Intergenic disease-associated regions are abundant in novel transcripts.

BACKGROUND: Genotyping of large populations through genome-wide association studies (GWAS) has successfully identified many genomic variants associated with traits or disease risk. Unexpectedly, a large proportion of GWAS single nucleotide polymorphisms (SNPs) and associated haplotype blocks are in intronic and intergenic regions, hindering their functional evaluation. While some of these risk-susceptibility regions encompass cis-regulatory sites, their transcriptional potential has never been systematically explored. RESULTS: To detect rare tissue-specific expression, we employed the transcript-enrichment method CaptureSeq on 21 human tissues to identify 1775 multi-exonic transcripts from 561 intronic and intergenic haploblocks associated with 392 traits and diseases, covering 73.9 Mb (2.2%) of the human genome. We show that a large proportion (85%) of disease-associated haploblocks express novel multi-exonic non-coding transcripts that are tissue-specific and enriched for GWAS SNPs as well as epigenetic markers of active transcription and enhancer activity. Similarly, we captured transcriptomes from 13 melanomas, targeting nine melanoma-associated haploblocks, and characterized 31 novel melanoma-specific transcripts that include fusion proteins, novel exons and non-coding RNAs, one-third of which showed allelically imbalanced expression. CONCLUSIONS: This resource of previously unreported transcripts in disease-associated regions ( http://gwas-captureseq.dingerlab.org ) should provide an important starting point for the translational community in search of novel biomarkers, disease mechanisms, and drug targets.

Gonadotropin suppression in men leads to a reduction in claudin-11 at the Sertoli cell tight junction.

STUDY QUESTION: Are Sertoli cell tight junctions (TJs) disrupted in men undergoing hormonal contraception? SUMMARY ANSWER: Localization of the key Sertoli cell TJ protein, claudin-11, was markedly disrupted by 8 weeks of gonadotropin suppression, the degree of which was related to the extent of adluminal germ cell suppression. WHAT IS KNOWN ALREADY: Sertoli cell TJs are vital components of the blood-testis barrier (BTB) that sequester developing adluminal meiotic germ cells and spermatids from the vascular compartment. Claudin-11 knockout mice are infertile; additionally claudin-11 is spatially disrupted in chronically gonadotropin-suppressed rats coincident with a loss of BTB function, and claudin-11 is disorganized in various human testicular disorders. These data support the Sertoli cell TJ as a potential site of hormonal contraceptive action. STUDY DESIGN, SIZE, DURATION: BTB proteins were assessed by immunohistochemistry (n = 16 samples) and mRNA (n = 18 samples) expression levels in available archived testis tissue from a previous study of 22 men who had undergone 8 weeks of gonadotropin suppression and for whom meiotic and post-meiotic germ cell numbers were available. The gonadotropin suppression regimens were (i) testosterone enanthate (TE) plus the GnRH antagonist, acyline (A); (ii) TE + the progestin, levonorgestrel, (LNG); (iii) TE + LNG + A or (iv) TE + LNG + the 5α-reductase inhibitor, dutasteride (D). A control group consisted of seven additional men, with three archived samples available for this study. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Immunohistochemical localization of claudin-11 (TJ) and other junctional type markers [ZO-1 (cytoplasmic plaque), ß-catenin (adherens junction), connexin-43 (gap junction), vinculin (ectoplasmic specialization) and ß-actin (cytoskeleton)] and quantitative PCR was conducted using matched frozen testis tissue. MAIN RESULTS AND THE ROLE OF CHANCE: Claudin-11 formed a continuous staining pattern at the BTB in control men. Regardless of gonadotropin suppression treatment, claudin-11 localization was markedly disrupted and was broadly associated with the extent of meiotic/post-meiotic germ cell suppression; claudin-11 staining was (i) punctate (i.e. 'spotty' appearance) at the basal aspect of tubules when the average numbers of adluminal germ cells were <15% of control, (ii) presented as short fragments with cytoplasmic extensions when numbers were 15-25% of control or (iii) remained continuous when numbers were >40% of control. Changes in localization of connexin-43 and vinculin were also observed (smaller effects than for claudin-11) but ZO-1, ß-catenin and ß-actin did not differ, compared with control. LIMITATIONS, REASONS FOR CAUTION: Claudin-11 was the only Sertoli cell TJ protein investigated, but it is considered to be the most pivotal of constituent proteins given its known implication in infertility and BTB function. We were limited to testis samples which had been gonadotropin-suppressed for 8 weeks, shorter than the 74-day spermatogenic wave, which may account for the heterogeneity in claudin-11 and germ cell response observed among the men. Longer suppression (12-24 weeks) is known to suppress germ cells further and claudin-11 disruption may be more uniform, although we could not access such samples. WIDER IMPLICATIONS OF THE FINDINGS: These findings are important for our understanding of the sites of action of male hormonal contraception, because they suggest that BTB function could be ablated following long-term hormone suppression treatment. STUDY FUNDING/COMPETING INTERESTS: National Health and Medical Research Council (Australia) Program Grants 241000 and 494802; Research Fellowship 1022327 (to R.I.M.) and the Victorian Government's Operational Infrastructure Support Program. None of the authors have any conflicts to disclose. TRIAL REGISTRATION NUMBER: Not applicable.

Long noncoding RNAs and the genetics of cancer.

Cancer is a disease of aberrant gene expression. While the genetic causes of cancer have been intensively studied, it is becoming evident that a large proportion of cancer susceptibility cannot be attributed to variation in protein-coding sequences. This is highlighted by genome-wide association studies in cancer that reveal that more than 80% of cancer-associated SNPs occur in noncoding regions of the genome. In this review, we posit that a significant fraction of the genetic aetiology of cancer is exacted by noncoding regulatory sequences, particularly by long noncoding RNAs (lncRNAs). Recent studies indicate that several cancer risk loci are transcribed into lncRNAs and these transcripts play key roles in tumorigenesis. We discuss the epigenetic and other mechanisms through which lncRNAs function and how they contribute to each stage of cancer progression, understanding of which will be crucial for realising new opportunities in cancer diagnosis and treatment. Long noncoding RNAs play important roles in almost every aspect of cell biology from nuclear organisation and epigenetic regulation to post-transcriptional regulation and splicing, and we link these processes to the hallmarks and genetics of cancer. Finally, we highlight recent progress and future potential in the application of lncRNAs as therapeutic targets and diagnostic markers.

Growth phase-dependent expression and degradation of histones in the thermophilic archaeon Thermococcus zilligii.

HTz is a member of the archaeal histone family. The archaeal histones have primary sequences and structural similarity to the eukaryal histone fold domain, and are thought to resemble the archetypal ancestor of the eukaryal nucleosome core histones. The effects of growth phase on the total soluble proteins from Thermococcus zilligii, isolated after various stages of growth from mid-logarithmic to late stationary phase, were examined by denaturing polyacrylamide gel electrophoresis. On entry into stationary phase, at least 11 proteins were detected that changed considerably in level. One of these proteins was identified by Western hybridization as HTz. The level of HTz decreased dramatically as cells entered stationary phase, and it could not be detected by late stationary phase. Unexpectedly, the Western hybridization detected a second protein, with an estimated molecular mass of approximately 14 kDa, which paralleled the decrease in level of HTz. Native purified HTz was shown to retain complete activity after prolonged incubation at the growth temperature of the organism, suggesting that the decrease in HTz was a specific cell-regulated process. Analysis of native purified HTz by electrospray ionization mass spectrometry revealed the molecular masses of HTz1 and HTz2 to be 7204 +/- 3 Da and 7016 +/- 3 Da respectively. The only non-covalent species that was detected corresponded to the molecular mass of an HTz1-HTz2 heterodimer. Northern analyses of T. zilligii total RNA with an htz1 gene probe indicated a rapid decrease in expression of htz1 with progression of the growth phase, and complete repression of htz1 transcript synthesis by late logarithmic phase. Three proteins that changed in level with growth phase were identified by N-terminal sequence analysis. The first was homologous to a hypothetical protein conserved in all Archaea sequenced to date, the second to the Sac10b family of archaeal DNA-binding proteins and the third to the C-terminal region of the leucine-responsive regulatory family of DNA-binding proteins (LRPs).

Identification of archaeal genes encoding a novel stationary phase-response protein.

A novel stationary phase-response protein has been identified in the acid-soluble protein extract of the thermophilic archaeon, Thermococcus zilligii. N-Terminal sequencing data were used to identify likely genes for homologues of the protein in the complete genome sequences of various archaeal species. The corresponding genes were identified and analysed. The genes encode a protein ranging from 83 to 92 amino acids in length, with a calculated pI ranging from 4.6 to 9.7. The amino acid sequences of the genes were highly conserved, even between members belonging to the different archaeal kingdoms. The computed secondary structure of the protein indicates it consists of four large helical regions separated by short coiled regions. We propose this protein as a candidate regulator of gene expression in stationary phase.