Showing papers by "Phillip A. Sharp published in 2008"
TL;DR: It is shown that mice deficient for miR-17 approximately 92 die shortly after birth with lung hypoplasia and a ventricular septal defect, and a link between the oncogenic properties of miR the 1792 and its functions during B lymphopoiesis and lung development is suggested.
1,551 citations
TL;DR: It is found that states of increased proliferation are associated with widespread reductions in the 3′UTR-based regulatory capacity of mRNAs, which is a characteristic of gene expression during immune cell activation and correlates with proliferation across diverse cell types and tissues.
Abstract: Messenger RNA (mRNA) stability, localization, and translation are largely determined by sequences in the 3' untranslated region (3'UTR). We found a conserved increase in expression of mRNAs terminating at upstream polyadenylation sites after activation of primary murine CD4+ T lymphocytes. This program, resulting in shorter 3'UTRs, is a characteristic of gene expression during immune cell activation and correlates with proliferation across diverse cell types and tissues. Forced expression of full-length 3'UTRs conferred reduced protein expression. In some cases the reduction in protein expression could be reversed by deletion of predicted microRNA target sites in the variably included region. Our data indicate that gene expression is coordinately regulated, such that states of increased proliferation are associated with widespread reductions in the 3'UTR-based regulatory capacity of mRNAs.
1,284 citations
TL;DR: Evidence of widespread divergent transcription at protein-encoding gene promoters is presented and it is suggested that Divergent transcription over short distances is common for active promoters and may help promoter regions maintain a state poised for subsequent regulation.
Abstract: Transcription initiation by RNA polymerase II (RNAPII) is thought to occur unidirectionally from most genes. Here, we present evidence of widespread divergent transcription at protein-encoding gene promoters. Transcription start site-associated RNAs (TSSa-RNAs) nonrandomly flank active promoters, with peaks of antisense and sense short RNAs at 250 nucleotides upstream and 50 nucleotides downstream of TSSs, respectively. Northern analysis shows that TSSa-RNAs are subsets of an RNA population 20 to 90 nucleotides in length. Promoter-associated RNAPII and H3K4-trimethylated histones, transcription initiation hallmarks, colocalize at sense and antisense TSSa-RNA positions; however, H3K79-dimethylated histones, characteristic of elongating RNAPII, are only present downstream of TSSs. These results suggest that divergent transcription over short distances is common for active promoters and may help promoter regions maintain a state poised for subsequent regulation.
881 citations
TL;DR: It is shown that let-7 functionally inhibits non-small cell tumor development and was more potent in lung cancer cell lines harboring oncogenic K-Ras mutations than in lines with other mutations.
Abstract: Many microRNAs (miRNAs) target mRNAs involved in processes aberrant in tumorigenesis, such as proliferation, survival, and differentiation. In particular, the let-7 miRNA family has been proposed to function in tumor suppression, because reduced expression of let-7 family members is common in non-small cell lung cancer (NSCLC). Here, we show that let-7 functionally inhibits non-small cell tumor development. Ectopic expression of let-7g in K-RasG12D-expressing murine lung cancer cells induced both cell cycle arrest and cell death. In tumor xenografts, we observed significant growth reduction of both murine and human non-small cell lung tumors when overexpression of let-7g was induced from lentiviral vectors. In let-7g expressing tumors, reductions in Ras family and HMGA2 protein levels were detected. Importantly, let-7g-mediated tumor suppression was more potent in lung cancer cell lines harboring oncogenic K-Ras mutations than in lines with other mutations. Ectopic expression of K-RasG12D largely rescued let-7g mediated tumor suppression, whereas ectopic expression of HMGA2 was less effective. Finally, in an autochthonous model of NSCLC in the mouse, let-7g expression substantially reduced lung tumor burden.
857 citations
TL;DR: The fact that macrophages respond more acutely to a hepcidin challenge is fully consistent with their central role in maintaining body iron homeostasis as well as the fact that the duodenum appears to be less sensitive to this initial rise in hePCidin levels.
Abstract: Background and aims: Reticulo-endothelial macrophages together with duodenal enterocytes coordinate body iron homeostasis. The aim of this study was to investigate the regulatory actions of the hormone hepcidin on ferroportin expression in these two cell types. Methods: We investigated the in vitro effects of hepcidin in well-characterised human cell culture models of macrophages (differentiated THP-1 cells) and intestinal epithelial cells (Caco-2 cells). The in vivo effects of hepcidin were also investigated in mice injected with a synthetic hepcidin peptide. Results: Exposure to hepcidin (presented either as conditioned medium from interleukin-6-stimulated HuH7 cells or as a synthetic peptide) resulted in a rapid (within 4 h) decrease in ferroportin expression in THP-1 macrophages but had no effect on ferroportin levels in Caco-2 cells. To determine whether these rapid effects of hepcidin were also evident in vivo we injected mice with a synthetic hepcidin peptide. Four hours post-injection, ferroportin levels in the macrophage-rich red pulp of the spleen were decreased significantly and the hepcidintreated mice developed hypoferraemia. Interestingly, in the same mice there was no effect of hepcidin on duodenal ferroportin protein expression or duodenal iron transport. Conclusions: These data suggests that the rapid response to hepcidin is cell type and tissue specific. Upon its release, hepcidin initially targets macrophage iron recycling. The duodenum appears to be less sensitive to this initial rise in hepcidin levels. We believe the fact that macrophages respond more acutely to a hepcidin challenge is fully consistent with their central role in maintaining body iron homeostasis.
138 citations
TL;DR: The data suggests that Dcytb plays a physiological role in both iron and copper uptake, through divalent metal transporter 1 (DMT1) and copper transporter 1, respectively, and shows that 59Fe uptake was significantly enhanced in D Cytb‐EGFP expressing MDCK cells which endogenously express DMT1.
98 citations
TL;DR: Cassettes incorporating various combinations of reporter genes, miRNA-based RNAi (including two shRNA constructs at once), and oncogenes are described and the delivery of effective RNA interference in cells in culture, efficient transduction into hematopoietic stem cells with cell-type-specific knockdown in their progeny, and rapid generation of regulated shRNAs in transgenic mice are demonstrated.
Abstract: We report a system for Cre-regulated expression of RNA interference in vivo. Expression cassettes comprise selectable and FACS-sortable markers in tandem with additional marker genes and shRNAs in the antisense orientation. The cassettes are flanked by tandem LoxP sites arranged so that Cre expression inverts the marker-shRNA construct, allowing its regulated expression (and, at the same time, deletes the original selection/marker genes). The cassettes can be incorporated into retroviral or lentiviral vectors and delivered to cells in culture or used to generate transgenic mice. We describe cassettes incorporating various combinations of reporter genes, miRNA-based RNAi (including two shRNA constructs at once), and oncogenes and demonstrate the delivery of effective RNA interference in cells in culture, efficient transduction into hematopoietic stem cells with cell-type-specific knockdown in their progeny, and rapid generation of regulated shRNA knockdown in transgenic mice. These vector systems allow regulated combinatorial manipulation (both overexpression and loss of function) of gene expression in multiple systems in vitro and in vivo.
75 citations
TL;DR: This year, the Lasker Foundation recognizes Victor Ambros, Gary Ruvkun, and David Baulcombe for their pioneering work elucidating the role of short RNA species in the posttranscriptional regulation of eukaryotic gene expression.
69 citations
TL;DR: Analysis of the (poly)phenols in these aqueous extracts suggested that dicaffeoylquinic acids and flavanols may be particularly important in producing these effects, suggesting that this merits evaluation in a clinical study.
67 citations
TL;DR: In this paper, the authors used various analytical techniques, and the final step was a human study to measure the effect of a candidate compound on nonheme iron absorption using radioiron.
Abstract: In this research, our aim was to isolate and characterize the substance known as "meat factor," which is reported to enhance nonheme iron absorption. We used various analytical techniques, and the final step was a human study to measure the effect of a candidate compound on iron absorption. Lean beef was selected for study, as it is known to increase nonheme iron absorption. Cooked ground beef was homogenized and aliquots were taken through a simulated gastric and intestinal digestion. This was followed by purification using fast protein liquid chromatography. The fractions were collected and applied to a Caco-2 cell system designed to measure iron absorption using radioiron. Fractions with an enhancing effect were analyzed by mass spectrometry, nuclear magnetic resonance, and HPLC, and a proposed empirical formula was obtained for the substance in the most active fraction (C(8)H(20) NO(6)P). Tandem mass spectrometry was used to identify the compound as L-alpha-glycerophosphocholine (L-alpha) by comparing the spectra against authentic material. We added a commercially available food grade source of L-alpha to vegetarian lasagna, with and without 100 mg ascorbic acid (a known enhancer of nonheme iron absorption), at the same enhancer:iron molar ratio (2:1), and fed meals to 13 women of child-bearing age with low iron stores. The nonheme iron was labeled with stable isotopes of iron to provide a total dose per meal of 10 mg iron, and absorption was measured from erythrocyte incorporation. Nonheme iron absorption from lasagna was increased by the addition of either ascorbic acid (P = 0.010) or L-alpha (P = 0.023). We have identified L-alpha as a component of muscle tissue that enhances nonheme iron absorption, and this finding provides new opportunities for iron fortification of foods.
39 citations
TL;DR: This study suggests that endogenous siRNAs cooperate with chromatin factors, either C. elegans ortholog of acute lymphoblastic leukemia-1 (ALL-1)-fused gene from chromosome 10 (AF10), ZFP-1, or tumor suppressor Rb, to regulate overlapping sets of genes and predicts a large role for RNAi-based chromatin silencing in control of gene expression in C. Chengans.
Abstract: In Caenorhabditis elegans, a vast number of endogenous short RNAs corresponding to thousands of genes have been discovered recently. This finding suggests that these short interfering RNAs (siRNAs) may contribute to regulation of many developmental and other signaling pathways in addition to silencing viruses and transposons. Here, we present a microarray analysis of gene expression in RNA interference (RNAi)-related mutants rde-4, zfp-1, and alg-1 and the retinoblastoma (Rb) mutant lin-35. We found that a component of Dicer complex RDE-4 and a chromatin-related zinc finger protein ZFP-1, not implicated in endogenous RNAi, regulate overlapping sets of genes. Notably, genes a) up-regulated in the rde-4 and zfp-1 mutants and b) up-regulated in the lin-35(Rb) mutant, but not the down-regulated genes are highly represented in the set of genes with corresponding endogenous siRNAs (endo-siRNAs). Our study suggests that endogenous siRNAs cooperate with chromatin factors, either C. elegans ortholog of acute lymphoblastic leukemia-1 (ALL-1)-fused gene from chromosome 10 (AF10), ZFP-1, or tumor suppressor Rb, to regulate overlapping sets of genes and predicts a large role for RNAi-based chromatin silencing in control of gene expression in C. elegans.
TL;DR: The Monte Carlo simulation was used to show that a significant relationship exists between fragile sites and rearrangements found at amniocentesis as well as between fragile Sites and rearranges found in spontaneous abortions, stillborns and newborns, and the levels of statistical significance differ from Hecht’s results.
Abstract: ber of breaks. Such conclusions assume that each chromosome band has the same length. Nevertheless, we know that all the chromosome bands do not have the same length and since we can assume that the chance of having a break in a band is dependent of its length it would have been more accurate to determine the probability of a relationship between fragile sites and constitutional rearrangements according to the relative length of the bands known to contain a fragile site. We already used such an approach to show that a relationship exists between fragile sites and structural rearrangements in cancer (De Braekeleer et al. 1984). In order to avoid multiple comparison between bands we used the Monte Carlo simulation (Rubinstein 1981). This technique has been described in detail elsewhere (De Braekeleer et al. 1984). In brief, we randomly simulated the same total number of breaks than observed and compared the random distribution with the actual observed distribution. A chi-square test is then performed and the P value determined. We retracted information concerning the number of breakpoints and their localization into bands from Hecht’s papers. Our results show that a significant relationship exists between fragile sites and rearrangements found at amniocentesis as well as between fragile sites and rearrangements found in spontaneous abortions, stillborns and newborns (Table 1). However, the levels of statistical significance differ from Hecht’s results. With the exception of fragile sites class V and amniocentesis where a significant relation was shown to exist, we could not find any other significant relation when considering each class of fragile site separately, which is in agreement with Hecht’s results (Table 1). Further studies are needed to assess if fragile sites are predisposing to meiotic chromosome breakage and per se to structural rearrangements. Marc De Braekeleer, M.D.
TL;DR: The Keystone Symposium on RNAi, microRNA and non-coding RNA convened on March 25–30 at Whistler Resort inWhistler, British Columbia, Canada, with researchers with backgrounds in different biochemical disciplines exchanging ideas on short RNAs and their roles in a host of biological processes.
Abstract: The Keystone Symposium on RNAi, microRNA and non-coding RNA convened on March 25–30 at Whistler Resort in Whistler, British Columbia, Canada. Researchers with backgrounds in different biochemical disciplines came together to exchange ideas on short RNAs and their roles in a host of biological processes.
TL;DR: SNPs in the TNF α gene that are associated with an increased risk of developing IDA during malaria-endemic areas are identified and identified in this issue of Blood.