Genomic insights into host and parasite interactions during intracellular infection by Toxoplasma gondii.

To gain insights into the molecular interactions of an intracellular pathogen and its host cell, we studied the gene expression and chromatin states of human fibroblasts infected with the Apicomplexan parasite Toxoplasma gondii. We show a striking activation of host cell genes that regulate a number of cellular processes, some of which are protective of the host cell, others likely to be advantageous to the pathogen. The simultaneous capture of host and parasite genomic information allowed us to gain insights into the regulation of the T. gondii genome. We show how chromatin accessibility and transcriptional profiling together permit novel annotation of the parasite's genome, including more accurate mapping of known genes and the identification of new genes and cis-regulatory elements. Motif analysis reveals not only the known T. gondii AP2 transcription factor-binding site but also a previously-undiscovered candidate TATA box-containing motif at one-quarter of promoters. By inferring the transcription factor and upstream cell signaling responses involved in the host cell, we can use genomic information to gain insights into T. gondii's perturbation of host cell physiology. Our resulting model builds on previously-described human host cell signalling responses to T. gondii infection, linked to induction of specific transcription factors, some of which appear to be solely protective of the host cell, others of which appear to be co-opted by the pathogen to enhance its own survival.

Identifying synergistic high-order 3D chromatin conformations from genome-scale nanopore concatemer sequencing.

High-order three-dimensional (3D) interactions between more than two genomic loci are common in human chromatin, but their role in gene regulation is unclear. Previous high-order 3D chromatin assays either measure distant interactions across the genome or proximal interactions at selected targets. To address this gap, we developed Pore-C, which combines chromatin conformation capture with nanopore sequencing of concatemers to profile proximal high-order chromatin contacts at the genome scale. We also developed the statistical method Chromunity to identify sets of genomic loci with frequencies of high-order contacts significantly higher than background ('synergies'). Applying these methods to human cell lines, we found that synergies were enriched in enhancers and promoters in active chromatin and in highly transcribed and lineage-defining genes. In prostate cancer cells, these included binding sites of androgen-driven transcription factors and the promoters of androgen-regulated genes. Concatemers of high-order contacts in highly expressed genes were demethylated relative to pairwise contacts at the same loci. Synergies in breast cancer cells were associated with tyfonas, a class of complex DNA amplicons. These results rigorously link genome-wide high-order 3D interactions to lineage-defining transcriptional programs and establish Pore-C and Chromunity as scalable approaches to assess high-order genome structure.

SMITE: an R/Bioconductor package that identifies network modules by integrating genomic and epigenomic information.

BACKGROUND: The molecular assays that test gene expression, transcriptional, and epigenetic regulation are increasingly diverse and numerous. The information generated by each type of assay individually gives an insight into the state of the cells tested. What should be possible is to add the information derived from separate, complementary assays to gain higher-confidence insights into cellular states. At present, the analysis of multi-dimensional, massive genome-wide data requires an initial pruning step to create manageable subsets of observations that are then used for integration, which decreases the sizes of the intersecting data sets and the potential for biological insights. Our Significance-based Modules Integrating the Transcriptome and Epigenome (SMITE) approach was developed to integrate transcriptional and epigenetic regulatory data without a loss of resolution. RESULTS: SMITE combines p-values by accounting for the correlation between non-independent values within data sets, allowing genes and gene modules in an interaction network to be assigned significance values. The contribution of each type of genomic data can be weighted, permitting integration of individually under-powered data sets, increasing the overall ability to detect effects within modules of genes. We apply SMITE to a complex genomic data set including the epigenomic and transcriptomic effects of Toxoplasma gondii infection on human host cells and demonstrate that SMITE is able to identify novel subnetworks of dysregulated genes. Additionally, we show that SMITE outperforms Functional Epigenetic Modules (FEM), the current paradigm of using the spin-glass algorithm to integrate gene expression and epigenetic data. CONCLUSIONS: SMITE represents a flexible, scalable tool that allows integration of transcriptional and epigenetic regulatory data from genome-wide assays to boost confidence in finding gene modules reflecting altered cellular states.

Genome-wide assays that identify and quantify modified cytosines in human disease studies.

The number of different assays that has been published to study DNA methylation is extensive, complemented by recently described assays that test modifications of cytosine other than the most abundant 5-methylcytosine (5mC) variant. In this review, we describe the considerations involved in choosing how to study 5mC throughout the genome, with an emphasis on the common application of testing for epigenetic dysregulation in human disease. While microarray studies of 5mC continue to be commonly used, these lack the additional qualitative information from sequencing-based approaches that is increasingly recognized to be valuable. When we test the representation of functional elements in the human genome by several current assay types, we find that no survey approach interrogates anything more than a small minority of the nonpromoter cis-regulatory sites where DNA methylation variability is now appreciated to influence gene expression and to be associated with human disease. However, whole-genome bisulphite sequencing (WGBS) adds a substantial representation of loci at which DNA methylation changes are unlikely to be occurring with transcriptional consequences. Our assessment is that the most effective approach to DNA methylation studies in human diseases is to use targeted bisulphite sequencing of the cis-regulatory loci in a cell type of interest, using a capture-based or comparable system, and that no single design of a survey approach will be suitable for all cell types.

Akt inhibitors MK-2206 and nelfinavir overcome mTOR inhibitor resistance in diffuse large B-cell lymphoma.

PURPOSE: The mTOR pathway is constitutively activated in diffuse large B-cell lymphoma (DLBCL). mTOR inhibitors have activity in DLBCL, although response rates remain low. We evaluated DLBCL cell lines with differential resistance to the mTOR inhibitor rapamycin: (i) to identify gene expression profile(s) (GEP) associated with resistance to rapamycin, (ii) to understand mechanisms of rapamycin resistance, and (iii) to identify compounds likely to synergize with mTOR inhibitor. EXPERIMENTAL DESIGN: We sought to identify a GEP of mTOR inhibitor resistance by stratification of eight DLBCL cell lines with respect to response to rapamycin. Then, using pathway analysis and connectivity mapping, we sought targets likely accounting for this resistance and compounds likely to overcome it. We then evaluated two compounds thus identified for their potential to synergize with rapamycin in DLBCL and confirmed mechanisms of activity with standard immunoassays. RESULTS: We identified a GEP capable of reliably distinguishing rapamycin-resistant from rapamycin-sensitive DLBCL cell lines. Pathway analysis identified Akt as central to the differentially expressed gene network. Connectivity mapping identified compounds targeting Akt as having a high likelihood of reversing the GEP associated with mTOR inhibitor resistance. Nelfinavir and MK-2206, chosen for their Akt-inhibitory properties, yielded synergistic inhibition of cell viability in combination with rapamycin in DLBCL cell lines, and potently inhibited phosphorylation of Akt and downstream targets of activated mTOR. CONCLUSIONS: GEP identifies DLBCL subsets resistant to mTOR inhibitor therapy. Combined targeting of mTOR and Akt suppresses activation of key components of the Akt/mTOR pathway and results in synergistic cytotoxicity. These findings are readily adaptable to clinical trials.

HDAC inhibitors and decitabine are highly synergistic and associated with unique gene-expression and epigenetic profiles in models of DLBCL.

Interactions between histone deacetylase inhibitors (HDACIs) and decitabine were investigated in models of diffuse large B-cell lymphoma (DLBCL). A number of cell lines representing both germinal center B-like and activated B-cell like DLBCL, patient-derived tumor cells and a murine xenograft model were used to study the effects of HDACIs and decitabine in this system. All explored HDACIs in combination with decitabine produced a synergistic effect in growth inhibition and induction of apoptosis in DLBCL cells. This effect was time dependent, mediated via caspase-3 activation, and resulted in increased levels of acetylated histones. Synergy in inducing apoptosis was confirmed in patient-derived primary tumor cells treated with panobinostat and decitabine. Xenografting experiments confirmed the in vitro activity and tolerability of the combination. We analyzed the molecular basis for this synergistic effect by evaluating gene-expression and methylation patterns using microarrays, with validation by bisulfite sequencing. These analyses revealed differentially expressed genes and networks identified by each of the single treatment conditions and by the combination therapy to be unique with few overlapping genes. Among the genes uniquely altered by the combination of panobinostat and decitabine were VHL, TCEB1, WT1, and DIRAS3.

Endoscopic fluorescence mapping of the left atrium: a novel experimental approach for high resolution endocardial mapping in the intact heart.

BACKGROUND: Despite the availability of several mapping technologies for investigating the electrophysiologic mechanisms of atrial fibrillation (AF), an experimental tool enabling high-resolution mapping of electrical impulses on the endocardial surface of the intact left atrium is lacking. OBJECTIVE: The purpose of this report is to present a new optical mapping approach implementing a steerable cardio-endoscope in isolated hearts. METHODS: The system consists of a direct or side-view endoscope coupled to a 532-nm excitation laser for illumination and a CCD camera for imaging of potentiometric dye fluorescence (di-4-ANEPPS, 80 x 80 pixels, 200-800 frames/s). The cardio-endoscope was aimed successively at diverse posterior left atrial locations to obtain high-resolution movies of electrical wave propagation and detailed endocardial anatomic features in the presence and absence of atrial stretch. RESULTS: We present several examples of high-resolution endoscopic posterior left atrial recordings of wave propagation patterns during both sinus rhythm and AF with signal-to-noise ratio similar to conventional optical mapping systems. We demonstrate the endoscope's ability to visualize highly organized AF sources (rotors) at specific locations on the posterior left atrium and posterior left atrium-pulmonary vein junctions. We present video images of waves emanating from such sources as they propagate into pectinate muscles in the left atrial appendage. In particular, we demonstrate this approach is ideally suited for studying the effects of atrial stretch on AF dynamics. CONCLUSION: In isolated hearts, cardio-endoscopic optical mapping of electrical activity should enable comprehensive evaluation of AF activity in the posterior left atrium, the role of local anatomy on AF dynamics, and the efficacy of pharmacologic and ablative interventions.