Gene regulatory programs in distinct cell types are maintained in large part through the cell-type-specific binding of transcription factors (TFs). The determinants of TF binding include direct DNA ...sequence preferences, DNA sequence preferences of cofactors, and the local cell-dependent chromatin context. To explore the contribution of DNA sequence signal, histone modifications, and DNase accessibility to cell-type-specific binding, we analyzed 286 ChIP-seq experiments performed by the ENCODE Consortium. This analysis included experiments for 67 transcriptional regulators, 15 of which were profiled in both the GM12878 (lymphoblastoid) and K562 (erythroleukemic) human hematopoietic cell lines. To model TF-bound regions, we trained support vector machines (SVMs) that use flexible k-mer patterns to capture DNA sequence signals more accurately than traditional motif approaches. In addition, we trained SVM spatial chromatin signatures to model local histone modifications and DNase accessibility, obtaining significantly more accurate TF occupancy predictions than simpler approaches. Consistent with previous studies, we find that DNase accessibility can explain cell-line-specific binding for many factors. However, we also find that of the 10 factors with prominent cell-type-specific binding patterns, four display distinct cell-type-specific DNA sequence preferences according to our models. Moreover, for two factors we identify cell-specific binding sites that are accessible in both cell types but bound only in one. For these sites, cell-type-specific sequence models, rather than DNase accessibility, are better able to explain differential binding. Our results suggest that using a single motif for each TF and filtering for chromatin accessible loci is not always sufficient to accurately account for cell-type-specific binding profiles.
GS-5734 is a monophosphate prodrug of an adenosine nucleoside analog that showed therapeutic efficacy in a non-human primate model of Ebola virus infection. It has been administered under ...compassionate use to two Ebola patients, both of whom survived, and is currently in Phase 2 clinical development for treatment of Ebola virus disease. Here we report the antiviral activities of GS-5734 and the parent nucleoside analog across multiple virus families, providing evidence to support new indications for this compound against human viruses of significant public health concern.
The transcription factor Foxp3 is indispensible for the differentiation and function of regulatory T cells (T(reg) cells). To gain insights into the molecular mechanisms of Foxp3-mediated gene ...expression, we purified Foxp3 complexes and explored their composition. Biochemical and mass-spectrometric analyses revealed that Foxp3 forms multiprotein complexes of 400-800 kDa or larger and identified 361 associated proteins, ∼30% of which were transcription related. Foxp3 directly regulated expression of a large proportion of the genes encoding its cofactors. Some transcription factor partners of Foxp3 facilitated its expression. Functional analysis of the cooperation of Foxp3 with one such partner, GATA-3, provided additional evidence for a network of transcriptional regulation afforded by Foxp3 and its associates to control distinct aspects of T(reg) cell biology.
Post‐transcriptional regulation by microRNAs and siRNAs depends not only on characteristics of individual binding sites in target mRNA molecules, but also on system‐level properties such as overall ...molecular concentrations. We hypothesize that an intracellular pool of microRNAs/siRNAs faced with a larger number of available predicted target transcripts will downregulate each individual target gene to a lesser extent. To test this hypothesis, we analyzed mRNA expression change from 178 microRNA and siRNA transfection experiments in two cell lines. We find that downregulation of particular genes mediated by microRNAs and siRNAs indeed varies with the total concentration of available target transcripts. We conclude that to interpret and design experiments involving gene regulation by small RNAs, global properties, such as target mRNA abundance, need to be considered in addition to local determinants. We propose that analysis of microRNA/siRNA targeting would benefit from a more quantitative definition, rather than simple categorization of genes as ‘target’ or ‘not a target.’ Our results are important for understanding microRNA regulation and may also have implications for siRNA design and small RNA therapeutics.
Epstein-Barr virus (EBV), which is associated with multiple human tumors, persists as a minichromosome in the nucleus of B lymphocytes and induces malignancies through incompletely understood ...mechanisms. Here, we present a large-scale functional genomic analysis of EBV. Our experimentally generated nucleosome positioning maps and viral protein binding data were integrated with over 700 publicly available high-throughput sequencing data sets for human lymphoblastoid cell lines mapped to the EBV genome. We found that viral lytic genes are coexpressed with cellular cancer-associated pathways, suggesting that the lytic cycle may play an unexpected role in virus-mediated oncogenesis. Host regulators of viral oncogene expression and chromosome structure were identified and validated, revealing a role for the B cell-specific protein Pax5 in viral gene regulation and the cohesin complex in regulating higher order chromatin structure. Our findings provide a deeper understanding of latent viral persistence in oncogenesis and establish a valuable viral genomics resource for future exploration.
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► The EBV transcriptome and epigenome were analyzed by systems approaches ► Viral lytic genes are coexpressed with cellular cancer-associated pathways ► B cell-specific Pax5 protein regulates viral oncoprotein expression ► CTCF-cohesin mediate long-distance DNA interactions important for latency regulation
Immunodeficiency dramatically increases susceptibility to cancer as a result of reduced immune surveillance and enhanced opportunities for virus-mediated oncogenesis. Although AIDS-related lymphomas ...(ARLs) are frequently associated with known oncogenic viruses, many cases contain no known transforming virus. To discover novel transforming viruses, we profiled a set of ARL samples using whole transcriptome sequencing. We determined that Epstein-Barr virus (EBV) was the only virus detected in the tumor samples of this cohort, suggesting that if unidentified pathogens exist in this disease, they are present in <10% of cases or undetectable by our methods. To evaluate the role of EBV in ARL pathogenesis, we analyzed viral gene expression and found highly heterogeneous patterns of viral transcription across samples. We also found significant heterogeneity of viral antigen expression across a large cohort, with many patient samples presenting with restricted type I viral latency, indicating that EBV latency proteins are under increased immunosurveillance in the post-combined antiretroviral therapies era. Furthermore, EBV infection of lymphoma cells in HIV-positive individuals was associated with a distinct host gene expression program. These findings provide insight into the joint host-virus regulatory network of primary ARL tumor samples and expand our understanding of virus-associated oncogenesis. Our findings may also have therapeutic implications, as treatment may be personalized to target specific viral and virus-associated host processes that are only present in a subset of patients.
•We evaluated a cohort of ARL samples for known and novel tumor viruses, revealing the oncogenic herpesvirus EBV as the sole detected infectious agent.•Heterogeneous viral gene expression suggests that variable host immunosurveillance of EBV latency may influence lymphomagenesis.
While the recognition of genomic islands can be a powerful mechanism for identifying genes that distinguish related bacteria, few methods have been developed to identify them specifically. Rather, ...identification of islands often begins with cataloging individual genes likely to have been recently introduced into the genome; regions with many putative alien genes are then examined for other features suggestive of recent acquisition of a large genomic region. When few phylogenetic relatives are available, the identification of alien genes relies on their atypical features relative to the bulk of the genes in the genome. The weakness of these 'bottom-up' approaches lies in the difficulty in identifying robustly those genes which are atypical, or phylogenetically restricted, due to recent foreign ancestry. Herein, we apply an alternative 'top-down' approach where bacterial genomes are recursively divided into progressively smaller regions, each with uniform composition. In this way, large chromosomal regions with atypical features are identified with high confidence due to the simultaneous analysis of multiple genes. This approach is based on a generalized divergence measure to quantify the compositional difference between segments in a hypothesis-testing framework. We tested the proposed genome island prediction algorithm on both artificial chimeric genomes and genuine bacterial genomes.
Regulatory T (Treg) cells, which suppress autoimmunity and other inflammatory states, are characterized by a distinct set of genetic elements controlling their gene expression. However, the extent of ...genetic and associated epigenetic variation in the Treg cell lineage and its possible relation to disease states in humans remain unknown. We explored evolutionary conservation of regulatory elements and natural human inter-individual epigenetic variation in Treg cells to identify the core transcriptional control program of lineage specification. Analysis of single nucleotide polymorphisms in core lineage-specific enhancers revealed disease associations, which were further corroborated by high-resolution genotyping to fine map causal polymorphisms in lineage-specific enhancers. Our findings suggest that a small set of regulatory elements specify the Treg lineage and that genetic variation in Treg cell-specific enhancers may alter Treg cell function contributing to polygenic disease.
► A group of non-membrane spanning tyrosine kinases contain one or more heme-binding CP motifs. ► Protein tyrosine kinases Jak2 and Src are regulated by heme. ► Purified Jak2 and Src can bind to heme ...directly. ► Heme binding to Jak2 and Src can change their conformation.
Protein tyrosine kinases play key roles in many molecular and cellular processes in diverse living organisms. Their proper functioning is crucial for the normal growth, development, and health in humans, whereas their dysfunction can cause serious diseases, including various cancers. As such, intense studies have been performed to understand the molecular mechanisms by which the activities of protein tyrosine kinases are regulated in mammalian cells. Particularly, small molecules that can modulate the activity of tyrosine kinases are of great importance for discovering therapeutic drug candidates for numerous diseases. Notably, heme cannot only serve as a prosthetic group for hemoglobins and enzymes, but it also is a small signaling molecule that can control the activity of diverse signaling and regulatory proteins. Using a computational search, we found that a group of non-membrane spanning tyrosine kinases contains one or more CP motifs that can potentially bind to heme and mediate heme regulation. We then used experimental approaches to determine whether heme can affect the activity of any of these tyrosine kinases. We found that heme indeed affects the phosphorylation of key tyrosine residues in Jak2 and Src, and is therefore able to modulate Jak2 and Src activity. Further experiments showed that Jak2 and Src bind to heme and that the presence of heme alters the sensitivity of Jak2 and Src to trypsin digestion. These results suggest that heme actively interacts with Jak2 and Src and alters their conformation.
Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of ...EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of enhancer-promoter interactions by viral transcription factors.
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