ALBERT

Description: When messenger RNA splicing occurs co-transcriptionally, the potential for kinetic control based on transcription dynamics is widely recognized. Indeed, perturbation studies have reported that when transcription kinetics are perturbed genetically or pharmacologically splice patterns may change. However, whether kinetic control is contributing to the control of splicing within the normal range of physiological conditions remains unknown. We examined if the kinetic determinants for co-transcriptional splicing (CTS) might be reflected in the structure and expression patterns of the genome and epigenome. To identify and then quantitatively relate multiple, simultaneous CTS determinants, we constructed a scalable mathematical model of the kinetic interplay of RNA synthesis and CTS and parameterized it with diverse next generation sequencing (NGS) data. We thus found a variety of CTS determinants encoded in vertebrate genomes and epigenomes, and that these combine variously for different groups of genes such as housekeeping versus regulated genes. Together, our findings indicate that the kinetic basis of splicing is functionally and physiologically relevant, and may meaningfully inform the analysis of genomic and epigenomic data to provide insights that are missed when relying on statistical approaches alone.

Description: Background: Macrophage activation by lipopolysaccharide and adenosine triphosphate (ATP) has been studied extensively because this model system mimics the physiological context of bacterial infection and subsequent inflammatory responses. Previous studies on macrophages elucidated the biological roles of caspase-1 in post-translational activation of interleukin-1β and interleukin-18 in inflammation and apoptosis. However, the results from these studies focused only on a small number of factors. To better understand the host response, we have performed a high-throughput study of Kdo2-lipid A (KLA)-primed macrophages stimulated with ATP. Results: The study suggests that treating mouse bone marrow-derived macrophages with KLA and ATP produces ‘synergistic’ effects that are not seen with treatment of KLA or ATP alone. The synergistic regulation of genes related to immunity, apoptosis and lipid metabolism is observed in a time-dependent manner. The synergistic effects are produced by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and activator protein (AP)-1 through regulation of their target cytokines. The synergistically regulated cytokines then activate signal transducer and activator of transcription (STAT) factors that result in enhanced immunity, apoptosis and lipid metabolism; STAT1 enhances immunity by promoting anti-microbial factors; and STAT3 contributes to downregulation of cell cycle and upregulation of apoptosis. STAT1 and STAT3 also regulate glycerolipid and eicosanoid metabolism, respectively. Further, western blot analysis for STAT1 and STAT3 showed that the changes in transcriptomic levels were consistent with their proteomic levels. In summary, this study shows the synergistic interaction between the toll-like receptor and purinergic receptor signaling during macrophage activation on bacterial infection. Availability: Time-course data of transcriptomics and lipidomics can be queried or downloaded from http://www.lipidmaps.org . Contact: shankar@ucsd.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Vascular endothelial growth factor A (VEGF-A) is a master regulator of angiogenesis, vascular development and function. In this study we investigated the transcriptional regulation of VEGF-A-responsive genes in primary human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) using genome-wide global run-on sequencing (GRO-Seq). We demonstrate that half of VEGF-A-regulated gene promoters are characterized by a transcriptionally competent paused RNA polymerase II (Pol II). We show that transition into productive elongation is a major mechanism of gene activation of virtually all VEGF-regulated genes, whereas only ~40% of the genes are induced at the level of initiation. In addition, we report a comprehensive chromatin interaction map generated in HUVECs using tethered conformation capture (TCC) and characterize chromatin interactions in relation to transcriptional activity. We demonstrate that sites of active transcription are more likely to engage in chromatin looping and cell type-specific transcriptional activity reflects the boundaries of chromatin interactions. Furthermore, we identify large chromatin compartments with a tendency to be coordinately transcribed upon VEGF-A stimulation. We provide evidence that these compartments are enriched for clusters of regulatory regions such as super-enhancers and for disease-associated single nucleotide polymorphisms (SNPs). Collectively, these findings provide new insights into mechanisms behind VEGF-A-regulated transcriptional programs in endothelial cells.

Description: Global run-on sequencing (GRO-seq) is a recent addition to the series of high-throughput sequencing methods that enables new insights into transcriptional dynamics within a cell. However, GRO-sequencing presents new algorithmic challenges, as existing analysis platforms for ChIP-seq and RNA-seq do not address the unique problem of identifying transcriptional units de novo from short reads located all across the genome. Here, we present a novel algorithm for de novo transcript identification from GRO-sequencing data, along with a system that determines transcript regions, stores them in a relational database and associates them with known reference annotations. We use this method to analyze GRO-sequencing data from primary mouse macrophages and derive novel quantitative insights into the extent and characteristics of non-coding transcription in mammalian cells. In doing so, we demonstrate that Vespucci expands existing annotations for mRNAs and lincRNAs by defining the primary transcript beyond the polyadenylation site. In addition, Vespucci generates assemblies for un-annotated non-coding RNAs such as those transcribed from enhancer-like elements. Vespucci thereby provides a robust system for defining, storing and analyzing diverse classes of primary RNA transcripts that are of increasing biological interest.