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Biology subjects

Wiles, E. T.

Publications and source records attributed to Wiles, E. T..

6 recordsLinked to original sources

Capsular Polysaccharide Safeguards a Prophage-Bacterium Symbiosis by Preventing Collateral Attack and Promoting Viral Transmission

Many symbioses exist along a continuum from cooperation to conflict. Latent bacterial viruses known as prophages embody this duality. Acting as both partner and predator, they can enhance bacterial fitness while retaining the capacity to kill their hosts through lytic replication. Yet what determines the balance between cooperation and conflict in prophage-bacterium symbioses, and how these associations avoid collapse, remains poorly characterized. To identify mechanisms that stabilize prophag-bacterium partnerships, we used experimental evolution to perturb a natural association through repeated cycles of transmission and reinfection. This perturbation consistently selected mutant hosts lacking capsular polysaccharide production and exposed a hidden conflict that proved detrimental to both partners. During lytic outbreaks, host cells suffered lethal collateral prophage attack while dispersing virions became entrapped on neighboring cells and lysis debris, severely restricting transmission. Genetic and imaging-based studies revealed that capsular polysaccharides suppress these maladaptive interactions by limiting phage readsorption at the cell surface. We term this host-mediated safeguard the Hyperion effect, after the Greek Titan of light. Hyperion interactions enable phages to radiate outward from host populations, thereby averting collateral attack and promoting viral transmission. Our findings demonstrate that conflicts between prophages and their hosts can extend beyond individual cells to whole populations, with consequences that scale to shape patterns of prophage spread and microbial community assembly. More broadly, our work illustrates how mutually beneficial prophage-bacterium interactions can arise not only through cooperation, but also through the suppression of mutually detrimental conflicts.

microbiology↗

Phollow: Visualizing Gut Bacteriophage Transmission within Microbial Communities and Living Animals

Bacterial viruses (known as "phages") shape the ecology and evolution of microbial communities, making them promising targets for microbiome engineering. However, knowledge of phage biology is constrained because it remains difficult to study phage transmission dynamics within multi-member communities and living animal hosts. We therefore created "Phollow": a live imaging-based approach for tracking phage replication and spread in situ with single-virion resolution. Combining Phollow with optically transparent zebrafish enabled us to directly visualize phage outbreaks within the vertebrate gut. We observed that virions can be rapidly taken up by intestinal tissues, including by enteroendocrine cells, and quickly disseminate to extraintestinal sites, including the liver and brain. Moreover, antibiotics trigger waves of interbacterial transmission leading to sudden shifts in spatial organization and composition of defined gut communities. Phollow ultimately empowers multiscale investigations connecting phage transmission to transkingdom interactions that have the potential to open new avenues for viral-based microbiome therapies.

microbiology↗

The ACF Chromatin Remodeling Complex is Essential for Polycomb Repression

Establishing and maintaining appropriate gene repression is critical for the health and development of multicellular organisms. Histone H3 lysine 27 (H3K27) methylation is a chromatin modification associated with repressed facultative heterochromatin, but the mechanism of this repression remains unclear. We used a forward genetic approach to identify genes involved in transcriptional silencing of H3K27-methylated chromatin in the filamentous fungus Neurospora crassa. We found that the N. crassa homologs of ISWI (NCU03875) and ACF (NCU00164) are required for repression of a subset of H3K27-methylated genes and that they form an ACF chromatin remodeling complex. This N. crassa ACF complex interacts with chromatin throughout the genome, yet association with facultative heterochromatin is specifically promoted by the H3K27 methyltransferase, SET-7. H3K27-methylated genes that are upregulated when iswi or acf1 are deleted show a downstream shift of the +1 nucleosome, suggesting that proper nucleosome positioning is critical for repression of facultative heterochromatin. Our findings support a direct role for the ACF complex in Polycomb repression.

genetics↗

Identification of a PRC2 accessory subunit required for subtelomeric H3K27 methylation in Neurospora

Polycomb repressive complex 2 (PRC2) catalyzes methylation of histone H3 lysine 27 (H3K27) in genomic regions of most eukaryotes and is critical for maintenance of the associated transcriptional repression. However, the mechanisms that shape the distribution of H3K27 methylation, such as recruitment of PRC2 to chromatin and/or stimulation of PRC2 activity, are unclear. Here, using a forward genetic approach in the model organism Neurospora crassa, we identified two alleles of a gene, NCU04278, encoding an unknown PRC2 accessory subunit (PAS). Loss of PAS resulted in losses of H3K27 methylation concentrated near the chromosome ends and derepression of a subset of associated subtelomeric genes. Immunoprecipitation followed by mass spectrometry confirmed reciprocal interactions between PAS and known PRC2 subunits, and sequence similarity searches demonstrated that PAS is not unique to N. crassa. PAS homologs likely influence the distribution of H3K27 methylation, and underlying gene repression, in a variety of fungal lineages.

molecular biology↗

Rapid and Inexpensive Preparation of Genome-Wide Nucleosome Footprints from Model and Non-Model Organisms

Eukaryotic DNA is packaged into nucleosomes, the smallest repeating unit of chromatin. The positions of nucleosomes determine the relative accessibility of genomic DNA. Several protocols exist for mapping nucleosome positions in eukaryotic genomes in order to study the relationship between chromatin structure and DNA-dependent processes. These nucleosome mapping protocols can be laborious and, at minimum, require two to three days to isolate nucleosome-protected DNA fragments. We have developed a streamlined protocol for mapping nucleosomes from S. cerevisiae liquid culture or from patches on solid agar. This method isolates nucleosome-sized footprints in three hours using 1.5 ml tubes with minimal chemical waste. We validate that these footprints match those produced by previously published methods and we demonstrate that our protocol works for N. crassa and S. pombe. A slightly modified protocol can be used for isolation of nucleosome-protected DNA fragments from a variety of wild fungal specimens thereby providing a simple, easily multiplexed and unified strategy to map nucleosome positions in model and non-model fungi. Finally, we demonstrate recovery of nucleosome footprints from the diploid myeloid leukemia cell line PLB-985 in less than three hours using an abbreviated version of the same protocol. With reduced volume and incubation times and a streamlined workflow, the described method should be compatible with high-throughput, automated creation of MNase-seq libraries. We believe this simple validated method for rapidly producing sequencing-ready nucleosome footprints from a variety of organisms will make nucleosome mapping studies widely accessible to researchers globally.

genetics↗

Evolutionarily ancient BAH-PHD protein mediates Polycomb silencing

Methylation of histone H3 lysine 27 (H3K27) is widely recognized as a transcriptionally repressive chromatin modification but the mechanism of repression remains unclear. We devised and implemented a forward genetic scheme to identify factors required for H3K27 methylation-mediated silencing in the filamentous fungus Neurospora crassa and identified a bromo-adjacent homology (BAH)-plant homeodomain (PHD)-containing protein, EPR-1 (Effector of Polycomb Repression 1; NCU07505). EPR-1 associates with H3K27 methylation in vivo and in vitro, and loss of EPR-1 de-represses H3K27-methylated genes without loss of H3K27 methylation. EPR-1 is not fungal-specific; orthologs of EPR-1 are present in a diverse array of eukaryotic lineages, suggesting an ancestral EPR-1 was a component of a primitive Polycomb repression pathway. SignificancePolycomb group (PcG) proteins are employed by a wide variety of eukaryotes for the maintenance of gene repression. Polycomb repressive complex 2 (PRC2), a multimeric complex of PcG proteins, catalyzes the methylation of histone H3 lysine 27 (H3K27). In the filamentous fungus, Neurospora crassa, H3K27 methylation represses scores of genes, despite the absence of canonical H3K27 methylation effectors that are present in plants and animals. We report the identification and characterization of an H3K27 methylation effector, EPR-1, in N. crassa and demonstrate its widespread presence and early eukaryotic origins with phylogenetic analyses. These findings indicate that an ancient EPR-1 may have been part of a nascent Polycomb repression system in eukaryotes.

molecular biology↗