Biology subjects

Rhodes, D.

Publications and source records attributed to Rhodes, D..

10 recordsLinked to original sources

bioRxiv2022

Effects of breast fibroepithelial tumor associated retinoic acid receptor alpha ligand binding domain mutations on receptor function and retinoid signaling

Point mutations in the ligand binding domain of retinoic acid receptor alpha (RAR) have been implicated in breast fibroepithelial tumors development. However, their role in the tumorigenesis of solid tumors is currently unknown. In this study, using a combination of biochemical and cellular assays, we evaluated the functional consequences of known tumor associated RAR mutations on retinoic acid signaling. All of the clinically associated mutants tested showed diminished transcriptional activities compared to wild type RAR. These mutants also exhibited a dominant negative effect, an activity which has previously been linked to developmental defects and tumor formation in mice. X-ray crystallography showed that mutants remain relatively intact structurally and the loss of transcriptional activity is due to altered co-activator recruitment. In agreement with our biochemical analyses, transcriptomics and cell growth analysis showed that the mutant RAR proteins confer resistance to growth inhibition in the presence of its ligand in phyllodes tumor cells. Although the mutations impair the receptor responses to retinoic acid, certain mutant RAR are partially reactivatable with alternative synthetic agonists. Our data provide insights into the mechanisms by which RAR mutations impact tumorigenesis.

cancer biology↗

bioRxiv2022

Stitched peptides as potential cell permeable inhibitors of oncogenic DAXX protein

The death domain associated protein 6 (DAXX) is frequently upregulated in a number of common cancers where its suppression has been linked to reduced tumour progression. As a master regulator protein, with >70 reported protein interaction partners, the role of DAXX in its oncogenecity remains unclear. We designed and developed a set of novel stapled/stitched peptides that target a surface on the N-terminal helical bundle domain of DAXX which is the anchor-point for binding to multiple interaction partners (including Rassf1C, P53, Mdm2 and ATRX) and also for the auto regulation of the DAXX N-terminal SUMO interaction motif (SIM). We demonstrate that these peptides bind to and inhibit DAXX with an affinity higher than those reported for the known interaction partners and release the auto-inhibited SIM for interaction with SUMO-1. NanoBret assays show that the peptides enter cells and that their intracellular concentrations remain at nanomolar levels even after 24 hours, without causing membrane perturbation. Together our data suggest that these peptides are both tools for probing the molecular interactions of DAXX and potential precursors to the development of therapeutics.

biochemistry↗

bioRxiv2022

The columnar structure of human telomeric chromatin suggests mechanisms for telomere maintenance

Telomeres, the ends of eukaryotic chromosomes, play pivotal roles in ageing and cancer and are targets of DNA damage and response. However, little is known about the structure and organization of telomeric chromatin at the molecular level. We used electron microscopy and single-molecule magnetic tweezers to characterize well-defined telomeric chromatin fibers of kilobasepair length. The cryo-EM structure of the compact telomeric tetranucleosome revealed a novel columnar folding, unusually short nucleosome repeat length of [~]132bp and the role of the histone N-terminal tails in stabilizing this structure. This is the first near-high resolution structure of chromatin with a native DNA sequence. The columnar structure exposes the DNA, making them susceptible to DNA damage. The telomeric tetranucleosome also exists in an alternative well-defined state, with one nucleosome open, accessible to protein factors. This suggests that protein factors, which plays a role in maintaining telomeres, can bind to telomeric chromatin in its compact heterochromatic form. The features of the telomeric chromatin structure reveals important insights of significant relevance for telomere function in vivo that provides information on mechanisms of nucleosome recognition by chromatin factors

molecular biology↗

bioRxiv2020

KIAA1841, a novel SANT and BTB domain-containing protein, inhibits class switch recombination

Class switch recombination (CSR) enables B cells to produce different immunoglobulin isotypes and mount an effective immune response against pathogens. Timely resolution of CSR prevents damage due to an uncontrolled and prolonged immune response. While many positive regulators of CSR have been described, negative regulators of CSR are relatively unknown. Using a shRNA library screen in a mouse B cell line, we have identified the novel protein KIAA1841 (NM_027860) as a negative regulator of CSR. KIAA1841 is an uncharacterized protein of 82kD containing SANT and BTB domains. The BTB domain of KIAA1841 exhibited characteristic properties such as self-dimerization and interaction with co-repressor proteins. Overexpression of KIAA1841 inhibited CSR in primary mouse splenic B cells, and inhibition of CSR is dependent on the BTB domain while the SANT domain is largely dispensable. Thus, we have identified a new member of the BTB family that serves as a negative regulator of CSR.

molecular biology↗

bioRxiv2020

Human AKTIP interacts with ESCRT proteins and functions at the midbody in cytokinesis

To complete mitosis, the intercellular bridge that links daughter cells needs to be cleaved. This abscission step is carried out by the sequential recruitment of ESCRT proteins at the midbody. We report here that a new factor, named AKTIP, works in association with ESCRTs. We find that AKTIP binds to the ESCRT I subunit VPS28, and show by high resolution microscopy that AKTIP forms a ring in the dark zone of the intercellular bridge. This ring is positioned in between the circular structures formed by ESCRTs type III. Functionally, we observe that the reduction of AKTIP impinges on the recruitment of the ESCRT III member IST1 at the midbody and causes abscission defects. Taken together, these data indicate that AKTIP is a new factor that contributes to the formation of the ESCRT complex at the midbody and is implicated in the performance of the ESCRT machinery during cytokinetic abscission.

cell biology↗

bioRxiv2019

The Human Telomeric Nucleosome Displays Distinct Structural and Dynamic Properties

Telomeres protect the ends of our chromosomes and are key to maintaining genomic integrity during cell division and differentiation. However, our knowledge of telomeric chromatin and nucleosome structure at the molecular level is limited. Here, we aimed to define the structure, dynamics as well as properties in solution of the human telomeric nucleosome. We first determined the 2.2 [A] crystal structure of a human telomeric nucleosome core particle (NCP) containing 145 bp DNA, which revealed the same helical path for the DNA as well as symmetric stretching in both halves of the NCP as that of the 145 bp 601 NCP. In solution, the telomeric nucleosome exhibited a less stable and a markedly more dynamic structure compared to NCPs containing DNA positioning sequences. These observations provide molecular insights into how telomeric DNA forms nucleosomes and chromatin and advance our understanding of the unique biological role of telomeres.

biochemistry↗

bioRxiv2019

DEK influences the trade-off between growth and arrest via H2A.Z-nucleosomes in Arabidopsis

The decision of whether to grow and proliferate or to restrict growth and develop resilience to stress is a key biological trade-off. In plants, constitutive growth results in increased sensitivity to environmental stress1,2. The underlying mechanisms controlling this decision are however not well understood. We used temperature as a cue to discover regulators of this process in plants, as it both enhances growth and development rates within a specific range and is also a stress at extremes. We found that the conserved chromatin-associated protein DEK plays a central role in balancing the response between growth and arrest in Arabidopsis, and it does this via H2A.Z-nucleosomes. DEK target genes show two distinct categories of chromatin architecture based on the distribution of H2A.Z in +1 nucleosome and gene body, and these predict induction or repression by DEK. We show that these chromatin signatures of DEK target genes are conserved in human cells, suggesting that DEK may act through an evolutionarily conserved mechanism to control the balance between growth and arrest in plants and animals.

plant biology↗

bioRxiv2019

Variation across 141,456 human exomes and genomes reveals the spectrum of loss-of-function intolerance across human protein-coding genes

Genetic variants that inactivate protein-coding genes are a powerful source of information about the phenotypic consequences of gene disruption: genes critical for an organisms function will be depleted for such variants in natural populations, while non-essential genes will tolerate their accumulation. However, predicted loss-of-function (pLoF) variants are enriched for annotation errors, and tend to be found at extremely low frequencies, so their analysis requires careful variant annotation and very large sample sizes1. Here, we describe the aggregation of 125,748 exomes and 15,708 genomes from human sequencing studies into the Genome Aggregation Database (gnomAD). We identify 443,769 high-confidence pLoF variants in this cohort after filtering for sequencing and annotation artifacts. Using an improved human mutation rate model, we classify human protein-coding genes along a spectrum representing tolerance to inactivation, validate this classification using data from model organisms and engineered human cells, and show that it can be used to improve gene discovery power for both common and rare diseases.

genomics↗

bioRxiv2018

Multiple Duration Priors Within and Across the Senses

Perception can be understood as an active process in which sensory samples are combined with prior expectations to shape perceptual content. A prominent example of the influence of priors on perception is that manually reproduced temporal durations are biased towards the mean of previously experienced durations. However, little is known about how prior expectations are acquired and maintained in environments in which multiple competing cues may indicate whether a given prior should be applied in that specific context. We tested whether human participants could acquire and maintain multiple priors for duration, dependent on the sensory signal in which the duration was presented. Human participants were presented with visual flashes or auditory tones, high or low pitch tones, or white noise versus pure tone audio. In each case, the presented duration on a given trial was drawn from a distribution that was, on average, shorter for tones than for flashes, or vice versa. Our participants timing reports were consistent with having acquired distinct duration priors dependent on the sensory signal in which the duration was presented (e.g. auditory or visual). Moreover, this was true whether signals differed across, or within, sensory modality. We account for our findings within a Bayesian framework in which duration priors are iteratively updated depending on determination of a common or distinct origin between successive events. Overall, these results show that the human brain can acquire and maintain multiple perceptual priors based on differences in stimulus properties both within and across the senses.

animal behavior and cognition↗

bioRxiv2018

FlowPot axenic plant growth system for microbiota research

The presence of resident microbiota on and inside plants is hypothesized to influence many phenotypic attributes of the host. Likewise, host factors and microbe-microbe interactions are believed to influence microbial community assembly. Rigorous testing of these hypotheses necessitates the ability to grow plants in the absence or presence of resident or defined microbiota. To enable such experiments, we developed the scalable and inexpensive FlowPot growth platform. FlowPots have a sterile peat substrate amenable to colonization by microbiota, and the platform supports growth of the model plant Arabidopsis thaliana in the absence or presence of soil-derived microbial communities. Mechanically, the FlowPot system is unique in that it allows for total-saturation of the sterile substrate by \"flushing\" with water and/or nutrient solution via an irrigation port. The irrigation port also facilitates passive drainage of the substrate, preventing root anoxia. Materials to construct an individual FlowPot total [~]$2. A simple experiment with 12 FlowPots requires [~]4.5 h of labor following peat and seed sterilization. Plants are grown on FlowPots within a standard tissue culture microbox after inoculation, thus the Flowpot system is modular and does not require a sterile growth chamber. Here, we provide a detailed assembly and microbiota inoculation protocol for the FlowPot system. Collectively, this standardized suite of tools and colonization protocols empowers the plant microbiome research community to conduct harmonized experiments to elucidate the rules microbial community assembly, the impact of microbiota on host phenotypes, and mechanisms by which host factors influence the structure and function of plant microbiota.

plant biology↗