A simple and robust method for connecting small-molecule drugs using gene-expression signatures

Background

Interaction of a drug or chemical with a biological system can result in a gene-expression profile or signature characteristic of the event. Using a suitably robust algorithm these signatures can potentially be used to connect molecules with similar pharmacological or toxicological properties by gene expression profile. Lamb et al first proposed the Connectivity Map [Lamb et al (2006), Science 313, 1929–1935] to make successful connections among small molecules, genes, and diseases using genomic signatures.

Results

Here we have built on the principles of the Connectivity Map to present a simpler and more robust method for the construction of reference gene-expression profiles and for the connection scoring scheme, which importantly allows the valuation of statistical significance of all the connections observed. We tested the new method with two randomly generated gene signatures and three experimentally derived gene signatures (for HDAC inhibitors, estrogens, and immunosuppressive drugs, respectively). Our testing with this method indicates that it achieves a higher level of specificity and sensitivity and so advances the original method.

Conclusion

The method presented here not only offers more principled statistical procedures for testing connections, but more importantly it provides effective safeguard against false connections at the same time achieving increased sensitivity. With its robust performance, the method has potential use in the drug development pipeline for the early recognition of pharmacological and toxicological properties in chemicals and new drug candidates, and also more broadly in other 'omics sciences.

Restricted gene flow in fragmented populations of a wind-pollinated tree

Fragmentation of natural populations can have negative effects at the genetic level, thus threatening their evolutionary potential. Many of the negative genetic impacts of population fragmentation can be ameliorated by gene flow and it has been suggested that in wind-pollinated tree species, high or even increased levels of gene flow are a feature of fragmented populations, although several studies have disputed this. We have used a combination of nuclear microsatellites and allele-specific PCR (AS-PCR) analysis of chloroplast single nucleotide polymorphisms (SNPs) to examine the levels and patterns of genetic diversity and population differentiation in fragmented populations of juniper (Juniperus communis) in Ireland and inform conservation programs for the species. Significant population differentiation was found for both chloroplast and nuclear markers, indicating restricted gene flow, particularly over larger geographic scales. For conservation purposes, the existence of genetically distinct clusters and geographically localised chloroplast haplotypes suggests that the concept of provenance should be taken into account when formulating augmentation or reintroduction strategies. Furthermore, the potential lack of seed dispersal and seedling establishment means that ex-situ approaches to seed and seedling management may have to be considered.

Cataract surgery induces retinal pro-inflammatory gene expression and protein secretion

To investigate the effect(s) of cataract surgery on the expression of pro-inflammatory genes and proteins in the retina using an experimental rodent model. An extracapsular lens extraction was performed in one eye of C57BL/6 mice (n=24); the contralateral unoperated eyes (n =24) as well as eyes from unoperated animals (n = 9) served as controls. The neurosensory retina and retinal pigment epithelium (RPE)/choroid were collected postoperatively. Expression of genes involved in the acute inflammatory/ injury response, including IL-1ß, fibroblast growth factor, transforming growth factor ß, chemokine CCL2, SDF-1, and complements C3, C4, and factor B (CFB), were examined by real-time PCR and, selectively, by immunohistochemistry. The expression of IL-1 ß and CCL2 genes was markedly upregulated (>0-fold, P >0.01) in the neurosensory retina 30 minutes postoperatively and maintained for the 2-week postoperative period of observation; IL-1 ß expression was also upregulated in RPE/choroid. The expression of complement C3 (>-fold) and CFB (>0-fold) genes in the neurosensory retina was also significantly upregulated (P

Hyperacetylation in prostate cancer induces cell cycle aberrations, chromatin reorganization and altered gene expression profiles.

Histone acetylation is a fundamental mechanism in the regulation of local chromatin conformation and gene expression. Research has focused on the impact of altered epigenetic environments on the expression of specific genes and their pathways. However, changes in histone acetylation also have a global impact on the cell. In this study we used digital texture analysis to assess global chromatin patterns following treatment with trichostatin A (TSA) and have observed significant alterations in the condensation and distribution of higher-order chromatin, which were associated with altered gene expression profiles in both immortalised normal PNT1A prostate cell line and androgen-dependent prostate cancer cell line LNCaP. Furthermore, the extent of TSA-induced disruption was both cell cycle and cell line dependent. This was illustrated by the identification of sub-populations of prostate cancer cells expressing high levels of H3K9 acetylation in the G2/M phase of the cell cycle that were absent in normal cell populations. In addition, the analysis of enriched populations of G1 cells showed a global decondensation of chromatin exclusively in normal cells.

Cannabinoid Receptor influences Chromatin Remodelling in Mouse Spermatids by affecting Content of Transition Protein 2 mRNA and Histone Replacement

Marijuana smokers and animals treated with ?9-tetrahydrocannabinol, THC, the principal component of marijuana, show alterations of sperm morphology suggesting a role for cannabinoids in sperm differentiation and/or maturation. Since the cannabinoid receptor 1 (CNR1) activation appears to play a pivotal role in spermiogenesis, the developmental stage where DNA is remodeled, we hypothesized that CNR1 receptors might also influence chromatin quality in sperm. We used Cnr1 null mutant (Cnr1-/-) mice to study the possible role of endocannabinoids on sperm chromatin during spermiogenesis. We demonstrated that CNR1 activation regulated chromatin remodeling of spermatids by either increasing Tnp2 levels or enhancing histone displacement. Comparative analysis of WT, Cnr1+/- and Cnr1-/- animals suggested the possible occurrence of haploinsufficiency for Tnp2 turnover control by CNR1, while histone displacement was disrupted to a lesser extent. Further, flow cytometry analysis demonstrated that the genetic loss of Cnr1 decreased sperm chromatin quality and was associated with sperm DNA fragmentation. This damage increased during epididymal transit, from caput to cauda. Collectively, our results show that the expression/activity of CNR1 controls the physiological alterations of DNA structure during spermiogenic maturation and epididymal transit. Given the deleterious effects of sperm DNA damage on male fertility, we suggest that the reproductive function of marijuana users may also be impaired by deregulation of the endogenous endocannabinoid system.

Lack of horizontal gene transfer of methicillin-resitance genetic determinants from PBP2a-positive, coagulase-negative Staphylococci to methicillin-sensitive Staphylococcus aureus using transcutaneous electric nerve stimulation (TENS),

Previous research shows that approximately half of the coagulase-negative staphylococci (CNS) isolated from patients in the intensive care unit (ICU) at Belfast City Hospital were resistant to methicillin. The presence of this relatively high proportion of methicillin-resistance genetic material gives rise to speculation that these organisms may act as potential reservoirs of methicillinresistance genetic material to methicillin-sensitive Staphylococcus aureus (MSSA). Mechanisms of horizontal gene transfer from PBP2a-positive CNS to MSSA, potentially transforming MSSA to MRSA, aided by electroporation-type activities such as transcutaneous electrical nerve stimulation (TENS), should be considered. Methicillin-resistant CNS (MR-CNS) isolates are collected over a two-month period from a variety of clinical specimen types, particularly wound swabs. The species of all isolates are confirmed, as well as their resistance to oxacillin by standard disc diffusion assays. In addition, MSSA isolates are collected over the same period and confirmed as PBP2a-negative. Electroporation experiments are designed to mimic the time/voltage combinations used commonly in the clinical application of TENS. No transformed MRSA were isolated and all viable S. aureus cells remained susceptible to oxacillin and PBP2a-negative. Experiments using MSSA pre-exposed to sublethal concentrations of oxacillin (0.25 µg/mL) showed no evidence of methicillin gene transfer and the generation of an MRSA. The study showed no evidence of horizontal transfer of methicillin resistance genetic material from MR-CNS to MSSA. These data support the belief that TENS and the associated time/voltage combinations used do not increase conjugational transposons or facilitate horizontal gene transfer from MR-CNS to MSSA.

De novo generation of a non-segmented negative strand RNA virus with a bicistronic gene

Reverse genetics has facilitated the use of non-segmented negative strand RNA viruses (NNSV) as vectors. Currently, heterologous gene expression necessitates insertion of extra-numeral transcription units (ENTUs), which may alter the NNSV polar transcription gradient and attenuate growth relative to wildtype (Wt). We hypothesized that rescuing recombinant Sendai Virus (rSeV) with a bicistronic gene might circumvent this attenuation but still allow heterologous open reading frame (ORF) expression. Therefore, we used a 9-nucleotide sequence previously described with internal ribosome entry site (IRES) activity, which, when constructed as several repeats, synergistically increased the level of expression of the second cistron [Chappell, S.A., Edelman, G.M., Mauro, V.P., 2000. A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity. Proc. Natl. Acad. Sci. U.S.A. 97, 1536-1541]. We inserted the Renilla luciferase (rLuc) ORF, preceded by 1, 3 or 7 IRES copies, downstream of the SeV N ORF in an infectious clone. Corresponding rSeVs were successfully rescued. Interestingly, bicistronic rSeVs grew as fast as or faster than Wt rSeV. Furthermore, SeV gene transcription downstream of the N/rLuc gene was either equivalent to, or slightly enhanced, compared to Wt rSeV. Importantly, all rSeV/rLuc viruses efficiently expressed rLuc. IRES repetition increased rLuc expression at a multiplicity of infection of 0.1, although without evidence of synergistic enhancement. In conclusion, our approach provides a novel way of insertion and expression of foreign genes in NNSVs. (C) 2008 Elsevier B.V. All rights reserved.

Murine model of autosomal dominant retinitis pigmentosa generated by targeted deletion at codon 307 of the rds-peripherin gene

We introduced a targeted single base deletion at codon 307 of the rds-peripherin gene in mice, similar mutations being known to cause autosomal dominant retinitis pigmentosa (RP) in man. Histopathological and electroretinographic analysis indicate that the retinopathy in mice homozygous for the codon 307 mutation appears more rapid than that in the naturally occurring null mutant, the rds(-/-) mouse, suggesting that the rds-307 mutation displays a dominant negative phenotype in combination with that due to haplosufficiency. RP is the most prevalent cause of registered visual handicap in those of working age in developed countries, the 50 or so mutations so far identified within the RDS-peripherin gene accounting for up to 10% of dominant cases of the disease. Given the sequence homologies that exist between the murine rds-peripherin and the human RDS-peripherin gene, this disease model, the first to be generated for peripherin-based RP using gene targeting techniques, should in principle be of value in the work-up in mice of therapeutics capable of targeting transcripts derived from the human gene.

Protein arginine-methyltransferase-dependent oncogenesis

Enzymes that mediate reversible epigenetic modifications have not only been recognized as key in regulating gene expression(1) and oncogenesis(2,3), but also provide potential targets for molecular therapy(4). Although the methylation of arginine 3 of histone 4 ( H4R3) by protein arginine methyltransferase 1 ( PRMT1) is a critical modification for active chromatin(5,6) and prevention of heterochromatin spread(7), there has been no direct evidence of any role of PRMTs in cancer. Here, we show that PRMT1 is an essential component of a novel Mixed Lineage Leukaemia ( MLL) oncogenic transcriptional complex with both histone acetylation and H4R3 methylation activities, which also correlate with the expression of critical MLL downstream targets. Direct fusion of MLL with PRMT1 or Sam68, a bridging molecule in the complex for PRMT1 interaction, could enhance self-renewal of primary haematopoietic cells. Conversely, specific knockdown of PRMT1 or Sam68 expression suppressed MLL-mediated transformation. This study not only functionally dissects the oncogenic transcriptional machinery associated with an MLL fusion complex, but also uncovers-for the first time-an essential function of PRMTs in oncogenesis and reveals their potential as novel therapeutic targets in human cancer.

A GREM1 gene variant associates with diabetic nephropathy

Gremlin, a cell growth and differentiation factor, promotes the development of diabetic nephropathy in animal models, but whether GREM1 gene variants associate with diabetic nephropathy is unknown. We comprehensively screened the 5' upstream region (including the predicted promoter), all exons, intron-exon boundaries, complete untranslated regions, and the 3' region downstream of the GREM1 gene. We identified 31 unique variants, including 24 with a minor allele frequency exceeding 5%, and 9 haplotype-tagging single nucleotide polymorphisms (htSNPs). We selected one additional variant that we predicted to alter transcription factor binding. We genotyped 709 individuals with type 1 diabetes of whom 267 had nephropathy (cases) and 442 had no evidence of kidney disease (controls). Three individual SNPs significantly associated with nephropathy at the 5% level, and two remained significant after adjustment for multiple testing. Subsequently, we genotyped a replicate population comprising 597 cases and 502 controls: this population supported an association with one of the SNPs (rs1129456; P = 0.0003). Combined analysis, adjusted for recruitment center (n = 8), suggested that the T allele conferred greater odds of nephropathy (OR 1.69; 95% CI 1.36 to 2.11). In summary, the GREM1 variant rs1129456 associates with diabetic nephropathy, perhaps explaining some of the genetic susceptibility to this condition.

Genome-wide association identifies a common variant in the reelin gene that increases the risk of schizophrenia only in women

Sex differences in schizophrenia are well known, but their genetic basis has not been identified. We performed a genome-wide association scan for schizophrenia in an Ashkenazi Jewish population using DNA pooling. We found a female-specific association with rs7341475, a SNP in the fourth intron of the reelin ( RELN) gene (p = 2.9 x 10(-5) in women), with a significant gene-sex effect (p = 1.8 x 10(-4)). We studied rs7341475 in four additional populations, totaling 2,274 cases and 4,401 controls. A significant effect was observed only in women, replicating the initial result (p = 2.1 x 10(-3) in women; p = 4.2 x 10(-3) for gene-sex interaction). Based on all populations the estimated relative risk of women carrying the common genotype is 1.58 (p = 8.8 x 10(-7); p = 1.6 x 10(-5) for gene-sex interaction). The female-specific association between RELN and schizophrenia is one of the few examples of a replicated sex-specific genetic association in any disease.

Activated oncogenic pathways and therapeutic targets in extranodal nasal-type NK/T cell lymphoma revealed by gene expression profiling

We performed comprehensive genome-wide gene expression profiling (GEP) of extranodal nasal-type natural killer/T-cell lymphoma (NKTL) using formalin-fixed, paraffin-embedded tissue (n = 9) and NK cell lines (n = 5) in comparison with normal NK cells, with the objective of understanding the oncogenic pathways involved in the pathogenesis of NKTL and to identify potential therapeutic targets. Pathway and network analysis of genes differentially expressed between NKTL and normal NK cells revealed significant enrichment for cell cycle-related genes and pathways, such as PLK1, CDK1, and Aurora-A. Furthermore, our results demonstrated a pro-proliferative and anti-apoptotic phenotype in NKTL characterized by activation of Myc and nuclear factor kappa B (NF-kappa B), and deregulation of p53. In corroboration with GEP findings, a significant percentage of NKTLs (n = 33) overexpressed c-Myc (45.4%), p53 (87.9%), and NF-kappa B p50 (67.7%) on immunohistochemistry using a tissue microarray containing 33 NKTL samples. Notably, overexpression of survivin was observed in 97% of cases. Based on our findings, we propose a model of NKTL pathogenesis where deregulation of p53 together with activation of Myc and NF-kappa B, possibly driven by EBV LMP-1, results in the cumulative up-regulation of survivin. Down-regulation of survivin with Terameprocol (EM-1421, a survivin inhibitor) results in reduced cell viability and increased apoptosis in tumour cells, suggesting that targeting survivin may be a potential novel therapeutic strategy in NKTL. Copyright (C) 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.