Showing papers by "Phillip A. Sharp published in 2010"

Histone H3K27ac separates active from poised enhancers and predicts developmental state

Abstract: Developmental programs are controlled by transcription factors and chromatin regulators, which maintain specific gene expression programs through epigenetic modification of the genome. These regulatory events at enhancers contribute to the specific gene expression programs that determine cell state and the potential for differentiation into new cell types. Although enhancer elements are known to be associated with certain histone modifications and transcription factors, the relationship of these modifications to gene expression and developmental state has not been clearly defined. Here we interrogate the epigenetic landscape of enhancer elements in embryonic stem cells and several adult tissues in the mouse. We find that histone H3K27ac distinguishes active enhancers from inactive/poised enhancer elements containing H3K4me1 alone. This indicates that the amount of actively used enhancers is lower than previously anticipated. Furthermore, poised enhancer networks provide clues to unrealized developmental programs. Finally, we show that enhancers are reset during nuclear reprogramming.

MicroRNA sponges: Progress and possibilities

Abstract: The microRNA (miRNA) “sponge” method was introduced three years ago as a means to create continuous miRNA loss of function in cell lines and transgenic organisms. Sponge RNAs contain complementary binding sites to a miRNA of interest, and are produced from transgenes within cells. As with most miRNA target genes, a sponge's binding sites are specific to the miRNA seed region, which allows them to block a whole family of related miRNAs. This transgenic approach has proven to be a useful tool to probe miRNA functions in a variety of experimental systems. Here we will discuss the ways sponge and related constructs can be optimized and review recent applications of this method with particular emphasis on stable expression in cancer studies and in transgenic animals.

Emerging Roles for Natural MicroRNA Sponges

Abstract: Recently, a non-coding RNA expressed from a human pseudogene was reported to regulate the corresponding protein-coding mRNA by acting as a decoy for microRNAs (miRNAs) that bind to common sites in the 3′ untranslated regions (UTRs). It was proposed that competing for miRNAs might be a general activity of pseudogenes. This study raises questions about the potential ability of thousands of non-coding transcripts to interact with miRNAs and influence the expression of miRNA target genes. Three years ago, artificial miRNA decoys termed ‘miRNA sponges’ were introduced as a means to create loss-of-function phenotypes for miRNA families in cell culture and in virally infected tissue and transgenic animals. Given the efficacy of miRNA sponges expressed from stable chromosomal insertions, it seemed plausible that natural non-coding RNAs might have evolved to sequence-specifically sequester miRNAs. The first such endogenous sponge RNA was discovered in plants and found to attenuate a miRNA-mediated response to an environmental stress. More recently, a viral non-coding RNA was observed to sequester and promote the degradation of a cellular miRNA in infected primate cells. In this review we discuss the potential and proven roles for endogenous miRNA sponges and consider some criteria for screening candidate sponge RNAs.

c-Myc regulates transcriptional pause release

Abstract: Recruitment of the RNA polymerase II (Pol II) transcription initiation apparatus to promoters by specific DNA-binding transcription factors is well recognized as a key regulatory step in gene expression. We report here that promoter-proximal pausing is a general feature of transcription by Pol II in mammalian cells and thus an additional step where regulation of gene expression occurs. This suggests that some transcription factors recruit the transcription apparatus to promoters, whereas others effect promoter-proximal pause release. Indeed, we find that the transcription factor c-Myc, a key regulator of cellular proliferation, plays a major role in Pol II pause release rather than Pol II recruitment at its target genes. We discuss the implications of these results for the role of c-Myc amplification in human cancer.

Intestinal iron absorption: regulation by dietary & systemic factors.

Abstract: Iron is an essential trace metal in human metabolism. However, imbalances in iron homeostasis are prevalent worldwide and have detrimental effects on human health. Humans do not have the ability to remove excess iron and therefore iron homeostasis is maintained by regulating the amount of iron entering the body from the diet. Iron is present in the human diet in number of different forms, including heme (from meat) and a variety of non-heme iron compounds. While heme is absorbed intact, the bioavailability of non-heme iron varies greatly depending on dietary composition. A number of dietary components are capable of interacting with iron to regulate its solubility and oxidation state. Interestingly, there is an emerging body of evidence suggesting that some nutrients also have direct effects on the expression and function of enterocyte iron transporters. In addition to dietary factors, body iron status is a major determinant of iron absorption. The roles of these important dietary and systemic factors in regulating iron absorption will be discussed in this review.

Effect of nanoparticle conjugation on gene silencing by RNA interference.

Abstract: RNA interference (RNAi) is a cellular process whereby the silencing of a particular gene is mediated by short RNAs (siRNAs). Although siRNAs have great therapeutic potential, cellular delivery has been a challenge. Nanoparticle-siRNA conjugates have emerged as potential delivery vehicles; however, reports describing the effects of nanoparticle conjugation on RISC incorporation and subsequent gene silencing have been mixed. In this report, we have systematically evaluated the effect of siRNA coupling strategies using a model nanoparticle system with varying conjugation schemes. We show that the accessibility of the siRNA linked to the nanoparticle and the lability of the cross-linker are critical for efficient gene knockdown.

Inhibitory effect of calcium on non-heme iron absorption may be related to translocation of DMT-1 at the apical membrane of enterocytes

Abstract: Many studies show that calcium reduces iron absorption from single meals, but the underlying mechanism is not known. We tested the hypothesis that calcium alters the expression and/or functionality of iron transport proteins. Differentiated Caco-2 cells were treated with ferric ammonium citrate and calcium chloride, and ferritin, DMT-1, and ferroportin were quantified in whole-cell lysate and cell-membrane fractions. Calcium attenuated the iron-induced increase in cell ferritin levels in a dose-dependent manner; a significant decrease was seen at calcium concentrations of 1.25 and 2.5 mM but was only evident after a 16-24 h incubation period. Calcium and iron treatments decreased DMT-1 protein in Caco-2 cell membranes, although total DMT-1 in whole cell lysates was unchanged by either iron or calcium. No change was seen in ferroportin expression. Our data suggest that calcium reduces iron bioavailability by decreasing DMT-1 expression at the apical cell membrane, thereby downregulating iron transport into the cell.

Transferrin receptor 2 is crucial for iron sensing in human hepatocytes

Abstract: Hepcidin expression in vivo is regulated in proportion to iron status (i.e., increased by iron loading and decreased in iron deficiency). However, in vitro studies with hepatoma cell lines often show an inverse relationship between iron status and hepcidin expression. Here, we investigated possible molecular mechanisms responsible for the differences in iron sensing between hepatoma cell lines and human primary hepatocytes. RNA was collected from primary human hepatocytes, and HepG2 and HuH7 hepatoma cells were treated with either transferrin-bound and non-transferrin-bound iron. Expression of hepcidin, transferrin receptor 2, HFE, and hemojuvelin were quantified by real-time PCR. Hepcidin expression was increased in primary human hepatocytes following 24-h exposure to holoferric transferrin. In contrast, hepcidin mRNA levels in hepatoma cells were decreased by transferrin. Hepcidin expression was positively correlated with transferrin receptor 2 mRNA levels in primary human hepatocytes. Compared with primary hepatocytes, transferrin receptor 2 expression was significantly lower in hepatoma cell lines; furthermore, there was no correlation between transferrin receptor 2 and hepcidin mRNA levels in either HepG2 or HuH7 cells. Taken together our data suggest that transferrin receptor 2 is a likely candidate to explain the differences in iron sensing between hepatoma cell lines and primary human hepatocytes.

Development of a Modified Caco-2 Cell Model System for Studying Iron Availability in Eggs

Abstract: A modified Caco-2 cell model system was developed for studying iron availability in mixtures of fresh and/or cooked foods subjected to a simulated gastrointestinal digestion. The effect of combining foods containing high levels of ascorbic acid with cooked eggs on ferritin expression in the cells was measured. There was no detectable increase in ferritin with eggs alone, indicating that none of the iron was available for uptake into the cells, but when mixed with orange juice or salad (lettuce, tomatoes, and red pepper) in ratios similar to those found in meals, there was a significant increase in ferritin concentration (p = 0.0012 and p = 9.2 x 10(-10), respectively); the enhancing effect of salad was greater than orange juice (p = 0.028). These results suggest that the iron in eggs will be more readily absorbed when consumed with foods high in ascorbic acid.

Anthocyanin-rich berry-extract treatment decreases expression of dietary glucose transporter genes in human intestinal Caco-2 cells

Abstract: Berries are a rich source of polyphenolic compounds such as flavonoids, including anthocyanins, with wide-ranging potential therapeutic effects 1. It has been demonstrated previously that intestinal uptake of dietary glucose is impeded by the presence of polyphenolic compounds; however, little is known regarding the genes involved in this mechanism 2. The aim of this study was to investigate the effects of an anthocyanin-rich berry-extract on dietary glucose transporting genes in Caco-2 cells. Human intestinal Caco-2 cells were cultured for 19 d and were then treated for 16 h with an anthocyanin-rich berry-extract (OptiBerry; InterHealth Nutraceuticals, Benicia, CA, USA) at a final concentration of 0.5% (w/v). Subsequently mRNA was isolated and used for the quantitative real-time polymerase chain reaction (qRT–PCR), using 18S and GAPDH as housekeeping genes. Gene expression data are expressed as means ( SEM) relative expression ratios of control; n = 5/6. The expression of GLUT2 (apical/basolateral monosaccharide transporter), GLUT5 (apical fructose transporter) and SGLT1 (apical sodium/glucose co-transporter) was decreased by the berry-extract treatment. GLUT2, GLUT5 and SGLT1 expression as a ratio of the control was as follows: GLUT2 (0.20 0.02; P = 1.9 · 10 ), GLUT5 (0.57 0.08; P = 2.5 · 10 ) and SGLT1 (0.45 0.03; P = 3.1 · 10 ). GLUT2, GLUT5 and SGLT1 have previously been identified as therapeutic targets for dysregulated glucose metabolism. In this study, the expression of these genes in Caco-2 cells was decreased by treatment with an anthocyanin-rich berry-extract. Studies are in progress to investigate the biological relevance of the observed effects in relation to berry consumption and the absorption of dietary sugar.

Development of an in vitro Caco-2 cell model system to measure iron availability from foods and meals

Abstract: An in vitro Caco-2–simulated digestion intestinal cell model has been developed to assess Fe availability by measuring ferritin formation in Caco-2 cells exposed to dried samples subjected to simulated gastrointestinal digestion. The technique was modified to study foods and meals as eaten using ferritin as a surrogate marker of Fe availability. Two separate methods were used to study the effects of an Fe absorption enhancer and inhibitor on ferritin formation in Caco-2 cells. Ferritin is the main storage protein for Fe, and specifically Fe + in enterocytes, and is well established as a surrogate marker of cellular Fe status. Ascorbic acid, an Fe absorption enhancer, present in orange juice or an ascorbic acid-rich salad, was subjected to an in vitro simulated digestion in conjunction with cooked egg, a rich source of poorly-available Fe. The digestate was applied to Caco-2 cells (using a dialysis membrane to protect the cells from digestive enzymes and food particles) for 2 h and ferritin formation was measured after 24 h. The effect of orange juice and also of freeze–thawing of the digestate before adding to the cell model (to facilitate timing of the experimental work) on the solubility of Fe was also studied. Ca, an Fe absorption inhibitor, was added to the cell culture media in the presence and absence of Fe and ferritin formation measured. The presence of ascorbic acid in the digestate following in vitro digestion increased the formation of ferritin in the Caco-2 cells as well as the concentration of soluble Fe in the digestate. Freezing the digestate also caused an increase, possibly as a result of rupture of cell walls increasing release of Fe into solution. In contrast to this finding, supplementation of the cell culture media with Ca suppressed the formation of ferritin, indicating a reduction in Fe transport into the cell. These results agree with published in vivo data and support the use of Caco-2 cells as a model of the epithelium of the small intestine and also the use of ferritin as a surrogate marker of Fe availability. Furthermore, the results show that digesting an ascorbic acid-rich food in conjunction with egg will increase Fe availability.

Flavonoid-rich-berry-extract treatment decreases the expression of DMT1 and functionally-similar metal transporter genes in human intestinal Caco-2 cells

Abstract: F. Alzaid, K. Pourvali, C. I. Lin, M. Arno, E. Aldecoa-Otalora Astarloa, P. A. Sharp, C. Hogstrand, P. W. Emery, D. Bagchi, V. R. Preedy and H. Wiseman Nutritional Sciences Division, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK and Department of Pharmacological and Pharmaceutical Sciences, University of Houston College of Pharmacy, Houston, Texas 77204, USA

Regulation of Synaptic Structure and Function by FMRP-Associated MicroRNAs miR-125b and miR-132

Abstract: MicroRNAs (miRNAs) are noncoding RNAs that suppress translation of specific mRNAs. The miRNA machinery interacts with fragile X mental retardation protein (FMRP), which functions as translational repressor. We show that miR-125b and miR-132, as well as several other miRNAs, are associated with FMRP in mouse brain. miR-125b and miR-132 had largely opposing effects on dendritic spine morphology and synaptic physiology in hippocampal neurons. FMRP knockdown ameliorates the effect of miRNA overexpression on spine morphology. We identified NMDA receptor subunit NR2A as a target of miR-125b and show that NR2A mRNA is specifically associated with FMRP in brain. In hippocampal neurons, NR2A expression is negatively regulated through its 3' UTR by FMRP, miR-125b, and Argonaute 1. Regulation of NR2A 3'UTR by FMRP depends in part on miR-125b. Because NMDA receptor subunit composition profoundly affects synaptic plasticity, these observations have implications for the pathophysiology of fragile X syndrome, in which plasticity is altered.

Methods and compositions for increasing the activity of inhibitory rna

Abstract: The invention provides methods for increasing the activity of an inhibitory RNA (RNAi) in a subject requiring administering one or more poly-ADP-ribose polymerase (PARP) inhibitors and/or one or more PARG activators to the subject. The invention also provides methods for increasing the activity of an inhibitory RNA in a cell or cell population requiring contacting a cell or cell population with one or more PARP inhibitors and/or one or more PARG activators. The invention further provides compositions and kits containing one or more PARP inhibitors and/or one or more PARG activators.

Flavonoid-rich berry-extract influences expression of genes in the iron-uptake pathway 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 impair Fe absorption and reduce non-haem-Fe transport across Caco-2 cell monolayers. Previously, we demonstrated that a flavonoidrich berry-extract decreases the expression of the divalent metal ion transporter (DMT1) and the haemochromatosis (HFE) genes, involved in the Fe-uptake pathway. The present study investigated the influence of this berry-extract on the gene expression and corresponding protein abundance of the apical ferrireductase, duodenal cytochrome B (DCYTB), the basolateral Fe efflux protein, ferroportin (FPN), the basolateral ferroxidase, hephaestin (HEPH) and DMT1 and HFE, which co-ordinate intestinal Fe transport. Human intestinal Caco-2 cells, cultured for 21 d, were then treated for 16 h with an anthocyanin-rich berry-extract (OptiBerry; InterHealth Nutraceuticals, Benicia, CA, USA) at a final concentration of 0.5% (w/v). RNA and protein were isolated for quantitative RT–PCR and western blotting, respectively. All gene expression data were normalised to 18S and GAPDH as housekeeping genes and presented as mean normalised expression ratio and SEM. All protein quantity data were normalised per mg of protein and presented as mean integral/{g protein and SEM. Statistical significance was determined by Student’s unpaired t-test with significance indicated at P £ 0.05. Following treatment with the berry-extract, there were significant decreases in DMT1 (mean 0.63 (SEM 0.07), p £ 0.039, n 6) and HFE (mean 0.52 (SEM 0.05), P £ 0.0001, n 6) mRNA expression. The abundance of the proteins HEPH and FPN, involved in the basolateral release of Fe, was significantly increased (Fig. 1). These results indicate that berry-flavonoids influence the expression of components of the Fe-uptake pathway. Studies are in progress to investigate the biological relevance of the observed effects in relation to berry consumption and the bioavailability of dietary Fe.

Compositions et procédés pour traiter le cancer et moduler la formation de granules de stress

Abstract: L'invention concerne des procedes pour traiter ou reduire la probabilite de developpement d'une affection et/ou d'un cancer associe aux granules de stress par administration d'un ou plusieurs inhibiteurs de poly-ADP-ribose polymerase (PARP), d'un ou plusieurs activateurs de PARP, d'un ou plusieurs activateurs de poly-ADP-ribose glycosylase (PARG) et/ou d'un ou plusieurs activateurs de poly-ADP-ribose glycohydrolase ARH3. L'invention concerne egalement des procedes correspondants de reduction de la formation des granules de stress et/ou de la proliferation dans une cellule ou une population de cellules. L'invention concerne en outre des procedes d'augmentation du nombre de granules de stress et de la proliferation dans une cellule ou une population de cellules par administration d'un ou plusieurs activateurs de PARP, d'un ou plusieurs inhibiteurs de PARP, d'un ou plusieurs inhibiteurs de PARG et/ou d'un ou plusieurs inhibiteurs de ARH3. L'invention concerne de surcroit des procedes pour cribler des agents permettant de traiter ou de reduire la probabilite de developpement d'une affection et/ou d'un cancer associe aux granules de stress, et des procedes pour determiner la propension au developpement d'une affection et/ou d'un cancer associe aux granules de stress, ainsi que des compositions et des necessaires contenant un ou plusieurs inhibiteurs de PARP, un ou plusieurs activateurs de PARP, un ou plusieurs activateurs de PARG et/ou d'un ou plusieurs activateurs de ARH3.

Flavonoid-rich berry-extract treatment influences expression of genes in the copper-uptake pathway 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 chelate Cu + and are known to alter the uptake of metal ions in human intestinal Caco-2 cells. However, little is known about the effects of dietary polyphenols on the expression of genes involved in the Cu-uptake pathway in the human intestine. The present study investigated the influence of a flavonoid-rich berry-extract on the expression of the following genes which co-ordinate the intestinal uptake of Cu: the cell surface metalloreductase (DCYTB); the Cu importers, divalent metal ion transporter (DMT1) and Cu transporter 1 (CTR1); the intracellular Cu chaperone (HAH1) and metallothionein (MT); the Cu transporting ATPases (ATP7A and ATP7B). 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). RNA was isolated for quantitative RT–PCR. All gene expression data were normalised to 18S and GAPDH as housekeeping genes and presented as mean normalised expression ratios SEM. Statistical significance was determined by Student’s t test with significance indicated at P £ 0.05 (n 12). Following treatment with the berry extract there were significant decreases in DMT1 (0.73 0.08, P<0.04), CTR1 (0.67 0.06, P<0.01), HAH1 (0.82 0.06, P<0.03) and ATP7B (0.72 0.05, P<0.001) mRNA expression (Fig. 1). The mRNA expression of the other genes did not change significantly in response to the berry-extract treatment. These results indicate that berry flavonoids influence the expression of components of the Cu-uptake pathway. Studies are in progress to investigate the biological relevance of the observed effects in relation to berry consumption and the bioavailability of dietary Cu.