High-throughput characterization of mutations in genes that drive clonal evolution using multiplex adaptome capture sequencing.

Understanding how cells are likely to evolve can guide medical interventions and bioengineering efforts that must contend with unwanted mutations. The adaptome of a cell-the neighborhood of genetic changes that are most likely to drive adaptation in a given environment-can be mapped by tracking rare beneficial variants during the early stages of clonal evolution. We used multiplex adaptome capture sequencing (mAdCap-seq), a procedure that combines unique molecular identifiers and hybridization-based enrichment, to characterize mutations in eight Escherichia coli genes known to be under selection in a laboratory environment.

Global Transcriptional Response of to Formaldehyde Stress Expands the Role of EfgA and Is Distinct from Antibiotic Translational Inhibition.

The potency and indiscriminate nature of formaldehyde reactivity upon biological molecules make it a universal stressor. However, some organisms such as possess means to rapidly and effectively mitigate formaldehyde-induced damage. EfgA is a recently identified formaldehyde sensor predicted to halt translation in response to elevated formaldehyde as a means to protect cells.

Evolution of satellite plasmids can prolong the maintenance of newly acquired accessory genes in bacteria.

Transmissible plasmids spread genes encoding antibiotic resistance and other traits to new bacterial species. Here we report that laboratory populations of Escherichia coli with a newly acquired IncQ plasmid often evolve 'satellite plasmids' with deletions of accessory genes and genes required for plasmid replication. Satellite plasmids are molecular parasites: their presence reduces the copy number of the full-length plasmid on which they rely for their continued replication.

Control of Lineage-Specific Gene Expression by Functionalized gRNA Barcodes.

Lineage tracking delivers essential quantitative insight into dynamic, probabilistic cellular processes, such as somatic tumor evolution and differentiation. Methods for high diversity lineage quantitation rely on sequencing a population of DNA barcodes. However, manipulation of specific individual lineages is not possible with this approach.

Directed evolution of Escherichia coli with lower-than-natural plasmid mutation rates.

Unwanted evolution of designed DNA sequences limits metabolic and genome engineering efforts. Engineered functions that are burdensome to host cells and slow their replication are rapidly inactivated by mutations, and unplanned mutations with unpredictable effects often accumulate alongside designed changes in large-scale genome editing projects. We developed a directed evolution strategy, Periodic Reselection for Evolutionarily Reliable Variants (PResERV), to discover mutations that prolong the function of a burdensome DNA sequence in an engineered organism.

Specificity of genome evolution in experimental populations of evolved at different temperatures.

Isolated populations derived from a common ancestor are expected to diverge genetically and phenotypically as they adapt to different local environments. To examine this process, 30 populations of were evolved for 2,000 generations, with six in each of five different thermal regimes: constant 20 °C, 32 °C, 37 °C, 42 °C, and daily alternations between 32 °C and 42 °C. Here, we sequenced the genomes of one endpoint clone from each population to test whether the history of adaptation in different thermal regimes was evident at the genomic level.

Tempo and mode of genome evolution in a 50,000-generation experiment.

Adaptation by natural selection depends on the rates, effects and interactions of many mutations, making it difficult to determine what proportion of mutations in an evolving lineage are beneficial. Here we analysed 264 complete genomes from 12 Escherichia coli populations to characterize their dynamics over 50,000 generations. The populations that retained the ancestral mutation rate support a model in which most fixed mutations are beneficial, the fraction of beneficial mutations declines as fitness rises, and neutral mutations accumulate at a constant rate.

Detecting rare structural variation in evolving microbial populations from new sequence junctions using breseq.

New mutations leading to structural variation (SV) in genomes-in the form of mobile element insertions, large deletions, gene duplications, and other chromosomal rearrangements-can play a key role in microbial evolution. Yet, SV is considerably more difficult to predict from short-read genome resequencing data than single-nucleotide substitutions and indels (SN), so it is not yet routinely identified in studies that profile population-level genetic diversity over time in evolution experiments. We implemented an algorithm for detecting polymorphic SV as part of the breseq computational pipeline.

Identifying structural variation in haploid microbial genomes from short-read resequencing data using breseq.

Background: Mutations that alter chromosomal structure play critical roles in evolution and disease, including in the origin of new lifestyles and pathogenic traits in microbes. Large-scale rearrangements in genomes are often mediated by recombination events involving new or existing copies of mobile genetic elements, recently duplicated genes, or other repetitive sequences. Most current software programs for predicting structural variation from short-read DNA resequencing data are intended primarily for use on human genomes.

Large chromosomal rearrangements during a long-term evolution experiment with Escherichia coli.

Unlabelled: Large-scale rearrangements may be important in evolution because they can alter chromosome organization and gene expression in ways not possible through point mutations. In a long-term evolution experiment, twelve Escherichia coli populations have been propagated in a glucose-limited environment for over 25 years. We used whole-genome mapping (optical mapping) combined with genome sequencing and PCR analysis to identify the large-scale chromosomal rearrangements in clones from each population after 40,000 generations.

Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq.

Next-generation DNA sequencing (NGS) can be used to reconstruct eco-evolutionary population dynamics and to identify the genetic basis of adaptation in laboratory evolution experiments. Here, we describe how to run the open-source breseq computational pipeline to identify and annotate genetic differences found in whole-genome and whole-population NGS data from haploid microbes where a high-quality reference genome is available. These methods can also be used to analyze mutants isolated in genetic screens and to detect unintended mutations that may occur during strain construction and genome editing.

Recursive genomewide recombination and sequencing reveals a key refinement step in the evolution of a metabolic innovation in Escherichia coli.

Evolutionary innovations often arise from complex genetic and ecological interactions, which can make it challenging to understand retrospectively how a novel trait arose. In a long-term experiment, Escherichia coli gained the ability to use abundant citrate (Cit(+)) in the growth medium after ∼31,500 generations of evolution. Exploiting this previously untapped resource was highly beneficial: later Cit(+) variants achieve a much higher population density in this environment.

ChIP-seq defined genome-wide map of TGFβ/SMAD4 targets: implications with clinical outcome of ovarian cancer.

Deregulation of the transforming growth factor-β (TGFβ) signaling pathway in epithelial ovarian cancer has been reported, but the precise mechanism underlying disrupted TGFβ signaling in the disease remains unclear. We performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) to investigate genome-wide screening of TGFβ-induced SMAD4 binding in epithelial ovarian cancer. Following TGFβ stimulation of the A2780 epithelial ovarian cancer cell line, we identified 2,362 SMAD4 binding loci and 318 differentially expressed SMAD4 target genes.

Aberrant TGFβ/SMAD4 signaling contributes to epigenetic silencing of a putative tumor suppressor, RunX1T1 in ovarian cancer.

Aberrant TGFβ signaling pathway may alter the expression of down-stream targets and promotes ovarian carcinogenesis. However, the mechanism of this impairment is not fully understood. Our previous study has identified RunX1T1 as a putative SMAD4 target in an immortalized ovarian surface epithelial cell line, IOSE.

Epigenetic silencing mediated through activated PI3K/AKT signaling in breast cancer.

Trimethylation of histone 3 lysine 27 (H3K27me3) is a critical epigenetic mark for the maintenance of gene silencing. Additional accumulation of DNA methylation in target loci is thought to cooperatively support this epigenetic silencing during tumorigenesis. However, molecular mechanisms underlying the complex interplay between the two marks remain to be explored.

Epigenetic influences of low-dose bisphenol A in primary human breast epithelial cells.

Substantial evidence indicates that exposure to bisphenol A (BPA) during early development may increase breast cancer risk later in life. The changes may persist into puberty and adulthood, suggesting an epigenetic process being imposed in differentiated breast epithelial cells. The molecular mechanisms by which early memory of BPA exposure is imprinted in breast progenitor cells and then passed onto their epithelial progeny are not well understood.

Estrogen-mediated epigenetic repression of large chromosomal regions through DNA looping.

The current concept of epigenetic repression is based on one repressor unit corresponding to one silent gene. This notion, however, cannot adequately explain concurrent silencing of multiple loci observed in large chromosome regions. The long-range epigenetic silencing (LRES) can be a frequent occurrence throughout the human genome.

Promoter hypermethylation of FBXO32, a novel TGF-beta/SMAD4 target gene and tumor suppressor, is associated with poor prognosis in human ovarian cancer.

Resistance to TGF-beta is frequently observed in ovarian cancer, and disrupted TGF-beta/SMAD4 signaling results in the aberrant expression of downstream target genes in the disease. Our previous study showed that ADAM19, a SMAD4 target gene, is downregulated through epigenetic mechanisms in ovarian cancer with aberrant TGF-beta/SMAD4 signaling. In this study, we investigated the mechanism of downregulation of FBXO32, another SMAD4 target gene, and the clinical significance of the loss of FBXO32 expression in ovarian cancer.

Epigenetic repression of microRNA-129-2 leads to overexpression of SOX4 oncogene in endometrial cancer.

Genetic amplification, mutation, and translocation are known to play a causal role in the upregulation of an oncogene in cancer cells. Here, we report an emerging role of microRNA, the epigenetic deregulation of which may also lead to this oncogenic activation. SOX4, an oncogene belonging to the SRY-related high mobility group box family, was found to be overexpressed (P < 0.

Xenoestrogen-induced epigenetic repression of microRNA-9-3 in breast epithelial cells.

Early exposure to xenoestrogens may predispose to breast cancer risk later in adult life. It is likely that long-lived, self-regenerating epithelial progenitor cells are more susceptible to these exposure injuries over time and transmit the injured memory through epigenetic mechanisms to their differentiated progeny. Here, we used progenitor-containing mammospheres as an in vitro exposure model to study this epigenetic effect.

Methylation analysis by microarray.

Differential methylation hybridization (DMH) is a high-throughput DNA methylation screening tool that utilizes methylation-sensitive restriction enzymes to profile methylated fragments by hybridizing them to a CpG island microarray. This array contains probes spanning all the 27,800 islands annotated in the UCSC Genome Browser. Herein we describe a DMH protocol with clearly identified quality control points.

Breast cancer-associated fibroblasts confer AKT1-mediated epigenetic silencing of Cystatin M in epithelial cells.

The interplay between histone modifications and promoter hypermethylation provides a causative explanation for epigenetic gene silencing in cancer. Less is known about the upstream initiators that direct this process. Here, we report that the Cystatin M (CST6) tumor suppressor gene is concurrently down-regulated with other loci in breast epithelial cells cocultured with cancer-associated fibroblasts (CAF).

Differential methylation hybridization: profiling DNA methylation with a high-density CpG island microarray.

Differential methylation hybridization (DMH) is a high-throughput DNA methylation screening tool that utilizes methylation-sensitive restriction enzymes to profile methylated fragments by hybridizing them to a CpG island microarray. This array contains probes spanning all the 27,800 islands annotated in the UCSC Genome Browser. Herein we describe a revised DMH protocol with clearly identified quality control points.

Aberrant transforming growth factor beta1 signaling and SMAD4 nuclear translocation confer epigenetic repression of ADAM19 in ovarian cancer.

Transforming growth factor-beta (TGF-beta)/SMAD signaling is a key growth regulatory pathway often dysregulated in ovarian cancer and other malignancies. Although loss of TGF-beta-mediated growth inhibition has been shown to contribute to aberrant cell behavior, the epigenetic consequence(s) of impaired TGF-beta/SMAD signaling on target genes is not well established. In this study, we show that TGF-beta1 causes growth inhibition of normal ovarian surface epithelial cells, induction of nuclear translocation SMAD4, and up-regulation of ADAM19 (a disintegrin and metalloprotease domain 19), a newly identified TGF-beta1 target gene.