Biology subjects

Brem, R.

Publications and source records attributed to Brem, R..

6 recordsLinked to original sources

bioRxiv2026

Genomic signatures of selection in the mound-building mouse Mus spicilegus

The steppe mouse Mus spicilegus is distinguished by several unusual phenotypes: animals of this species build complex mounds from brush and dirt, and this behavior as well as the pace of development is regulated by season of birth. The genetic and evolutionary basis of species-defining traits in Mus spicilegus is unknown. To discover genomic clues to the mechanisms at play, we took a molecular-evolution approach. Sequence-based tests of protein-coding exons, and of brain-active cis-regulatory regions, revealed a striking excess of evidence for evolutionary acceleration in the M. spicilegus genome relative to those of closely related species. Transcriptional analyses also revealed unique expression programs in the M. spicilegus brain, which could be traced in part to cis-regulatory sequence variation. Hit loci from our molecular-evolution tests tended to be active in the cerebral cortices and hypothalamus and have annotated functions in neuronal development, neural plasticity, or endocrine signaling. In many cases, their orthologs in other animal systems had been implicated in neoteny/the pace of maturation, unusual building behaviors, and autism. Together, our data indicate that M. spicilegus has undergone divergence in brain-active genes to an extent far more than its relatives. We interpret the affected genes under a model in which they mediate the seasonal capacity by M. spicilegus for protracted maturation and mound-building. Thus our work provides an evolutionary interpretation of, and catalog of candidate determinants for, shifts in motivated behavior and development in this intriguing mouse species.

evolutionary biology↗

bioRxiv2024

Inferring the composition of a mixed culture of natural microbial isolates by deep sequencing

Next generation sequencing has unlocked a wealth of genotype information for microbial populations, but phenotyping remains a bottleneck for exploiting this information, particularly for pathogens that are difficult to manipulate. Here, we establish a method for high-throughput phenotyping of mixed cultures, in which the pattern of naturally occurring single-nucleotide polymorphisms in each isolate is used as intrinsic barcodes which can be read out by sequencing. We demonstrate that our method can correctly deconvolute strain proportions in simulated mixed-strain pools. As an experimental test of our method, we perform whole genome sequencing of 66 natural isolates of the thermally dimorphic pathogenic fungus Coccidioides posadasii and infer the strain compositions for large mixed pools of these strains after competition at 37{degrees}C and room temperature. We validate the results of these selection experiments by recapitulating the temperature-specific enrichment results in smaller pools. Additionally, we demonstrate that strain fitness estimated by our method can be used as a quantitative trait for genome-wide association studies. We anticipate that our method will be broadly applicable to natural populations of microbes and allow high-throughput phenotyping to match the rate of genomic data acquisition. Author summaryThe diversity of the gene pool in natural populations encodes a wealth of information about its molecular biology. This is an especially valuable resource for non-model organisms, from humans to many microbial pathogens, lacking traditional genetic approaches. An effective method for reading out this population genetic information is a genome wide association study (GWAS) which searches for genotypes correlated with a phenotype of interest. With the advent of cheap genotyping, high throughput phenotyping is the primary bottleneck for GWAS, particularly for microbes that are difficult to manipulate. Here, we take advantage of the fact that the naturally occurring genetic variation within each individual strain can be used as an intrinsic barcode, which can be used to read out relative abundance of each strain as a quantitative phenotype from a mixed culture. Coccidioides posadasii, the causative agent of Valley Fever, is a fungal pathogen that must be manipulated under biosafety level 3 conditions, precluding many high-throughput phenotyping approaches. We apply our method to pooled competitions of C. posadasii strains at environmental and host temperatures. We identify robustly growing and temperature-sensitive strains, confirm these inferences in validation pooled growth experiments, and successfully demonstrate their use in GWAS.

genetics↗

bioRxiv2022

A natural variation-based screen in mouse cells reveals USF2 as a regulator of the DNA damage response and cellular senescence

Cellular senescence is a program of cell cycle arrest, apoptosis resistance, and cytokine release induced by stress exposure in metazoan cells. Landmark studies in laboratory mice have characterized a number of master senescence regulators, including p16INK4a, p21, NF-kB, p53, and C/EBP{beta}. To discover other molecular players in senescence, we developed a screening approach to harness the evolutionary divergence between mouse species. We found that primary cells from the Mediterranean mouse Mus spretus, when treated with DNA damage to induce senescence, produced less cytokine and had less-active lysosomes than cells from laboratory M. musculus. We used allele-specific expression profiling to catalog senescence-dependent cis-regulatory variation between the species at thousands of genes. We then tested for correlation between these expression changes and interspecies sequence variants in the binding sites of transcription factors. Among the emergent candidate senescence regulators, we chose a little-studied cell cycle factor, USF2, for molecular validation. In acute irradiation experiments, cells lacking USF2 had compromised DNA damage repair and response. Longer-term senescent cultures without USF2 mounted an exaggerated senescence regulatory program--shutting down cell cycle and DNA repair pathways, and turning up cytokine expression, more avidly than wild-type. We interpret these findings under a model of pro-repair, anti-senescence regulatory function by USF2. Our study affords new insights into the mechanisms by which cells commit to senescence, and serves as a validated proof of concept for natural variation-based regulator screens.

genomics↗

bioRxiv2019

Musashi expression in intestinal stem cells attenuates radiation-induced decline in intestinal homeostasis and survival in Drosophila

Exposure to genotoxic stress by environmental agents or treatments, such as radiation therapy, can diminish health span and accelerate aging. We have developed a Drosophila melanogaster model to study the molecular effects of radiation-induced damage and repair. Utilizing a quantitative intestinal permeability assay, we performed an unbiased GWAS screen (using [~]150 strains from the DGRP reference panel) to search for natural genetic variants that regulate radiation-induced gut permeability. From this screen, we identified the RNA binding protein, Musashi (msi).Msi overexpression promoted intestinal stem cell proliferation, which increased survival after irradiation and rescued radiation-induced gut permeability. We identified that a novel role of Msi in ISC proliferation a novel role for Msi in enhancing ISC function following radiation-induced gut damage, which identifies Msi as a potential therapeutic target.

molecular biology↗

bioRxiv2017

Transposon insertional mutagenesis in Saccharomyces uvarum reveals trans-acting effects influencing species dependent essential genes

To understand how complex genetic networks perform and regulate diverse cellular processes, the function of each individual component must be defined. Comprehensive phenotypic studies of mutant alleles have been successful in model organisms in determining what processes depend on the normal function of a gene. These results are often ported to newly sequenced genomes by using sequence homology. However, sequence similarity does not always mean identical function or phenotype, suggesting that new methods are required to functionally annotate newly sequenced species. We have implemented comparative analysis by high-throughput experimental testing of gene dispensability in Saccharomyces uvarum, a sister species of S. cerevisiae. We created haploid and heterozygous diploid Tn7 insertional mutagenesis libraries in S. uvarum to identify species dependent essential genes, with the goal of detecting genes with divergent functions and/or different genetic interactions. Comprehensive gene dispensability comparisons with S. cerevisiae predicted diverged dispensability at 12% of conserved orthologs, and validation experiments confirmed 22 differentially essential genes. Surprisingly, despite their differences in essentiality, these genes were capable of cross-species complementation, demonstrating that trans-acting factors that are background-dependent contribute to differential gene essentiality. This study demonstrates that direct experimental testing of gene disruption phenotypes across species can inform comparative genomic analyses and improve gene annotation. Our method can be widely applied in microorganisms to further our understanding of genome evolution.

genomics↗

bioRxiv2017

Functional genomics of lipid metabolism in the oleaginous yeast Rhodosporidium toruloides

The basidomycete yeast Rhodosporidium toruloides (a.k.a. Rhodotorula toruloides) accumulates high concentrations of lipids and carotenoids from diverse carbon sources. It has great potential as a model for the cellular biology of lipid droplets and for sustainable chemical production. We developed a method for high-throughput genetics (RB-TDNAseq), using sequence-barcoded Agrobacterium tumefaciens T-DNA insertions into the R. toruloides genome. We identified 1337 putative essential genes with low T-DNA insertion rates. We functionally profiled genes required for fatty acid catabolism and lipid accumulation, validating results with 35 targeted deletion strains. We found that both mitochondrial and peroxisomal enzymes were required for growth on fatty acids, with different peroxisomal enzymes required on different fatty acids. We identified a high-confidence set of 150 genes affecting lipid accumulation, including genes with predicted function in signaling cascades, gene expression, protein modification and vesicular trafficking, autophagy, amino acid synthesis and tRNA modification, as well as genes of unknown function. These results greatly advance our understanding of lipid metabolism in this oleaginous species, identify key biological processes to be further explored and optimized for production of lipid-based bioproducts, and demonstrate a general approach for barcoded mutagenesis that should enable functional genomics in diverse fungi.

systems biology↗