A Rapid Spin Column-Based Method to Enrich Pathogen Transcripts from Eukaryotic Host Cells Prior to Sequencing.

When analyzing pathogen transcriptomes during the infection of host cells, the signal-to-background (pathogen-to-host) ratio of nucleic acids (NA) in infected samples is very small. Despite the advancements in next-generation sequencing, the minute amount of pathogen NA makes standard RNA-seq library preps inadequate for effective gene-level analysis of the pathogen in cases with low bacterial loads. In order to provide a more complete picture of the pathogen transcriptome during an infection, we developed a novel pathogen enrichment technique, which can enrich for transcripts from any cultivable bacteria or virus, using common, readily available laboratory equipment and reagents. To evenly enrich for pathogen transcripts, we generate biotinylated pathogen-targeted capture probes in an enzymatic process using the entire genome of the pathogen as a template. The capture probes are hybridized to a strand-specific cDNA library generated from an RNA sample. The biotinylated probes are captured on a monomeric avidin resin in a miniature spin column, and enriched pathogen-specific cDNA is eluted following a series of washes. To test this method, we performed an in vitro time-course infection using Klebsiella pneumoniae to infect murine macrophage cells. K. pneumoniae transcript enrichment efficiency was evaluated using RNA-seq. Bacterial transcripts were enriched up to ~400-fold, and allowed the recovery of transcripts from ~2000-3600 genes not observed in untreated control samples. These additional transcripts revealed interesting aspects of K. pneumoniae biology including the expression of putative virulence factors and the expression of several genes responsible for antibiotic resistance even in the absence of drugs.

The Modifier of Transcription 1 (Mot1) ATPase and Spt16 Histone Chaperone Co-regulate Transcription through Preinitiation Complex Assembly and Nucleosome Organization.

Modifier of transcription 1 (Mot1) is a conserved and essential Swi2/Snf2 ATPase that can remove TATA-binding protein (TBP) from DNA using ATP hydrolysis and in so doing exerts global effects on transcription. Spt16 is also essential and functions globally in transcriptional regulation as a component of the facilitates chromatin transcription (FACT) histone chaperone complex. Here we demonstrate that Mot1 and Spt16 regulate a largely overlapping set of genes in Saccharomyces cerevisiae. As expected, Mot1 was found to control TBP levels at co-regulated promoters. In contrast, Spt16 did not affect TBP recruitment. On a global scale, Spt16 was required for Mot1 promoter localization, and Mot1 also affected Spt16 localization to genes. Interestingly, we found that Mot1 has an unanticipated role in establishing or maintaining the occupancy and positioning of nucleosomes at the 5' ends of genes. Spt16 has a broad role in regulating chromatin organization in gene bodies, including those nucleosomes affected by Mot1. These results suggest that the large scale overlap in Mot1 and Spt16 function arises from a combination of both their unique and shared functions in transcription complex assembly and chromatin structure regulation.

Transcriptomic Analysis of Yersinia enterocolitica Biovar 1B Infecting Murine Macrophages Reveals New Mechanisms of Extracellular and Intracellular Survival.

Yersinia enterocolitica is typically considered an extracellular pathogen; however, during the course of an infection, a significant number of bacteria are stably maintained within host cell vacuoles. Little is known about this population and the role it plays during an infection. To address this question and to elucidate the spatially and temporally dynamic gene expression patterns of Y. enterocolitica biovar 1B through the course of an in vitro infection, transcriptome sequencing and differential gene expression analysis of bacteria infecting murine macrophage cells were performed under four distinct conditions. Bacteria were first grown in a nutrient-rich medium at 26 °C to establish a baseline of gene expression that is unrelated to infection. The transcriptomes of these bacteria were then compared to bacteria grown in a conditioned cell culture medium at 37 °C to identify genes that were differentially expressed in response to the increased temperature and medium but not in response to host cells. Infections were then performed, and the transcriptomes of bacteria found on the extracellular surface and intracellular compartments were analyzed individually. The upregulated genes revealed potential roles for a variety of systems in promoting intracellular virulence, including the Ysa type III secretion system, the Yts2 type II secretion system, and the Tad pilus. It was further determined that mutants of each of these systems had decreased virulence while infecting macrophages. Overall, these results reveal the complete set of genes expressed by Y. enterocolitica in response to infection and provide the groundwork for future virulence studies.

Measuring chromatin interaction dynamics on the second time scale at single-copy genes.

The chromatin immunoprecipitation (ChIP) assay is widely used to capture interactions between chromatin and regulatory proteins, but it is unknown how stable most native interactions are. Although live-cell imaging suggests short-lived interactions at tandem gene arrays, current methods cannot measure rapid binding dynamics at single-copy genes. We show, by using a modified ChIP assay with subsecond temporal resolution, that the time dependence of formaldehyde cross-linking can be used to extract in vivo on and off rates for site-specific chromatin interactions varying over a ~100-fold dynamic range. By using the method, we show that a regulatory process can shift weakly bound TATA-binding protein to stable promoter interactions, thereby facilitating transcription complex formation. This assay provides an approach for systematic, quantitative analyses of chromatin binding dynamics in vivo.

Genome-wide analysis of functional sirtuin chromatin targets in yeast.

BACKGROUND: The sirtuins are a conserved family of NAD⁺-dependent histone/protein deacetylases that regulate numerous cellular processes, including heterochromatin formation and transcription. Multiple sirtuins are encoded by each eukaryotic genome, raising the possibility of cooperativity or functional overlap. The scope and variety of chromatin binding sites of the sirtuins in any specific organism remain unclear. RESULTS: Here we utilize the ChIP-seq technique to identify and functionally characterize the genome-wide targets of the sirtuins, Sir2, Hst1 to Hst4, and the DNA binding partner of Hst1, Sum 1, in Saccharomyces cerevisiae. Unexpectedly, Sir2, Hst1 and Sum1, but not the other sirtuins, exhibit co-enrichment at several classes of chromatin targets. These include telomeric repeat clusters, tRNA genes, and surprisingly, the open reading frames (ORFs) of multiple highly expressed RNA polymerase II-transcribed genes that function in processes such as fermentation, glycolysis, and translation. Repression of these target genes during the diauxic shift is specifically dependent on Sir2/Hst1/Sum1 binding to the ORF and sufficiently high intracellular NAD⁺ concentrations. Sir2 recruitment to the ORFs is independent of the canonical SIR complex and surprisingly requires Sum1. The shared Sir2/Hst1/Sum1 targets also significantly overlap with condensin and cohesin binding sites, where Sir2, Hst1, and Sum1 were found to be important for condensin and cohesin deposition, suggesting a possible mechanistic link between metabolism and chromatin architecture during the diauxic shift. CONCLUSIONS: This study demonstrates the existence of overlap in sirtuin function, and advances our understanding of conserved sirtuin-regulated functions, including the regulation of glycolytic gene expression and condensin loading.

RNA synthesis precision is regulated by preinitiation complex turnover.

TATA-binding protein (TBP) nucleates the assembly of the transcription preinitiation complex (PIC), and although TBP can bind promoters with high stability in vitro, recent results establish that virtually the entire TBP population is highly dynamic in yeast nuclei in vivo. This dynamic behavior is surprising in light of models that posit that a stable TBP-containing scaffold facilitates transcription reinitiation at active promoters. The dynamic behavior of TBP is a consequence of the enzymatic activity of the essential Snf2/Swi2 ATPase Mot1, suggesting that ensuring a highly mobile TBP population is critical for transcriptional regulation on a global scale. Here high-resolution tiling arrays were used to define how perturbed TBP dynamics impact the precision of RNA synthesis in Saccharomyces cerevisiae. We find that Mot1 plays a broad role in establishing the precision and efficiency of RNA synthesis: In mot1-42 cells, RNA length changes were observed for 713 genes, about twice the number observed in set2Δ cells, which display a previously reported propensity for spurious initiation within open reading frames. Loss of Mot1 led to both aberrant transcription initiation and termination, with prematurely terminated transcripts representing the largest class of events. Genetic and genomic analyses support the conclusion that these effects on RNA length are mechanistically tied to dynamic TBP occupancies at certain types of promoters. These results suggest a new model whereby dynamic disassembly of the PIC can influence productive RNA synthesis.