Enhanced bovine genome annotation through integration of transcriptomics and epi-transcriptomics datasets facilitates genomic biology.

BACKGROUND: The accurate identification of the functional elements in the bovine genome is a fundamental requirement for high-quality analysis of data informing both genome biology and genomic selection. Functional annotation of the bovine genome was performed to identify a more complete catalog of transcript isoforms across bovine tissues. RESULTS: A total of 160,820 unique transcripts (50% protein coding) representing 34,882 unique genes (60% protein coding) were identified across tissues. Among them, 118,563 transcripts (73% of the total) were structurally validated by independent datasets (PacBio isoform sequencing data, Oxford Nanopore Technologies sequencing data, de novo assembled transcripts from RNA sequencing data) and comparison with Ensembl and NCBI gene sets. In addition, all transcripts were supported by extensive data from different technologies such as whole transcriptome termini site sequencing, RNA Annotation and Mapping of Promoters for the Analysis of Gene Expression, chromatin immunoprecipitation sequencing, and assay for transposase-accessible chromatin using sequencing. A large proportion of identified transcripts (69%) were unannotated, of which 86% were produced by annotated genes and 14% by unannotated genes. A median of two 5' untranslated regions were expressed per gene. Around 50% of protein-coding genes in each tissue were bifunctional and transcribed both coding and noncoding isoforms. Furthermore, we identified 3,744 genes that functioned as noncoding genes in fetal tissues but as protein-coding genes in adult tissues. Our new bovine genome annotation extended more than 11,000 annotated gene borders compared to Ensembl or NCBI annotations. The resulting bovine transcriptome was integrated with publicly available quantitative trait loci data to study tissue-tissue interconnection involved in different traits and construct the first bovine trait similarity network. CONCLUSIONS: These validated results show significant improvement over current bovine genome annotations.

Jagged-1 is induced by mTOR inhibitors in renal cancer cells through an Akt/ALK5/Smad4-dependent mechanism.

The mammalian target of rapamycin (mTOR) plays an important role in the aggressiveness and therapeutic resistance of many cancers. Targeting mTOR continues to be under clinical investigation for cancer therapy. Despite the notable clinical success of mTOR inhibitors in extending the overall survival of patients with certain malignancies including metastatic renal cell carcinomas (RCCs), the overall impact of mTOR inhibitors on cancers has been generally disappointing and attributed to various compensatory responses. Here we provide the first report that expression of the Notch ligand Jagged-1 (JAG1), which is associated with aggressiveness of RCCs, is induced by several inhibitors of mTOR (rapamycin (Rap), BEZ235, KU-0063794) in human clear cell RCC (ccRCC) cells. Using both molecular and chemical inhibitors of PI3K, Akt, and TGF-ß signaling, we provide evidence that the induction of JAG1 expression by mTOR inhibitors in ccRCC cells depends on the activation of Akt and occurs through an ALK5 kinase/Smad4-dependent mechanism. Furthermore, we show that mTOR inhibitors activate Notch1 and induce the expression of drivers of epithelial-mesenchymal transition, notably Hic-5 and Slug. Silencing JAG1 with selective shRNAs blocked the ability of KU-0063794 and Rap to induce Hic-5 in ccRCC cells. Moreover, Rap enhanced TGF-ß-induced expression of Hic-5 and Slug, both of which were repressed in JAG1-silenced ccRCC cells. Silencing JAG1 selectively decreased the motility of ccRCC cells treated with Rap or TGF-ß1. Moreover, inhibition of Notch signaling with γ-secretase inhibitors enhanced or permitted mTOR inhibitors to suppress the motility of ccRCC cells. We suggest targeting JAG1 may enhance therapeutic responses to mTOR inhibitors in ccRCCs.

Hypoxia represses early responses of prostate and renal cancer cells to YM155 independent of HIF-1α and HIF-2α.

The imidazolium compound Sepantronium Bromide (YM155) successfully promotes tumor regression in various pre-clinical models but has shown modest responses in human clinical trials. We provide evidence to support that the hypoxic milieu of tumors may limit the clinical usefulness of YM155. Hypoxia (1% O2) strongly (>16-fold) represses the cytotoxic activity of YM155 on prostate and renal cancer cells in vitro. Hypoxia also represses all early signaling responses associated with YM155, including activation of AMPK and retinoblastoma protein (Rb), inactivation of the mechanistic target of rapamycin complex 1 (mTORC1), inhibition of phospho-ribosomal protein S6 (rS6), and suppression of the expression of Cyclin Ds, Mcl-1 and Survivin. Cells pre-incubated with hypoxia for 24 â€‹h are desensitized to YM155 even when they are treated with YM155 under atmospheric oxygen conditions, supporting that cells at least temporarily retain hypoxia-induced resistance to YM155. We tested the role of hypoxia-inducible factor (HIF)-1α and HIF-2α in the hypoxia-induced resistance to YM155 by comparing responses of YM155 in VHL-proficient versus VHL-deficient RCC4 and 786-O renal cancer cells and silencing HIF expression in PC-3 prostate cancer cells. Those studies suggested that hypoxia-induced resistance to YM155 occurs independent of HIF-1α and HIF-2α. Moreover, the hypoxia mimetics deferoxamine and dimethyloxalylglycine, which robustly induce HIF-1α levels in PC-3 â€‹cells under atmospheric oxygen, did not diminish their early cellular responses to YM155. Collectively, our data support that hypoxia induces resistance of cells to YM155 through a HIF-1α and HIF-2α-independent mechanism. We hypothesize that a hypothetical hypoxia-inducer factor (HIF-X) represses early signaling responses to YM155.

A Signaling Network Controlling Androgenic Repression of c-Fos Protein in Prostate Adenocarcinoma Cells.

The transcription factor c-Fos controls many important cellular processes, including cell growth and apoptosis. c-Fos expression is rapidly elevated in the prostate upon castration-mediated androgen withdrawal through an undefined mechanism. Here we show that androgens (5α-dihydrotestosterone and R1881) suppress c-Fos protein and mRNA expression induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) or EGF in human prostate cancer (PCa) cell lines. Such suppression transpires through a transcriptional mechanism, predominantly at the proximal serum response element of the c-fos promoter. We show that androgen signaling suppresses TPA-induced c-Fos expression through repressing a PKC/MEK/ERK/ELK-1 signaling pathway. Moreover, our results support the hypothesis that p38(MAPK), PI3K, and PKCδ are involved in the androgenic regulation of c-Fos through controlling MEK/ERK. Stable silencing of c-Fos and PKCδ with shRNAs suggests that R1881 promotes cell death induced by low-dose TPA through a mechanism that is dependent on both PKCδ and loss of c-Fos expression. Reciprocally, loss of either PKCδ or c-Fos activates p38(MAPK) while suppressing the activation of ERK1/2. We also provide the first demonstration that R1881 permits cell death induced by low-dose TPA in the LNCaP androgen-dependent PCa cell line and that TPA-induced cell death is independent of exogenous androgen in the castration-resistant variants of LNCaP, C4-2 and C4-2B. Acquisition of androgen-independent killing by TPA correlates with activation of p38(MAPK), suppression of ERK1/2, and loss of c-Fos. These results provide new insights into androgenic control of c-Fos and use of PKC inhibitors in PCa therapy.