Showing papers by "Phillip A. Sharp published in 2005"

TAZ, a transcriptional modulator of mesenchymal stem cell differentiation.

Abstract: Mesenchymal stem cells (MSCs) are a pluripotent cell type that can differentiate into several distinct lineages. Two key transcription factors, Runx2 and peroxisome proliferator-activated receptor gamma (PPARgamma), drive MSCs to differentiate into either osteoblasts or adipocytes, respectively. How these two transcription factors are regulated in order to specify these alternate cell fates remains a pivotal question. Here we report that a 14-3-3-binding protein, TAZ (transcriptional coactivator with PDZ-binding motif), coactivates Runx2-dependent gene transcription while repressing PPARgamma-dependent gene transcription. By modulating TAZ expression in model cell lines, mouse embryonic fibroblasts, and primary MSCs in culture and in zebrafish in vivo, we observed alterations in osteogenic versus adipogenic potential. These results indicate that TAZ functions as a molecular rheostat that modulates MSC differentiation.

Transcriptional silencing of a transgene by RNAi in the soma of C. elegans

Abstract: The silencing of transgene expression at the level of transcription in the soma of Caenorhabditis elegans through an RNAi-dependent pathway has not been previously characterized. Most gene silencing due to RNAi in C. elegans occurs at the post-transcriptional level. We observed transcriptional silencing when worms containing the elt-2::gfp/LacZ transgene were fed RNA produced from the commonly used L4440 vector. The transgene and the vector share plasmid backbone sequences. This transgene silencing depends on multiple RNAi pathway genes, including dcr-1, rde-1, rde-4, and rrf-1. Unlike post-transcriptional gene silencing in worms, elt-2::gfp/LacZ silencing is dependent on the PAZ-PIWI protein Alg-1 and on the HP1 homolog Hpl-2. The latter is a chromatin silencing factor, and expression of the transgene is inhibited at the level of intron-containing precursor mRNA. This inhibition is accompanied by a decrease in the acetylation of histones associated with the transgene. This transcriptional silencing in the soma can be distinguished from transgene silencing in the germline by its inability to be transmitted across generations and its dependence on the rde-1 gene. We therefore define this type of silencing as RNAi-induced Transcriptional Gene Silencing (RNAi-TGS). Additional chromatin-modifying components affecting RNAi-TGS were identified in a candidate RNAi screen.

Characterization of a highly variable eutherian microRNA gene.

Abstract: Mouse microRNAs (miRNAs) miR-290-miR295 are encoded by a cluster of partially homologous pre-miRNA hairpins and are likely to be functionally important in embryonic stem (ES) cells and preimplantation embryos. We present evidence that a spliced, capped, and polyadenylated primary transcript spans this entire Early Embryonic microRNA Cluster (EEmiRC). Partial Drosha processing yields additional large nuclear RNA intermediates. A conserved promoter element containing a TATA-box directs EEmiRC transcription. Sequence analysis shows that the EEmiRC transcription unit is remarkably variable and can only be identified bioinformatically in placental (eutherian) mammals. Consistent with eutherian-specific function, EEmiRC is expressed in trophoblastic stem (TS) cells. When analyzing evolutionary and functional relationships, the organization of the entire miRNA loci should be considered in addition to the mature miRNA sequences. Application of this concept suggests that EEmiRC is a recently acquired rapidly evolving gene important for eutherian development.

The octamer binding transcription factor Oct-1 is a stress sensor

Abstract: The POU-domain transcription factor Oct-1 is widely expressed in adult tissues and has been proposed to regulate a large group of target genes. Microarray expression profiling was used to evaluate gene expression changes in Oct-1-deficient mouse fibroblasts. A number of genes associated with cellular stress exhibited altered expression. Consistent with this finding, Oct-1-deficient fibroblasts were hypersensitive to gamma radiation, doxorubicin, and hydrogen peroxide and harbored elevated reactive oxygen species. Expression profiling identified a second group of genes dysregulated in Oct-1-deficient fibroblasts following irradiation, including many associated with oxidative and metabolic stress. A number of these genes contain octamer sequences in their immediate 5' regulatory regions, some of which are conserved in human. These results indicate that Oct-1 modulates the activity of genes important for the cellular response to stress.

The usefulness of in vitro models to predict the bioavailability of iron and zinc: A consensus statement from the HarvestPlus expert consultation

Abstract: A combination of dietary and host-related factors determines iron and zinc absorption, and several in vitro methods have been developed as preliminary screening tools for assessing bioavailability. An expert committee has reviewed evidence for their usefulness and reached a consensus. Dialyzability (with and without simulated digestion) gives some useful information but cannot predict the correct magnitude of response and may sometimes predict the wrong direction of response. Caco-2 cell systems (with and without simulated digestion) have been developed for iron availability, but the magnitude of different effects does not always agree with results obtained in human volunteers, and the data for zinc are too limited to draw conclusions about the validity of the method. Caco-2 methodologies vary significantly between laboratories and require experienced technicians and good quality cell culture facilities to obtain reproducible results. Algorithms can provide semi-quantitative information enabling diets to be classified as high, moderate, or low bioavailability. While in vitro methods can be used to generate ideas and develop hypotheses, they cannot be used alone for important decisions concerning food fortification policy, selection of varieties for plant breeding programs, or for new product development in the food industry. Ultimately human studies are required for such determinations.

Negative regulation of nuclear divisions in Caenorhabditis elegans by retinoblastoma and RNA interference-related genes.

Abstract: Short RNA regulatory molecules, microRNAs, and short interfering RNAs participate in a range of developmental gene networks by base-pairing with their target sequences. Consistent with these findings, genes required for the biogenesis and function of short interfering RNAs and microRNAs, dicer (dcr-1 in Caenorhabditis elegans) and argonaute homologs, are essential for development in diverse organisms, including C. elegans. We demonstrate that genes required for the function of short RNAs synergize with the retinoblastoma tumor suppressor homolog lin-35 in negative regulation of the nuclear divisions in the intestine of C. elegans. The level of cyclin E (cye-1) expression is critical for nuclear divisions in the intestine and is elevated in double mutants in lin-35 and RNA interference pathway genes. We propose that RNA interference-related pathways cooperate with retinoblastoma in transcriptional repression of endogenous genes, an example being cyclin E.

1918 flu and responsible science.

Abstract: T he influenza pandemic of 1918 is estimated to have caused 50 million deaths worldwide; 675,000 in the United States. The reconstruction of the 1918 virus by the synthesis of all eight subunits and the generation of infectious virus are described on p. 77 of this issue,[*][1] and the sequences of the final three gene segments of the virus are described in a concurrent Nature paper.[†][2] Predictably, but alarmingly, this virus is more lethal to mice than are other influenza strains, suggesting that this property of the 1918 virus has been recovered in the published sequence. The good news is that we now have the sequence of this virus, perhaps permitting the development of new therapies and vaccines to protect against another such pandemic. The concern is that a terrorist group or a careless investigator could convert this new knowledge into another pandemic. Should the sequence of the 1918 virus have been published, given its potential use by terrorists? The dual-use nature of biological information has been debated widely since September 11, 2001. In 2003, a committee of the U.S. National Academies chaired by Gerald Fink considered this issue, weighing the benefits against the risks of restricting the publication of such biological information. They outlined the tradeoff between erring on the side of prudence, thus potentially hindering the progress of critical science, and erring on the side of disclosure, thus potentially aiding terrorists. The U.S. National Science Advisory Board for Biosecurity (NSABB) was established to advise governmental agencies and the scientific community on policies relative to public disclosure. This board has begun to deliberate, but the questions are complex, as typified by these papers on the 1918 virus. It is reassuring that the NSABB was asked to consider these papers before publication and concluded that the scientific benefit of the future use of this information far outweighs the potential risk of misuse. People may be reassured that the system is working, because agencies representing the public, the scientific community, and the publishing journals were involved in the decision. ![Figure][3] CREDIT: COURTESY OF THE NATIONAL MUSEUM OF HEALTH AND MEDICINE, ARMED FORCES INSTITUTE OF PATHOLOGY, WASHINGTON, D.C. (NCP 1603) I firmly believe that allowing the publication of this information was the correct decision in terms of both national security and public health. It is impossible to forecast how scientific observations might stimulate others to create new treatments or procedures to control future pandemics. For example, in the Nature article, sequence comparisons suggest that the 1918 virus was generated not by incremental changes in the polymerase genes, but by the movement of these genes, in total, from an avian source into a human influenza virus. The availability of these sequences will permit identification of their avian origin and should show why this particular set of genes was selected. Similarly, the results in the Science article suggest that the cleavage of a protein on the surface of the 1918 virus, a step critical for virulent infection, may occur by a previously unknown mechanism—a hint that could lead to new drugs for inhibiting this step and thus preventing future pandemic eruptions. Influenza is highly infectious, and a new strain could spread around the world in a matter of months, if not weeks. The public needs confidence that the 1918 virus will not escape from research labs. All of the described experiments were done in a Biosafety Level 3 laboratory, a high-containment environment recommended by the U.S. Centers for Disease Control and Prevention and the National Institutes of Health on an interim basis, whose use should become a permanent requirement for such experiments. Current evidence suggests that some available drugs and possible future vaccines could suppress infections by the 1918 virus. Given the prospect of another natural influenza pandemic, the recent decision by the U.S. administration to stockpile antivirals for influenza treatment seems wise. Finally, although a sequence of the 1918 virus has been determined and is highly virulent in mice, this may not be the specific form of the virus that caused the pandemic of 1918. An article in the same issue of Nature [‡][4] reports the existence of sequence variation in a natural population of influenza virus; yet we have only one sequence for the 1918 pandemic strain, and the reconstructed virus described in the Science article was built into the backbone of a laboratory strain. Because a pandemic infection is dependent on many unknown properties, there is no certainty that the reconstructed 1918 virus is capable of causing a pandemic. [1]: #fn-1 [2]: #fn-2 [3]: pending:yes [4]: #fn-3

Methods and options for estimating iron and zinc bioavailability using Caco-2 cell models: Benefits and limitations

Abstract: Micronutrient deficiencies are prevalent worldwide and have detrimental effects on human health. Complex interactions between micronutrients and other dietary components largely determine micronutrient bioavailability, and understanding these interactions is key to improving micronutrient status. A number of in vitro and in vivo methodologies are available for assessing micronutrient bioavailability. The purpose of this review is to highlight the usefulness of one of the in vitro models, the Caco-2 cell, as a predictive tool for human micronutrient bioavailability. The review focuses on current methods used with the Caco-2 cell line, their benefits and limitations, and the possibilities for the future development of this model.

Herpesviruses throw a curve ball: new insights into microRNA biogenesis and evolution.

Abstract: Validation and comparison of previously unknown microRNA genes in related herpesviruses yielded several surprises, most notably in regard to viral evolution and microRNA biogenesis. An explosion of literature has recently appeared describing the identification and mechanism of action of microRNAs (miRNAs)—small RNA regulators of gene expression in plant and animal cells.

Modulator of Mesenchymal Stem Cell Differentiation

Abstract: Mesenchymal stem cells (MSCs) are a pluripotent cell type that can differentiate into several distinct lineages. Two key transcription factors, Runx2 and peroxisome proliferator–activated receptor g (PPARg), drive MSCs to differentiate into either osteoblasts or adipocytes, respectively. How these two transcription factors are regulated in order to specify these alternate cell fates remains a pivotal question. Here we report that a 14-3-3–binding protein, TAZ (transcriptional coactivator with PDZ-binding motif), coactivates Runx2-dependent gene transcription while repressing PPARg-dependent gene transcription. By modulating TAZ expression in model cell lines, mouse embryonic fibroblasts, and primary MSCs in culture and in zebrafish in vivo, we observed alterations in osteogenic versus adipogenic potential. These results indicate that TAZ functions as a molecular rheostat that modulates MSC differentiation. Pluripotent MSCs can differentiate into several distinct cell types, including osteoblasts and adipocytes (1, 2). Two key transcription factors, Runx2 (also called Cbfa1 or Pebp2aA) and PPARg, drive MSCs to differentiate into either osteoblasts or adipocytes, respectively

Iron uptake by rat proximal colon is increased in animals fed an iron deficient diet

Abstract: The typical western diet provides 10 mg of iron each day. However only 10% of this is absorbed in the duodenum (i.e. 1mg/day), meaning that 90% of our daily intake reaches the distal small intestine and colon. It is presumed that this excess iron is simply excreted in the faeces. However, a recent report has suggested that the proximal colon might have some iron transport capacity (Bougle et al. 2002). In the present study we have investigated this possibility by measuring iron flux across the proximal colonic mucosa in animals fed either an iron replete (control) or an iron deficient (FeD) diet. In parallel studies the effect of dietary iron on the expression of the intestinal iron transporters DMT1 and IREG1 has also been determined. All studies were carried out on male Wistar rats (250g). Rats were fed diets containing 44mg Fe/Kg (control) or <0.5mg/Kg (FeD) for 14 days prior to experimentation. For in vivo iron uptake studies, animals were anaesthetised with intraperitoneal pentobarbitone sodium (60 mg/Kg body weight) and 0.2mM 59Fe2+ (complexed with 4mM ascorbate) was instilled into a tied-off segment of proximal colon. Blood samples were removed after 20, 40 and 60 min via a femoral artery cannula to determine iron transfer into the blood. Tissue iron uptake was measured at the end of each experiment by gamma counting of the colonic segments. In a separate group of animals the mucosa was isolated and used as a source of membrane protein and total RNA for analysis of iron transporter expression by Western blotting and RT-PCR respectively. Data are mean ± SEM. Statistical analysis was performed using Student’s unpaired t-test. Iron uptake was significantly increased in FeD group (162.4 ± 36.1 pmoles/mg dry wt tissue n=6) compared with control (71.4 ± 18.3 pmoles/mg dry wt tissue, p<0.05 n=6). This corresponded with a significant increase in DMT1 mRNA and protein in the FeD group. Interestingly, there was no difference in either iron efflux into the blood or in IREG1 expression in the two animal groups. These data indicate that the proximal colon has some capacity to absorb iron from the intestinal lumen.However, rather than transferring the iron into the blood for physiological utilisation, it is retained within the colonic mucosa. The physiological rationale for such a mechanism requires further investigation.