Tyson A. Clark

HITS-CLIP yields genome-wide insights into brain alternative RNA processing

Protein–RNA interactions have critical roles in all aspects of gene expression. However, applying biochemical methods to understand such interactions in living tissues has been challenging. Here we develop a genome-wide means of mapping protein–RNA binding sites in vivo, by high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). HITS-CLIP analysis of the neuron-specific splicing factor Nova revealed extremely reproducible RNA-binding maps in multiple mouse brains. These maps provide genome-wide in vivo biochemical footprints confirming the previous prediction that the position of Nova binding determines the outcome of alternative splicing; moreover, they are sufficiently powerful to predict Nova action de novo. HITS-CLIP revealed a large number of Nova–RNA interactions in 3′ untranslated regions, leading to the discovery that Nova regulates alternative polyadenylation in the brain. HITS-CLIP, therefore, provides a robust, unbiased means to identify functional protein–RNA interactions in vivo.

Direct detection of DNA methylation during single-molecule, real-time sequencing.

We describe the direct detection of DNA methylation, without bisulfite conversion, through single-molecule, real-time (SMRT) sequencing. In SMRT sequencing, DNA polymerases catalyze the incorporation of fluorescently labeled nucleotides into complementary nucleic acid strands. The arrival times and durations of the resulting fluorescence pulses yield information about polymerase kinetics and allow direct detection of modified nucleotides in the DNA template, including N6-methyladenine, 5-methylcytosine and 5-hydroxymethylcytosine. Measurement of polymerase kinetics is an intrinsic part of SMRT sequencing and does not adversely affect determination of primary DNA sequence. The various modifications affect polymerase kinetics differently, allowing discrimination between them. We used these kinetic signatures to identify adenine methylation in genomic samples and found that, in combination with circular consensus sequencing, they can enable single-molecule identification of epigenetic modifications with base-pair resolution. This method is amenable to long read lengths and will likely enable mapping of methylation patterns in even highly repetitive genomic regions.

Nova regulates brain-specific splicing to shape the synapse

Alternative RNA splicing greatly increases proteome diversity and may thereby contribute to tissue-specific functions. We carried out genome-wide quantitative analysis of alternative splicing using a custom Affymetrix microarray to assess the role of the neuronal splicing factor Nova in the brain. We used a stringent algorithm to identify 591 exons that were differentially spliced in the brain relative to immune tissues, and 6.6% of these showed major splicing defects in the neocortex of Nova2−/− mice. We tested 49 exons with the largest predicted Nova-dependent splicing changes and validated all 49 by RT-PCR. We analyzed the encoded proteins and found that all those with defined brain functions acted in the synapse (34 of 40, including neurotransmitter receptors, cation channels, adhesion and scaffold proteins) or in axon guidance (8 of 40). Moreover, of the 35 proteins with known interaction partners, 74% (26) interact with each other. Validating a large set of Nova RNA targets has led us to identify a multi-tiered network in which Nova regulates the exon content of RNAs encoding proteins that interact in the synapse.

Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array

BackgroundAlternative splicing is a mechanism for increasing protein diversity by excluding or including exons during post-transcriptional processing. Alternatively spliced proteins are particularly relevant in oncology since they may contribute to the etiology of cancer, provide selective drug targets, or serve as a marker set for cancer diagnosis. While conventional identification of splice variants generally targets individual genes, we present here a new exon-centric array (GeneChip Human Exon 1.0 ST) that allows genome-wide identification of differential splice variation, and concurrently provides a flexible and inclusive analysis of gene expression.ResultsWe analyzed 20 paired tumor-normal colon cancer samples using a microarray designed to detect over one million putative exons that can be virtually assembled into potential gene-level transcripts according to various levels of prior supporting evidence. Analysis of high confidence (empirically supported) transcripts identified 160 differentially expressed genes, with 42 genes occupying a network impacting cell proliferation and another twenty nine genes with unknown functions. A more speculative analysis, including transcripts based solely on computational prediction, produced another 160 differentially expressed genes, three-fourths of which have no previous annotation. We also present a comparison of gene signal estimations from the Exon 1.0 ST and the U133 Plus 2.0 arrays.Novel splicing events were predicted by experimental algorithms that compare the relative contribution of each exon to the cognate transcript intensity in each tissue. The resulting candidate splice variants were validated with RT-PCR. We found nine genes that were differentially spliced between colon tumors and normal colon tissues, several of which have not been previously implicated in cancer. Top scoring candidates from our analysis were also found to substantially overlap with EST-based bioinformatic predictions of alternative splicing in cancer.ConclusionDifferential expression of high confidence transcripts correlated extremely well with known cancer genes and pathways, suggesting that the more speculative transcripts, largely based solely on computational prediction and mostly with no previous annotation, might be novel targets in colon cancer. Five of the identified splicing events affect mediators of cytoskeletal organization (ACTN1, VCL, CALD1, CTTN, TPM1), two affect extracellular matrix proteins (FN1, COL6A3) and another participates in integrin signaling (SLC3A2). Altogether they form a pattern of colon-cancer specific alterations that may particularly impact cell motility.

Discovery of tissue-specific exons using comprehensive human exon microarrays

BackgroundHigher eukaryotes express a diverse population of messenger RNAs generated by alternative splicing. Large-scale methods for monitoring gene expression must adapt in order to accurately detect the transcript variation generated by this splicing.ResultsWe have designed a high-density oligonucleotide microarray with probesets for more than one million annotated and predicted exons in the human genome. Using these arrays and a simple algorithm that normalizes exon signal to signal from the gene as a whole, we have identified tissue-specific exons from a panel of 16 different normal adult tissues. RT-PCR validation confirms approximately 86% of the predicted tissue-enriched probesets. Pair-wise comparisons between the tissues suggest that as many as 73% of detected genes are differentially alternatively spliced. We also demonstrate how an inclusive exon microarray can be used to discover novel alternative splicing events. As examples, 17 new tissue-specific exons from 11 genes were validated by RT-PCR and sequencing.ConclusionIn conjunction with a conceptually simple algorithm, comprehensive exon microarrays can detect tissue-specific alternative splicing events. Our data suggest significant expression outside of known exons and well annotated genes and a high frequency of alternative splicing events. In addition, we identified and validated a number of novel exons with tissue-specific splicing patterns. The tissue map data will likely serve as a valuable source of information on the regulation of alternative splicing.

Aberrant alternative splicing and extracellular matrix gene expression in mouse models of myotonic dystrophy

The common form of myotonic dystrophy (DM1) is associated with the expression of expanded CTG DNA repeats as RNA (CUGexp RNA). To test whether CUGexp RNA creates a global splicing defect, we compared the skeletal muscle of two mouse models of DM1, one expressing a CTGexp transgene and another homozygous for a defective muscleblind 1 (Mbnl1) gene. Strong correlation in splicing changes for ∼100 new Mbnl1-regulated exons indicates that loss of Mbnl1 explains >80% of the splicing pathology due to CUGexp RNA. In contrast, only about half of mRNA-level changes can be attributed to loss of Mbnl1, indicating that CUGexp RNA has Mbnl1-independent effects, particularly on mRNAs for extracellular matrix proteins. We propose that CUGexp RNA causes two separate effects: loss of Mbnl1 function (disrupting splicing) and loss of another function that disrupts extracellular matrix mRNA regulation, possibly mediated by Mbnl2. These findings reveal unanticipated similarities between DM1 and other muscular dystrophies.

Evaluation of Genetic Variation Contributing to Differences in Gene Expression between Populations

Gene expression is a complex quantitative trait partially regulated by genetic variation in DNA sequence. Population differences in gene expression could contribute to some of the observed differences in susceptibility to common diseases and response to drug treatments. We characterized gene expression in the full set of HapMap lymphoblastoid cell lines derived from individuals of European and African ancestry for 9156 transcript clusters (gene-level) evaluated with the Affymetrix GeneChip Human Exon 1.0 ST Array. Gene expression was found to differ significantly between these samples for 383 transcript clusters. Biological processes including ribosome biogenesis and antimicrobial humoral response were found to be enriched in these differential genes, suggesting their possible roles in contributing to the population differences at a higher level than that of mRNA expression and in response to environmental information. Genome-wide association studies for local or distant genetic variants that correlate with the differentially expressed genes enabled identification of significant associations with one or more single-nucleotide polymorphisms (SNPs), consistent with the hypothesis that genetic factors and not simply population identity or other characteristics (age of cell lines, length of culture, etc.) contribute to differences in gene expression in these samples. Our results provide a comprehensive view of the genes differentially expressed between populations and the enriched biological processes involved in these genes. We also provide an evaluation of the contributions of genetic variation and nongenetic factors to the population differences in gene expression.

Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing

Single-molecule real-time (SMRT) DNA sequencing allows the systematic detection of chemical modifications such as methylation but has not previously been applied on a genome-wide scale. We used this approach to detect 49,311 putative 6-methyladenine (m6A) residues and 1,407 putative 5-methylcytosine (m5C) residues in the genome of a pathogenic Escherichia coli strain. We obtained strand-specific information for methylation sites and a quantitative assessment of the frequency of methylation at each modified position. We deduced the sequence motifs recognized by the methyltransferase enzymes present in this strain without prior knowledge of their specificity. Furthermore, we found that deletion of a phage-encoded methyltransferase-endonuclease (restriction-modification; RM) system induced global transcriptional changes and led to gene amplification, suggesting that the role of RM systems extends beyond protecting host genomes from foreign DNA.

Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine.

We describe strand-specific, base-resolution detection of 5-hydroxymethylcytosine (5-hmC) in genomic DNA with single-molecule sensitivity, combining a bioorthogonal, selective chemical labeling method of 5-hmC with single-molecule, real-time (SMRT) DNA sequencing. The chemical labeling not only allows affinity enrichment of 5-hmC–containing DNA fragments but also enhances the kinetic signal of 5-hmC during SMRT sequencing. We applied the approach to sequence 5-hmC in a genomic DNA sample with high confidence.

Single-molecule sequencing to track plasmid diversity of hospital-associated carbapenemase-producing Enterobacteriaceae

Single-molecule sequencing of bacteria at the NIH Clinical Center documents diverse plasmids encoding antibiotic resistance and their transfer between microbes. How Antibiotic Resistance Spreads Among Bacteria Antibiotic-resistant microbes are spreading at an alarming rate in health care facilities throughout the world. Conlan et al. use a new DNA sequencing method to take a close look at one way in which antibiotic resistance spreads. With single-molecule sequencing, the authors completely characterized individual plasmids, the circular bits of DNA that carry the genes for antibiotic resistance in bacteria. They focused on resistance to the carbapenems, a class of antibiotics that is often used for infections that do not respond to more conventional antimicrobial agents. By using this approach in their microbial surveillance program at the NIH Clinical Center, the authors found evidence that plasmids carrying carbapenemase genes moved from one microbial species to another within the hospital environment. They also used the technique to test hypotheses about patient-to-patient transmission and to characterize a previously undescribed carbapenemase-encoding plasmid carried by diverse bacterial species that could cause dangerous clinical infections. Public health officials have raised concerns that plasmid transfer between Enterobacteriaceae species may spread resistance to carbapenems, an antibiotic class of last resort, thereby rendering common health care–associated infections nearly impossible to treat. To determine the diversity of carbapenemase-encoding plasmids and assess their mobility among bacterial species, we performed comprehensive surveillance and genomic sequencing of carbapenem-resistant Enterobacteriaceae in the National Institutes of Health (NIH) Clinical Center patient population and hospital environment. We isolated a repertoire of carbapenemase-encoding Enterobacteriaceae, including multiple strains of Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, Enterobacter cloacae, Citrobacter freundii, and Pantoea species. Long-read genome sequencing with full end-to-end assembly revealed that these organisms carry the carbapenem resistance genes on a wide array of plasmids. K. pneumoniae and E. cloacae isolated simultaneously from a single patient harbored two different carbapenemase-encoding plasmids, indicating that plasmid transfer between organisms was unlikely within this patient. We did, however, find evidence of horizontal transfer of carbapenemase-encoding plasmids between K. pneumoniae, E. cloacae, and C. freundii in the hospital environment. Our data, including full plasmid identification, challenge assumptions about horizontal gene transfer events within patients and identify possible connections between patients and the hospital environment. In addition, we identified a new carbapenemase-encoding plasmid of potentially high clinical impact carried by K. pneumoniae, E. coli, E. cloacae, and Pantoea species, in unrelated patients and in the hospital environment.