Biology subjects

Ganley, A. R. D.

Publications and source records attributed to Ganley, A. R. D..

6 recordsLinked to original sources

bioRxiv2026

Coupling cell differentiation to dewetting can explain villus elongation

During vertebrate development, the flat surface of the gut epithelium undergoes a dramatic transformation into densely packed arrays of finger-like projections called intestinal villi. Recent studies show that the villus formation relies on a tissue dewetting process, in which mesenchymal tissues buckle the overlying epithelial layer into periodic folds. However, the mechanisms driving subsequent elongation of these folds into finger-like villi remain largely unexplored. Here, we propose a simple mechanism for villus elongation that couples tissue dewetting to cell differentiation, which emerged as a repeated outcome of multiple independent simulations of an evolutionary-developmental Cellular Potts Model. In this mechanism, a liquid-like mesenchymal tissue continuously differentiates into a solid-like mesenchymal tissue at the interface between them. This differentiation drives the liquid-like tissue to continuously retract from the solid-like tissue in the opposite direction of the interface through dewetting, ultimately creating a finger-like projection. A merit of our proposed mechanism is that it only requires two tissues with different viscosities, high surface tension, and cell differentiation. We develop a simplified phase-field model to determine exactly how villus morphology depends on these three requirements. Since these requirements are satisfied not only in intestinal villi but also in many other developing tissues, we propose that the same mechanism could also drive the elongation of other tissues.

developmental biology↗

bioRxiv2022

Tissue-Specific Control of Ribosomal RNA Synthesis Revealed by Transcription Factor Profiling

Ribosomal RNAs (rRNAs) are the most abundant cellular RNAs, and their synthesis from rDNA repeats by RNA Polymerase I accounts for the bulk of all transcription. Despite substantial variation in rRNA transcription rates across cell types, little is known about cell-type-specific factors that bind rDNA and regulate rRNA transcription to meet tissue-specific needs. Using hematopoiesis as a model system, we mapped about 2200 ChIP-Seq datasets for 250 transcription factors (TFs) and chromatin proteins to human and mouse rDNA, and identified robust binding of multiple TF families to canonical TF motifs on rDNA. Using a 47S-FISH-Flow assay developed for nascent rRNA quantification, we demonstrated that targeted degradation of CEBPA (C/EBP alpha), a critical hematopoietic TF with conserved rDNA binding, caused rapid reduction in rRNA transcription due to reduced Pol I occupancy. Our work identifies numerous potential rRNA regulators, and provides a template for dissection of TF roles in rRNA transcription. HIGHLIGHTSO_LIMultiple cell-type-specific transcription factors (TFs) bind canonical motifs on rDNA. C_LIO_LIThe hematopoietic TF CEBPA binds to active rDNA alleles at a conserved site. C_LIO_LICEBPA promotes Polymerase I occupancy and rRNA transcription in myeloid progenitors. C_LIO_LIWe present 47S-FISH-Flow, a sensitive assay to quantify nascent rRNA. C_LI

molecular biology↗

bioRxiv2021

A new method for determining ribosomal DNA copy number shows differences between Saccharomyces cerevisiae populations

Ribosomal DNA genes (rDNA) encode the major ribosomal RNAs (rRNA) and in eukaryotic genomes are typically present as one or more arrays of tandem repeats. Species have characteristic rDNA copy numbers, ranging from tens to thousands of copies, with the number thought to be redundant for rRNA production. However, the tandem rDNA repeats are prone to recombination-mediated changes in copy number, resulting in substantial intra-species copy number variation. There is growing evidence that these copy number differences can have phenotypic consequences. However, we lack a comprehensive understanding of what determines rDNA copy number, how it evolves, and what the consequences are, in part because of difficulties in quantifying copy number. Here, we developed a genomic sequence read approach that estimates rDNA copy number from the modal coverage of the rDNA and whole genome to help overcome limitations in quantifying copy number with existing mean coverage-based approaches. We validated our method using strains of the yeast Saccharomyces cerevisiae with previously-determined rDNA copy numbers, and then applied our pipeline to investigate rDNA copy number in a global sample of 788 yeast isolates. We found that wild yeast have a mean copy number of 92, consistent with what is reported for other fungi but much lower than in laboratory strains. We also show that different populations have different rDNA copy numbers. These differences can partially be explained by phylogeny, but other factors such as environment are also likely to contribute to population differences in copy number. Our results demonstrate the utility of the modal coverage method, and highlight the high level of rDNA copy number variation within and between populations. Author summaryThe ribosomal RNA gene repeats (rDNA) form large tandem repeat arrays in most eukaryote genomes. Their tandem arrangement makes the rDNA prone to copy number variation, and there is increasing evidence that this copy number variation has phenotypic consequences. However, difficulties in measuring rDNA copy number hamper investigation into rDNA copy number dynamics and their significance. Here we developed a novel bioinformatics method for measuring rDNA copy number from whole genome sequence data that is based on the modal sequence read coverage. We established parameters for optimal performance of the method and validated it using yeast strains of known rDNA copy numbers. We then applied the method to a dataset of almost 800 global yeast isolates and demonstrate that yeast populations have different rDNA copy numbers that partially correlate with phylogeny. Our work provides a simple and accurate method for determining rDNA copy number that leverages the growing number of whole genome datasets, and highlights the dynamic nature of rDNA copy number.

genomics↗

bioRxiv2020

A simple, cheap, and robust protocol for the identification of mating type in Saccharomyces cerevisiae

Saccharomyces cerevisiae is an exceptional genetic system, with genetic crosses facilitated by its ability to be maintained in haploid and diploid forms. Such crosses are straightforward if the mating type/ploidy of the strains are known. Several techniques can be used to determine mating type (or ploidy), but all have limitations. Here we validate a simple, cheap and robust method to rapidly identify S. cerevisiae mating types. When cells of opposite mating type are mixed in liquid media, they "creep" up the culture vessel sides, a phenotype that can be easily detected visually. In contrast, mixtures of the same mating type or with a diploid simply settle out. The phenotype is robust to different media, cell densities, temperatures and strains, and is observable for several days. Microscopy suggests that cell aggregation during mating is responsible for the phenotype. Yeast knockout collection analysis identified 107 genes required for the creeping phenotype, with these being enriched for mating-specific genes. Surprisingly, the RIM101 signalling pathway was strongly represented. We propose that RIM101 signalling regulates aggregation as part of a wider, previously-unrecognized role in mating. The simplicity and robustness of this method makes it ideal for routine verification of S. cerevisiae mating type, with future studies required to verify its molecular basis.

microbiology↗

bioRxiv2018

The conservation landscape of the human ribosomal RNA gene repeats

Ribosomal RNA gene repeats (rDNA) encode ribosomal RNA, a major component of ribosomes. Ribosome biogenesis is central to cellular metabolic regulation, and several diseases are associated with rDNA dysfunction, notably cancer, However, its highly repetitive nature has severely limited characterization of the elements responsible for rDNA function. Here we make use of phylogenetic footprinting to provide a comprehensive list of novel, potentially functional elements in the human rDNA. Complete rDNA sequences for six non-human primate species were constructed using de novo whole genome assemblies. These new sequences were used to determine the conservation profile of the human rDNA, revealing 49 conserved regions in the rDNA intergenic spacer (IGS). To provide insights into the potential roles of these conserved regions, the conservation profile was integrated with functional genomics datasets. We find two major zones that contain conserved elements characterised by enrichment of transcription-associated chromatin factors, and transcription. Conservation of some IGS transcripts in the apes underpins the potential functional significance of these transcripts and the elements controlling their expression. Our results characterize the conservation landscape of the human IGS, and suggest that noncoding transcription and chromatin elements are conserved and important features of this unique genomic region.

genomics↗

bioRxiv2018

Contrasting patterns of coding and flanking region evolution in mammalian keratin associated protein-1 genes

DNA repeats are common elements in eukaryotic genomes, and their multi-copy nature provides the opportunity for genetic exchange. This exchange can produce altered evolutionary patterns, including concerted evolution where within genome repeat copies are more similar to each other than to orthologous repeats in related species. Here we investigated the genetic architecture of the keratin-associated protein (KAP) gene family, KRTAP1. This family encodes proteins that are important components of hair and wool in mammals, and the genes are present in tandem copies. Comparison of KRTAP1 gene repeats from species across the mammalian phylogeny shows strongly contrasting evolutionary patterns between the coding regions, which have a concerted evolution pattern, and the flanking regions, which have a normal, radiating pattern of evolution. This dichotomy in evolutionary pattern transitions abruptly at the start and stop codons, and we show it is not the result of purifying selection acting to maintain species-specific protein sequences, nor of codon adaptation or reverse transcription of KRTAP1-n mRNA. Instead, the results are consistent with short-tract gene conversion events coupled with selection for these events in the coding region driving the contrasting evolutionary patterns found in the KRTAP1 repeats. Our work shows the power that repeat recombination has to complement selection and finely tune the sequences of repetitive genes. Interplay between selection and recombination may be a more common mechanism than currently appreciated for achieving specific adaptive outcomes in the many eukaryotic multi-gene families, and our work argues for greater emphasis on exploring the sequence structures of these families.

genetics↗