A molecular threshold for effector CD8(+) T cell differentiation controlled by transcription factors Blimp-1 and T-bet.

T cell responses are guided by cytokines that induce transcriptional regulators, which ultimately control differentiation of effector and memory T cells. However, it is unknown how the activities of these molecular regulators are coordinated and integrated during the differentiation process. Using genetic approaches and transcriptional profiling of antigen-specific CD8(+) T cells, we reveal a common program of effector differentiation that is regulated by IL-2 and IL-12 signaling and the combined activities of the transcriptional regulators Blimp-1 and T-bet. The loss of both T-bet and Blimp-1 leads to abrogated cytotoxic function and ectopic IL-17 production in CD8(+) T cells. Overall, our data reveal two major overlapping pathways of effector differentiation governed by the availability of Blimp-1 and T-bet and suggest a model for cytokine-induced transcriptional changes that combine, quantitatively and qualitatively, to promote robust effector CD8(+) T cell differentiation.

MTR3D: identifying regions within protein tertiary structures under purifying selection.

The identification of disease-causal variants is non-trivial. By mapping population variation from over 448,000 exome and genome sequences to over 81,000 experimental structures and homology models of the human proteome, we have calculated both regional intolerance to missense variation (Missense Tolerance Ratio, MTR), using a sliding window of 21-41 codons, and introduce a new 3D spatial intolerance to missense variation score (3D Missense Tolerance Ratio, MTR3D), using spheres of 5-8 Å. We show that the MTR3D is less biased by regions with limited data and more accurately identifies regions under purifying selection than estimates relying on the sequence alone. Intolerant regions were highly enriched for both ClinVar pathogenic and COSMIC somatic missense variants (Mann-Whitney U test P < 2.2 × 10-16). Further, we combine sequence- and spatial-based scores to generate a consensus score, MTRX, which distinguishes pathogenic from benign variants more accurately than either score separately (AUC = 0.85). The MTR3D server enables easy visualisation of population variation, MTR, MTR3D and MTRX scores across the entire gene and protein structure for >17,000 human genes and >42,000 alternative alternate transcripts, including both Ensembl and RefSeq transcripts. MTR3D is freely available by user-friendly web-interface and API at http://biosig.unimelb.edu.au/mtr3d/.

Insights from Spatial Measures of Intolerance to Identifying Pathogenic Variants in Developmental and Epileptic Encephalopathies.

Developmental and epileptic encephalopathies (DEEs) are a group of epilepsies with early onset and severe symptoms that sometimes lead to death. Although previous work successfully discovered several genes implicated in disease outcomes, it remains challenging to identify causative mutations within these genes from the background variation present in all individuals due to disease heterogeneity. Nevertheless, our ability to detect possible pathogenic variants has continued to improve as in silico predictors of deleteriousness have advanced. We investigate their use in prioritising likely pathogenic variants in epileptic encephalopathy patients' whole exome sequences. We showed that the inclusion of structure-based predictors of intolerance improved upon previous attempts to demonstrate enrichment within epilepsy genes.

SATB1 ensures appropriate transcriptional programs within naïve CD8+ T cells.

Special AT-binding protein 1 (SATB1) is a chromatin-binding protein that has been shown to be a key regulator of T-cell development and CD4+ T-cell fate decisions and function. The underlying function for SATB1 in peripheral CD8+ T-cell differentiation processes is largely unknown. To address this, we examined SATB1-binding patterns in naïve and effector CD8+ T cells demonstrating that SATB1 binds to noncoding regulatory elements linked to T-cell lineage-specific gene programs, particularly in naïve CD8+ T cells. We then assessed SATB1 function using N-ethyl-N-nitrosourea-mutant mice that exhibit a point mutation in the SATB1 DNA-binding domain (termed Satb1m1Anu/m1Anu ). Satb1m1Anu/m1Anu mice exhibit diminished SATB1-binding, naïve, Satb1m1Anu/m1Anu CD8+ T cells exhibiting transcriptional and phenotypic characteristics reminiscent of effector T cells. Upon activation, the transcriptional signatures of Satb1m1Anu/m1Anu and wild-type effector CD8+ T cells converged. While there were no overt differences, primary respiratory infection of Satb1m1Anu/m1Anu mice with influenza A virus (IAV) resulted in a decreased proportion and number of IAV-specific CD8+ effector T cells recruited to the infected lung when compared with wild-type mice. Together, these data suggest that SATB1 has a major role in an appropriate transcriptional state within naïve CD8+ T cells and ensures appropriate CD8+ T-cell effector gene expression upon activation.

CD4+ T help promotes influenza virus-specific CD8+ T cell memory by limiting metabolic dysfunction.

There is continued interest in developing novel vaccine strategies that induce establish optimal CD8+ cytotoxic T lymphocyte (CTL) memory for pathogens like the influenza A viruses (IAVs), where the recall of IAV-specific T cell immunity is able to protect against serologically distinct IAV infection. While it is well established that CD4+ T cell help is required for optimal CTL responses and the establishment of memory, when and how CD4+ T cell help contributes to determining the ideal memory phenotype remains unclear. We assessed the quality of IAV-specific CD8+ T cell memory established in the presence or absence of a concurrent CD4+ T cell response. We demonstrate that CD4+ T cell help appears to be required at the initial priming phase of infection for the maintenance of IAV-specific CTL memory, with "unhelped" memory CTL exhibiting intrinsic dysfunction. High-throughput RNA-sequencing established that distinct transcriptional signatures characterize the helped vs. unhelped IAV-specific memory CTL phenotype, with the unhelped set showing a more "exhausted T cell" transcriptional profile. Moreover, we identify that unhelped memory CTLs exhibit defects in a variety of energetic pathways, leading to diminished spare respiratory capacity and diminished capacity to engage glycolysis upon reactivation. Hence, CD4+ T help at the time of initial priming promotes molecular pathways that limit exhaustion by channeling metabolic processes essential for the rapid recall of memory CD8+ T cells.

A saturating mutagenesis CRISPR-Cas9-mediated functional genomic screen identifies cis- and trans-regulatory elements of Oct4 in murine ESCs.

Regulatory elements (REs) consist of enhancers and promoters that occupy a significant portion of the noncoding genome and control gene expression programs either in cis or in trans Putative REs have been identified largely based on their regulatory features (co-occupancy of ESC-specific transcription factors, enhancer histone marks, and DNase hypersensitivity) in mouse embryonic stem cells (mESCs). However, less has been established regarding their regulatory functions in their native context. We deployed cis- and trans-regulatory elements scanning through saturating mutagenesis and sequencing (ctSCAN-SMS) to target elements within the ∼12-kb cis-region (cis-REs; CREs) of the Oct4 gene locus, as well as genome-wide 2,613 high-confidence trans-REs (TREs), in mESCs. ctSCAN-SMS identified 10 CREs and 12 TREs as novel candidate REs of the Oct4 gene in mESCs. Furthermore, deletions of these candidate REs confirmed that the majority of the REs are functionally active, and CREs are more active than TREs in controlling Oct4 gene expression. A subset of active CREs and TREs physically interact with the Oct4 promoter to varying degrees; specifically, a greater number of active CREs, compared with active TREs, physically interact with the Oct4 promoter. Moreover, comparative genomics analysis reveals that a greater number of active CREs than active TREs are evolutionarily conserved between mice and primates, including humans. Taken together, our study demonstrates the reliability and robustness of ctSCAN-SMS screening to identify critical REs and investigate their roles in the regulation of transcriptional output of a target gene (in this case Oct4) in their native context.

Early T-BET Expression Ensures an Appropriate CD8+ Lineage-Specific Transcriptional Landscape after Influenza A Virus Infection.

Virus infection triggers large-scale changes in the phenotype and function of naive CD8+ T cells, resulting in the generation of effector and memory T cells that are then critical for immune clearance. The T-BOX family of transcription factors (TFs) are known to play a key role in T cell differentiation, with mice deficient for the TF T-BET (encoded by Tbx21) unable to generate optimal virus-specific effector responses. Although the importance of T-BET in directing optimal virus-specific T cell responses is accepted, the precise timing and molecular mechanism of action remains unclear. Using a mouse model of influenza A virus infection, we demonstrate that although T-BET is not required for early CD8+ T cell activation and cellular division, it is essential for early acquisition of virus-specific CD8+ T cell function and sustained differentiation and expansion. Whole transcriptome analysis at this early time point showed that Tbx21 deficiency resulted in global dysregulation in early programming events with inappropriate lineage-specific signatures apparent with alterations in the potential TF binding landscape. Assessment of histone posttranslational modifications within the Ifng locus demonstrated that Tbx21 -/- CD8+ T cells were unable to activate "poised" enhancer elements compared with wild-type CD8+ T cells, correlating with diminished Ifng transcription. In all, these data support a model whereby T-BET serves to promote appropriate chromatin remodeling at specific gene loci that underpins appropriate CD8+ T cell lineage-specific commitment and differentiation.

Unique Transcriptional Architecture in Airway Epithelial Cells and Macrophages Shapes Distinct Responses following Influenza Virus Infection Ex Vivo.

Airway epithelial cells and macrophages differ markedly in their responses to influenza A virus (IAV) infection. To investigate transcriptional responses underlying these differences, purified subsets of type II airway epithelial cells (ATII) and alveolar macrophages (AM) recovered from the lungs of mock- or IAV-infected mice at 9 h postinfection were subjected to RNA sequencing. This time point was chosen to allow for characterization of cell types first infected with the virus inoculum, prior to multicycle virus replication and the infiltration of inflammatory cells into the airways. In the absence of infection, AM predominantly expressed genes related to immunity, whereas ATII expressed genes consistent with their physiological roles in the lung. Following IAV infection, AM almost exclusively activated cell-intrinsic antiviral pathways that were dependent on interferon (IFN) regulatory factor 3/7 (IRF3/7) and/or type I IFN signaling. In contrast, IAV-infected ATII activated a broader range of physiological responses, including cell-intrinsic antiviral pathways, which were both independent of and dependent on IRF3/7 and/or type I IFN. These data suggest that transcriptional profiles hardwired during development are a major determinant underlying the different responses of ATII and AM to IAV infection.IMPORTANCE Airway epithelial cells (AEC) and airway macrophages (AM) represent major targets of influenza A virus (IAV) infection in the lung, yet the two cell types respond very differently to IAV infection. We have used RNA sequencing to define the host transcriptional responses in each cell type under steady-state conditions as well as following IAV infection. To do this, different cell subsets isolated from the lungs of mock- and IAV-infected mice were subjected to RNA sequencing. Under steady-state conditions, AM and AEC express distinct transcriptional activities, consistent with distinct physiological roles in the airways. Not surprisingly, these cells also exhibited major differences in transcriptional responses following IAV infection. These studies shed light on how the different transcriptional architectures of airway cells from two different lineages drive transcriptional responses to IAV infection.

Dynamic regulation of permissive histone modifications and GATA3 binding underpin acquisition of granzyme A expression by virus-specific CD8(+) T cells.

Numerous studies have focused on the molecular regulation of perforin (PFP) and granzyme B (GZMB) expression by activated cytotoxic T lymphocytes (CTLs), but little is known about the molecular factors that underpin granzyme A (GZMA) expression. In vitro activation of naïve CD8(+) T cells, in the presence of IL-4, enhanced STAT6-dependent GZMA expression and was associated with GATA3 binding and enrichment of transcriptionally permissive histone posttranslational modifications (PTMs) across the Gzma gene locus. While GZMA expression by effector influenza A virus specific CTLs was also associated with a similar permissive epigenetic signature, memory CTL lacked enrichment of permissive histone PTMs at the Gzma locus, although this was restored within recalled secondary effector CTLs. Importantly, GZMA expression by virus-specific CTLs was associated with GATA3 binding at the Gzma locus, and independent of STAT6-mediated signaling. This suggests regulation of GZMA expression is underpinned by differentiation-dependent regulation of chromatin composition at the Gzma locus and that, given GATA3 is key for CTL differentiation in response to infection, GATA3 expression is regulated by a distinct, IL-4 independent, signaling pathway. Overall, this study provides insights into the molecular mechanisms that control transcription of Gzma during virus-induced CD8(+) T-cell differentiation.

Identification of genes differentially expressed in menstrual breakdown and repair.

STUDY QUESTION: Does the changing molecular profile of the endometrium during menstruation correlate with the histological profile of menstruation. SUMMARY ANSWER: We identified several genes not previously associated with menstruation; on Day 2 of menstruation (early-menstruation), processes related to inflammation are predominantly up-regulated and on Day 4 (late-menstruation), the endometrium is predominantly repairing and regenerating. WHAT IS KNOWN ALREADY: Menstruation is induced by progesterone withdrawal at the end of the menstrual cycle and involves endometrial tissue breakdown, regeneration and repair. Perturbations in the regulation of menstruation may result in menstrual disorders including abnormal uterine bleeding. STUDY DESIGN, SIZE DURATION: Endometrial samples were collected by Pipelle biopsy on Days 2 (n = 9), 3 (n = 9) or 4 (n = 6) of menstruation. PARTICIPANTS/MATERIALS, SETTING, METHODS: RNA was extracted from endometrial biopsies and analysed by genome wide expression Illumina Sentrix Human HT12 arrays. Data were analysed using 'Remove Unwanted Variation-inverse (RUV-inv)'. Ingenuity pathway analysis (IPA) and the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 were used to identify canonical pathways, upstream regulators and functional gene clusters enriched between Days 2, 3 and 4 of menstruation. Selected individual genes were validated by quantitative PCR. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, 1753 genes were differentially expressed in one or more comparisons. Significant canonical pathways, gene clusters and upstream regulators enriched during menstrual bleeding included those associated with immune cell trafficking, inflammation, cell cycle regulation, extracellular remodelling and the complement and coagulation cascade. We provide the first evidence for a role for glutathione-mediated detoxification (glutathione-S-transferase mu 1 and 2; GSTM1 and GSTM2) during menstruation. The largest number of differentially expressed genes was between Days 2 and 4 of menstruation (n = 1176). We identified several genes not previously associated with menstruation including lipopolysaccharide binding protein, serpin peptidase inhibitor, clade B (ovalbumin), member 3 (SERPINB3) and -4 (SERPINB4), interleukin-17C (IL17C), V-set domain containing T-cell activation inhibitor 1 (VTCN1), proliferating cell nuclear antigen factor (KIAA0101/PAF), trefoil factor 3 (TFF3), laminin alpha 2 (LAMA2) and serine peptidase inhibitor, Kazal type 1 (SPINK1). Genes related to inflammatory processes were up-regulated on Day 2 (early-menstruation), and those associated with endometrial repair and regeneration were up-regulated on Day 4 (late-menstruation). LIMITATIONS, REASONS FOR CAUTION: Participants presented with a variety of endometrial pathologies related to bleeding status and other menstrual characteristics. These variations may also have influenced the menstrual process. WIDER IMPLICATIONS OF THE FINDINGS: The temporal molecular profile of menstruation presented in this study identifies a number of genes not previously associated with the menstrual process. Our findings provide valuable insight into the menstrual process and may present novel targets for therapeutic intervention in cases of endometrial dysfunction. LARGE SCALE DATA: All microarray data have been deposited in the public data repository Gene Expression Omnibus (GSE86003). STUDY FUNDING AND COMPETING INTERESTS: Funding for this work was provided by a National Health and Medical Research Council of Australia (NHMRC) Project Grant APP1008553 to M.H., P.R. and J.G. M.H. is supported by an NHMRC Practitioner Fellowship. P.P. is supported by a NHMRC Early Career Fellowship. The authors have no conflict of interest to declare.

Id2-mediated inhibition of E2A represses memory CD8+ T cell differentiation.

The transcription factor inhibitor of DNA binding (Id)2 modulates T cell fate decisions, but the molecular mechanism underpinning this regulation is unclear. In this study we show that loss of Id2 cripples effector differentiation and instead programs CD8(+) T cells to adopt a memory fate with increased Eomesodermin and Tcf7 expression. We demonstrate that Id2 restrains CD8(+) T cell memory differentiation by inhibiting E2A-mediated direct activation of Tcf7 and that Id2 expression level mirrors T cell memory recall capacity. As a result of the defective effector differentiation, Id2-deficient CD8(+) T cells fail to induce sufficient Tbx21 expression to generate short-lived effector CD8(+) T cells. Our findings reveal that the Id2/E2A axis orchestrates T cell differentiation through the induction or repression of downstream transcription factors essential for effector and memory T cell differentiation.

Epigenetic and transcriptional regulation of CCL17 production by glucocorticoids in arthritis.

Glucocorticoids (GCs) are potent anti-inflammatory agents and are broadly used in treating rheumatoid arthritis (RA) patients, albeit with adverse side effects associated with long-term usage. The negative consequences of GC therapy provide an impetus for research into gaining insights into the molecular mechanisms of GC action. We have previously reported that granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced CCL17 has a non-redundant role in inflammatory arthritis. Here, we provide molecular evidence that GCs can suppress GM-CSF-mediated upregulation of IRF4 and CCL17 expression via downregulating JMJD3 expression and activity. In mouse models of inflammatory arthritis, GC treatment inhibited CCL17 expression and ameliorated arthritic pain-like behavior and disease. Significantly, GC treatment of RA patient peripheral blood mononuclear cells ex vivo resulted in decreased CCL17 production. This delineated pathway potentially provides new therapeutic options for the treatment of many inflammatory conditions, where GCs are used as an anti-inflammatory drug but without the associated adverse side effects.

Targeting BMI-1 to deplete antibody-secreting cells in autoimmunity.

Objectives: B cells drive the production of autoreactive antibody-secreting cells (ASCs) in autoimmune diseases such as Systemic Lupus Erythematosus (SLE) and Sjögren's syndrome, causing long-term organ damage. Current treatments for antibody-mediated autoimmune diseases target B cells or broadly suppress the immune system. However, pre-existing long-lived ASCs are often refractory to treatment, leaving a reservoir of autoreactive cells that continue to produce antibodies. Therefore, the development of novel treatment methods targeting ASCs is vital to improve patient outcomes. Our objective was to test whether targeting the epigenetic regulator BMI-1 could deplete ASCs in autoimmune conditions in vivo and in vitro. Methods: Use of a BMI-1 inhibitor in both mouse and human autoimmune settings was investigated. Lyn -/- mice, a model of SLE, were treated with the BMI-1 small molecule inhibitor PTC-028, before assessment of ASCs, serum antibody and immune complexes. To examine human ASC survival, a novel human fibroblast-based assay was established, and the impact of PTC-028 on ASCs derived from Sjögren's syndrome patients was evaluated. Results: BMI-1 inhibition significantly decreased splenic and bone marrow ASCs in Lyn -/- mice. The decline in ASCs was linked to aberrant cell cycle gene expression and led to a significant decrease in serum IgG3, immune complexes and anti-DNA IgG. PTC-028 was also efficacious in reducing ex vivo plasma cell survival from both Sjögren's syndrome patients and age-matched healthy donors. Conclusion: These data provide evidence that inhibiting BMI-1 can deplete ASC in a variety of contexts and thus BMI-1 is a viable therapeutic target for antibody-mediated autoimmune diseases.

Active maintenance of CD8+ T cell naivety through regulation of global genome architecture.

The differentiation of naive CD8+ T lymphocytes into cytotoxic effector and memory CTL results in large-scale changes in transcriptional and phenotypic profiles. Little is known about how large-scale changes in genome organization underpin these transcriptional programs. We use Hi-C to map changes in the spatial organization of long-range genome contacts within naive, effector, and memory virus-specific CD8+ T cells. We observe that the architecture of the naive CD8+ T cell genome is distinct from effector and memory genome configurations, with extensive changes within discrete functional chromatin domains associated with effector/memory differentiation. Deletion of BACH2, or to a lesser extent, reducing SATB1 DNA binding, within naive CD8+ T cells results in a chromatin architecture more reminiscent of effector/memory states. This suggests that key transcription factors within naive CD8+ T cells act to restrain T cell differentiation by actively enforcing a unique naive chromatin state.

Active maintenance of CD8+ T cell naïvety through regulation of global genome architecture.

The differentiation of naïve CD8+ cytotoxic T lymphocytes (CTLs) into effector and memory states results in large scale changes in transcriptional and phenotypic profiles. Little is known about how large-scale changes in genome organisation reflect or underpin these transcriptional programs. We utilised Hi-C to map changes in the spatial organisation of long-range genome contacts within naïve, effector and memory virus-specific CD8+ T cells. We observed that the architecture of the naive CD8+ T cell genome was distinct from effector and memory genome configurations with extensive changes within discrete functional chromatin domains. However, deletion of the BACH2 or SATB1 transcription factors was sufficient to remodel the naïve chromatin architecture and engage transcriptional programs characteristic of differentiated cells. This suggests that the chromatin architecture within naïve CD8+ T cells is preconfigured to undergo autonomous remodelling upon activation, with key transcription factors restraining differentiation by actively enforcing the unique naïve chromatin state.

Chromatin alternates between A and B compartments at kilobase scale for subgenic organization.

Nuclear compartments are prominent features of 3D chromatin organization, but sequencing depth limitations have impeded investigation at ultra fine-scale. CTCF loops are generally studied at a finer scale, but the impact of looping on proximal interactions remains enigmatic. Here, we critically examine nuclear compartments and CTCF loop-proximal interactions using a combination of in situ Hi-C at unparalleled depth, algorithm development, and biophysical modeling. Producing a large Hi-C map with 33 billion contacts in conjunction with an algorithm for performing principal component analysis on sparse, super massive matrices (POSSUMM), we resolve compartments to 500 bp. Our results demonstrate that essentially all active promoters and distal enhancers localize in the A compartment, even when flanking sequences do not. Furthermore, we find that the TSS and TTS of paused genes are often segregated into separate compartments. We then identify diffuse interactions that radiate from CTCF loop anchors, which correlate with strong enhancer-promoter interactions and proximal transcription. We also find that these diffuse interactions depend on CTCF's RNA binding domains. In this work, we demonstrate features of fine-scale chromatin organization consistent with a revised model in which compartments are more precise than commonly thought while CTCF loops are more protracted.

The ENCODE Uniform Analysis Pipelines.

The Encyclopedia of DNA elements (ENCODE) project is a collaborative effort to create a comprehensive catalog of functional elements in the human genome. The current database comprises more than 19000 functional genomics experiments across more than 1000 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the Homo sapiens and Mus musculus genomes. All experimental data, metadata, and associated computational analyses created by the ENCODE consortium are submitted to the Data Coordination Center (DCC) for validation, tracking, storage, and distribution to community resources and the scientific community. The ENCODE project has engineered and distributed uniform processing pipelines in order to promote data provenance and reproducibility as well as allow interoperability between genomic resources and other consortia. All data files, reference genome versions, software versions, and parameters used by the pipelines are captured and available via the ENCODE Portal. The pipeline code, developed using Docker and Workflow Description Language (WDL; https://openwdl.org/) is publicly available in GitHub, with images available on Dockerhub (https://hub.docker.com), enabling access to a diverse range of biomedical researchers. ENCODE pipelines maintained and used by the DCC can be installed to run on personal computers, local HPC clusters, or in cloud computing environments via Cromwell. Access to the pipelines and data via the cloud allows small labs the ability to use the data or software without access to institutional compute clusters. Standardization of the computational methodologies for analysis and quality control leads to comparable results from different ENCODE collections - a prerequisite for successful integrative analyses.

The ENCODE Uniform Analysis Pipelines.

The Encyclopedia of DNA elements (ENCODE) project is a collaborative effort to create a comprehensive catalog of functional elements in the human genome. The current database comprises more than 19000 functional genomics experiments across more than 1000 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the Homo sapiens and Mus musculus genomes. All experimental data, metadata, and associated computational analyses created by the ENCODE consortium are submitted to the Data Coordination Center (DCC) for validation, tracking, storage, and distribution to community resources and the scientific community. The ENCODE project has engineered and distributed uniform processing pipelines in order to promote data provenance and reproducibility as well as allow interoperability between genomic resources and other consortia. All data files, reference genome versions, software versions, and parameters used by the pipelines are captured and available via the ENCODE Portal. The pipeline code, developed using Docker and Workflow Description Language (WDL; https://openwdl.org/) is publicly available in GitHub, with images available on Dockerhub (https://hub.docker.com), enabling access to a diverse range of biomedical researchers. ENCODE pipelines maintained and used by the DCC can be installed to run on personal computers, local HPC clusters, or in cloud computing environments via Cromwell. Access to the pipelines and data via the cloud allows small labs the ability to use the data or software without access to institutional compute clusters. Standardization of the computational methodologies for analysis and quality control leads to comparable results from different ENCODE collections - a prerequisite for successful integrative analyses.

PyMS: a Python toolkit for processing of gas chromatography-mass spectrometry (GC-MS) data. Application and comparative study of selected tools.

BACKGROUND: Gas chromatography-mass spectrometry (GC-MS) is a technique frequently used in targeted and non-targeted measurements of metabolites. Most existing software tools for processing of raw instrument GC-MS data tightly integrate data processing methods with graphical user interface facilitating interactive data processing. While interactive processing remains critically important in GC-MS applications, high-throughput studies increasingly dictate the need for command line tools, suitable for scripting of high-throughput, customized processing pipelines. RESULTS: PyMS comprises a library of functions for processing of instrument GC-MS data developed in Python. PyMS currently provides a complete set of GC-MS processing functions, including reading of standard data formats (ANDI- MS/NetCDF and JCAMP-DX), noise smoothing, baseline correction, peak detection, peak deconvolution, peak integration, and peak alignment by dynamic programming. A novel common ion single quantitation algorithm allows automated, accurate quantitation of GC-MS electron impact (EI) fragmentation spectra when a large number of experiments are being analyzed. PyMS implements parallel processing for by-row and by-column data processing tasks based on Message Passing Interface (MPI), allowing processing to scale on multiple CPUs in distributed computing environments. A set of specifically designed experiments was performed in-house and used to comparatively evaluate the performance of PyMS and three widely used software packages for GC-MS data processing (AMDIS, AnalyzerPro, and XCMS). CONCLUSIONS: PyMS is a novel software package for the processing of raw GC-MS data, particularly suitable for scripting of customized processing pipelines and for data processing in batch mode. PyMS provides limited graphical capabilities and can be used both for routine data processing and interactive/exploratory data analysis. In real-life GC-MS data processing scenarios PyMS performs as well or better than leading software packages. We demonstrate data processing scenarios simple to implement in PyMS, yet difficult to achieve with many conventional GC-MS data processing software. Automated sample processing and quantitation with PyMS can provide substantial time savings compared to more traditional interactive software systems that tightly integrate data processing with the graphical user interface.

Detection of Chimeric Cellular: HIV mRNAs Generated Through Aberrant Splicing in HIV-1 Latently Infected Resting CD4+ T Cells.

Latent HIV-1 provirus in infected individuals on suppressive therapy does not always remain transcriptionally silent. Both HIV-1 LTR and human gene promoter derived transcriptional events can contribute HIV-1 sequences to the mRNA produced in the cell. In addition, chimeric cellular:HIV mRNA can arise through readthrough transcription and aberrant splicing. Using target enrichment coupled to the Illumina Mi-Seq and PacBio RS II platforms, we show that 3' LTR activation is frequent in latently infected cells from both the CCL19-induced primary cell model of HIV-1 latency as well as ex vivo samples. In both systems of latent HIV-1 infection, we detected several chimeric species that were generated via activation of a cryptic splice donor site in the 5' LTR of HIV-1. Aberrant splicing involving the major HIV-1 splice donor sites, SD1 and SD4 disrupts post-transcriptional processing of the gene in which HIV-1 is integrated. In the primary cell model of HIV-1 latency, Tat-encoding sequences are incorporated into the chimeric mRNA transcripts through the use of SD4. Our study unravels clues to the characteristics of HIV-1 integrants that promote formation of chimeric cellular:HIV mRNA and improves the understanding of the HIV-1 RNA footprint in latently infected cells.