Showing papers by "Phillip A. Sharp published in 2011"

MicroRNAs can generate thresholds in target gene expression

Abstract: Alexander van Oudenaarden and colleagues examine microRNA-mediated regulation of gene expression using single-cell measurements of a target gene's expression. They find that microRNAs can repress gene expression either as a switch or through fine-tuning and that the strength of repression can vary widely between cells.

Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs

Abstract: MicroRNAs (miRNAs) are 19-22-nucleotide noncoding RNAs that post-transcriptionally regulate mRNA targets. We have identified endogenous miRNA binding sites in mouse embryonic stem cells (mESCs), by performing photo-cross-linking immunoprecipitation using antibodies to Argonaute (Ago2) followed by deep sequencing of RNAs (CLIP-seq). We also performed CLIP-seq in Dicer⁻/⁻ mESCs that lack mature miRNAs, allowing us to define whether the association of Ago2 with the identified sites was miRNA dependent. A significantly enriched motif, GCACUU, was identified only in wild-type mESCs in 3' untranslated and coding regions. This motif matches the seed of a miRNA family that constitutes ~68% of the mESC miRNA population. Unexpectedly, a G-rich motif was enriched in sequences cross-linked to Ago2 in both the presence and absence of miRNAs. Expression analysis and reporter assays confirmed that the seed-related motif confers miRNA-directed regulation on host mRNAs and that the G-rich motif can modulate this regulation.

Antisense RNA polymerase II divergent transcripts are P-TEFb dependent and substrates for the RNA exosome

Abstract: Divergent transcription occurs at the majority of RNA polymerase II (RNAPII) promoters in mouse embryonic stem cells (mESCs), and this activity correlates with CpG islands. Here we report the characterization of upstream antisense transcription in regions encoding transcription start site associated RNAs (TSSa-RNAs) at four divergent CpG island promoters: Isg20l1, Tcea1, Txn1, and Sf3b1. We find that upstream antisense RNAs (uaRNAs) have distinct capped 5′ termini and heterogeneous nonpolyadenylated 3′ ends. uaRNAs are short-lived with average half-lives of 18 minutes and are present at 1–4 copies per cell, approximately one RNA per DNA template. Exosome depletion stabilizes uaRNAs. These uaRNAs are probably initiation products because their capped termini correlate with peaks of paused RNAPII. The pausing factors NELF and DSIF are associated with these antisense polymerases and their sense partners. Knockdown of either NELF or DSIF results in an increase in the levels of uaRNAs. Consistent with P-TEFb controlling release from pausing, treatment with its inhibitor, flavopiridol, decreases uaRNA and nascent mRNA transcripts with similar kinetics. Finally, Isg20l1 induction reveals equivalent increases in transcriptional activity in sense and antisense directions. Together these data show divergent polymerases are regulated after P-TEFb recruitment with uaRNA levels controlled by the exosome.

Mir-290–295 deficiency in mice results in partially penetrant embryonic lethality and germ cell defects

Abstract: Mir-290 through mir-295 (mir-290–295) is a mammalian-specific microRNA (miRNA) cluster that, in mice, is expressed specifically in early embryos and embryonic germ cells. Here, we show that mir-290–295 plays important roles in embryonic development as indicated by the partially penetrant lethality of mutant embryos. In addition, we show that in surviving mir-290–295-deficient embryos, female but not male fertility is compromised. This impairment in fertility arises from a defect in migrating primordial germ cells and occurs equally in male and female mutant animals. Male mir-290–295−/− mice, due to the extended proliferative lifespan of their germ cells, are able to recover from this initial germ cell loss and are fertile. Female mir-290–295−/− mice are unable to recover and are sterile, due to premature ovarian failure.

tRNAs Marked with CCACCA Are Targeted for Degradation

Abstract: The CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase] adds CCA to the 3' ends of transfer RNAs (tRNAs), a critical step in tRNA biogenesis that generates the amino acid attachment site. We found that the CCA-adding enzyme plays a key role in tRNA quality control by selectively marking structurally unstable tRNAs and tRNA-like small RNAs for degradation. Instead of adding CCA to the 3' ends of these transcripts, CCA-adding enzymes from all three kingdoms of life add CCACCA. In addition, hypomodified mature tRNAs are subjected to CCACCA addition as part of a rapid tRNA decay pathway in vivo. We conjecture that CCACCA addition is a universal mechanism for controlling tRNA levels and preventing errors in translation.

Promoting Convergence in Biomedical Science

Abstract: The next challenge for biomedical research will be to solve problems of highly complex and integrated biological systems within the human body. Predictive models of these systems in either normal or disease states are beyond the capability of current knowledge and technology.

A Latent Pro-Survival Function for the Mir-290-295 Cluster in Mouse Embryonic Stem Cells

Abstract: MicroRNAs (miRNAs) post-transcriptionally regulate the expression of thousands of distinct mRNAs. While some regulatory interactions help to maintain basal cellular functions, others are likely relevant in more specific settings, such as response to stress. Here we describe such a role for the mir-290-295 cluster, the dominant miRNA cluster in mouse embryonic stem cells (mESCs). Examination of a target list generated from bioinformatic prediction, as well as expression data following miRNA loss, revealed strong enrichment for apoptotic regulators, two of which we validated directly: Caspase 2, the most highly conserved mammalian caspase, and Ei24, a p53 transcriptional target. Consistent with these predictions, mESCs lacking miRNAs were more likely to initiate apoptosis following genotoxic exposure to gamma irradiation or doxorubicin. Knockdown of either candidate partially rescued this pro-apoptotic phenotype, as did transfection of members of the mir290-295 cluster. These findings were recapitulated in a specific mir-290-295 deletion line, confirming that they reflect miRNA functions at physiological levels. In contrast to the basal regulatory roles previously identified, the pro-survival phenotype shown here may be most relevant to stressful gestations, where pro-oxidant metabolic states induce DNA damage. Similarly, this cluster may mediate chemotherapeutic resistance in a neoplastic context, making it a useful clinical target.

Nanoparticle-mediated delivery of siRNA targeting Parp1 extends survival of mice bearing tumors derived from Brca1-deficient ovarian cancer cells

Abstract: Inhibition of the DNA repair enzyme poly(ADP-ribose) polymerase 1 (PARP1) with small molecules has been shown to be an effective treatment for ovarian cancer with BRCA mutations. Here, we report the in vivo administration of siRNA to Parp1 in mouse models of ovarian cancer. A unique member of the lipid-like materials known as lipidoids is shown to deliver siRNA to disseminated murine ovarian carcinoma allograft tumors following intraperitoneal (i.p.) injection. siParp1 inhibits cell growth, primarily by induction of apoptosis, in Brca1-deficient cells both in vitro and in vivo. Additionally, the treatment extends the survival of mice bearing tumors derived from Brca1-deficient ovarian cancer cells but not from Brca1 wild-type cells, confirming the proposed mechanism of synthetic lethality. Because there are 17 members of the Parp family, the inherent complementarity of RNA affords a high level of specificity for therapeutically addressing Parp1 in the context of impaired homologous recombination.

MicroRNAs can generate thresholds in target gene expression

Abstract: Alexander van Oudenaarden and colleagues examine microRNA-mediated regulation of gene expression using single-cell measurements of a target gene's expression. They find that microRNAs can repress gene expression either as a switch or through fine-tuning and that the strength of repression can vary widely between cells.

Hypoxia inhibits hepcidin expression in HuH7 hepatoma cells via decreased SMAD4 signaling

Abstract: Hepcidin negatively regulates systemic iron homeostasis in response to inflammation and elevated serum iron. Conversely, hepcidin expression is diminished in response to hypoxia, oxidative stress, and increased erythropoietic demand, though the molecular intermediates involved are incompletely understood. To address this, we have investigated hypoxic hepcidin regulation in HuH7 hepatoma cells either cultured alone or cocultured with activated THP-1 macrophages. HuH7 hepcidin mRNA expression was determined using quantitative polymerase chain reaction (Q-PCR). Hepcidin promoter activity was measured using luciferase reporter constructs containing a 0.9 kb fragment of the wild-type human hepcidin promoter, and constructs containing mutations in bone morphogenetic protein (BMP)/SMAD4, signal transducer and activator of transcription 3 (STAT3), CCAAT/enhancer-binding protein (C/EBP), and E-box-responsive elements. Hepatic expression of bone morphogenetic proteins BMP2 and BMP6 and the BMP inhibitor noggin was determined using Q-PCR, and the protein expression of hemojuvelin (HJV), pSMAD 1/5/8, and SMAD4 was determined by western blotting. Following exposure to hypoxia or H2O2, hepcidin mRNA expression and promoter activity increased in HuH7 cells monocultures but were decreased in HuH7 cells cocultured with THP-1 macrophages. This repression was attenuated by mutation of the BMP/SMAD4-response element, suggesting that modulation of SMAD signaling mediated the response to hypoxia. No changes in hepatocyte BMP2, BMP6 or noggin mRNA, or protein expression of HJV or pSMAD 1/5/8 were detected. However, treatment with hypoxia caused a marked decrease in nuclear and cytosolic SMAD4 protein and SMAD4 mRNA expression in cocultured HuH7 cells. Together these data indicate that hypoxia represses hepcidin expression through inhibition of BMP/SMAD signaling.

Genome-wide impact of a recently expanded microRNA cluster in mouse

Abstract: Variations in microRNA (miRNA) gene and/or target repertoire are likely to be key drivers of phenotypic differences between species. To better understand these changes, we developed a computational method that identifies signatures of species-specific target site gain and loss associated with miRNA acquisition. Interestingly, several of the miRNAs implicated in mouse 3′ UTR evolution derive from a single rapidly expanded rodent-specific miRNA cluster. Located in the intron of Sfmbt2, a maternally imprinted polycomb gene, these miRNAs (referred to as the Sfmbt2 cluster) are expressed in both embryonic stem cells and the placenta. One abundant miRNA from the cluster, miR-467a, functionally overlaps with the mir-290-295 cluster in promoting growth and survival of mouse embryonic stem cells. Predicted novel targets of the remaining cluster members are enriched in pathways regulating cell survival. Two relevant species-specific target candidates, Lats2 and Dedd2, were validated in cultured cells. We suggest that the rapid evolution of the Sfmbt2 cluster may be a result of intersex conflict for growth regulation in early mammalian development and could provide a general model for the genomic response to acquisition of miRNAs and similar regulatory factors.

Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs

Abstract: MicroRNAs (miRNAs) are 19-22-nucleotide noncoding RNAs that post-transcriptionally regulate mRNA targets. We have identified endogenous miRNA binding sites in mouse embryonic stem cells (mESCs), by performing photo-cross-linking immunoprecipitation using antibodies to Argonaute (Ago2) followed by deep sequencing of RNAs (CLIP-seq). We also performed CLIP-seq in Dicer⁻/⁻ mESCs that lack mature miRNAs, allowing us to define whether the association of Ago2 with the identified sites was miRNA dependent. A significantly enriched motif, GCACUU, was identified only in wild-type mESCs in 3' untranslated and coding regions. This motif matches the seed of a miRNA family that constitutes ~68% of the mESC miRNA population. Unexpectedly, a G-rich motif was enriched in sequences cross-linked to Ago2 in both the presence and absence of miRNAs. Expression analysis and reporter assays confirmed that the seed-related motif confers miRNA-directed regulation on host mRNAs and that the G-rich motif can modulate this regulation.

tRNAs Marked with CCACCA Are Targeted for Degradation

Abstract: Transfer RNAs with unstable acceptor stems can be tagged by CCA-adding enzymes and targeted for destruction by 3′-5′ exonucleases. The CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase] adds CCA to the 3′ ends of transfer RNAs (tRNAs), a critical step in tRNA biogenesis that generates the amino acid attachment site. We found that the CCA-adding enzyme plays a key role in tRNA quality control by selectively marking structurally unstable tRNAs and tRNA-like small RNAs for degradation. Instead of adding CCA to the 3′ ends of these transcripts, CCA-adding enzymes from all three kingdoms of life add CCACCA. In addition, hypomodified mature tRNAs are subjected to CCACCA addition as part of a rapid tRNA decay pathway in vivo. We conjecture that CCACCA addition is a universal mechanism for controlling tRNA levels and preventing errors in translation.

Q&A: Phillip Sharp on biomedical convergence.

Abstract: Upgrading therapeutics calls for tighter links across scientific and engineering disciplines.

Flavonoid-rich berry-extract treatment decreases glucose uptake in human intestinal Caco-2 cells

Abstract: Berries are a rich dietary source of bioactive polyphenols, including flavonoids, such as anthocyanins. Dietary flavonoids are known to decrease the expression of the facilitative GLUT2 and the sodium-glucose cotransporter (SGLT1) genes, the two main glucose transporters in human subjects’ intestinal Caco-2 cells. Acute inhibitory effects on glucose absorption have previously been observed in the presence of flavonoids, however, little is known regarding genomic effects on uptake. The present study investigated the influence of a flavonoid-rich berry-extract on glucose uptake in human subject’s intestinal Caco-2 cell monolayers. Human subject’s intestinal Caco-2 cells, cultured for 19 d, were treated, acutely (15 min) or chronically (16 h) with a flavonoid-rich berry-extract (OptiBerry; InterHealth Nutraceuticals, Benicia, CA, USA) at a final concentration of 0.125% (w/v) then subjected to radiolabelled glucose for 2 min, in the presence or absence of Na. Uptake was determined by liquid scintillation counting of cell lysate. Data were normalised to cell protein concentration and presented as uptake as a percentage of control. Statistical significance was determined by Student’s t-test (P £ 0.05; n 12). Results indicated that berry flavonoids significantly decrease glucose uptake in human subject’s intestinal Caco-2 cells both acutely and chronically. These inhibitory effects are evident in both Na -dependent and Na -independent glucose uptake pathways. Studies are in progress to investigate the biological relevance of the observed effects in relation to berry consumption and glucose metabolism. Potentially, such berry-extracts may be useful in the dietary modulation of postprandial glucose homeostasis.

Effect of quercetin on non-haem iron transport in human intestinal Caco-2 cells

Abstract: Non-haem iron bioavailability is regulated by a number of dietary components including, polyphenols which are thought to act through chelation of iron. The low solubility of iron/polyphenol complexes is regarded as the primary reason for reduced bioavailabilty. However, recent studies by our group and others suggest that the effects of polyphenols of iron absorption are complex and may involve not only control of iron solubility but also include nutrigenomic effects on iron transporter expression . In this present study we investigated the influence of quercetin, the most abundant flavonol in the diet on iron bioavailability by measuring transepithelial iron transport using radioactive Fe. The chronic effects of quercetin were assessed in fully differentiated Caco2 (intestinal) cells exposed for 24 h to quercetin (0, 10mM, and 100mM). In addition, the acute effects of polyphenols on iron bioavailability were also investigated. In these studies quercetin was added along with Fe at the start of the experimental period. Iron uptake was measured as the cellular accumulation of Fe over a 20 min time course. Efflux of iron from the cells into the basolateral medium was measured after 120 min. Data are mean S.E.M. of 5 observations in each group. Statistical analysis was carried out using One-way ANOVA. In the acute setting, the initial rate of iron uptake across the apical membrane of Caco-2 cells was significantly increased by the addition of quercetin (control, 5.7 0.9; 10mM quercetin, 11.0 1.6; 100mM quercetin 16.7 3.8 nmol/mg protein/20 min; P<0.03). In contrast, iron transport across the basolateral membrane was significantly decreased (control, 5.2 1.6; 10mM quercetin, 2.8 0.6; 100mM quercetin 0.12 0.03 nmol/mg protein/120 min; P<0.03). These data are in agreement with the reported effects of grape-seed polyphenols on iron transport and indicate that polyphenols may potentiate iron uptake across the apical membrane but act intracellularly to inhibit iron efflux via ferroportin. The mechanisms involved remain unresolved. Chronic exposure to quercetin for 24 h had no significant effect on iron uptake (P = 0.1), but iron efflux into the basolateral medium was significantly decreased (control, 1.0 0.3; 10mM quercetin, 0.7 0.1; 100mM quercetin 0.08 0.03 nmol/mg protein/120 min; P<0.002). These findings are consistent with our previous observation that quercetin significantly decreases the expression of the basolateral iron transporter ferroportin and its partner ferroxidase hephaestin .

MicroRNA expression in human intestinal Caco-2 cells in response to a flavonoid-rich berry extract

Abstract: MicroRNAs (miRNAs) are short non-coding RNAs which regulate gene expression post-transcriptionally by triggering mRNA degradation or translation repression. Little is known about the role of miRNAs in the gastrointestinal tract. They may be instrumental in regulating gene expression in intestinal epithelial cells, particularly in response to nutrients. The present study investigated miRNA expression in response to a flavonoid-rich berry extract in human intestinal Caco-2 cell monolayers. Human intestinal Caco-2 cells, cultured for 19 d, were treated for 16 h with a flavonoid-rich berry extract (Optiberry; Interhealth Nutraceuticals, Benicia, CA, USA) at a final concentration of 0.125% (w/v). Subsequently miRNA was extracted, pooled and used for the miRNA microarray analysis (Affymetrix1; UK) and validated by quantitative RT-PCR. MiRNA microarray data was analysed using the bioinformatic TargetScanHuman 5.1 database, which predicts miRNA targets. The results showed that 162 miRNA species were detected (P<0.01). Using quantile normalisation, 2 miRNAs were up-regulated and 27 were down-regulated in response to the berry extract. Previously we have shown that incubation with berry extract decreases expression of the intestinal sugar transporter GLUT2 in Caco-2 cells. Bioinformatic analysis identified nine of these differentially expressed miRNAs as potential regulators of the intestinal sugar transporter, GLUT2. Validation by qRT-PCR showed that two of these miRNAs, let-7a (P = 0.001) and miR-106b (P<0.001) were significantly up-regulated in Caco-2 cells in response to berry flavonoids. Studies are in progress, using RNA interference techniques, to investigate whether let-7a and miR-106b directly regulate GLUT2 expression.