Genome Sequencing in Microfabricated High-density Picolitre Reactors

Nature. Sep, 2005  |  Pubmed ID: 16056220

The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.

Genome Sequencing and Analysis Reveals Possible Determinants of Staphylococcus Aureus Nasal Carriage

BMC Genomics. 2008  |  Pubmed ID: 18808706

Nasal carriage of Staphylococcus aureus is a major risk factor in clinical and community settings due to the range of etiologies caused by the organism. We have identified unique immunological and ultrastructural properties associated with nasal carriage isolates denoting a role for bacterial factors in nasal carriage. However, despite extensive molecular level characterizations by several groups suggesting factors necessary for colonization on nasal epithelium, genetic determinants of nasal carriage are unknown. Herein, we have set a genomic foundation for unraveling the bacterial determinants of nasal carriage in S. aureus.

Four Histone Variants Mark the Boundaries of Polycistronic Transcription Units in Trypanosoma Brucei

Genes & Development. May, 2009  |  Pubmed ID: 19369410

Unusually for a eukaryote, genes transcribed by RNA polymerase II (pol II) in Trypanosoma brucei are arranged in polycistronic transcription units. With one exception, no pol II promoter motifs have been identified, and how transcription is initiated remains an enigma. T. brucei has four histone variants: H2AZ, H2BV, H3V, and H4V. Using chromatin immunoprecipitation (ChIP) and sequencing (ChIP-seq) to examine the genome-wide distribution of chromatin components, we show that histones H4K10ac, H2AZ, H2BV, and the bromodomain factor BDF3 are enriched up to 300-fold at probable pol II transcription start sites (TSSs). We also show that nucleosomes containing H2AZ and H2BV are less stable than canonical nucleosomes. Our analysis also identifies >60 unexpected TSS candidates and reveals the presence of long guanine runs at probable TSSs. Apparently unique to trypanosomes, additional histone variants H3V and H4V are enriched at probable pol II transcription termination sites. Our findings suggest that histone modifications and histone variants play crucial roles in transcription initiation and termination in trypanosomes and that destabilization of nucleosomes by histone variants is an evolutionarily ancient and general mechanism of transcription initiation, demonstrated in an organism in which general pol II transcription factors have been elusive.

Distinct Factors Control Histone Variant H3.3 Localization at Specific Genomic Regions

Cell. Mar, 2010  |  Pubmed ID: 20211137

The incorporation of histone H3 variants has been implicated in the epigenetic memory of cellular state. Using genome editing with zinc-finger nucleases to tag endogenous H3.3, we report genome-wide profiles of H3 variants in mammalian embryonic stem cells and neuronal precursor cells. Genome-wide patterns of H3.3 are dependent on amino acid sequence and change with cellular differentiation at developmentally regulated loci. The H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for localization of H3.3 at telomeres and many transcription factor binding sites. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Atrx is required for Hira-independent localization of H3.3 at telomeres and for the repression of telomeric RNA. Our data demonstrate that multiple and distinct factors are responsible for H3.3 localization at specific genomic locations in mammalian cells.

Transcriptome-wide Identification of RNA-binding Protein and MicroRNA Target Sites by PAR-CLIP

Cell. Apr, 2010  |  Pubmed ID: 20371350

RNA transcripts are subject to posttranscriptional gene regulation involving hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) expressed in a cell-type dependent fashion. We developed a cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs. The crosslinked sites are revealed by thymidine to cytidine transitions in the cDNAs prepared from immunopurified RNPs of 4-thiouridine-treated cells. We determined the binding sites and regulatory consequences for several intensely studied RBPs and miRNPs, including PUM2, QKI, IGF2BP1-3, AGO/EIF2C1-4 and TNRC6A-C. Our study revealed that these factors bind thousands of sites containing defined sequence motifs and have distinct preferences for exonic versus intronic or coding versus untranslated transcript regions. The precise mapping of binding sites across the transcriptome will be critical to the interpretation of the rapidly emerging data on genetic variation between individuals and how these variations contribute to complex genetic diseases.

Genome-wide Analysis of MRNA Abundance in Two Life-cycle Stages of Trypanosoma Brucei and Identification of Splicing and Polyadenylation Sites

Nucleic Acids Research. Aug, 2010  |  Pubmed ID: 20385579

Transcription of protein-coding genes in trypanosomes is polycistronic and gene expression is primarily regulated by post-transcriptional mechanisms. Sequence motifs in the untranslated regions regulate mRNA trans-splicing and RNA stability, yet where UTRs begin and end is known for very few genes. We used high-throughput RNA-sequencing to determine the genome-wide steady-state mRNA levels ('transcriptomes') for approximately 90% of the genome in two stages of the Trypanosoma brucei life cycle cultured in vitro. Almost 6% of genes were differentially expressed between the two life-cycle stages. We identified 5' splice-acceptor sites (SAS) and polyadenylation sites (PAS) for 6959 and 5948 genes, respectively. Most genes have between one and three alternative SAS, but PAS are more dispersed. For 488 genes, SAS were identified downstream of the originally assigned initiator ATG, so a subsequent in-frame ATG presumably designates the start of the true coding sequence. In some cases, alternative SAS would give rise to mRNAs encoding proteins with different N-terminal sequences. We could identify the introns in two genes known to contain them, but found no additional genes with introns. Our study demonstrates the usefulness of the RNA-seq technology to study the transcriptional landscape of an organism whose genome has not been fully annotated.

Whole-exome Sequencing-based Discovery of STIM1 Deficiency in a Child with Fatal Classic Kaposi Sarcoma

The Journal of Experimental Medicine. Oct, 2010  |  Pubmed ID: 20876309

Classic Kaposi sarcoma (KS) is exceedingly rare in children from the Mediterranean Basin, despite the high prevalence of human herpesvirus-8 (HHV-8) infection in this region. We hypothesized that rare single-gene inborn errors of immunity to HHV-8 may underlie classic KS in childhood. We investigated a child with no other unusually severe infectious or tumoral phenotype who died from disseminated KS at two years of age. Whole-exome sequencing in the patient revealed a homozygous splice-site mutation in STIM1, the gene encoding stromal interaction molecule 1, which regulates store-operated Ca(2+) entry. STIM1 mRNA splicing, protein production, and Ca(2+) influx were completely abolished in EBV-transformed B cell lines from the patient, but were rescued by the expression of wild-type STIM1. Based on the previous discovery of STIM1 deficiency in a single family with a severe T cell immunodeficiency and the much higher risk of KS in individuals with acquired T cell deficiencies, we conclude that STIM1 T cell deficiency precipitated the development of lethal KS in this child upon infection with HHV-8. Our report provides the first evidence that isolated classic KS in childhood may result from single-gene defects and provides proof-of-principle that whole-exome sequencing in single patients can decipher the genetic basis of rare inborn errors.

ETV1 is a Lineage Survival Factor That Cooperates with KIT in Gastrointestinal Stromal Tumours

Nature. Oct, 2010  |  Pubmed ID: 20927104

Gastrointestinal stromal tumour (GIST) is the most common human sarcoma and is primarily defined by activating mutations in the KIT or PDGFRA receptor tyrosine kinases. KIT is highly expressed in interstitial cells of Cajal (ICCs)-the presumed cell of origin for GIST-as well as in haematopoietic stem cells, melanocytes, mast cells and germ cells. Yet, families harbouring germline activating KIT mutations and mice with knock-in Kit mutations almost exclusively develop ICC hyperplasia and GIST, suggesting that the cellular context is important for KIT to mediate oncogenesis. Here we show that the ETS family member ETV1 is highly expressed in the subtypes of ICCs sensitive to oncogenic KIT mediated transformation, and is required for their development. In addition, ETV1 is universally highly expressed in GISTs and is required for growth of imatinib-sensitive and resistant GIST cell lines. Transcriptome profiling and global analyses of ETV1-binding sites suggest that ETV1 is a master regulator of an ICC-GIST-specific transcription network mainly through enhancer binding. The ETV1 transcriptional program is further regulated by activated KIT, which prolongs ETV1 protein stability and cooperates with ETV1 to promote tumorigenesis. We propose that GIST arises from ICCs with high levels of endogenous ETV1 expression that, when coupled with an activating KIT mutation, drives an oncogenic ETS transcriptional program. This differs from other ETS-dependent tumours such as prostate cancer, melanoma and Ewing sarcoma where genomic translocation or amplification drives aberrant ETS expression. It also represents a novel mechanism of oncogenic transcription factor activation.

Suppression of Inflammation by a Synthetic Histone Mimic

Nature. Dec, 2010  |  Pubmed ID: 21068722

Interaction of pathogens with cells of the immune system results in activation of inflammatory gene expression. This response, although vital for immune defence, is frequently deleterious to the host due to the exaggerated production of inflammatory proteins. The scope of inflammatory responses reflects the activation state of signalling proteins upstream of inflammatory genes as well as signal-induced assembly of nuclear chromatin complexes that support mRNA expression. Recognition of post-translationally modified histones by nuclear proteins that initiate mRNA transcription and support mRNA elongation is a critical step in the regulation of gene expression. Here we present a novel pharmacological approach that targets inflammatory gene expression by interfering with the recognition of acetylated histones by the bromodomain and extra terminal domain (BET) family of proteins. We describe a synthetic compound (I-BET) that by 'mimicking' acetylated histones disrupts chromatin complexes responsible for the expression of key inflammatory genes in activated macrophages, and confers protection against lipopolysaccharide-induced endotoxic shock and bacteria-induced sepsis. Our findings suggest that synthetic compounds specifically targeting proteins that recognize post-translationally modified histones can serve as a new generation of immunomodulatory drugs.

Transcriptome-wide Sequencing Reveals Numerous APOBEC1 MRNA-editing Targets in Transcript 3' UTRs

Nature Structural & Molecular Biology. Feb, 2011  |  Pubmed ID: 21258325

Apolipoprotein B-editing enzyme, catalytic polypeptide-1 (APOBEC1) is a cytidine deaminase initially identified by its activity in converting a specific cytidine (C) to uridine (U) in apolipoprotein B (apoB) mRNA transcripts in the small intestine. Editing results in the translation of a truncated apoB isoform with distinct functions in lipid transport. To address the possibility that APOBEC1 edits additional mRNAs, we developed a transcriptome-wide comparative RNA sequencing (RNA-Seq) screen. We identified and validated 32 previously undescribed mRNA targets of APOBEC1 editing, all of which are located in AU-rich segments of transcript 3' untranslated regions (3' UTRs). Further analysis established several characteristic sequence features of editing targets, which were predictive for the identification of additional APOBEC1 substrates. The transcriptomics approach to RNA editing presented here dramatically expands the list of APOBEC1 mRNA editing targets and reveals a novel cellular mechanism for the modification of transcript 3' UTRs.

Identifying MRNA Editing Deaminase Targets by RNA-Seq

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21370044

RNA editing deaminases act on a variety of targets in different organisms. A number of such enzymes have been shown to act on mRNA, with the resultant nucleotide changes modifying a transcript's information content. Though the deaminase activity of mRNA editing enzymes is readily demonstrated in vitro, identifying their physiological targets has proved challenging. Recent advances in ultra high-throughput sequencing technologies have allowed for whole transcriptome sequencing and expression profiling (RNA-Seq). We have developed a system to identify novel mRNA editing deamination targets based on comparative analysis of RNA-Seq data. The efficacy and utility of this approach is demonstrated for APOBEC1, a cytidine deaminase with a known and well-characterized mRNA editing target in the mammalian small intestine.

Recognition of a Mononucleosomal Histone Modification Pattern by BPTF Via Multivalent Interactions

Cell. May, 2011  |  Pubmed ID: 21596426

Little is known about how combinations of histone marks are interpreted at the level of nucleosomes. The second PHD finger of human BPTF is known to specifically recognize histone H3 when methylated on lysine 4 (H3K4me2/3). Here, we examine how additional heterotypic modifications influence BPTF binding. Using peptide surrogates, three acetyllysine ligands are indentified for a PHD-adjacent bromodomain in BPTF via systematic screening and biophysical characterization. Although the bromodomain displays limited discrimination among the three possible acetyllysines at the peptide level, marked selectivity is observed for only one of these sites, H4K16ac, in combination with H3K4me3 at the mononucleosome level. In support, these two histone marks constitute a unique trans-histone modification pattern that unambiguously resides within a single nucleosomal unit in human cells, and this module colocalizes with these marks in the genome. Together, our data call attention to nucleosomal patterning of covalent marks in dictating critical chromatin associations.

Transcriptional Profiling of Psoriasis Using RNA-seq Reveals Previously Unidentified Differentially Expressed Genes

The Journal of Investigative Dermatology. Jan, 2012  |  Pubmed ID: 21850022

Environmental DNA-Encoded Antibiotics Fasamycins A and B Inhibit FabF in Type II Fatty Acid Biosynthesis

Journal of the American Chemical Society. Feb, 2012  |  Pubmed ID: 22224500

In a recent study of polyketide biosynthetic gene clusters cloned directly from soil, we isolated two antibiotics, fasamycins A and B, which showed activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. To identify the target of the fasamycins, mutants with elevated fasamycin A minimum inhibitory concentrations were selected from a wild-type culture of E. faecalis OG1RF. Next-generation sequencing of these mutants, in conjunction with in vitro biochemical assays, showed that the fasamycins inhibit FabF of type II fatty acid biosynthesis (FASII). Candidate gene overexpression studies also showed that fasamycin resistance is conferred by fabF overexpression. On the basis of comparisons with known FASII inhibitors and in silico docking studies, the chloro-gem-dimethyl-anthracenone substructure seen in the fasamycins is predicted to represent a naturally occurring FabF-specific antibiotic pharmacophore. Optimization of this pharmacophore should yield FabF-specific antibiotics with increased potencies and differing spectra of activity. This study demonstrates that culture-independent antibiotic discovery methods have the potential to provide access to novel metabolites with modes of action that differ from those of antibiotics currently in clinical use.