Predicting gene ontology annotations of orphan GWAS genes using protein-protein interactions

Background: The number of genome-wide association studies (GWAS) has increased rapidly in the past couple of years, resulting in the identification of genes associated with different diseases. The next step in translating these findings into biomedically useful information is to find out the mechanism of the action of these genes. However, GWAS studies often implicate genes whose functions are currently unknown; for example, MYEOV, ANKLE1, TMEM45B and ORAOV1 are found to be associated with breast cancer, but their molecular function is unknown. Results: We carried out Bayesian inference of Gene Ontology (GO) term annotations of genes by employing the directed acyclic graph structure of GO and the network of protein-protein interactions (PPIs). The approach is designed based on the fact that two proteins that interact biophysically would be in physical proximity of each other, would possess complementary molecular function, and play role in related biological processes. Predicted GO terms were ranked according to their relative association scores and the approach was evaluated quantitatively by plotting the precision versus recall values and F-scores (the harmonic mean of precision and recall) versus varying thresholds. Precisions of similar to 58% and similar to 40% for localization and functions respectively of proteins were determined at a threshold of similar to 30 (top 30 GO terms in the ranked list). Comparison with function prediction based on semantic similarity among nodes in an ontology and incorporation of those similarities in a k nearest neighbor classifier confirmed that our results compared favorably. Conclusions: This approach was applied to predict the cellular component and molecular function GO terms of all human proteins that have interacting partners possessing at least one known GO annotation. The list of predictions is available at http://severus.dbmi.pitt.edu/engo/GOPRED.html. We present the algorithm, evaluations and the results of the computational predictions, especially for genes identified in GWAS studies to be associated with diseases, which are of translational interest.

Trans-spliced Heat Shock Protein 90 Modulates Encystation in Giardia lamblia

Background: Hsp90 from Giardia lamblia is expressed by splicing of two independently transcribed RNA molecules, coded by genes named HspN and HspC located 777 kb apart. The reasons underlying such unique trans-splicing based generation of GlHsp90 remain unclear. Principle Finding: In this study using mass-spectrometry we identify the sequence of the unique, junctional peptide contributed by the 5' UTR of HspC ORF. This peptide is critical for the catalytic function of Hsp90 as it harbours an essential ``Arg'' in its sequence. We also show that full length GlHsp90 possesses all the functional hall marks of a canonical Hsp90 including its ability to bind and hydrolyze ATP. Using qRT-PCR as well as western blotting approach we find the reconstructed Hsp90 to be induced in response to heat shock. On the contrary we find GlHsp90 to be down regulated during transition from proliferative trophozoites to environmentally resistant cysts. This down regulation of GlHsp90 appears to be mechanistically linked to the encystation process as we find pharmacological inhibition of GlHsp90 function to specifically induce encystation. Significance: Our results implicate the trans-spliced GlHsp90 from Giardia lamblia to regulate an essential stage transition in the life cycle of this important human parasite.

Role of DNA sequence based structural features of promoters in transcription initiation and gene expression

D Regulatory information for transcription initiation is present in a stretch of genomic DNA, called the promoter region that is located upstream of the transcription start site (TSS) of the gene. The promoter region interacts with different transcription factors and RNA polymerase to initiate transcription and contains short stretches of transcription factor binding sites (TFBSs), as well as structurally unique elements. Recent experimental and computational analyses of promoter sequences show that they often have non-B-DNA structural motifs, as well as some conserved structural properties, such as stability, bendability, nucleosome positioning preference and curvature, across a class of organisms. Here, we briefly describe these structural features, the differences observed in various organisms and their possible role in regulation of gene expression.

Multi-Temporal Satellite Image Analysis Using Gene Expression Programming

This paper discusses an approach for river mapping and flood evaluation to aid multi-temporal time series analysis of satellite images utilizing pixel spectral information for image classification and region-based segmentation to extract water covered region. Analysis of Moderate Resolution Imaging Spectroradiometer (MODIS) satellite images is applied in two stages: before flood and during flood. For these images the extraction of water region utilizes spectral information for image classification and spatial information for image segmentation. Multi-temporal MODIS images from ``normal'' (non-flood) and flood time-periods are processed in two steps. In the first step, image classifiers such as artificial neural networks and gene expression programming to separate the image pixels into water and non-water groups based on their spectral features. The classified image is then segmented using spatial features of the water pixels to remove the misclassified water region. From the results obtained, we evaluate the performance of the method and conclude that the use of image classification and region-based segmentation is an accurate and reliable for the extraction of water-covered region.

Ontology analysis of global gene expression differences of human bone marrow stromal cells cultured on 3D scaffolds or 2D films

Differences in gene expression of human bone marrow stromal cells (hBMSCs) during culture in three-dimensional (3D) nanofiber scaffolds or on two-dimensional (2D) films were investigated via pathway analysis of microarray mRNA expression profiles. Previous work has shown that hBMSC culture in nanofiber scaffolds can induce osteogenic differentiation in the absence of osteogenic supplements (OS). Analysis using ontology databases revealed that nanofibers and OS regulated similar pathways and that both were enriched for TGF-beta and cell-adhesion/ECM-receptor pathways. The most notable difference between the two was that nanofibers had stronger enrichment for cell-adhesion/ECM-receptor pathways. Comparison of nanofibers scaffolds with flat films yielded stronger differences in gene expression than comparison of nanofibers made from different polymers, suggesting that substrate structure had stronger effects on cell function than substrate polymer composition. These results demonstrate that physical (nanofibers) and biochemical (OS) signals regulate similar ontological pathways, suggesting that these cues use similar molecular mechanisms to control hBMSC differentiation. Published by Elsevier Ltd.

Methylation Silencing of ULK2, an Autophagy Gene, Is Essential for Astrocyte Transformation and Tumor Growth

Glioblastoma (GBM) is the most aggressive type of brain tumor and shows very poor prognosis. Here, using genome-wide methylation analysis, we show that G-CIMP+ and G-CIMP-subtypes enrich distinct classes of biological processes. One of the hypermethylated genes in GBM, ULK2, an upstream autophagy inducer, was found to be down-regulated in GBM. Promoter hypermethylation of ULK2 was confirmed by bisulfite sequencing. GBM and glioma cell lines had low levels of ULK2 transcripts, which could be reversed upon methylation inhibitor treatment. ULK2 promoter methylation and transcript levels showed significant negative correlation. Ectopic overexpression of ULK2-induced autophagy, which further enhanced upon nutrient starvation or temozolomide chemotherapy. ULK2 also inhibited the growth of glioma cells, which required autophagy induction as kinase mutant of ULK2 failed to induce autophagy and inhibit growth. Furthermore, ULK2 induced autophagy and inhibited growth in Ras-transformed immortalized Baby Mouse Kidney (iBMK) ATG5(+/+) but not in autophagy-deficient ATG5(-/-) cells. Growth inhibition due to ULK2 induced high levels of autophagy under starvation or chemotherapy utilized apoptotic cell death but not at low levels of autophagy. Growth inhibition by ULK2 also appears to involve catalase degradation and reactive oxygen species generation. ULK2 overexpression inhibited anchorage independent growth, inhibited astrocyte transformation in vitro and tumor growth in vivo. Of all autophagy genes, we found ULK2 and its homologue ULK1 were only down-regulated in all grades of glioma. Thus these results altogether suggest that inhibition of autophagy by ULK1/2 down-regulation is essential for glioma development.

A Novel Mutation in the Transglutaminase-1 Gene in an Autosomal Recessive Congenital Ichthyosis Patient

Structure-function implication on a novel homozygous Trp250/Gly mutation of transglutaminase-1 (TGM1) observed in a patient of autosomal recessive congenital ichthyosis is invoked from a bioinformatics analysis. Structural consequences of this mutation are hypothesized in comparison to homologous enzyme human factor XIIIA accepted as valid in similar structural analysis and are projected as guidelines for future studies at an experimental level on TGM1 thus mutated.

Co-liposomes comprising a lipidated multivalent RGD-peptide and a cationic gemini cholesterol induce selective gene transfection in alpha v beta 3 and alpha v beta 5 integrin receptor-rich cancer cells

The alpha v beta 3 and alpha v beta 5 integrins, transmembrane glycoprotein receptors, are over-expressed in numerous tumors and in endothelial cells that constitute tumor blood vessels. As this protein selectively binds to the Arg-Gly-Asp (RGD) sequence containing peptides, it is an attractive way to target tumors. Herein we have developed novel formulations for integrin mediated selective gene delivery. These formulations are composed of a novel palmitoylated tetrameric RGD containing scaffold (named RAFT-RGD), cationic gemini cholesterol (GL5) and a natural helper lipid 1,2-dioleoyl-L-alpha-glycero-3-phosphatidylethanolamine (DOPE). We have optimized a co-liposomal formulation to introduce the multivalent RGD-containing macromolecule in GL5: DOPE (GL5D) mixture to produce GL5D-RGD. We have unambiguously shown the selectivity of these formulations towards cancer cells that over express alpha v beta 3 and alpha v beta 5 integrins. Two reporter plasmids, pEGFP-C3 and PGL-3, were employed for the transfection experiments and it was shown that GL5D-RGD Liposomes increased exclusively the transfection in alpha v beta 3 and alpha v beta 5 overexpressing HeLa cells.

Cancer gene signatures in risk stratification: use in personalized medicine

Cancer is a complex disease which arises due to a series of genetic changes related to cell division and growth control. Cancer remains the second leading cause of death in humans next to heart diseases. As a testimony to our progress in understanding the biology of cancer and developments in cancer diagnosis and treatment methods, the overall median survival time of all cancers has increased six fold one year to six years during the last four decades. However, while the median survival time has increased dramatically for some cancers like breast and colon, there has been only little change for other cancers like pancreas and brain. Further, not all patients having a single type of tumour respond to the standard treatment. The differential response is due to genetic heterogeneity which exists not only between tumours, which is called intertumour heterogeneity, but also within individual tumours, which is called intratumoural heterogeneity. Thus it becomes essential to personalize the cancer treatment based on a specific genetic change in a given tumour. It is also possible to stratify cancer patients into low- and high-risk groups based on expression changes or alterations in a group of genes gene signatures and choose a more suitable mode of therapy. It is now possible that each tumour can be analysed using various high-throughput methods like gene expression profiling and next-generation sequencing to identify its unique fingerprint based on which a personalized or tailor-made therapy can be developed. Here, we review the important progress made in the recent years towards personalizing cancer treatment with the use of gene signatures.

The polycomb group gene EMF2B is essential for maintenance of floral meristem determinacy in rice

Polycomb Repressive Complex 2 (PRC2) represses the transcriptional activity of target genes through trimethylation of lysine 27 of histone H3. The functions of plant PRC2 have been chiefly described in Arabidopsis, but specific functions in other plant species, especially cereals, are still largely unknown. Here we characterize mutants in the rice EMF2B gene, an ortholog of the Arabidopsis EMBRYONIC FLOWER2 (EMF2) gene. Loss of EMF2B in rice results in complete sterility, and mutant flowers have severe floral organ defects and indeterminacy that resemble loss-of-function mutants in E-function floral organ specification genes. Transcriptome analysis identified the E-function genes OsMADS1, OsMADS6 and OsMADS34 as differentially expressed in the emf2b mutant compared with wild type. OsMADS1 and OsMADS6, known to be required for meristem determinacy in rice, have reduced expression in the emf2b mutant, whereas OsMADS34 which interacts genetically with OsMADS1 was ectopically expressed. Chromatin immunoprecipitation for H3K27me3 followed by quantitative (q)RT-PCR showed that all three genes are presumptive targets of PRC2 in the meristem. Therefore, in rice, and possibly other cereals, PRC2 appears to play a major role in floral meristem determinacy through modulation of the expression of E-function genes.

Histone H3K9 acetylation level modulates gene expression and may affect parasite growth in human malaria parasite Plasmodium falciparum

Three-dimensional positioning of the nuclear genome plays an important role in the epigenetic regulation of genes. Although nucleographic domain compartmentalization in the regulation of epigenetic state and gene expression is well established in higher organisms, it remains poorly understood in the pathogenic parasite Plasmodium falciparum. In the present study, we report that two histone tail modifications, H3K9Ac and H3K14Ac, are differentially distributed in the parasite nucleus. We find colocalization of active gene promoters such as Tu1 (tubulin-1 expressed in the asexual stages) with H3K9Ac marks at the nuclear periphery. By contrast, asexual stage inactive gene promoters such as Pfg27 (gametocyte marker) and Pfs28 (ookinete marker) occupy H3K9Ac devoid zones at the nuclear periphery. The histone H3K9 is predominantly acetylated by the PCAF/GCN5 class of lysine acetyltransferases, which is well characterized in the parasite. Interestingly, embelin, a specific inhibitor of PCAF/GCN5 family histone acetyltransferase, selectively decreases total H3K9Ac acetylation levels (but not H3K14Ac levels) around the var gene promoters, leading to the downregulation of var gene expression, suggesting interplay among histone acetylation status, as well as subnuclear compartmentalization of different genes and their activation in the parasites. Finally, we found that embelin inhibited parasitic growth at the low micromolar range, raising the possibility of using histone acetyltransferases as a target for antimalarial therapy.

Genome wide chromatin occupancy of mrhl RNA and its role in gene regulation in mouse spermatogonial cells

Mrhl RNA is a nuclear lncRNA encoded in the mouse genome and negatively regulates Wnt signaling in spermatogonial cells through p68/Ddx5 RNA helicase. Mrhl RNA is present in the chromatin fraction of mouse spermatogonial Gc1-Spg cells and genome wide chromatin occupancy of mrhl RNA by ChOP (Chromatin oligo affinity precipitation) technique identified 1370 statistically significant genomic loci. Among these, genes at 37 genomic loci also showed altered expression pattern upon mrhl RNA down regulation which are referred to as GRPAM (Genes Regulated by Physical Association of Mrhl RNA). p68 interacted with mrhl RNA in chromatin at these GRPAM loci. p68 silencing drastically reduced mrhl RNA occupancy at 27 GRPAM loci and also perturbed the expression of GRPAM suggesting a role for p68 mediated mrhl RNA occupancy in regulating GRPAM expression. Wnt3a ligand treatment of Gc1-Spg cells down regulated mrhl RNA expression and also perturbed expression of these 27 GRPAM genes that included genes regulating Wnt signaling pathway and spermatogenesis, one of them being Sox8, a developmentally important transcription factor. We also identified interacting proteins of mrhl RNA associated chromatin fraction which included Pc4, a chromatin organizer protein and hnRNP A/B and hnRNP A2/B1 which have been shown to be associated with lincRNA-Cox2 function in gene regulation. Our findings in the Gc1-Spg cell line also correlate with the results from analysis of mouse testicular tissue which further highlights the in vivo physiological significance of mrhl RNA in the context of gene regulation during mammalian spermatogenesis.

Efficacious Gene Silencing in Serum and Significant Apoptotic Activity Induction by Survivin Downregulation Mediated by New Cationic Gemini Tocopheryl Lipids

Nonviral gene delivery offers cationic liposomes as promising instruments for the delivery of double-stranded RNA (ds RNA) molecules for successful sequence-specific gene silencing (RNA interference). The efficient delivery of siRNA (small interfering RNA) to cells while avoiding unexpected side effects is an important prerequisite for the exploitation of the power of this excellent tool. We present here six new tocopherol based cationic gemini lipids, which induce substantial gene knockdown without any obvious cytotoxicity. All the efficient coliposomal formulations derived from each of these geminis and a helper lipid, dioleoylphosphatidylethanolamine (DOPE), were well characterized using physical methods such as atomic force microscopy (AFM) and dynamic light scattering (DLS). Zeta potential measurements were conducted to estimate the surface charge of these formulations. Flow cytometric analysis showed that the optimized coliposomal formulations could transfect anti-GFP siRNA efficiently in three different GFP expressing cell lines, viz., HEK 293T, HeLa, and Caco-2, significantly better than a potent commercial standard Lipofectamine 2000 (L2K) both in the absence and in the presence of serum (FBS). Notably, the knockdown activity of coliposomes of gemini lipids was not affected even in the presence of serum (10% and 50% FBS) while it dropped down for L2K significantly. Observations under a fluorescence microscope, RT-PCR, and Western blot analysis substantiated the flow cytometry results. The efficient cellular entry of labeled siRNA in GFP expressing cells as evidenced from confocal microscopy put forward these gemini lipids among the potent lipidic carriers for siRNA. The efficient transfection capabilities were also profiled in a more relevant fashion while performing siRNA transfections against survivin (an anti-apoptotic protein) which induced substantial apoptosis. Furthermore, the survivin downregulation improved the therapeutic efficacy levels of an anticancer drug, doxorubicin, significantly. In short, the new tocopherol based gemini lipids appear to be highly promising for achieving siRNA mediated gene knockdown in various cell lines.

alpha-Tocopherol derived lipid dimers as efficient gene transfection agents. Mechanistic insights into lipoplex internalization and therapeutic induction of apoptotic activity

In this report, we present cationic dimeric (gemini) lipids for significant plasmid DNA (pDNA) delivery to different cell lines without any marked toxicity in the presence of serum. Six gemini lipids based on alpha-tocopherol were synthesized, which differed in their spacer chain lengths. Each of these gemini lipids mixed with a helper lipid, 1,2-dioleoyl phosphatidyl ethanolamine (DOPE), was capable of forming stable aqueous suspensions. These co-liposomal systems were examined for their potential to transfect pEGFP-C3 plasmid DNA into nine cell lines of different origins. The transfection efficacies noticed in terms of EGFP expression levels using flow cytometry were well corroborated using independent fluorescence microscopy studies. Significant EGFP expression levels were reported using the gemini co-liposomes, which counted significantly better than one well known commercial formulation, Lipofectamine 2000 (L2 K). Transfection efficacies were also analyzed in terms of the degree of intracellular delivery of labeled plasmid DNA (pDNA) using confocal microscopy, which revealed an efficient internalization in the presence of serum. The cell viability assays performed using optimized formulations demonstrated no significant toxicity towards any of the cell lines used in the study. We also had a look at the lipoplex internalization pathway to profile the uptake characteristics. A caveolae/lipid raft route was attributed to their excellent gene transfection capabilities. The study was further advanced by using a therapeutic p53-EGFP-C3 plasmid and the apoptotic activity was observed using FACS and growth inhibition assay.