Biology subjects

Carlton, P. M.

Publications and source records attributed to Carlton, P. M..

8 recordsLinked to original sources

bioRxiv2026

Multifaceted and evolutionarily dynamic interactions between Caenorhabditis elegans SPO-11 and its cofactors ensure proper formation of meiotic DNA double-strand breaks

DNA double-strand breaks (DSBs) generated during meiotic prophase by the topoisomerase-like protein SPO11 are essential to create crossovers between homologous chromosomes. Since crossovers are required to biorient chromosomes at the first meiotic division, DSB formation is essential for meiosis in most sexually-reproducing organisms. Since excess DSBs have the potential to destabilize the genome, SPO-11 activity must be strictly regulated by many cofactors. Recent studies have established that SPO11 must dimerize to cut DNA, whereas soluble SPO11 and SPO11-TOPOVIBL complexes are predominantly monomeric (1-3). This contrast suggested that a major role of SPO11 cofactors could be to promote SPO11 dimerization, through means such as increasing local concentration or co-orienting SPO11 protomers. However, the mechanism of this regulation is not well-understood. Here, by taking advantage of phylogenomic analysis in the nematode genus Caenorhabditis, we show that the conserved cofactor DSB-1Rec114 evolved to replace TOPOVIBL function in C. elegans. We provide genetic and biochemical evidence that multiple interactions between SPO-11 and DSB-1 stabilize protein complex formation and promote SPO-11 dimerization. Our results shed light on the regulatory mechanism of programmed DSB formation, which ensures crossover formation and meiotic chromosome segregation while protecting genomic stability. Significance StatementProgrammed DNA double-strand breaks catalyzed by SPO11 are essential for meiosis, but how SPO11 and its cofactors cooperate to cut DNA is not understood. SPO11 only cuts DNA as a homodimer, but soluble SPO11, with or without its core component TOPOVIBL, is predominantly monomeric. We show here that DSB-1, a conserved cofactor of C. elegans SPO-11, has evolved to replace TOPOVIBL to make direct, multifaceted interactions with SPO-11. We provide evidence that DSB-1 simultaneously binds both SPO-11 protomers, and this binding is critical for DNA cleavage, implying a major role of DSB-1 in promoting SPO-11 dimerization. Our phylogenetic analysis also highlights the evolutionary flexibility of a conserved, essential protein complex after the loss of one of its members, TOPOVIBL.

cell biology↗

bioRxiv2026

ZHP-2/HEI10 generates CO precursors and triggers procrossover factor coarsening and CO formation through Polo kinase recruitment

During meiosis, programmed DNA double-strand breaks (DSBs) are repaired with the homolog to form crossovers (COs) essential for chromosome segregation, or noncrossovers (nCOs) to restore genomic integrity. Only a few recombination intermediates will become COs and these sites are progressively enriched in proCO factors at the expense of nCO sites, referred to as coarsening. Here, we show that in C. elegans this process is engineered by ZHP-2/HEI10, a conserved E3 ligase that has functions at early and late stages of CO formation. Early on, ZHP-2-mediated loss of RMH-1/RMI1 and HIM-6/BLM from strand exchange intermediates is required for joint molecule formation and nCO repair. In zhp-2 mutants, proCO factors accumulate at HR sites, leading to defects in CO formation and CO spacing. We find that Polo kinase recruitment to a ZHP-2 at CO precursors signals an end to further DSB formation, and triggers proCO factor coarsening and CO formation. We propose that ZHP-2 disassembles proCO factors from recombination intermediates until it is inactivated at CO precursors through Polo kinase recruitment, leading to stabilization of proCO factors, and resolution of joint molecules as crossovers. Our study reveals that ZHP-2/HEI10 is the central component of a Polo kinase-driven switch that initiates proCO factor coarsening and execution of crossing over.

cell biology↗

bioRxiv2024

Length-sensitive partitioning of meiotic chromosomes in Caenorhabditis elegans senses proximity and number of crossover sites

Sensing and control of size is critical for cellular function and survival. A striking example of size sensing occurs during meiosis in the nematode Caenorhabditis elegans. C. elegans chromosomes compare the lengths of the two chromosome "arms" demarcated by the position of their single off-center crossover, and differentially modify these arms to ensure that sister chromatid cohesion is lost specifically on the shorter arm in the first meiotic division, while the longer arm maintains cohesion until the second division. While many of the downstream steps leading to cohesion loss have been characterized, the length sensing process itself remains poorly understood. Here, we have used cytological visualization of the short arm, combined with quantitative microscopy, live imaging, and simulations, to investigate the principles underlying length-sensitive chromosome partitioning. By quantitatively analyzing short arm designation patterns on fusion chromosomes carrying multiple crossovers, we develop a model in which a short arm-determining factor originates at crossover designation sites, diffuses within the phase-separated synaptonemal complex, and accumulates within crossover-bounded chromosome segments. We demonstrate experimental support for a critical assumption of this model, that crossovers act as boundaries to diffusion within the synaptonemal complex. Further, we develop a discrete simulation based on our results that recapitulates a wide variety of observed partitioning outcomes in both wild type and previously reported mutants. Our results suggest that the concentration of a diffusible factor is used as a proxy for chromosome length, enabling the correct designation of short and long arms and proper segregation of chromosomes.

cell biology↗

bioRxiv2022

Phosphoregulation of DSB-1 mediates control of meiotic double-strand break activity

In the first meiotic cell division, proper segregation of chromosomes in most organisms depends on chiasmata, exchanges of continuity between homologous chromosomes that originate from the repair of programmed double-strand breaks (DSBs) catalyzed by the Spo11 endonuclease. Since DSBs can lead to irreparable damage in germ cells, while chromosomes lacking DSBs also lack chiasmata, the number of DSBs must be carefully regulated, so as to be neither too high nor too low. Here, we show that in Caenorhabditis elegans, meiotic DSB levels are controlled by the phosphoregulation of DSB-1, a homolog of the yeast Spo11 cofactor Rec114, by the opposing activities of PP4PPH-4.1 phosphatase and ATRATL-1 kinase. Increased DSB-1 phosphorylation in pph-4.1 mutants correlates with reduction in DSB formation, while prevention of DSB-1 phosphorylation drastically increases the number of meiotic DSBs both in pph-4.1 mutants as well as in the wild type background. C. elegans and its close relatives also possess a diverged paralog of DSB-1, called DSB-2, and loss of dsb-2 is known to reduce DSB formation in oocytes with increasing age. We show that the proportion of the phosphorylated, and thus inactivated, form of DSB-1 increases with age and also upon loss of DSB-2, while non-phosphorylatable DSB-1 rescues the age-dependent decrease in DSBs in dsb-2 mutants. These results suggest that DSB-2 evolved in part to compensate for the inactivation of DSB-1 through phosphorylation, to maintain levels of DSBs in older animals. Our work shows that PP4PPH-4.1, ATRATL-1, and DSB-2 act in concert with DSB-1 to promote optimal DSB levels throughout the reproductive lifespan.

cell biology↗

bioRxiv2020

Phosphoregulation of HORMA domain protein HIM-3 promotes asymmetric synaptonemal complex disassembly in meiotic prophase in C. elegans

In the two cell divisions of meiosis, diploid genomes are reduced into complementary haploid sets through the discrete, two-step removal of chromosome cohesion, a task carried out in most eukaryotes by protecting cohesion at the centromere until the second division. In eukaryotes without defined centromeres, however, alternative strategies have been innovated. The best-understood of these is that used by the nematode Caenorhabditis elegans, where upon division of the chromosome into two segments or arms by the single off-center crossover, several chromosome-associated proteins or post-translational modifications become specifically partitioned to either the short or long arm, where they affect the timing of cohesion loss through as-yet unknown mechanisms. Here, we investigate the meiotic axis HORMA-domain protein HIM-3 and show that it becomes phosphorylated at its C-terminus, within the conserved “closure motif” region bound by the related HORMA-domain proteins HTP-1 and HTP-2. Binding of HTP-2 is abrogated by phosphorylation of the closure motif in in vitro assays, strongly suggesting that in vivo phosphorylation of HIM-3 likely modulates the hierarchical structure of the chromosome axis. Phosphorylation of HIM-3 only occurs on synapsed chromosomes, and similarly to previously-described phosphorylated proteins of the synaptonemal complex, becomes restricted to the short arm after designation of crossover recombination sites. Regulation of HIM-3 phosphorylation status is required for timely disassembly of synaptonemal complex central elements from the long arm, and is also required for proper timing of HTP-1 and HTP-2 dissociation from the short arm. Phosphorylation of HIM-3 thus plays a role in establishing the identity of short and long arms, thereby contributing to the robustness of the two-step chromosome segregation.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2020

Sequential enrichment at the nuclear periphery of H2A.Zac and H3K9me2 accompanies pluripotency loss in human embryonicstem cells

During the transition from pluripotency to a lineage-committed state, chromatin undergoes large-scale changes in structure to effect the required changes to the transcriptional program. This involves covalent modification of histone tails, replacement of histone variants, and alteration in the subnuclear position of genes, including associations with the nuclear periphery. Here, using high-resolution microscopy and quantitative image analysis, we surveyed a panel of histone variants and covalent modifications for changes in nuclear periphery association during differentiation of human embryonic stem cells to a trophoblast-like lineage. This differentiation process is rapid and homogeneous, facilitating the use of a relatively fine timecourse (12h, 24h, and 48h post-initiation) to enable detection of transient changes. With this scheme, we detected two modifications with significant changes in enrichment at the nuclear periphery: acetylation of histone variant H2A.Z, and dimethylation of histone H3 at lysine 9. We show that these chromatin marks increase specifically at the nuclear periphery in a sequential, complementary manner, with a H2A.Z acetylation preceding H3K9 dimethylation. The increase of H3K9 dimethylation occurred coincidentally with but independently of accumulation of Lamin A, since Lamin A-/- hES cells showed no changes in the localization pattern of H3K9 dimethylation. Inhibition of histone deacetylases led to persistent and increased H2A.Z acetylation at the periphery, and failure to differentiate. Our results show that a concerted dynamic change in the nature of peripheral chromatin is required for differentiation into the trophoblast state.

cell biology↗

bioRxiv2018

The conserved phosphatase GSP-2/PP1 promotes germline immortality via small RNA-mediated genome silencing during meiosis

Genomic silencing can promote germ cell immortality, or transgenerational maintenance of the germ line, via mechanisms that may occur during mitosis or meiosis. Here we report that the gsp-2 PP1/Glc7 phosphatase promotes germ cell immortality. We identified a separation-of-function allele of C. elegans GSP-2 that caused a meiosis-specific chromosome segregation defect and defects in transgenerational small RNA-induced genome silencing. GSP-2 is recruited to meiotic chromosomes by LAB-1, which also promoted germ cell immortality. Sterile gsp-2 and lab-1 mutant adults displayed germline degeneration, univalents and histone phosphorylation defects in oocytes, similar to small RNA genome silencing mutants. Epistasis and RNA analysis suggested that GSP-2 functions downstream of small RNAs. We conclude that a meiosis-specific function of GSP-2/LAB-1 ties small RNA-mediated silencing of the epigenome to germ cell immortality. Given that hemizygous genetic elements can drive transgenerational epigenomic silencing, and given that LAB-1 promotes pairing of homologous chromosomes and localizes to the interface between homologous chromosomes during pachytene, we suggest that discontinuities at this interface could promote nuclear silencing in a manner that depends on GSP-2.\n\nAuthor SummaryThe germ line of an organism is considered immortal in its capacity to give rise to an unlimited number of future generations. To protect the integrity of the germ line, mechanisms act to suppress the accumulation of transgenerational damage to the genome or epigenome. Loss of germ cell immortality can result from mutations that disrupt the small RNA-mediated silencing pathway that helps to protect the integrity of the epigenome. Here we report for the first time that the C. elegans protein phosphatase GSP-2 that promotes core chromosome biology functions during meiosis is also required for germ cell immortality. Specifically, we identified a partial loss of function allele of gsp-2 that exhibits defects in meiotic chromosome segregation and is also dysfunctional for transgenerational small RNA-mediated genome silencing. Our results are consistent with a known role of Drosophila Protein Phosphatase 1 in heterochromatin silencing, and point to a meiotic phosphatase function that is relevant to germ cell immortality, conceivably related to its roles in chromosome pairing or sister chromatid cohesion.

genetics↗

bioRxiv2017

Phosphorylation of the synaptonemal complex protein SYP-1 promotes meiotic chromosome segregation

Chromosomes that have undergone crossing-over in meiotic prophase must maintain sister chromatid cohesion somewhere along their length between the first and second meiotic divisions. While many eukaryotes use the centromere as a site to maintain cohesion, the holocentric organism C. elegans instead creates two chromosome domains of unequal length termed the short arm and long arm, which become the first and second site of cohesion loss at meiosis I and II. The mechanisms that confer distinct functions to the short and long arm domains remain poorly understood. Here, we show that phosphorylation of the synaptonemal complex protein SYP-1 is required to create these domains. Once crossovers are made, phosphorylated SYP-1 and PLK-2 become cooperatively confined to short arms and guide phosphorylated histone H3 and the chromosomal passenger complex to the site of meiosis I cohesion loss. Our results show that PLK-2 and phosphorylated SYP-1 ensure creation of the short arm subdomain, promoting disjunction of chromosomes in meiosis I.

cell biology↗