John C. Obenauer

Scansite 2.0: proteome-wide prediction of cell signaling interactions using short sequence motifs

Scansite identifies short protein sequence motifs that are recognized by modular signaling domains, phosphorylated by protein Ser/Thr- or Tyr-kinases or mediate specific interactions with protein or phospholipid ligands. Each sequence motif is represented as a position-specific scoring matrix (PSSM) based on results from oriented peptide library and phage display experiments. Predicted domain-motif interactions from Scansite can be sequentially combined, allowing segments of biological pathways to be constructed in silico. The current release of Scansite, version 2.0, includes 62 motifs characterizing the binding and/or substrate specificities of many families of Ser/Thr- or Tyr-kinases, SH2, SH3, PDZ, 14-3-3 and PTB domains, together with signature motifs for PtdIns(3,4,5)P(3)-specific PH domains. Scansite 2.0 contains significant improvements to its original interface, including a number of new generalized user features and significantly enhanced performance. Searches of all SWISS-PROT, TrEMBL, Genpept and Ensembl protein database entries are now possible with run times reduced by approximately 60% when compared with Scansite version 1.0. Scansite 2.0 allows restricted searching of species-specific proteins, as well as isoelectric point and molecular weight sorting to facilitate comparison of predictions with results from two-dimensional gel electrophoresis experiments. Support for user-defined motifs has been increased, allowing easier input of user-defined matrices and permitting user-defined motifs to be combined with pre-compiled Scansite motifs for dual motif searching. In addition, a new series of Sequence Match programs for non-quantitative user-defined motifs has been implemented. Scansite is available via the World Wide Web at http://scansite.mit.edu.

The genetic basis of early T-cell precursor acute lymphoblastic leukaemia.

Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL.

Large-scale sequence analysis of avian influenza isolates.

The spread of H5N1 avian influenza viruses (AIVs) from China to Europe has raised global concern about their potential to infect humans and cause a pandemic. In spite of their substantial threat to human health, remarkably little AIV whole-genome information is available. We report here a preliminary analysis of the first large-scale sequencing of AIVs, including 2196 AIV genes and 169 complete genomes. We combine this new information with public AIV data to identify new gene alleles, persistent genotypes, compensatory mutations, and a potential virulence determinant.

Persistent Host Markers in Pandemic and H5N1 Influenza Viruses

ABSTRACT Avian influenza viruses have adapted to human hosts, causing pandemics in humans. The key host-specific amino acid mutations required for an avian influenza virus to function in humans are unknown. Through multiple-sequence alignment and statistical testing of each aligned amino acid, we identified markers that discriminate human influenza viruses from avian influenza viruses. We applied strict thresholds to select only markers which are highly preserved in human influenza virus isolates over time. We found that a subset of these persistent host markers exist in all human pandemic influenza virus sequences from 1918, 1957, and 1968, while others are acquired as the virus becomes a seasonal influenza virus. We also show that human H5N1 influenza viruses are significantly more likely to contain the amino acid predominant in human strains for a few persistent host markers than avian H5N1 influenza viruses. This sporadic enrichment of amino acids present in human-hosted viruses may indicate that some H5N1 viruses have made modest adaptations to their new hosts in the recent past. The markers reported here should be useful in monitoring potential pandemic influenza viruses.

Globin Lentiviral Vector Insertions Can Perturb the Expression of Endogenous Genes in β-thalassemic Hematopoietic Cells

Although hematopoietic cell gene therapy using retroviral vectors has recently achieved success in clinical trials, safety issues regarding vector insertional mutagenesis have emerged. Vector insertion, resulting in transcriptional activation of proto-oncogenes, played a role in the development of lymphoid leukemia in an X-linked severe combined immunodeficiency trial, and caused myeloid clonal dominance in a trial for chronic granulomatous disease. These events have raised the question of whether gene therapy for other disorders such as beta-thalassemia and sickle cell disease may hold a similar risk. In this study, we prospectively evaluated whether gamma-globin lentiviral vectors containing enhancer elements from the beta-globin locus control region could alter the expression of genes near the vector insertion. We studied this question in primary, clonal murine beta-thalassemic erythroid cells, where globin regulatory elements are highly active. We found an overall incidence of perturbed expression in 28% of the transduced clones, with 11% of all genes contained within a 600-kilobase region surrounding the vector-insertion site demonstrating altered expression. This rate was higher than that observed for a lentiviral vector containing a viral long-terminal repeat (LTR). This is the first direct evidence that lentiviral vectors can cause insertional dysregulation of cellular genes at a frequent rate.Although hematopoietic cell gene therapy using retroviral vectors has recently achieved success in clinical trials, safety issues regarding vector insertional mutagenesis have emerged. Vector insertion, resulting in transcriptional activation of proto-oncogenes, played a role in the development of lymphoid leukemia in an X-linked severe combined immunodeficiency trial, and caused myeloid clonal dominance in a trial for chronic granulomatous disease. These events have raised the question of whether gene therapy for other disorders such as β-thalassemia and sickle cell disease may hold a similar risk. In this study, we prospectively evaluated whether γ-globin lentiviral vectors containing enhancer elements from the β-globin locus control region could alter the expression of genes near the vector insertion. We studied this question in primary, clonal murine β-thalassemic erythroid cells, where globin regulatory elements are highly active. We found an overall incidence of perturbed expression in 28% of the transduced clones, with 11% of all genes contained within a 600-kilobase region surrounding the vector-insertion site demonstrating altered expression. This rate was higher than that observed for a lentiviral vector containing a viral long-terminal repeat (LTR). This is the first direct evidence that lentiviral vectors can cause insertional dysregulation of cellular genes at a frequent rate.

Large-scale analysis of thermostable, mammalian proteins provides insights into the intrinsically disordered proteome

Intrinsically disordered proteins are predicted to be highly abundant and play broad biological roles in eukaryotic cells. In particular, by virtue of their structural malleability and propensity to interact with multiple binding partners, disordered proteins are thought to be specialized for roles in signaling and regulation. However, these concepts are based on in silico analyses of translated whole genome sequences, not on large-scale analyses of proteins expressed in living cells. Therefore, whether these concepts broadly apply to expressed proteins is currently unknown. Previous studies have shown that heat-treatment of cell extracts lead to partial enrichment of soluble, disordered proteins. On the basis of this observation, we sought to address the current dearth of knowledge about expressed, disordered proteins by performing a large-scale proteomics study of thermostable proteins isolated from mouse fibroblast cells. With the use of novel multidimensional chromatography methods and mass spectrometry, we identified a total of 1320 thermostable proteins from these cells. Further, we used a variety of bioinformatics methods to analyze the structural and biological properties of these proteins. Interestingly, more than 900 of these expressed proteins were predicted to be substantially disordered. These were divided into two categories, with 514 predicted to be predominantly disordered and 395 predicted to exhibit both disordered and ordered/folded features. In addition, 411 of the thermostable proteins were predicted to be folded. Despite the use of heat treatment (60 min at 98 degrees C) to partially enrich for disordered proteins, which might have been expected to select for small proteins, the sequences of these proteins exhibited a wide range of lengths (622 +/- 555 residues (average length +/- standard deviation) for disordered proteins and 569 +/- 598 residues for folded proteins). Computational structural analyses revealed several unexpected features of the thermostable proteins: (1) disordered domains and coiled-coil domains occurred together in a large number of disordered proteins, suggesting functional interplay between these domains; and (2) more than 170 proteins contained lengthy domains (>300 residues) known to be folded. Reference to Gene Ontology Consortium functional annotations revealed that, while disordered proteins play diverse biological roles in mouse fibroblasts, they do exhibit heightened involvement in several functional categories, including, cytoskeletal structure and cell movement, metabolic and biosynthetic processes, organelle structure, cell division, gene transcription, and ribonucleoprotein complexes. We believe that these results reflect the general properties of the mouse intrinsically disordered proteome (IDP-ome) although they also reflect the specialized physiology of fibroblast cells. Large-scale identification of expressed, thermostable proteins from other cell types in the future, grown under varied physiological conditions, will dramatically expand our understanding of the structural and biological properties of disordered eukaryotic proteins.

T Cell Receptor CDR3 Sequence but Not Recognition Characteristics Distinguish Autoreactive Effector and Foxp3+ Regulatory T Cells

The source, specificity, and plasticity of the forkhead box transcription factor 3 (Foxp3)(+) regulatory T (Treg) and conventional T (Tconv) cell populations active at sites of autoimmune pathology are not well characterized. To evaluate this, we combined global repertoire analyses and functional assessments of isolated T cell receptors (TCR) from TCRalpha retrogenic mice with autoimmune encephalomyelitis. Treg and Tconv cell TCR repertoires were distinct, and autoantigen-specific Treg and Tconv cells were enriched in diseased tissue. Autoantigen sensitivity and fine specificity of these cells intersected, implying that differences in responsiveness were not responsible for lineage specification. Notably, autoreactive Treg and Tconv cells could be fully distinguished by an acidic versus aliphatic variation at a single TCR CDR3 residue. Our results imply that ontogenically distinct Treg and Tconv cell repertoires with convergent specificities for autoantigen respond during autoimmunity and argue against more than limited plasticity between Treg and Tconv cells during autoimmune inflammation.

Genome-wide and single-cell analyses reveal a context dependent relationship between CBP recruitment and gene expression

Genome-wide distribution of histone H3K18 and H3K27 acetyltransferases, CBP (CREBBP) and p300 (EP300), is used to map enhancers and promoters, but whether these elements functionally require CBP/p300 remains largely uncertain. Here we compared global CBP recruitment with gene expression in wild-type and CBP/p300 double-knockout (dKO) fibroblasts. ChIP-seq using CBP-null cells as a control revealed nearby CBP recruitment for 20% of constitutively-expressed genes, but surprisingly, three-quarters of these genes were unaffected or slightly activated in dKO cells. Computationally defined enhancer-promoter-units (EPUs) having a CBP peak near the enhancer-like element were more predictive, with CBP/p300 deletion attenuating expression of 40% of such constitutively-expressed genes. Examining signal-responsive (Hypoxia Inducible Factor) genes showed that 97% were within 50 kilobases of an inducible CBP peak, and 70% of these required CBP/p300 for full induction. Unexpectedly, most inducible CBP peaks occurred near signal-nonresponsive genes. Finally, single-cell expression analysis revealed additional context dependence where some signal-responsive genes were not uniformly dependent on CBP/p300 in individual cells. While CBP/p300 was needed for full induction of some genes in single-cells, for other genes CBP/p300 increased the probability of maximal expression. Thus, target gene context influences the transcriptional requirement for CBP/p300, possibly by multiple mechanisms.

Fecal influenza in mammals: selection of novel variants

ABSTRACT In aquatic birds, influenza A viruses mainly replicate in the intestinal tract without significantly affecting the health of the host, but in mammals, they replicate in the respiratory tract and often cause disease. Occasionally, influenza viruses have been detected in stool samples of hospitalized patients and in rectal swabs of naturally or experimentally infected mammals. In this study, we compared the biological and molecular differences among four wild-type avian H1N1 influenza viruses and their corresponding fecal and lung isolates in DBA/2J and BALB/cJ mice. All fecal and lung isolates were more pathogenic than the original wild-type viruses, when inoculated into mice of both strains. The increased virulence was associated with the acquisition of genetic mutations. Most of the novel genotypes emerged as PB2E627K, HAF128V, HAF454L, or HAH300P variations, and double mutations frequently occurred in the same isolate. However, influenza virus strain- and host-specific differences were also observed in terms of selected variants. The avian H1N1 virus of shorebird origin appeared to be unique in its ability to rapidly adapt to BALB/cJ mice via the fecal route, compared to the adaptability of the H1N1 virus of mallard origin. Furthermore, a bimodal distribution in fecal shedding was observed in mice infected with the fecal isolates, while a normal distribution was observed after infection with the lung isolates or wild-type virus. Fecal isolates contained HA mutations that increased the activation pH of the HA protein. We conclude that influenza virus variants that emerge in fecal isolates in mammals might influence viral transmission, adaptation to mammals, and viral ecology or evolution.

Survival analysis of infected mice reveals pathogenic variations in the genome of avian H1N1 viruses

Most influenza pandemics have been caused by H1N1 viruses of purely or partially avian origin. Here, using Cox proportional hazard model, we attempt to identify the genetic variations in the whole genome of wild-type North American avian H1N1 influenza A viruses that are associated with their virulence in mice by residue variations, host origins of virus (Anseriformes-ducks or Charadriiformes-shorebirds), and host-residue interactions. In addition, through structural modeling, we predicted that several polymorphic sites associated with pathogenicity were located in structurally important sites, especially in the polymerase complex and NS genes. Our study introduces a new approach to identify pathogenic variations in wild-type viruses circulating in the natural reservoirs and ultimately to understand their infectious risks to humans as part of risk assessment efforts towards the emergence of future pandemic strains.