Biology subjects

Robertson, D.

Publications and source records attributed to Robertson, D..

9 recordsLinked to original sources

bioRxiv2026

A conserved mechanism for targeted cohesin loading organises specialised chromosomal domains

Cohesin organises chromosomes through loop extrusion, yet how cohesin loading is targeted to specific genomic regions to shape chromosome architecture remains poorly understood. Here, we identify a conserved mechanism that directs cohesin loading to specialised chromosomal domains in Schizosaccharomyces pombe. Mutation of a conserved interaction surface in the cohesin loader Scc4/Ssl3 abolishes cohesin enrichment at centromeres and ribosomal DNA while largely preserving cohesin levels along chromosome arms, demonstrating that domain-specific accumulation depends on targeted recruitment rather than global loading capacity. We further identify the nucleolar protein Dnt1 as a receptor for cohesin loading at rDNA and show that it is required for proper organisation of this domain. High-resolution MicroC-XL maps reveal that targeted cohesin loading positions loop-based interactions within pericentromeres and rDNA without disrupting larger-scale domain boundaries. Together, these findings show that domain-specific receptors acting through a conserved loader interface direct cohesin positioning and identify targeted loading as a conserved organising principle for specialised chromosomal domains.

cell biology↗

bioRxiv2026

Tracing the evolutionary histories of ultra-rare variants using variational dating of large ancestral recombination graphs

Ultra-rare variants dominate whole-genome sequencing datasets, yet their interpretation is limited by allele frequency, which provides little information at very low counts and is highly sensitive to uneven ancestry representation. Allele age offers an ancestry-agnostic alternative but existing methods do not scale to biobank-sized cohorts. Here we present a scalable variational algorithm for dating Ancestral Recombination Graphs (ARGs), implemented in tsdate, together with new distributed methods enabling practical biobank-scale ARG inference using tsinfer. Applied to 47,535 genomes from the Genomics England 100,000 Genomes Project, we infer contiguous ARGs spanning 206 Mb and estimate ages for 23.2 million variants, including 11.8 million singletons. ARG-based allele ages remain accurate under extreme sampling imbalance and, in real data, reveal signatures of purifying selection and clinically relevant heterogeneity among variants with identical observed frequencies. Estimates for recent mutations are precise only at large sample sizes, highlighting the information accessible in the haplotype structure of large datasets. Biobank-scale ARGs therefore enable robust, ancestry-agnostic age estimation for ultra-rare variation with broad utility for statistical and clinical genomics.

genetics↗

bioRxiv2023

A supernumerary synthetic chromosome in Komagataella phaffii as a repository for extraneous genetic material

BackgroundKomagataella phaffii (Pichia pastoris) is a methylotropic commercially important non-conventional species of yeast that grows in a fermentor to exceptionally high densities on simple media and secretes recombinant proteins efficiently. Genetic engineering strategies are being explored in this organism to facilitate cost-effective biomanufacturing. Small, stable artificial chromosomes in K. phaffii could offer unique advantages by accommodating multiple integrations of extraneous genes and their promoters without accumulating perturbations of native chromosomes or exhausting the availability of selection markers. ResultsHere, we describe a linear "nano"chromosome (of 15-25 kb) that, according to whole-genome sequencing, persists in K. phaffii over many generations with a copy number per cell of one, provided non-homologous end joining is compromised (by KU70-knockout). The nanochromosome includes a copy of the centromere from K. phaffii chromosome 3, a K. phaffii-derived autonomously replicating sequence on either side of the centromere, and a pair of K. phaffii-like telomeres. It contains, within its q arm, a landing zone in which genes of interest alternate with long (approx. 1-kb) non-coding DNA chosen to facilitate homologous recombination and serve as spacers. The landing zone can be extended along the nanochromosome, in an inch-worming mode of sequential gene integrations, accompanied by recycling of just two antibiotic-resistance markers. The nanochromosome was used to express PDI, a gene encoding protein disulfide isomerase. Co-expression with PDI allowed the production, from a genomically integrated gene, of secreted murine complement factor H, a plasma protein containing 40 disulfide bonds. As further proof-of-principle, we co-expressed, from a nanochromosome, both PDI and a gene for GFP-tagged human complement factor H under the control of PAOX1 and demonstrated that the secreted protein was active as a regulator of the complement system. ConclusionsWe have added K. phaffii to the list of organisms that can produce human proteins from genes carried on a stable, linear, artificial chromosome. We envisage using nanochromosomes as repositories for numerous extraneous genes, allowing intensive engineering of K. phaffii without compromising its genome or weakening the resulting strain.

bioengineering↗

bioRxiv2022

Enhancing adult neuroplasticity by epigenetic regulation of Parvalbumin-expressing GABAergic cells

Failure of inhibiting fear in response to harmless stimuli contributes to anxiety disorders. Extinction training only temporarily suppresses fear memories in adults, but it is highly effective in juveniles. GABAergic parvalbumin-positive (PV+) cells restrict plasticity in adult brains, thus increasing PV+ cell plasticity could promote the suppression of fear memories following extinction training in adults. Histone deacetylase 2 (Hdac2) restrains both structural and functional synaptic plasticity; however, whether and how Hdac2 controls adult PV+ cell plasticity is unknown. Here, we report that Hdac2 deletion or pharmacological inhibition in PV+ cells attenuate spontaneous recovery of fear memory after fear extinction learning in adults. These manipulations promote a temporally restricted downregulation of Acan, a critical perineuronal net component expressed exclusively by PV+ cells in medial prefrontal cortex. Finally, we show that Acan transient downregulation before extinction training but after fear memory acquisition is sufficient to reduce spontaneous fear memory recovery in wild-type mice.

neuroscience↗

bioRxiv2022

Condensin reorganizes centromeric chromatin during mitotic entry into a bipartite structure stabilized by cohesin

The Structural Maintenance of Chromosomes (SMC) complexes cohesin and condensin establish the 3D organization of mitotic chromosomes1-3. Cohesin is essential to maintain sister chromatid pairing until anaphase onset4, while condensin is important for mitotic centromere structure and elastic resistance to spindle forces5-8. Both complexes are also important to form productive kinetochore-spindle attachments6, 8, 9. How condensin and cohesin work together to shape the mitotic centromere to ensure faithful chromosome segregation remains unclear. Here we show by super-resolution imaging, Capture-C analysis and polymer modeling that vertebrate centromeres are partitioned into two distinct condensin-dependent subdomains during mitosis. This bipartite sub-structure is found in human, mouse and chicken centromeres and also in human neocentromeres devoid of satellite repeats, and is therefore a fundamental feature of vertebrate centromere identity. Super-resolution imaging reveals that bipartite centromeres assemble bipartite kinetochores with each subdomain capable of binding a distinct microtubule bundle. Cohesin helps to link the centromere subdomains, limiting their separation in response to mitotic spindle forces. In its absence, separated bipartite kinetochores frequently engage in merotelic spindle attachments. Consistently, uncoupling of centromere subdomains is a common feature of lagging chromosomes in cancer cells. The two-domain structure of vertebrate regional centromeres described here incorporates architectural roles for both condensin and cohesin and may have implications for avoiding chromosomal instability in cancer cells.

cell biology↗

bioRxiv2021

Eco1-dependent cohesin acetylation anchors chromatin loops and cohesion to define functional meiotic chromosome domains

Cohesin organizes the genome by forming intra-chromosomal loops and inter-sister chromatid linkages. During gamete formation by meiosis, chromosomes are reshaped to support crossover recombination and two consecutive rounds of chromosome segregation. Here we show that Eco1 acetyltransferase positions both chromatin loops and sister chromatid cohesion to organize meiotic chromosomes into functional domains in budding yeast. Eco1 acetylates the Smc3 cohesin subunit in meiotic S phase to establish chromatin boundaries, independently of DNA replication. Boundary formation by Eco1 is critical for prophase exit and for the maintenance of cohesion until meiosis II, but is independent of the ability of Eco1 to antagonize the cohesin-release factor, Wpl1. Conversely, prevention of cohesin release by Wpl1 is essential for centromeric cohesion, kinetochore monoorientation and co-segregation of sister chromatids in meiosis I. Our findings establish Eco1 as a key determinant of chromatin boundaries and cohesion positioning, revealing how local chromosome structuring directs genome transmission into gametes.

cell biology↗

bioRxiv2019

Evolutionary repair: changes in multiple functional modules allow meiotic cohesin to support mitosis

Different members of the same protein family often perform distinct cellular functions. How much are these differing functions due to changes in a proteins biochemical activity versus changes in other proteins? We asked how the budding yeast, Saccharomyces cerevisiae, evolves when forced to use the meiosis-specific kleisin, Rec8, instead of the mitotic kleisin, Scc1, during the mitotic cell cycle. This perturbation impairs sister chromosome linkage and reduces reproductive fitness by 45%. We evolved 15 populations for 1750 generations, substantially increasing their fitness, and analyzed their genotypes and phenotypes. We found no mutations in Rec8, but many populations had mutations in the transcriptional mediator complex, cohesin-related genes, and cell cycle regulators that induce S phase. These mutations improve sister chromosome cohesion and slow genome replication in Rec8-expressing cells. We conclude that changes in known and novel partners allow proteins to improve their ability to perform new functions.

evolutionary biology↗

bioRxiv2019

Convergent genes shape budding yeast pericentromeres

The 3D architecture of the genome governs its maintenance, expression and transmission. The conserved ring-shaped cohesin complex organises the genome by topologically linking distant loci on either a single DNA molecule or, after DNA replication, on separate sister chromatids to provide the cohesion that resists the pulling forces of spindle microtubules during mitosis1,2. Cohesin is highly enriched in specialized chromosomal domains surrounding centromeres, called pericentromeres3-7. However, the structural organisation of pericentromeres and implications for chromosome segregation are unknown. Here we report the 3D structure of budding yeast pericentromeres and establish the relationship between genome organisation and function. We find that convergent genes mark pericentromere borders and, together with core centromeres, define their structure and function by positioning cohesin. Centromeres load cohesin and convergent genes at pericentromere borders trap it. Each side of the pericentromere is organised into a looped conformation, with border convergent genes at the base. Microtubule attachment extends a single pericentromere loop, size-limited by convergent genes at its borders. Re-orienting genes at borders into a tandem configuration repositions cohesin, enlarges the pericentromere and impairs chromosome biorientation in mitosis. Thus, the linear arrangement of transcriptional units together with targeted cohesin loading at centromeres shapes pericentromeres into a structure competent for chromosome segregation during mitosis. Our results reveal the architecture of the chromosomal region within which kinetochores are embedded and the re-structuring caused by microtubule attachment. Furthermore, we establish a direct, causal relationship between 3D genome organization of a specific chromosomal domain and cellular function.

cell biology↗

bioRxiv2018

Initiation of droxidopa during hospital admission for management of refractory neurogenic orthostatic hypotension in severely ill patients

IntroductionOrthostatic hypotension (OH) is a common cause of hospitalization, particularly in the elderly. Hospitalized patients with OH are often severely ill, with complex medical comorbidities and high rates of disability. Droxidopa is a norepinephrine precursor approved for the treatment of neurogenic OH (nOH) associated with autonomic failure that is commonly used in the outpatient setting, but there is currently no data regarding the safety and efficacy of droxidopa initiation in medically complex patients.\n\nMethodsWe performed a retrospective review of patients started on droxidopa for refractory nOH while hospitalized at Vanderbilt University Medical Center between October 2014 and May 2017. Primary outcome measures were safety, change in physician global impression of illness severity from admission to discharge, and persistence on medication after 180-day follow-up.\n\nResultsA total of 20 patients were identified through chart review. Patients were medically complex with high rates of cardiovascular comorbidities and a diverse array of underlying autonomic diagnoses. Rapid titration of droxidopa was safe and well-tolerated in this cohort, with no cardiovascular events or new onset arrhythmias. Supine hypertension requiring treatment occurred in four patients. One death occurred during hospital admission due to organ failure associated with end-stage amyloidosis. Treating physicians noted improvements in presyncopal symptoms in 80% of patients. After 6 months, 13 patients (65%) continued on droxidopa therapy.\n\nConclusionIn a retrospective cohort of hospitalized, severely ill patients with refractory nOH, supervised rapid titration of droxidopa was safe and effective. Treatment persistence was high, suggesting that symptomatic benefit extended beyond acute intervention.

pharmacology and toxicology↗