Comparative transcriptomics of extreme phenotypes of human HIV-1 infection and SIV infection in sooty mangabey and rhesus macaque

High levels of HIV-1 replication during the chronic phase of infection usually correlate with rapid progression to severe immunodeficiency. However, a minority of highly viremic individuals remains asymptomatic and maintains high CD4⁺ T cell counts. This tolerant profile is poorly understood and reminiscent of the widely studied nonprogressive disease model of SIV infection in natural hosts. Here, we identify transcriptome differences between rapid progressors (RPs) and viremic nonprogressors (VNPs) and highlight several genes relevant for the understanding of HIV-1-induced immunosuppression. RPs were characterized by a specific transcriptome profile of CD4⁺ and CD8⁺ T cells similar to that observed in pathogenic SIV-infected rhesus macaques. In contrast, VNPs exhibited lower expression of interferon-stimulated genes and shared a common gene regulation profile with nonpathogenic SIV-infected sooty mangabeys. A short list of genes associated with VNP, including CASP1, CD38, LAG3, TNFSF13B, SOCS1, and EEF1D, showed significant correlation with time to disease progression when evaluated in an independent set of CD4⁺ T cell expression data. This work characterizes 2 minimally studied clinical patterns of progression to AIDS, whose analysis may inform our understanding of HIV pathogenesis.

Mechanistic aspects of NMD in human cells

Eukaryotic mRNAs with premature translation termination codons (PTCs) are recognized and degraded through a process termed nonsense-mediated mRNA decay (NMD). To get more insight into the recruitment of the central NMD factor UPF1 to target mRNAs, we mapped transcriptome-wide UPF1-binding sites by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP) in human cells and found that UPF1 preferentially associated with 3′ UTRs in translationally active cells but underwent significant redistribution toward coding regions (CDS) upon translation inhibition. This indicates that UPF1 binds RNA before translation and gets displaced from the CDS by translating ribosomes. Corroborated by RNA immunoprecipitation and by UPF1 cross-linking to long noncoding RNAs, our evidence for translation-independent UPF1-RNA interaction suggests that the triggering of NMD occurs after UPF1 binding to mRNA, presumably through activation of RNA-bound UPF1 by aberrant translation termination. Unlike in yeast, in mammalian cells NMD has been reported to be restricted to cap-binding complex (CBC)–bound mRNAs during the pioneer round of translation. However, we compared decay kinetics of two NMD reporter genes in mRNA fractions bound to either CBC or the eukaryotic initiation factor 4E (eIF4E) in human cells and show that NMD destabilizes eIF4E-bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event.

Mechanistic aspects of mRNA targeting for nonsense-mediated mRNA decay in human cells

The nonsense-mediated mRNA decay (NMD) pathway is best known as a translation-coupled quality control system that recognizes and degrades aberrant mRNAs with ORF-truncating premature termination codons (PTCs), but a more general role of NMD in posttranscriptional regulation of gene expression is indicated by transcriptome-wide mRNA profilings that identified a plethora of physiological mRNAs as NMD substrates. We try to decipher the mechanism of mRNA targeting to the NMD pathway in human cells. Recruitment of the conserved RNA-binding helicase UPF1 to target mRNAs has been reported to occur through interaction with release factors at terminating ribosomes, but evidence for translation-independent interaction of UPF1 with the 3’ untranslated region (UTR) of mRNAs has also been reported. We have transcriptome-wide determined the UPF1 binding sites by individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) in human cells, untreated or after inhibiting translation. We detected a strongly enriched association of UPF1 with 3’ UTRs in undisturbed, translationally active cells. After translation inhibition, a significant increase in UPF1 binding to coding sequence (CDS) was observed, indicating that UPF1 binds RNA before translation and gets displaced from the CDS by translating ribosomes. This suggests that the decision to trigger NMD occurs after association of UPF1 with mRNA, presumably through activation of RNA-bound UPF1 by aberrant translation termination. In a second recent study, we re-visited the reported restriction of NMD in mammals to the ‘pioneer round of translation’, i.e. to cap-binding complex (CBC)-bound mRNAs. The limitation of mammalian NMD to early rounds of translation would indicate a – from an evolutionary perspective – unexpected mechanistic difference to NMD in yeast and plants, where PTC-containing mRNAs seem to be available to NMD at each round of translation. In contrast to previous reports, our comparison of decay kinetics of two NMD reporter genes in mRNA fractions bound to either CBC or the eukaryotic initiation factor 4E (eIF4E) in human cells revealed that NMD destabilizes eIF4E-bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event.

Regulation of human (adrenal) androgen biosynthesis-New insights from novel throughput technology studies

Androgens are precursors for sex steroids and are predominantly produced in the human gonads and the adrenal cortex. They are important for intrauterine and postnatal sexual development and human reproduction. Although human androgen biosynthesis has been extensively studied in the past, exact mechanisms underlying the regulation of androgen production in health and disease remain vague. Here, the knowledge on human androgen biosynthesis and regulation is reviewed with a special focus on human adrenal androgen production and the hyperandrogenic disorder of polycystic ovary syndrome (PCOS). Since human androgen regulation is highly specific without a good animal model, most studies are performed on patients harboring inborn errors of androgen biosynthesis, on human biomaterials and human (tumor) cell models. In the past, most studies used a candidate gene approach while newer studies use high throughput technologies to identify novel regulators of androgen biosynthesis. Using genome wide association studies on cohorts of patients, novel PCOS candidate genes have been recently described. Variant 2 of the DENND1A gene was found overexpressed in PCOS theca cells and confirmed to enhance androgen production. Transcriptome profiling of dissected adrenal zones established a role for BMP4 in androgen synthesis. Similarly, transcriptome analysis of human adrenal NCI-H295 cells identified novel regulators of androgen production. Kinase p38α (MAPK14) was found to phosphorylate CYP17 for enhanced 17,20 lyase activity and RARB and ANGPTL1 were detected in novel networks regulating androgens. The discovery of novel players for androgen biosynthesis is of clinical significance as it provides targets for diagnostic and therapeutic use.

Mouse p53-deficient cancer models as platforms for obtaining genomic predictors of human cancer clinical outcomes

Mutations in the TP53 gene are very common in human cancers, and are associated with poor clinical outcome. Transgenic mouse models lacking the Trp53 gene or that express mutant Trp53 transgenes produce tumours with malignant features in many organs. We previously showed the transcriptome of a p53-deficient mouse skin carcinoma model to be similar to those of human cancers with TP53 mutations and associated with poor clinical outcomes. This report shows that much of the 682-gene signature of this murine skin carcinoma transcriptome is also present in breast and lung cancer mouse models in which p53 is inhibited. Further, we report validated gene-expression-based tests for predicting the clinical outcome of human breast and lung adenocarcinoma. It was found that human patients with cancer could be stratified based on the similarity of their transcriptome with the mouse skin carcinoma 682-gene signature. The results also provide new targets for the treatment of p53-defective tumours.

Identification and analysis of chromodomain-containing proteins encoded in the mouse transcriptome

The chromodomain is 40-50 amino acids in length and is conserved in a wide range of chromatic and regulatory proteins involved in chromatin remodeling. Chromodomain-containing proteins can be classified into families based on their broader characteristics, in particular the presence of other types of domains, and which correlate with different subclasses of the chromodomains themselves. Hidden Markov model (HMM)-generated profiles of different subclasses of chromodomains were used here to identify sequences encoding chromodomain-containing proteins in the mouse transcriptome and genome. A total of 36 different loci encoding proteins containing chromodomains, including 17 novel loci, were identified. Six of these loci (including three apparent pseudogenes, a novel HP1 ortholog, and two novel Msl-3 transcription factor-like proteins) are not present in the human genome, whereas the human genome contains four loci (two CDY orthologs and two apparent CDY pseuclogenes) that are not present in mouse. A number of these loci exhibit alternative splicing to produce different isoforms, including 43 novel variants, some of which lack the chromodomain. The likely functions of these proteins are discussed in relation to the known functions of other chromodomain-containing proteins within the same family.

Targeting a complex transcriptome: The construction of the mouse full-length cDNA encyclopedia

We report the construction of the mouse full-length cDNA encyclopedia, the most extensive view of a complex transcriptome, on the basis of preparing and sequencing 246 libraries. Before cloning, cDNAs were enriched in full-length by Cap-Trapper, and in most cases, aggressively subtracted/normalized. We have produced 1,442,236 successful 3'-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5' end reads, which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU), which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC), which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large. numbers of clusters (and TUs) of this project, which also include non-protein-coding RNAs, and the lower gene number estimation of genome annotations. Altogether, S'-end clusters identify regions that are potential promoters for 8637 known genes and S'-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete.

Impact of alternative initiation, splicing, and termination on the diversity of the mRNA transcripts encoded by the mouse transcriptome

We analyzed the FANTOM2 clone set of 60,770 RIKEN full-length mouse cDNA sequences and 44,122 public mRNA sequences. We developed a new computational procedure to identify and classify the forms of splice variation evident in this data set and organized the results into a publicly accessible database that can be used for future expression array construction, structural genomics, and analyses of the mechanism and regulation of alternative splicing. Statistical analysis shows that at least 41% and possibly as much as 60% of multiexon genes in mouse have multiple splice forms. Of the transcription units with multiple splice forms, 49% contain transcripts in which the apparent use of an alternative transcription start (stop) is accompanied by alternative splicing of the initial (terminal) exon. This implies that alternative transcription may frequently induce alternative splicing. The fact that 73% of all exons with splice variation fall within the annotated coding region indicates that most splice variation is likely to affect the protein form. Finally, we compared the set of constitutive (present in all transcripts) exons with the set of cryptic (present only in some transcripts) exons and found statistically significant differences in their length distributions, the nucleoticle distributions around their splice junctions, and the frequencies of occurrence of several short sequence motifs.

Antisense transcription in the mammalian transcriptome

Antisense transcription (transcription from the opposite strand to a protein-coding or sense strand) has been ascribed roles in gene regulation involving degradation of the corresponding sense transcripts (RNA interference), as well as gene silencing at the chromatin level. Global transcriptome analysis provides evidence that a large proportion of the genome can produce transcripts from both strands, and that antisense transcripts commonly link neighboring genes in complex loci into chains of linked transcriptional units. Expression profiling reveals frequent concordant regulation of sense/antisense pairs. We present experimental evidence that perturbation of an antisense RNA can alter the expression of sense messenger RNAs, suggesting that antisense transcription contributes to control of transcriptional outputs in mammals.

In vitro and in silico analysis of signal peptides from the human blood fluke, Schistosoma mansoni

Proteins secreted by and anchored on the surfaces of parasites are in intimate contact with host tissues. The transcriptome of infective cercariae of the blood fluke, Schistosoma mansoni, was screened using signal sequence trap to isolate cDNAs encoding predicted proteins with an N-terminal signal peptide. Twenty cDNA fragments were identified, most of which contained predicted signal peptides or transmembrane regions, including a novel putative seven-transmembrane receptor and a membrane-associated mitogen-activated protein kinase. The developmental expression pattern within different life-cycle stages ranged from ubiquitous to a transcript that was highly upregulated in the cercaria. A bioinformatics-based comparison of 100 signal peptides from each of schistosomes, humans, a parasitic nematode and Escherichia coli showed that differences in the sequence composition of signal peptides, notably the residues flanking the predicted cleavage site, might account for the negative bias exhibited in the processing of schistosome signal peptides in mammalian cells. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Retrotransposon expression profiling: Unveiling the hidden SINE transcriptome through Next-Generation Sequencing data analysis

Of the ~1.7 million SINE elements in the human genome, only a tiny number are estimated to be active in transcription by RNA polymerase (Pol) III. Tracing the individual loci from which SINE transcripts originate is complicated by their highly repetitive nature. By exploiting RNA-Seq datasets and unique SINE DNA sequences, we devised a bioinformatic pipeline allowing us to identify Pol III-dependent transcripts of individual SINE elements. When applied to ENCODE transcriptomes of seven human cell lines, this search strategy identified ~1300 Alu loci and ~1100 MIR loci corresponding to detectable transcripts, with ~120 and ~60 respectively Alu and MIR loci expressed in at least three cell lines. In vitro transcription of selected SINEs did not reflect their in vivo expression properties, and required the native 5’-flanking region in addition to internal promoter. We also identified a cluster of expressed AluYa5-derived transcription units, juxtaposed to snaR genes on chromosome 19, formed by a promoter-containing left monomer fused to an Alu-unrelated downstream moiety. Autonomous Pol III transcription was also revealed for SINEs nested within Pol II-transcribed genes raising the possibility of an underlying mechanism for Pol II gene regulation by SINE transcriptional units. Moreover the application of our bioinformatic pipeline to both RNA-seq data of cells subjected to an in vitro pro-oncogenic stimulus and of in vivo matched tumor and non-tumor samples allowed us to detect increased Alu RNA expression as well as the source loci of such deregulation. The ability to investigate SINE transcriptomes at single-locus resolution will facilitate both the identification of novel biologically relevant SINE RNAs and the assessment of SINE expression alteration under pathological conditions.

Comparative transcriptome analyses of Pseudomonas aeruginosa

One of the hallmarks of bacterial survival is their ability to adapt rapidly to changing environmental conditions. Niche adaptation is a response to the signals received that are relayed, often to regulators that modulate gene expression. In the post-genomic era, DNA microarrays are used to study the dynamics of gene expression on a global scale. Numerous studies have used Pseudomonas aeruginosa--a Gram-negative environmental and opportunistic human pathogenic bacterium--as the model organism in whole-genome transcriptome analysis. This paper reviews the transcriptome studies that have led to immense advances in our understanding of the biology of this intractable human pathogen. Comparative analysis of 23 P. aeruginosa transcriptome studies has led to the identification of a unique set of genes that are signal specific and a core set that is differentially regulated. The 303 genes in the core set are involved in bacterial homeostasis, making them attractive therapeutic targets.

An investigation of the molecular interactions between statins and bacterial pathogens, and their combined impact on the human immune system

Statins are a class of drug that inhibits cholesterol biosynthesis, and are used to treat patients with high serum cholesterol levels. They exert this function by competitively binding to the enzyme 3-hydroxy-3-methylglutaryl-CoenzymeA reductase (HMGR), which catalyses the formation of mevalonate, a rate-limiting step in cholesterol biosynthesis. In addition, statins have what are called “pleiotropic effects”, which include the reduction of inflammation, immunomodulation, and antimicrobial effects. Statins can also improve survival of patients with sepsis and pneumonia. Cystic fibrosis (CF) is the most common recessive inherited disease in the Caucasian population, which is characterised by factors including, but not limited to, excessive lung inflammation and increased susceptibility to infection. Therefore, the overall objective of this study was to examine the effects of statins on CFassociated bacterial pathogens and the host response. In this work, the prevalence of HMGR was examined in respiratory pathogens, and several CF-associated pathogens were found to possess homologues of this enzyme. HMGR homology was analysed in Staphylococcus aureus, Burkholderia cenocepacia and Streptococcus pneumoniae, and the HMGR of B. cenocepacia was found to have significant conservation to that of Pseudomonas mevalonii, which is the most widely-characterised bacterial HMGR. However, in silico analysis revealed that, unlike S. aureus and S. pneumoniae, B. cenocepacia did not possess homologues of other mevalonate pathway proteins, and that the HMGR of B. cenocepacia appeared to be involved in an alternative metabolic pathway. The effect of simvastatin was subsequently tested on the growth and virulence of S. aureus, B. cenocepacia and S. pneumoniae. Simvastatin inhibited the growth of all 3 species in a dose-dependent manner. In addition, statin treatment also attenuated biofilm formation of all 3 species, and reduced in vitro motility of S. aureus. Interestingly, simvastatin also increased the potency of the aminoglycoside antibiotic gentamicin against B. cenocepacia. The impact of statins was subsequently tested on the predominant CF-associated pathogen Pseudomonas aeruginosa, which does not possess a HMGR homologue. Mevastatin, lovastatin and simvastatin did not influence the growth of this species. However, sub-inhibitory statin concentrations reduced the swarming motility and biofilm formation of P. aeruginosa. The influence of statins was also examined on Type 3 toxin secretion, quorum sensing and chemotaxis, and no statin effect was observed on any of these phenotypes. Statins did not appear to have a characteristic effect on the P. aeruginosa transcriptome. However, a mutant library screen revealed that the effect of statins on P. aeruginosa biofilm was mediated through the PvrR regulator and the Cup fimbrial biosynthesis genes. Furthermore, proteomic analysis demonstrated that 6 proteins were reproducibly induced by simvastatin in the P. aeruginosa swarming cells. The effect of statins on the regulation of the host-P. aeruginosa immune response was also investigated. Statin treatment increased expression of the pro-inflammatory cytokines IL-8 and CCL20 in lung epithelial cells, but did not attenuate P. aeruginosa-mediated inflammatory gene induction. In fact, simvastatin and P. aeruginosa caused a synergistic effect on CCL20 expression. The expression of the transcriptional regulators KLF2 and KLF6 was also increased by statins and P. aeruginosa, with the induction of KLF6 by simvastatin proving to be a novel effect. Interestingly, both statins and P. aeruginosa were capable of inducing alternative splicing of KLF6. P. aeruginosa was found to induce KLF6 alternative splicing by way of the type 3 secreted toxin ExoS. In addition, a mechanistic role was elucidated for KLF6 in the lung, as it was determined that statin-mediated induction of this protein was responsible for the induction of the host response genes CCL20 and iNOS. Moreover, statin treatment caused a slight increase in infection-related cytotoxicity, and increased bacterial adhesion to cells. Taken together, these data demonstrate that statins can reduce the virulence of CFassociated bacterial pathogens and alter host response effectors. Furthermore, novel statin effectors were identified in both bacterial and host cells.

Comparative genome analysis of human bifidobacteria with commercial potential

This thesis describes two newly sequenced B. longum subsp. longum genomes and subsequent comparative analysis with publicly available B. longum subsp. longum, B. longum subsp. infantis and B. longum subsp. suis genomes (Chapter 2). The acquired data revealed a closed pan-genome for this bifidobacterial species and furthermore facilitated the definition of the B. longum core genome. The comparative analysis also highlights differences in the potential metabolic abilities of all three sub-species. Interestingly, phylogenetic analysis of the B. longum core genome indicated the existence of a novel B. longum subspecies. Characterisation of restriction-modification systems from two B. longum subsp. longum strains is described in Chapter 3. These defence mechanisms limit the uptake of genetic material, which was successfully demonstrated for some of the identified systems. When these systems were by-passed by methylation of DNA prior to the transformation procedure, the resulting transformation efficiency of both B. longum subsp. longum strains was increased to a level that allowed for the generation of mutants via homologous recombination. Arabinoxylan metabolism by B. longum subsp. longum NCIMB 8809 was investigated in Chapter 4 of this thesis. Transcriptome analysis allowed the identification of a number of genes involved in the degradation, uptake and utilisation of arabinoxylan. Biochemical analysis revealed that three of the identified genes encode arabinofuranosidase activity. Phenotypic assessment of a number of insertion mutants in genes identified by the transcriptome analysis revealed the essential role of two of these enzymes in arabinoxylan metabolism, and a third enzyme in the metabolism of debranched arabinan. Furthermore, this investigation revealed that B. longum subsp. longum NCIMB 8809 does not completely degrade arabinoxylan, but utilises the arabinose substitutions only, while leaving the xylan backbone untouched.Finally, Chapter 5 outlines that B. longum subsp. longum NCIMB 8809 is capable of removing ferulic and p-coumaric acid substitutions that originate from arabinoxylan. Analysis of the genome sequence led to the identification of a candidate gene for this activity, which was subsequently cloned and expressed in E. coli. Biochemical analysis revealed that the enzyme, designated here as FaeA, is indeed capable of releasing both ferulic and p-coumaric acid from arabinoxylan. Furthermore, it is shown that a derivative of B. longum subsp. longum NCIMB 8809 carrying an insertion mutation in faeA had lost the ability to release ferulic and p-coumaric acid from arabinoxylan, and that growth of this mutant strain is negatively affected when cultivated on growth-limiting levels of arabinoxylan.