The rate of transcription elongation plays an important role in the timing of expression of full-length transcripts as well as in the regulation of alternative splicing. In this study, we coupled Bru-seq technology with 5,6-dichlorobenzimidazole 1-b-D-ribofuranoside (DRB) to estimate the elongation rates of over 2000 individual genes in human cells. This technique, BruDRB-seq, revealed gene-specific differences in elongation rates with a median rate of around 1.5 kb/min. We found that genes with rapid elongation rates showed higher densities of H3K79me2 and H4K20me1 histone marks compared to slower elongating genes. Furthermore, high elongation rates had a positive correlation with gene length, low complexity DNA sequence, and distance from the nearest active transcription unit. Features that negatively correlated with elongation rate included the density of exons, long terminal repeats, GC content of the gene, and DNA methylation density in the bodies of genes. Our results suggest that some static gene features influence transcription elongation rates and that cells may alter elongation rates by epigenetic regulation. The BruDRB-seq technique offers new opportunities to interrogate mechanisms of regulation of transcription elongation.

Gene expression studies commonly examine total cellular RNA, which only provides information about its steady-state pool of RNA. It remains unclear whether differences in the steady-state reflects variable rates of transcription or RNA degradation. To specifically monitor RNA synthesis and degradation genome-wide, we developed Bru-Seq and BruChase-Seq. These assays are based on metabolic pulse-chase labeling of RNA using bromouridine (Bru). In Bru-Seq, recently labeled RNAs are sequenced to reveal spans of nascent transcription in the genome. In BruChase-Seq, cells are chased in uridine for different periods of time following Bru-labeling, allowing for the isolation of RNA populations of specific ages. Here we describe these methodologies in detail and highlight their usefulness in assessing RNA synthesis and stability as well as splicing kinetics with examples of specific genes from different human cell lines.

Steady-state gene expression is a coordination of synthesis and decay of RNA through epigenetic regulation, transcription factors, micro RNAs (miRNAs), and RNA-binding proteins. Here, we present bromouride labeling and sequencing (Bru-Seq) and bromouridine pulse-chase and sequencing (BruChase-Seq) to assess genomewide changes to RNA synthesis and stability in human fibroblasts at homeostasis and after exposure to the proinflammatory tumor necrosis factor (TNF). The inflammatory response in human cells involves rapid and dramatic changes in gene expression, and the Bru-Seq and BruChase-Seq techniques revealed a coordinated and complex regulation of gene expression both at the transcriptional and posttranscriptional levels. The combinatory analysis of both RNA synthesis and stability using Bru-Seq and BruChase-Seq allows for a much deeper understanding of mechanisms of gene regulation than afforded by the analysis of steady-state total RNA and should be useful in many biological settings.T he acute inflammatory response is critical for the defense against infections and in the healing of damaged tissues (1). The orchestration of the reprogramming of gene expression associated with the acute inflammatory response is complex and involves both transcriptional and posttranscriptional regulation (2-5). Conventional exploration of gene expression using total RNA does not fully capture this complexity because it does not provide insight into the contribution of nascent RNA synthesis or RNA decay to steady-state RNA changes. A number of different approaches have recently been developed to assess nascent RNA synthesis in cells such as global run-on and sequencing (GROSeq) (6), native elongating transcript sequencing (NET-Seq) (7), nascent RNA sequencing (Nascent-Seq) (8), and metabolic labeling of nascent RNA by using microarrays (9) or RNA-Seq (10, 11). By comparing the data obtained with metabolically labeled nascent RNA with the steady-state RNA levels, the rates of degradation of all transcripts can be computationally estimated. The stability of steady-state RNA can also be estimated from the decay rate of steady-state RNA after transcription inhibition (12)(13)(14) or by immunoprecipitation of metabolically labeled steady-state RNA after different chase periods (15, 16). These approaches work well when the system is at homeostasis, but not when conditions are altered by environmental stimuli or stress, such as the induction of the acute inflammatory response, when the rates of decay of transcripts are expected to change (10,11).In this study, we present Bru-Seq and BruChase-Seq based on bromouridine pulse labeling of nascent RNA followed by chases in uridine to obtain RNA populations of specific ages. The Brulabeled RNA is then immunocaptured followed by deep sequencing. These techniques allowed us to assess changes in the rates of both synthesis and degradation of RNA globally after the activation of the proinflammatory response by TNF. Our results provide a comprehensive and complex picture of the contribution of tr...

BackgroundIsobutanol is a promising next-generation biofuel with demonstrated high yield microbial production, but the toxicity of this molecule reduces fermentation volumetric productivity and final titer. Organic solvent tolerance is a complex, multigenic phenotype that has been recalcitrant to rational engineering approaches. We apply experimental evolution followed by genome resequencing and a gene expression study to elucidate genetic bases of adaptation to exogenous isobutanol stress.ResultsThe adaptations acquired in our evolved lineages exhibit antagonistic pleiotropy between minimal and rich medium, and appear to be specific to the effects of longer chain alcohols. By examining genotypic adaptation in multiple independent lineages, we find evidence of parallel evolution in marC, hfq, mdh, acrAB, gatYZABCD, and rph genes. Many isobutanol tolerant lineages show reduced RpoS activity, perhaps related to mutations in hfq or acrAB. Consistent with the complex, multigenic nature of solvent tolerance, we observe adaptations in a diversity of cellular processes. Many adaptations appear to involve epistasis between different mutations, implying a rugged fitness landscape for isobutanol tolerance. We observe a trend of evolution targeting post-transcriptional regulation and high centrality nodes of biochemical networks. Collectively, the genotypic adaptations we observe suggest mechanisms of adaptation to isobutanol stress based on remodeling the cell envelope and surprisingly, stress response attenuation.ConclusionsWe have discovered a set of genotypic adaptations that confer increased tolerance to exogenous isobutanol stress. Our results are immediately useful to further efforts to engineer more isobutanol tolerant host strains of E. coli for isobutanol production. We suggest that rpoS and post-transcriptional regulators, such as hfq, RNA helicases, and sRNAs may be interesting mutagenesis targets for future global phenotype engineering.

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Understanding the mechanisms by which organisms adapt to environmental conditions is a fundamental question for ecology and evolution. In this study, we evaluate changes in gene expression of a marine mollusc, the eastern oyster Crassostrea virginica, associated with the physico-chemical conditions and the levels of metals and other contaminants in their environment. The results indicate that transcript signatures can effectively disentangle the complex interactive gene expression responses to the environment and are also capable of disentangling the complex dynamic effects of environmental factors on gene expression. In this context, the mapping of environment to gene and gene to environment is reciprocal and mutually reinforcing. In general, the response of transcripts to the environment is driven by major factors known to affect oyster physiology such as temperature, pH, salinity, and dissolved oxygen, with pollutant levels playing a relatively small role, at least within the range of concentrations found in the studied oyster habitats. Further, the two environmental factors that dominate these effects (temperature and pH) interact in a dynamic and nonlinear fashion to impact gene expression. Transcriptomic data obtained in our study provide insights into the mechanisms of physiological responses to temperature and pH in oysters that are consistent with the known effects of these factors on physiological functions of ectotherms and indicate important linkages between transcriptomics and physiological outcomes. Should these linkages hold in further studies and in other organisms, they may provide a novel integrated approach for assessing the impacts of climate change, ocean acidification and anthropogenic contaminants on aquatic organisms via relatively inexpensive microarray platforms.

Racial/ethnic disparities in breast cancer mortality continue to widen but genomic studies rarely interrogate breast cancer in diverse populations. Through genome, exome, and RNA sequencing, we examined the molecular features of breast cancers using 194 patients from Nigeria and 1037 patients from The Cancer Genome Atlas (TCGA). Relative to Black and White cohorts in TCGA, Nigerian HR + /HER2 − tumors are characterized by increased homologous recombination deficiency signature, pervasive TP53 mutations, and greater structural variation—indicating aggressive biology. GATA3 mutations are also more frequent in Nigerians regardless of subtype. Higher proportions of APOBEC-mediated substitutions strongly associate with PIK3CA and CDH1 mutations, which are underrepresented in Nigerians and Blacks. PLK2, KDM6A, and B2M are also identified as previously unreported significantly mutated genes in breast cancer. This dataset provides novel insights into potential molecular mechanisms underlying outcome disparities and lay a foundation for deployment of precision therapeutics in underserved populations.

Triatoma brasiliensis is the most important autochthon vector of Trypanosoma cruzi in Brazil,where it is widely distributed in the semiarid areas of the Northeast. In order to advance the knowledge of the salivary biomolecules of Triatominae, a salivary gland cDNA library of T. brasiliensis was mass sequenced and analyzed. Polypeptides were sequenced by HPLC/Edman degradation experiments. 1,712 cDNA sequences were obtained and grouped in 786 clusters. The housekeeping category had 24.4% and 17.8% of the clusters and sequences, respectively. The putatively secreted category contained 47.1% of the clusters and 68.2% of the sequences. Finally, 28.5% of the clusters, containing 14% of all sequences, were classified as unknown. The sialoma of T. brasiliensis showed a high amount and great variety of different lipocalins (93.8% of secreted proteins). Remarkably, a great number of serine proteases that were not observed in previous blood-sucking sialotranscriptomes were found. Nine Kazal peptides were identified, among them one with high homology to the tabanid vasodilator vasotab, suggesting that the Triatoma vasodilator could be a Kazal protein.