A variant of the KLK4 gene is expressed as a cis sense-antisense chimeric transcript in prostate cancer cells

In humans, more than 30,000 chimeric transcripts originating from 23,686 genes have been identified. The mechanisms and association of chimeric transcripts arising from chromosomal rearrangements with cancer are well established, but much remains unknown regarding the biogenesis and importance of other chimeric transcripts that arise from nongenomic alterations. Recently, a SLC45A3–ELK4 chimera has been shown to be androgen-regulated, and is overexpressed in metastatic or high-grade prostate tumors relative to local prostate cancers. Here, we characterize the expression of a KLK4 cis sense–antisense chimeric transcript, and show other examples in prostate cancer. Using non-protein-coding microarray analyses, we initially identified an androgen-regulated antisense transcript within the 3′ untranslated region of the KLK4 gene in LNCaP cells. The KLK4 cis-NAT was validated by strand-specific linker-mediated RT-PCR and Northern blotting. Characterization of the KLK4 cis-NAT by 5′ and 3′ rapid amplification of cDNA ends (RACE) revealed that this transcript forms multiple fusions with the KLK4 sense transcript. Lack of KLK4 antisense promoter activity using reporter assays suggests that these transcripts are unlikely to arise from a trans-splicing mechanism. 5′ RACE and analyses of deep sequencing data from LNCaP cells treated ±androgens revealed six high-confidence sense–antisense chimeras of which three were supported by the cDNA databases. In this study, we have shown complex gene expression at the KLK4 locus that might be a hallmark of cis sense–antisense chimeric transcription.

Identification of a novel prostate cancer susceptibility variant in the KLK3 gene transcript

Abstract Genome-wide association studies (GWAS) have identified more than 30 prostate cancer (PrCa) susceptibility loci. One of these (rs2735839) is located close to a plausible candidate susceptibility gene, KLK3, which encodes prostate-specific antigen (PSA). PSA is widely used as a biomarker for PrCa detection and disease monitoring. To refine the association between PrCa and variants in this region, we used genotyping data from a two-stage GWAS using samples from the UK and Australia, and the Cancer Genetic Markers of Susceptibility (CGEMS) study. Genotypes were imputed for 197 and 312 single nucleotide polymorphisms (SNPs) from HapMap2 and the 1000 Genome Project, respectively. The most significant association with PrCa was with a previously unidentified SNP, rs17632542 (combined P = 3.9 × 10−22). This association was confirmed by direct genotyping in three stages of the UK/Australian GWAS, involving 10,405 cases and 10,681 controls (combined P = 1.9 × 10−34). rs17632542 is also shown to be associated with PSA levels and it is a non-synonymous coding SNP (Ile179Thr) in KLK3. Using molecular dynamic simulation, we showed evidence that this variant has the potential to introduce alterations in the protein or affect RNA splicing. We propose that rs17632542 may directly influence PrCa risk.

Cloning of a novel insulin-regulated ghrelin transcript in prostate cancer

Ghrelin is a multifunctional hormone, with roles in stimulating appetite and regulating energy balance, insulin secretion and glucose homeostasis. The ghrelin gene locus (GHRL) is highly complex and gives rise to a range of novel transcripts derived from alternative first exons and internally spliced exons. The wild-type transcript encodes a 117 amino acid preprohormone that is processed to yield the 28 amino acid peptide ghrelin. Here, we identified insulin-responsive transcription corresponding to cryptic exons in intron 2 of the human ghrelin gene. A transcript, termed in2c-ghrelin (intron 2-cryptic), was cloned from the testis and the LNCaP prostate cancer cell line. This transcript may encode an 83 AA preproghrelin isoform that codes for the ghrelin, but not obestatin. It is expressed in a limited number of normal tissues and in tumours of the prostate, testis, breast and ovary. Finally, we confirmed that in2c-ghrelin transcript expression, as well as the recently described in1-ghrelin transcript, is significantly upregulated by insulin in cultured prostate cancer cells. Metabolic syndrome and hyperinsulinaemia has been associated with prostate cancer risk and progression. This may be particularly significant after androgen deprivation therapy for prostate cancer, which induces hyperinsulinaemia, and this could contribute to castrate resistant prostate cancer growth. We have previously demonstrated that ghrelin stimulates prostate cancer cell line proliferation in vitro. This study is the first description of insulin regulation of a ghrelin transcript in cancer, and should provide further impetus for studies into the expression, regulation and function of ghrelin gene products.

High levels of BACH2 associated with lower levels of BCL2 transcript abundance in t(14;18)(q21;q34) translocation positive non-Hodgkin’s lymphoma

The t(14;18)(q21;q34) BCL2 translocation is a common genetic alteration in follicular and diffuse large B-cell lymphoma. However, it is not invariably associated with BCL2 gene overexpression due to undefined mechanisms that regulate expression from the proximal immunoglobulin heavy-chain (IgH) promoter. The BACH2 transcriptional repressor is able to modulate activity of this promoter. Here we have shown that, in tumor samples with BCL2 translocation, those with high levels of BACH2 had significantly lower BCL2 transcript abundance compared to those with low levels of BACH2. This indicates that BACH2 may be partially responsible for regulation of BCL2 expression from the t(14;18)(q21;q34) translocation.

Transcript and metabolite profiling for the evaluation of tobacco tree and poplar as feedstock for the bio-based industry

The global demand for food, feed, energy and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

The IFITM5 mutation c.-14C > T results in an elongated transcript expressed in human bone; And causes varying phenotypic severity of osteogenesis imperfecta type V

Background The genetic mutation resulting in osteogenesis imperfecta (OI) type V was recently characterised as a single point mutation (c.-14C > T) in the 5' untranslated region (UTR) of IFITM5, a gene encoding a transmembrane protein with expression restricted to skeletal tissue. This mutation creates an alternative start codon and has been shown in a eukaryotic cell line to result in a longer variant of IFITM5, but its expression has not previously been demonstrated in bone from a patient with OI type V. Methods Sanger sequencing of the IFITM5 5' UTR was performed in our cohort of subjects with a clinical diagnosis of OI type V. Clinical data was collated from referring clinicians. RNA was extracted from a bone sample from one patient and Sanger sequenced to determine expression of wild-type and mutant IFITM5. Results: All nine subjects with OI type V were heterozygous for the c.-14C > T IFITM5 mutation. Clinically, there was heterogeneity in phenotype, particularly in the manifestation of bone fragility amongst subjects. Both wild-type and mutant IFITM5 mRNA transcripts were present in bone. Conclusions The c.-14C > T IFITM5 mutation does not result in an RNA-null allele but is expressed in bone. Individuals with identical mutations in IFITM5 have highly variable phenotypic expression, even within the same family.

Identification of a novel fusion transcript between human relaxin-1 (RLN1) and human relaxin-2 (RLN2) in prostate cancer

Simultaneous expression of highly homologous RLN1 and RLN2 genes in prostate impairs their accurate delineation. We used PacBio SMRT sequencing and RNA-Seq in LNCaP cells in order to dissect the expression of RLN1 and RLN2 variants. We identified a novel fusion transcript comprising the RLN1 and RLN2 genes and found evidence of its expression in the normal and prostate cancer tissues. The RLN1-RLN2 fusion putatively encodes RLN2 isoform with the deleted secretory signal peptide. The identification of the fusion transcript provided information to determine unique RLN1-RLN2 fusion and RLN1 regions. The RLN1-RLN2 fusion was co-expressed with RLN1 in LNCaP cells, but the two gene products were inversely regulated by androgens. We showed that RLN1 is underrepresented in common PCa cell lines in comparison to normal and PCa tissue. The current study brings a highly relevant update to the relaxin field, and will encourage further studies of RLN1 and RLN2 in PCa and broader.

A linear programming approach for estimating the structure of a sparse linear genetic network from transcript profiling data

Background: A genetic network can be represented as a directed graph in which a node corresponds to a gene and a directed edge specifies the direction of influence of one gene on another. The reconstruction of such networks from transcript profiling data remains an important yet challenging endeavor. A transcript profile specifies the abundances of many genes in a biological sample of interest. Prevailing strategies for learning the structure of a genetic network from high-dimensional transcript profiling data assume sparsity and linearity. Many methods consider relatively small directed graphs, inferring graphs with up to a few hundred nodes. This work examines large undirected graphs representations of genetic networks, graphs with many thousands of nodes where an undirected edge between two nodes does not indicate the direction of influence, and the problem of estimating the structure of such a sparse linear genetic network (SLGN) from transcript profiling data. Results: The structure learning task is cast as a sparse linear regression problem which is then posed as a LASSO (l1-constrained fitting) problem and solved finally by formulating a Linear Program (LP). A bound on the Generalization Error of this approach is given in terms of the Leave-One-Out Error. The accuracy and utility of LP-SLGNs is assessed quantitatively and qualitatively using simulated and real data. The Dialogue for Reverse Engineering Assessments and Methods (DREAM) initiative provides gold standard data sets and evaluation metrics that enable and facilitate the comparison of algorithms for deducing the structure of networks. The structures of LP-SLGNs estimated from the INSILICO1, INSILICO2 and INSILICO3 simulated DREAM2 data sets are comparable to those proposed by the first and/or second ranked teams in the DREAM2 competition. The structures of LP-SLGNs estimated from two published Saccharomyces cerevisae cell cycle transcript profiling data sets capture known regulatory associations. In each S. cerevisiae LP-SLGN, the number of nodes with a particular degree follows an approximate power law suggesting that its degree distributions is similar to that observed in real-world networks. Inspection of these LP-SLGNs suggests biological hypotheses amenable to experimental verification. Conclusion: A statistically robust and computationally efficient LP-based method for estimating the topology of a large sparse undirected graph from high-dimensional data yields representations of genetic networks that are biologically plausible and useful abstractions of the structures of real genetic networks. Analysis of the statistical and topological properties of learned LP-SLGNs may have practical value; for example, genes with high random walk betweenness, a measure of the centrality of a node in a graph, are good candidates for intervention studies and hence integrated computational – experimental investigations designed to infer more realistic and sophisticated probabilistic directed graphical model representations of genetic networks. The LP-based solutions of the sparse linear regression problem described here may provide a method for learning the structure of transcription factor networks from transcript profiling and transcription factor binding motif data.

Fusion transcript loci share many genomic features with non-fusion loci

Background Fusion transcripts are found in many tissues and have the potential to create novel functional products. Here, we investigate the genomic sequences around fusion junctions to better understand the transcriptional mechanisms mediating fusion transcription/splicing. We analyzed data from prostate (cancer) cells as previous studies have shown extensively that these cells readily undergo fusion transcription. Results We used the FusionMap program to identify high-confidence fusion transcripts from RNAseq data. The RNAseq datasets were from our (N = 8) and other (N = 14) clinical prostate tumors with adjacent non-cancer cells, and from the LNCaP prostate cancer cell line that were mock-, androgen- (DHT), and anti-androgen- (bicalutamide, enzalutamide) treated. In total, 185 fusion transcripts were identified from all RNAseq datasets. The majority (76 %) of these fusion transcripts were ‘read-through chimeras’ derived from adjacent genes in the genome. Characterization of sequences at fusion loci were carried out using a combination of the FusionMap program, custom Perl scripts, and the RNAfold program. Our computational analysis indicated that most fusion junctions (76 %) use the consensus GT-AG intron donor-acceptor splice site, and most fusion transcripts (85 %) maintained the open reading frame. We assessed whether parental genes of fusion transcripts have the potential to form complementary base pairing between parental genes which might bring them into physical proximity. Our computational analysis of sequences flanking fusion junctions at parental loci indicate that these loci have a similar propensity as non-fusion loci to hybridize. The abundance of repetitive sequences at fusion and non-fusion loci was also investigated given that SINE repeats are involved in aberrant gene transcription. We found few instances of repetitive sequences at both fusion and non-fusion junctions. Finally, RT-qPCR was performed on RNA from both clinical prostate tumors and adjacent non-cancer cells (N = 7), and LNCaP cells treated as above to validate the expression of seven fusion transcripts and their respective parental genes. We reveal that fusion transcript expression is similar to the expression of parental genes. Conclusions Fusion transcripts maintain the open reading frame, and likely use the same transcriptional machinery as non-fusion transcripts as they share many genomic features at splice/fusion junctions.

A Transcript Cleavage Factor of Mycobacterium tuberculosis Important for Its Survival

After initiation of transcription, a number of proteins participate during elongation and termination modifying the properties of the RNA polymerase (RNAP). Gre factors are one such group conserved across bacteria. They regulate transcription by projecting their N-terminal coiled-coil domain into the active center of RNAP through the secondary channel and stimulating hydrolysis of the newly synthesized RNA in backtracked elongation complexes. Rv1080c is a putative gre factor (MtbGre) in the genome of Mycobacterium tuberculosis. The protein enhanced the efficiency of promoter clearance by lowering abortive transcription and also rescued arrested and paused elongation complexes on the GC rich mycobacterial template. Although MtbGre is similar in domain organization and shares key residues for catalysis and RNAP interaction with the Gre factors of Escherichia coli, it could not complement an E. coli gre deficient strain. Moreover, MtbGre failed to rescue E. coli RNAP stalled elongation complexes, indicating the importance of specific protein-protein interactions for transcript cleavage. Decrease in the level of MtbGre reduced the bacterial survival by several fold indicating its essential role in mycobacteria. Another Gre homolog, Rv3788 was not functional in transcript cleavage activity indicating that a single Gre is sufficient for efficient transcription of the M. tuberculosis genome.

The requirements for Ca2+, protein phosphorylation and concurrent protein synthesis for zeatin signaling of acidic chitinase transcript accumulation in Cucumis sativus L

We have previously reported that both Ca2+ and staurosporine-sensitive protein kinase(s) are involved in the cytokinin zeatin induction of cucumber chitinase activity and its protein content (Barwe et al. 2001). To further characterize signal transduction events involved in this cytokinin induction of chitinase gene expression, Northern hybridizations of total RNAs prepared from excised, dark-grown cucumber cotyledons treated with cytokinins and/or various agonists and antagonists of signal transduction components, were carried out using a cucumber acidic chitinase (CACHT) cDNA probe (Metraux et al. 1989). CACHT mRNA increased by approximately 5- to 6-fold in response to exogenous zeatin (Z), zeatin riboside (ZR), and benzyladenine (BA) treatment, but failed to accumulate in response to kinetin (K). Among the cytokinins tested, Z was most effective. The Z-induced accumulation of CACHT mRNA was inhibited by a plasma membrane Ca2+ channel blocker verapamil. Treatment of cotyledons with exogenous CaCl2 and calcium ionophore A23187 in the presence and absence of cytokinin enhanced CACHT mRNA accumulation. These two observations suggest the participation of extracellular calcium in signaling Z-induction. Furthermore, the presence of staurosporine (an inhibitor of protein kinase) in Z treatment reduced CACHT mRNA, suggesting the involvement of phosphorylation of one or more cellular proteins. In addition, we provide evidence that the Z-induction of CACHT mRNA is blocked by protein synthesis inhibitor cycloheximide treatment. Taken together, these results suggest that Ca2+ influx from extracellular space, protein phosphorylation, and concurrent protein synthesis events participate in cytokinin signaling during Z-induced CACHT transcript accumulation.

Identification of a core promoter and a novel isoform of the human TSCI gene transcript and structural comparison with mouse homolog

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder with loci on chromosome 9q34.12 (TSC1) and chromosome 16p13.3 (TSC2). Genes for both loci have been isolated and characterized. The promoters of both genes have not been characterized so far and little is known about the regulation of these genes. This study reports the characterization of the human TSC1 promoter region for the first time. We have identified a novel alternative isoform in the 5' untranslated region (UTR) of the TSC1 gene transcript involving exon 1. Alternative isoforms in the 5' UTR of the mouse Tsc1 gene transcript involving exon I and exon 2 have also been identified. We have identified three upstream open reading frames (uORFs) in the 5' UTR of the TSC1/Tsc1 gene. A comparative study of the 5' UTR of TSC1/Tsc1 gene has revealed that there is a high degree of similarity not only in the sequence but also in the splicing pattern of both human and mouse TSC1 genes. We have used PCR methodology to isolate approximately 1.6 kb genomic DNA 5' to the TSC1 cDNA. This sequence has directed a high level of expression of luciferase activity in both HeLa and HepG2 cells. Successive 5' and 3' deletion analysis has suggested that a -587 bp region, from position +77 to -510 from the transcription start site (TSS), contains the promoter activity. Interestingly, this region contains no consensus TATA box or CAAT box. However, a 521-bp fragment surrounding the TSS exhibits the characteristics of a CpG island which overlaps with the promoter region. The identification of the TSC1 promoter region will help in designing a suitable strategy to identify mutations in this region in patients who do not show any mutations in the coding regions. It will also help to study the regulation of the TSC1 gene and its role in tumorigenesis. (C) 2003 Elsevier B.V. All rights reserved.

Higher topoisomerase 2 alpha gene transcript levels predict better prognosis in GBM patients receiving temozolomide chemotherapy: identification of temozolomide as a TOP2A inhibitor

The search for molecular markers which predict response to chemotherapy is an important aspect of current neuro-oncology research. MGMT promoter methylation is the only proved marker of glioblastoma. The purpose of this study was to assess the effect of topoisomerase expression on glioblastoma survival and study the mechanisms involved. The transcript levels of all isoforms of the topoisomerase family in all grades of diffuse astrocytoma were assessed. A prospective study of patients with glioblastoma treated by a uniform treatment procedure was performed with the objective of correlating outcome with gene expression. The ability of TOP2A enzyme to relax the super coiled plasmid DNA in the presence of temozolomide was evaluated to assess its effect on TOP2A. The temozolomide cyctotoxicity of TOP2A-silenced U251 cells was assessed. The transcript levels of TOP2A, TOP2B, and TOP3A are upregulated significantly in GBM in comparison with lower grades of astrocytoma and normal brain samples. mRNA levels of TOP2A correlated significantly with survival of the patients. Higher TOP2A transcript levels in GBM patients predicted better prognosis (P = 0.043; HR = 0.889). Interestingly, we noted that temozolomide inhibited TOP2A activity in in-vitro enzyme assays. We also noted that siRNA knock down of TOP2A rendered a glioma cell line resistant to temozolomide chemotherapy. We demonstrated for the first time that temozolomide is also a TOP2A inhibitor and established that TOP2A transcript levels determine the chemosensitivity of glioblastoma to temozolomide therapy. Very high levels of TOP2A are a good prognostic indicator in GBM patients receiving temozolomide chemotherapy.