John T. Tossberg

Leveraging skewed transcript abundance by RNA-Seq to increase the genomic depth of the tree of life

Assembling the tree of life is a major goal of biology, but progress has been hindered by the difficulty and expense of obtaining the orthologous DNA required for accurate and fully resolved phylogenies. Next-generation DNA sequencing technologies promise to accelerate progress, but sequencing the genomes of hundreds of thousands of eukaryotic species remains impractical. Eukaryotic transcriptomes, which are smaller than genomes and biased toward highly expressed genes that tend to be conserved, could potentially provide a rich set of phylogenetic characters. We sampled the transcriptomes of 10 mosquito species by assembling 36-bp sequence reads into phylogenomic data matrices containing hundreds of thousands of orthologous nucleotides from hundreds of genes. Analysis of these data matrices yielded robust phylogenetic inferences, even with data matrices constructed from surprisingly few sequence reads. This approach is more efficient, data-rich, and economical than traditional PCR-based and EST-based methods and provides a scalable strategy for generating phylogenomic data matrices to infer the branches and twigs of the tree of life.

Expression and functions of long noncoding RNAs during human T helper cell differentiation

Long noncoding RNAs (lncRNAs) regulate an array of biological processes in cells and organ systems. Less is known about their expression and function in lymphocyte lineages. Here we have identified >2000 lncRNAs expressed in human T cell cultures and those which display a TH lineage specific pattern of expression and are intragenic or adjacent to TH lineage specific genes encoding proteins with immunologic functions. One lncRNA cluster selectively expressed by the effector TH2 lineage consists of four alternatively spliced transcripts that regulate expression of TH2 cytokines, IL-4, IL-5 and IL-13. Genes encoding this lncRNA cluster in humans overlap the RAD50 gene and thus are contiguous with the previously described TH2 locus control region (LCR) in the mouse. Given its genomic synteny with the TH2 LCR, we refer to this lncRNA cluster as TH2-LCR lncRNA.

Methotrexate Inhibits NF‐κB Activity Via Long Intergenic (Noncoding) RNA–p21 Induction

To determine interrelationships between the expression of long intergenic (noncoding) RNA–p21 (lincRNA‐p21), NF‐κB activity, and responses to methotrexate (MTX) in rheumatoid arthritis (RA) by analyzing patient blood samples and cell culture models.

Regulation of the Th1 genomic locus from Ifng through Tmevpg1 by T-bet.

Long noncoding RNAs (lncRNAs), critical regulators of protein-coding genes, are likely to be coexpressed with neighboring protein-coding genes in the genome. How the genome integrates signals to achieve coexpression of lncRNA genes and neighboring protein-coding genes is not well understood. The lncRNA Tmevpg1 (NeST, Ifng-AS1) is critical for Th1-lineage–specific expression of Ifng and is coexpressed with Ifng. In this study, we show that T-bet guides epigenetic remodeling of Tmevpg1 proximal and distal enhancers, leading to recruitment of stimulus-inducible transcription factors, NF-κB and Ets-1, to the locus. Activities of Tmevpg1-specific enhancers and Tmevpg1 transcription are dependent upon NF-κB. Thus, we propose that T-bet stimulates epigenetic remodeling of Tmevpg1-specific enhancers and Ifng-specific enhancers to achieve Th1-lineage–specific expression of Ifng.

Methotrexate increases expression of cell cycle checkpoint genes via JNK activation

OBJECTIVE To assess defects in expression of critical cell cycle checkpoint genes and proteins in patients with rheumatoid arthritis (RA) relative to presence or absence of methotrexate (MTX) treatment, and to investigate the role of JNK in induction of these genes by MTX. METHODS Flow cytometric analysis was used to quantify changes in levels of intracellular proteins, measure reactive oxygen species (ROS), and determine apoptosis in different lymphoid populations. Quantitative reverse transcription-polymerase chain reaction was used to identify changes in cell cycle checkpoint target genes. RESULTS RA patients expressed reduced baseline levels of MAPK9, TP53, CDKN1A, CDKN1B, CHEK2, and RANGAP1 messenger RNA (mRNA) and JNK total protein. The reduction in expression of mRNA for MAPK9, TP53, CDKN1A, and CDKN1B was greater in patients not receiving MTX than in those receiving low-dose MTX, with no difference in expression levels of CHEK2 and RANGAP1 mRNA between MTX-treated and non-MTX-treated patients. Further, JNK levels were inversely correlated with C-reactive protein levels in RA patients. In tissue culture, MTX induced expression of both p53 and p21 by JNK-2- and JNK-1-dependent mechanisms, respectively, while CHEK2 and RANGAP1 were not induced by MTX. MTX also induced ROS production, JNK activation, and sensitivity to apoptosis in activated T cells. Supplementation with tetrahydrobiopterin blocked these MTX-mediated effects. CONCLUSION Our findings support the notion that MTX restores some, but not all, of the proteins contributing to cell cycle checkpoint deficiencies in RA T cells, via a JNK-dependent pathway.

Gene-expression signatures: biomarkers toward diagnosing multiple sclerosis

Identification of biomarkers contributing to disease diagnosis, classification or prognosis could be of considerable utility. For example, primary methods to diagnose multiple sclerosis (MS) include magnetic resonance imaging and detection of immunological abnormalities in cerebrospinal fluid. We determined whether gene-expression differences in blood discriminated MS subjects from comparator groups, and identified panels of ratios that performed with varying degrees of accuracy depending upon complexity of comparator groups. High levels of overall accuracy were achieved by comparing MS with homogeneous comparator groups. Overall accuracy was compromised when MS was compared with a heterogeneous comparator group. Results, validated in independent cohorts, indicate that gene-expression differences in blood accurately exclude or include a diagnosis of MS and suggest that these approaches may provide clinically useful prediction of MS.

Expression of IL-33 and its epigenetic regulation in Multiple Sclerosis.

We examined the expression of IL‐33 as an indicator of an innate immune response in relapsing remitting MS (RRMS) and controls. We proposed a link between the expression of IL‐33 and IL‐33 regulated genes to histone deacetylase (HDAC) activity and in particular HDAC3, an enzyme that plays a role in the epigenetic regulation of a number genes including those which regulate inflammation.

Cutting Edge: Chronic NF-κB Activation in CD4+ T Cells in Rheumatoid Arthritis Is Genetically Determined by HLA Risk Alleles

Of identified genetic variants, HLA polymorphisms confer the greatest risk for developing autoimmune diseases, including rheumatoid arthritis (HLA-DRB1*04). There are strong influences of HLA polymorphisms on cell type–specific gene expression in B cells and monocytes. Their influence on gene expression in CD4+ T cells is not known. We determined transcript and proteins levels of target genes in lymphocyte/monocyte subsets in healthy controls and rheumatoid arthritis subjects as a function of HLA-DRB1*04 haplotype. We identified gene expression dependent on HLA-DRB1*04 genotype in CD4+ T cells. NF-κB activity in CD4+ T cells was also dependent on HLA-DRB1*04 genotype, and blocking HLA-DR inhibited NF-κB activity in CD4+ T cells and normalized gene expression, as did pharmacologic inhibition of NF-κB. We conclude that interactions between TCR and MHC class II encoded by HLA-DRB1*04 create a proinflammatory “hum” altering CD4+ T cell phenotype.

Using gene expression data to identify certain gastro-intestinal diseases

BackgroundInflammatory bowel diseases, ulcerative colitis and Crohn’s disease are considered to be of autoimmune origin, but the etiology of irritable bowel syndrome remains elusive. Furthermore, classifying patients into irritable bowel syndrome and inflammatory bowel diseases can be difficult without invasive testing and holds important treatment implications. Our aim was to assess the ability of gene expression profiling in blood to differentiate among these subject groups.MethodsTranscript levels of a total of 45 genes in blood were determined by quantitative real-time polymerase chain reaction (RT-PCR). We applied three separate analytic approaches; one utilized a scoring system derived from combinations of ratios of expression levels of two genes and two different support vector machines.ResultsAll methods discriminated different subject cohorts, irritable bowel syndrome from control, inflammatory bowel disease from control, irritable bowel syndrome from inflammatory bowel disease, and ulcerative colitis from Crohn’s disease, with high degrees of sensitivity and specificity.ConclusionsThese results suggest these approaches may provide clinically useful prediction of the presence of these gastro-intestinal diseases and syndromes.

Defective structural RNA processing in relapsing-remitting multiple sclerosis.

BackgroundSurveillance of integrity of the basic elements of the cell including DNA, RNA, and proteins is a critical element of cellular physiology. Mechanisms of surveillance of DNA and protein integrity are well understood. Surveillance of structural RNAs making up the vast majority of RNA in a cell is less well understood. Here, we sought to explore integrity of processing of structural RNAs in relapsing remitting multiple sclerosis (RRMS) and other inflammatory diseases.ResultsWe employed mononuclear cells obtained from subjects with RRMS and cell lines. We used quantitative-PCR and whole genome RNA sequencing to define defects in structural RNA surveillance and siRNAs to deplete target proteins. We report profound defects in surveillance of structural RNAs in RRMS exemplified by elevated levels of poly(A) + Y1-RNA, poly(A) + 18S rRNA and 28S rRNAs, elevated levels of misprocessed 18S and 28S rRNAs and levels of the U-class of small nuclear RNAs. Multiple sclerosis is also associated with genome-wide defects in mRNA splicing. Ro60 and La proteins, which exist in ribonucleoprotein particles and play different roles in quality control of structural RNAs, are also deficient in RRMS. In cell lines, silencing of the genes encoding Ro60 and La proteins gives rise to these same defects in surveillance of structural RNAs.ConclusionsOur results establish that profound defects in structural RNA surveillance exist in RRMS and establish a causal link between Ro60 and La proteins and integrity of structural RNAs.