Biology subjects

Brown, R. R.

Publications and source records attributed to Brown, R. R..

3 recordsLinked to original sources

bioRxiv2025

Regulation of X. laevis M18BP1 centromeric localization and CENP-A assembly

Eukaryotic chromosome segregation requires attachment of chromosomes to microtubules of the mitotic spindle through the kinetochore so that chromosomes can align and move in mitosis. Kinetochores are assembled on the centromere which is a unique chromatin domain that is epigenetically defined by the histone H3 variant CENtromere Protein A (CENP-A). During DNA replication CENP-A is equally divided between replicated chromatids and new CENP-A nucleosomes are re-assembled during the subsequent G1 phase of the cell cycle. How cells regulate the strict cell cycle timing of CENP-A assembly is a central question in the epigenetic maintenance of centromeres and kinetochores. One essential assembly factor for CENP-A nucleosomes is the Mis18 complex (Mis18, Mis18{beta}, and M18BP1) which is regulated in its localization to centromeres between metaphase and G1 when CENP-A assembly occurs. Here, we define a new regulatory mechanism that works through cell cycle dependent phosphorylation of Xenopus laevis M18BP1 between metaphase and interphase. This phosphoregulatory switch disrupts binding of M18BP1 to CENP-A nucleosomes in metaphase, and when relieved enables M18BP1 binding to CENP-A nucleosomes in interphase. We show that this phosphorylation dependent switching mechanism regulates CENP-A nucleosome assembly. We propose that the phospho-regulated binding of M18BP1 to CENP-A nucleosomes is an important control mechanism that restricts the timing of new CENP-A assembly.

cell biology↗

bioRxiv2021

DiMeLo-seq: a long-read, single-molecule method for mappingprotein-DNA interactions genome-wide

Molecular studies of genome regulation often rely on the ability to map where specific proteins interact with genomic DNA. Existing techniques for mapping protein-DNA interactions genome-wide rely on DNA amplification methods followed by sequencing with short reads, which dissociates joint binding information at neighboring sites, removes endogenous DNA methylation information, and precludes the ability to reliably map interactions in repetitive regions of the genome. To address these limitations, we created a new protein-DNA mapping method, called Directed Methylation with Long-read sequencing (DiMeLo-seq), which methylates DNA near each target proteins DNA binding site in situ, then leverages the ability to distinguish methylated and unmethylated bases on long, native DNA molecules using long-read, single-molecule sequencing technologies. We demonstrate the optimization and utility of this method by mapping the interaction sites of a variety of different proteins and histone modifications across the human genome, achieving a single-molecule binding site resolution of less than 200 bp. Furthermore, we mapped the positions of the centromeric histone H3 variant CENP-A in repetitive regions that are unmappable with short reads, while simultaneously analyzing endogenous CpG methylation and joint binding events on single molecules. DiMeLo-seq is a versatile method that can provide multimodal and truly genome-wide information for investigating protein-DNA interactions.

genomics↗

bioRxiv2020

TaqMan Array Cards enable monitoring of diverse enteric pathogens across environmental and host reservoirs

BackgroundMultiple bacteria, viruses, protists, and helminths cause enteric infections that greatly impact human health and wellbeing. These enteropathogens are transmitted via several pathways through human, animal, and environmental reservoirs. Individual quantitative PCR (qPCR) assays have been extensively used to detect enteropathogens within these types of samples, whereas the TaqMan Array Card (TAC) that allows simultaneous detection of multiple enteropathogens has only previously been validated in human clinical samples. MethodsHere, we performed a comprehensive double-blinded comparison of the performance of a custom TAC relative to standard qPCR for the detection of eight enteric targets, by using spiked samples, wastewater from Melbourne (Australia), and human, animal, and environmental samples from informal settlements in Suva, Fiji. FindingsBoth methods exhibited high and comparable specificity (TAC: 100%, qPCR: 94%), sensitivity (TAC: 92%; qPCR: 100%), and quantitation accuracy (TAC: 91%; qPCR: 99%) in non-inhibited sample matrices. PCR inhibitors substantially impacted detection via TAC, though this issue was alleviated by 10-fold sample dilution. Among samples from informal settlements, the two techniques were comparable for detection (89% agreement) and quantitation (R2 = 0.82). The TAC additionally included 38 other targets, enabling detection of diverse faecal pathogens and extensive environmental contamination that would be prohibitively labour intensive to assay by standard qPCR. InterpretationOverall, the two techniques produce comparable results across diverse sample types, with qPCR prioritising greater sensitivity and quantitation accuracy, and TAC trading small reductions in these for a cost-effective larger enteropathogen panel that enables a greater number of enteric pathogens to be analysed concurrently, which is beneficial given the abundance and variety of enteric pathogens in environments such as urban informal settlements. The ability to monitor multiple enteric pathogens across diverse reservoirs in turn allows better resolution of pathogen exposure pathways, and the design and monitoring of interventions to reduce pathogen load. FundingWellcome Trust Our Planet, Our Health program [OPOH grant 205222/Z/16/Z].

microbiology↗