play in genome function. In Drosophila, the Suppressor of Hairy wing [Su(Hw)] insulator is targeted to the nuclear lamina, preferentially localizes at euchromatin/heterochromatin boundaries, and is associated with the Gypsy retrotransposon. The properties that the insulator confers to these sites rely on the ability of the Su(Hw) protein to bind the DNA at specific sites and interact with Mod(mdg4)-67.2 and CP190 partner proteins. HP1 and insulator partner protein 1 (HIPP1) is a recently identified partner of Su(Hw), but how HIPP1 contributes to the function of Su(Hw) insulators has not yet been elucidated. Here, we find that mutations in the HIPP1 crotonase-like domain have no impact on the function of Su(Hw) enhancer-blocking activity but do exhibit an impaired ability to repair double-strand breaks. Additionally, we find that the overexpression of each HIPP1 and Su(Hw) causes defects in cell proliferation by limiting the progression of DNA replication. We also find that HIPP1 overexpression suppresses the Su(Hw) insulator enhancer-blocking function.

molecular biology↗

bioRxiv2019

Mutations in the Insulator Protein Suppressor of Hairy Wing Induce Genome Instability

Chromatin insulator proteins mediate the formation of contacts between distant insulator sites along chromatin fibers. Long-range contacts facilitate communication between regulatory sequences and gene promoters throughout the genome, allowing accurate gene transcription regulation during embryo development and cell differentiation. Lack of insulator function has detrimental effects often resulting in lethality. The Drosophila insulator protein Suppressor of Hairy wing [Su(Hw)] is not essential for viability, but plays a crucial role in female oogenesis. The mechanism(s) by which Su(Hw) promotes proper oogenesis remains unclear. To gain insight into the functional properties of chromatin insulators, we further characterize the oogenesis phenotypes of su(Hw) mutant females. We find that mutant egg chambers frequently display an irregular number of nurse cells, have poorly formed microtubule organization centers (MTOC) in the germarium, and show mislocalized Gurken (Grk) in later stages of oogenesis. Furthermore, eggshells produced by partially rescued su(Hw) mutant females exhibit dorsoventral patterning defects that are identical to defects found in spindle mutants or in piRNA pathway mutants. Further analysis reveals an excess of DNA damage in egg chambers, which is independent of activation of transposable elements, and that Gurken localization defects and oogenesis progression are partially rescued by mutations in mei-41 and chk1 genes. In addition, we show that Su(Hw) is required for chromosome integrity in dividing neuroblasts from larval brains. Together, these findings suggest that Su(Hw) plays a critical role in maintaining genome integrity during germline development in Drosophila females as well as in dividing somatic cells.

developmental biology↗

bioRxiv2018

Programmed Variations of Cytokinesis Contribute to Morphogenesis in the C. elegans embryo

While cytokinesis has been intensely studied, how it is executed during development is not well understood, despite a long-standing appreciation that various aspects of cytokinesis vary across cell and tissue types. To address this, we investigated cytokinesis during the invariant C. elegans embryo lineage and found several reproducibly altered parameters at different stages. During early divisions, furrow ingression asymmetry and midbody inheritance is consistent, suggesting specific regulation of these events. During morphogenesis, we find several unexpected alterations including migration of midbodies to the apical surface during epithelial polarization in different tissues. Aurora B kinase, which is essential for several aspects of cytokinesis, remains localized to the apical membrane after internalization of other midbody components. Inactivation of Aurora B causes cytokinesis failure, which disrupts polarization and tissue formation. Therefore, cytokinesis shows surprising diversity during development and is required during epithelial polarization to establish cellular architecture during morphogenesis.

cell biology↗