Martin Steffen

Measuring absolute expression with microarrays with a calibrated reference sample and an extended signal intensity range.

Gene expression ratios derived from spotted-glass microarray experiments have become invaluable to researchers by providing sensitive and comprehensive indicators of the molecular underpinnings of cell behaviors and states. However, several drawbacks to this form of data have been noted, including the inability to relate ratios to absolute expression levels or to compare experimental conditions not measured with the same control. In this study we demonstrate a method for overcoming these obstacles. First, instead of cohybridizing labeled experimental and control samples, we hybridize each sample against labeled oligos complementary to every microarray feature. Ratios between sample intensities and intensities of the oligo reference measure sample RNA levels on a scale that relates to their absolute abundance, instead of to the variable and unknown abundances of a cDNA reference. We demonstrate that results from this type of hybridization are accurate and retain absolute abundance information far better than conventional microarray ratios. Next, to ensure the accurate measurement of sample and oligo reference intensities, which may differ by several orders of magnitude, we use a linear regression algorithm, implemented in a freely available perl script, to combine the linear ranges of multiple scans taken at different scanner sensitivity settings onto an extended linear scale. We discuss future applications of our method to measure RNA expression on the absolute scale of number of transcripts per cell from any organism for which oligo-based spotted-glass microarrays are available.

Automated modelling of signal transduction networks

BackgroundIntracellular signal transduction is achieved by networks of proteins and small molecules that transmit information from the cell surface to the nucleus, where they ultimately effect transcriptional changes. Understanding the mechanisms cells use to accomplish this important process requires a detailed molecular description of the networks involved.ResultsWe have developed a computational approach for generating static models of signal transduction networks which utilizes protein-interaction maps generated from large-scale two-hybrid screens and expression profiles from DNA microarrays. Networks are determined entirely by integrating protein-protein interaction data with microarray expression data, without prior knowledge of any pathway intermediates. In effect, this is equivalent to extracting subnetworks of the protein interaction dataset whose members have the most correlated expression profiles.ConclusionWe show that our technique accurately reconstructs MAP Kinase signaling networks in Saccharomyces cerevisiae. This approach should enhance our ability to model signaling networks and to discover new components of known networks. More generally, it provides a method for synthesizing molecular data, either individual transcript abundance measurements or pairwise protein interactions, into higher level structures, such as pathways and networks.

The COMBREX Project: Design, Methodology, and Initial Results

Experimental data exists for only a vanishingly small fraction of sequenced microbial genes. This community page discusses the progress made by the COMBREX project to address this important issue using both computational and experimental resources.

Flap Endonuclease 1 Contributes to Telomere Stability

Telomere stability plays an important role in the preservation of genomic stability and is maintained through the coordinated actions of telomere-specific proteins and DNA repair and replication proteins [1, 2]. Flap endonuclease 1 (FEN1) is a protein that plays a role in lagging-strand DNA replication, base excision repair, homologous recombination, and reinitiation of stalled replication forks [3, 4]. Here, we demonstrate that FEN1 depletion leads to telomere dysfunction characterized by the presence of gammaH2AX and sister telomere loss. Expression of catalytically active telomerase, the reverse transcriptase that adds telomeric repeats to chromosome ends, was sufficient to rescue telomere dysfunction upon FEN1 depletion. Strikingly, FEN1 depletion exclusively abrogates telomeres replicated by lagging-strand DNA replication. Genetic rescue experiments utilizing FEN1 mutant proteins that retained the ability to localize to telomeric repeats revealed that FEN1s nuclease activity and ability to interact with the Werner protein (WRN) and telomere-binding protein (TRF2) were required for FEN1 activity at the telomere. Given FEN1s role in lagging-strand DNA replication and reinitiation of stalled replication forks, we propose that FEN1 contributes to telomere stability by ensuring efficient telomere replication.

Comparison of proteomic and transcriptomic profiles in the bronchial airway epithelium of current and never smokers.

Background Although prior studies have demonstrated a smoking-induced field of molecular injury throughout the lung and airway, the impact of smoking on the airway epithelial proteome and its relationship to smoking-related changes in the airway transcriptome are unclear. Methodology/Principal Findings Airway epithelial cells were obtained from never (n = 5) and current (n = 5) smokers by brushing the mainstem bronchus. Proteins were separated by one dimensional polyacrylamide gel electrophoresis (1D-PAGE). After in-gel digestion, tryptic peptides were processed via liquid chromatography/ tandem mass spectrometry (LC-MS/MS) and proteins identified. RNA from the same samples was hybridized to HG-U133A microarrays. Protein detection was compared to RNA expression in the current study and a previously published airway dataset. The functional properties of many of the 197 proteins detected in a majority of never smokers were similar to those observed in the never smoker airway transcriptome. LC-MS/MS identified 23 proteins that differed between never and current smokers. Western blotting confirmed the smoking-related changes of PLUNC, P4HB1, and uteroglobin protein levels. Many of the proteins differentially detected between never and current smokers were also altered at the level of gene expression in this cohort and the prior airway transcriptome study. There was a strong association between protein detection and expression of its corresponding transcript within the same sample, with 86% of the proteins detected by LC-MS/MS having a detectable corresponding probeset by microarray in the same sample. Forty-one proteins identified by LC-MS/MS lacked detectable expression of a corresponding transcript and were detected in ≤5% of airway samples from a previously published dataset. Conclusions/Significance 1D-PAGE coupled with LC-MS/MS effectively profiled the airway epithelium proteome and identified proteins expressed at different levels as a result of cigarette smoke exposure. While there was a strong correlation between protein and transcript detection within the same sample, we also identified proteins whose corresponding transcripts were not detected by microarray. This noninvasive approach to proteomic profiling of airway epithelium may provide additional insights into the field of injury induced by tobacco exposure.

COMBREX: a project to accelerate the functional annotation of prokaryotic genomes

COMBREX (http://combrex.bu.edu) is a project to increase the speed of the functional annotation of new bacterial and archaeal genomes. It consists of a database of functional predictions produced by computational biologists and a mechanism for experimental biochemists to bid for the validation of those predictions. Small grants are available to support successful bids.

Interaction of tau with the RNA-Binding Protein TIA1 Regulates tau Pathophysiology and Toxicity

Dendritic mislocalization of microtubule associated protein tau is a hallmark of tauopathies, but the role of dendritic tau is unknown. We now report that tau interacts with the RNA-binding protein (RBP) TIA1 in brain tissue, and we present the brain-protein interactome network for TIA1. Analysis of the TIA1 interactome in brain tissue from wild-type (WT) and tau knockout mice demonstrates that tau is required for normal interactions of TIA1 with proteins linked to RNA metabolism, including ribosomal proteins and RBPs. Expression studies show that tau regulates the distribution of TIA1, and tau accelerates stress granule (SG) formation. Conversely, TIA1 knockdown or knockout inhibits tau misfolding and associated toxicity in cultured hippocampal neurons, while overexpressing TIA1 induces tau misfolding and stimulates neurodegeneration. Pharmacological interventions that prevent SG formation also inhibit tau pathophysiology. These studies suggest that the pathophysiology of tauopathy requires an intimate interaction with RNA-binding proteins.

Suggestive Evidence for Darwinian Selection against Asparagine-Linked Glycans of Plasmodium falciparum and Toxoplasma gondii†

ABSTRACT We are interested in asparagine-linked glycans (N-glycans) of Plasmodium falciparum and Toxoplasma gondii, because their N-glycan structures have been controversial and because we hypothesize that there might be selection against N-glycans in nucleus-encoded proteins that must pass through the endoplasmic reticulum (ER) prior to threading into the apicoplast. In support of our hypothesis, we observed the following. First, in protists with apicoplasts, there is extensive secondary loss of Alg enzymes that make lipid-linked precursors to N-glycans. Theileria makes no N-glycans, and Plasmodium makes a severely truncated N-glycan precursor composed of one or two GlcNAc residues. Second, secreted proteins of Toxoplasma, which uses its own 10-sugar precursor (Glc3Man5GlcNAc2) and the host 14-sugar precursor (Glc3Man9GlcNAc2) to make N-glycans, have very few sites for N glycosylation, and there is additional selection against N-glycan sites in its apicoplast-targeted proteins. Third, while the GlcNAc-binding Griffonia simplicifolia lectin II labels ER, rhoptries, and surface of plasmodia, there is no apicoplast labeling. Similarly, the antiretroviral lectin cyanovirin-N, which binds to N-glycans of Toxoplasma, labels ER and rhoptries, but there is no apicoplast labeling. We conclude that possible selection against N-glycans in protists with apicoplasts occurs by eliminating N-glycans (Theileria), reducing their length (Plasmodium), or reducing the number of N-glycan sites (Toxoplasma). In addition, occupation of N-glycan sites is markedly reduced in apicoplast proteins versus some secretory proteins in both Plasmodium and Toxoplasma.

Changes in the N-Glycome, Glycoproteins with Asn-Linked Glycans, of Giardia lamblia with Differentiation from Trophozoites to Cysts

ABSTRACT Giardia lamblia is present in the intestinal lumen as a binucleate, flagellated trophozoite or a quadranucleate, immotile cyst. Here we used the plant lectin wheat germ agglutinin (WGA), which binds to the disaccharide di-N-acetyl-chitobiose (GlcNAc2), which is the truncated Asn-linked glycan (N-glycan) of Giardia, to affinity purify the N-glycomes (glycoproteins with N-glycans) of trophozoites and cysts. Fluorescent WGA bound to the perinuclear membranes, peripheral acidified vesicles, and plasma membranes of trophozoites. In contrast, WGA bound strongly to membranes adjacent to the wall of Giardia cysts and less strongly to the endoplasmic reticulum and acidified vesicles. WGA lectin-affinity chromatography dramatically enriched secreted and membrane proteins of Giardia, including proteases and acid phosphatases that retain their activities. With mass spectroscopy, we identified 91 glycopeptides with N-glycans and 194 trophozoite-secreted and membrane proteins, including 42 unique proteins. The Giardia oligosaccharyltransferase, which contains a single catalytic subunit, preferred N glycosylation sites with Thr to those with Ser in vivo but had no preference for flanking amino acids. The most-abundant glycoproteins in the N-glycome of trophozoites were lysosomal enzymes, folding-associated proteins, and unique transmembrane proteins with Cys-, Leu-, or Gly-rich repeats. We identified 157 secreted and membrane proteins in the Giardia cysts, including 20 unique proteins. Compared to trophozoites, cysts were enriched in Gly-rich repeat transmembrane proteins, cyst wall proteins, and unique membrane proteins but had relatively fewer Leu-rich repeat proteins, folding-associated proteins, and unique secreted proteins. In summary, there are major changes in the Giardia N-glycome with the differentiation from trophozoites to cysts.

Negative Elongation Factor (NELF) Coordinates RNA Polymerase II Pausing, Premature Termination, and Chromatin Remodeling to Regulate HIV Transcription

Background: Multiple mechanisms contribute to HIV latency, including NELF-mediated RNA polymerase II (RNAP II) pausing. Results: Paused RNAP II recruits a transcription termination factor and a transcriptional corepressor complex to the HIV promoter. Conclusion: Paused RNAP II couples premature transcription termination and chromatin remodeling to maintain HIV latency. Significance: Paused RNAP II may be targeted to purge latent HIV infection. A barrier to eradicating HIV infection is targeting and eliminating latently infected cells. Events that contribute to HIV transcriptional latency include repressive chromatin structure, transcriptional interference, the inability of Tat to recruit positive transcription factor b, and poor processivity of RNA polymerase II (RNAP II). In this study, we investigated mechanisms by which negative elongation factor (NELF) establishes and maintains HIV latency. Negative elongation factor (NELF) induces RNAP II promoter proximal pausing and limits provirus expression in HIV-infected primary CD4+ T cells. Decreasing NELF expression overcomes RNAP II pausing to enhance HIV transcription elongation in infected primary T cells, demonstrating the importance of pausing in repressing HIV transcription. We also show that RNAP II pausing is coupled to premature transcription termination and chromatin remodeling. NELF interacts with Pcf11, a transcription termination factor, and diminishing Pcf11 in primary CD4+ T cells induces HIV transcription elongation. In addition, we identify NCoR1-GPS2-HDAC3 as a NELF-interacting corepressor complex that is associated with repressed HIV long terminal repeats. We propose a model in which NELF recruits Pcf11 and NCoR1-GPS2-HDAC3 to paused RNAP II, reinforcing repression of HIV transcription and establishing a critical checkpoint for HIV transcription and latency.