The following alphabetical list represents papers published in 2005 with at least one Whitehead author (in red). Not all of this work was done at the Whitehead Institute. Some of these papers are collaborations with scientists elsewhere. The papers are gathered from PubMed and from Science Citation Index (also known as the Web of Science) Preceding the bibliography is an alphabetical list of the titles of the papers followed by the first author :
Aborn, J. H., El-Difrawy, S. A., Novotny, M., Gismondi, E. A., Lam, R., Matsudaira, P., McKenna, B. K., O'Neil, T., Streechon, P. and Ehrlich, D. J. (2005) A 768-lane microfabricated system for high-throughput DNA sequencing. Lab Chip. 5 669-74. A 768-lane DNA sequencing system based on microfluidic plates has been designed as a near-term successor to 96-lane capillary arrays. Electrophoretic separations are implemented for the first time in large-format (25 cm [times] 50 cm) microdevices, with the objective of proving realistic read length, parallelism, and the scaled sample requirements for long-read de novo sequencing. Two 384-lane plates are alternatively cycled between electrophoresis and regeneration via a robotic pipettor. A total of greater than 172000 bases, 99% accuracy (corresponding to quality score 20) is achieved for each iteration of a 384 lane plate. At current operating conditions, this implies a system throughput exceeding 4 megabases of raw sequence (Phred 20) per day on the new platform. Standard operation is at "1/32[times]" Sanger chemistry, equal to typical genome center operation on mature capillary array machines, and a 16-fold improvement in scaling relative to previous microfabricated devices. Experiments provide evidence that sample concentration can be further reduced to 1/256[times] Sanger chemistry in the microdevice. Life-testing indicates a usable life of >150 hours (more than 50 runs) for the 384 lane plates. The combined advances, particularly those in read length and sample requirement, directly address the cost model requirements for adaptation of the new technology as the next step beyond capillary array instruments. Full Text
Ackerman, K. G., Huang, H. L., Grasemann, H., Puma, C., Singer, J. B., Hill, A. E., Lander, E., Nadeau, J. H., Churchill, G. A., Drazen, J. M. and Beier, D. R. (2005) Interacting genetic loci cause airway hyperresponsiveness. Physiological Genomics. 21 105-111. component of asthma, and the genetic basis of this complex trait has remained elusive. We created recombinant congenic mice with increased naive AHR by serially backcrossing A/J mice (which have elevated naive AHR) with C57BL/6J mice and selecting for mice with an elevated naive AHR phenotype. The seventh backcross-generation hyperresponsive mice retained A/J loci in three regions. Quantitative trait linkage (QTL) analysis of 123 unselected N8 progeny demonstrated that the AHR phenotype was not associated with any single locus but was significantly associated with an interaction of loci on chromosomes 2 and 6. These findings were confirmed in an independent analysis of chromosome substitution strain mice. The identification of genomic regions containing loci causally associated with AHR and the demonstration that this trait requires their interaction have important implications for the dissection of the genetic etiology of asthma in humans.
Ali, S. M. and Sabatini, D. M. (2005) Structure of S6K1 determines if raptor-mTOR or rictor-mTOR phosphorylates its hydrophobic motif site. J Biol Chem.Apr 4; [Epub ahead of print] The mTOR protein kinase is the target of the immunosuppressive and anti-cancer drug rapamycin and is increasingly recognized as a key regulator of cell growth in mammals. S6 kinase 1 (S6K1) is the best-characterized effector of mTOR and its regulation serves as a model for mTOR signaling. Nutrients and growth factors activate S6K1 by inducing the phosphorylation of threonine 389 in the hydrophobic motif of S6K1. As phosphorylation of T389 is rapamycin sensitive and mTOR can phosphorylate the same site in vitro, it has been suggested that mTOR is the physiological T389 kinase. This proposal is not supported, however, by the existence of mutants of S6K1 that are phosphorylated in vivo on T389 in a rapamycin-resistant fashion. Here, we demonstrate that the raptor-mTOR complex phosphorylates the rapamycin-sensitive forms of S6K1 while the distinct rictor-mTOR complex phosphorylates the rapamycin-resistant mutants of S6K1. Phosphorylation of T389 by rictor-mTOR is independent of the TOS motif and depends on removal of the carboxy terminal domain of S6K1. Because many members of the AGC family of kinases lack an analogous domain, rictor-mTOR may phosphorylate the hydrophobic motifs of other kinases. PDF
Anwar, M. and Rousso, I. (2005) Atomic force microscopy with time resolution of microseconds. Applied Physics Letters. 86 The atomic force microscope (AFM) can acquire high-resolution images under nondestructive conditions albeit at a very poor temporal resolution. This work presents two AFM mapping techniques, stroboscopic, and continuous, for imaging rapid periodical processes with nanometer spatial resolution and microsecond time resolution. Application of these methods is demonstrated for imaging very rapid cyclic changes in the position of a microfabricated grid. Motion was resolved with 10 nm spatial resolution, and 5 and 25 mus temporal resolution using the stroboscopic and continuous mode, respectively. The proposed methods have the potential to image wide variety of rapid processes with unparalleled combination of lateral and temporal resolutions.
Axtell, M. J. and Bartel, D. P. (2005) Antiquity of MicroRNAs and Their Targets in Land Plants. Plant Cell. Apr 22; [Epub ahead of print] MicroRNAs (miRNAs) affect the morphology of flowering plants by the posttranscriptional regulation of genes involved in critical developmental events. Understanding the spatial and temporal dynamics of miRNA activity during development is therefore central for understanding miRNA functions. We describe a microarray suitable for detection of plant miRNAs. Profiling of Arabidopsis thaliana miRNAs during normal development extends previous expression analyses, highlighting differential expression of miRNA families within specific organs and tissue types. Comparison of our miRNA expression data with existing mRNA microarray data provided a global intersection of plant miRNA and mRNA expression profiles and revealed that tissues in which a given miRNA is highly expressed are unlikely to also show high expression of the corresponding targets. Expression profiling was also used in a phylogenetic survey to test the depth of plant miRNA conservation. Of the 23 families of miRNAs tested, expression of 11 was detected in a gymnosperm and eight in a fern, directly demonstrating that many plant miRNAs have remained essentially unchanged since before the emergence of flowering plants. We also describe an empirical strategy for detecting miRNA target genes from unsequenced transcriptomes and show that targets in nonflowering plants as deeply branching as ferns and mosses are homologous to the targets in Arabidopsis. Therefore, several individual miRNA regulatory circuits have ancient origins and have remained intact throughout the evolution and diversification of plants.PDF
Barrett, J. C., Fry, B., Maller, J. and Daly, M. J. (2005) Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics. 21 263-265. Research over the last few years has revealed significant haplotype structure in the human genome. The characterization of these patterns, particularly in the context of medical genetic association studies, is becoming a routine research activity. Haploview is a software package that provides computation of linkage disequilibrium statistics and population haplotype patterns from primary genotype data in a visually appealing and interactive interface.Full Text.
Baskerville, S. and Bartel, D. P. (2005) Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes. RNA. 11 241-7. MicroRNAs (miRNAs) are short endogenous RNAs known to post-transcriptionally repress gene expression in animals and plants. A microarray profiling survey revealed the expression patterns of 175 human miRNAs across 24 different human organs. Our results show that proximal pairs of miRNAs are generally coexpressed. In addition, an abrupt transition in the correlation between pairs of expressed miRNAs occurs at a distance of 50 kb, implying that miRNAs separated by <50 kb typically derive from a common transcript. Some microRNAs are within the introns of host genes. Intronic miRNAs are usually coordinately expressed with their host gene mRNA, implying that they also generally derive from a common transcript, and that in situ analyses of host gene expression can be used to probe the spatial and temporal localization of intronic miRNAs.Full Text.
BenPorath, I. and Weinberg, R. A. (2005) The signals and pathways activating cellular senescence. Int J Biochem Cell Biol. 37 961-76. Cellular senescence is a program activated by normal cells in response to various types of stress. These include telomere uncapping, DNA damage, oxidative stress, oncogene activity and others. Senescence can occur following a period of cellular proliferation or in a rapid manner in response to acute stress. Once cells have entered senescence, they cease to divide and undergo a series of dramatic morphologic and metabolic changes. Cellular senescence is thought to play an important role in tumor suppression and to contribute to organismal aging, but a detailed description of its physiologic occurrence in vivo is lacking. Recent studies have provided important insights regarding the manner by which different stresses and stimuli activate the signaling pathways leading to senescence. These studies reveal that a population of growing cells may suffer from a combination of different physiologic stresses acting simultaneously. The signaling pathways activated by these stresses are funneled to the p53 and Rb proteins, whose combined levels of activity determine whether cells enter senescence. Here we review recent advances in our understanding of the stimuli that trigger senescence, the molecular pathways activated by these stimuli, and the manner by which these signals determine the entry of a population of cells into senescence. Full Text
Bernstein, B. E., Kamal, M., Lindblad-Toh, K., Bekiranov, S., Bailey, D. K., Huebert, D. J., McMahon, S., Karlsson, E. K., Kulbokas, E. J., Gingeras, T. R., Schreiber, S. L. and Lander, E. S. (2005) Genomic maps and comparative analysis of histone modifications in human and mouse. Cell. 120 169-181. We mapped histone H3 lysine 4 di- and trimethylation and lysine 9/14 acetylation across the nonrepetitive portions of human chromosomes 21 and 22 and compared patterns of lysine 4 dimethylation for several orthologous human and mouse loci. Both chromosomes show punctate sites enriched for modified histones. Sites showing trimethylation correlate with transcription starts, while those showing mainly dimethylation occur elsewhere in the vicinity of active genes. Punctate methylation patterns are also evident at the cytokine and IL-4 receptor loci. The Hox clusters present a strikingly different picture, with broad lysine 4-methylated regions that overlay multiple active genes. We suggest these regions represent active chromatin domains required for the maintenance of Hox gene expression. Methylation patterns at orthologous loci are strongly conserved between human and mouse even though many methylated sites do not show sequence conservation notably higher than background. This suggests that the DNA elements that direct the methylation represent only a small fraction of the region or lie at some distance from the site. Full Text.
Button, T. M. M., Scourfield, J., Martin, N., Purcell, S. and McGuffin, P. (2005) Family dysfunction interacts with genes in the causation of antisocial symptoms. Behavior Genetics. 35 115-120. There is emerging evidence of gene-environment interaction effects on conduct problems, both from adoption studies and from a study using a measured genotype. An association between non-violent family dysfunction and conduct problems has also been reported, although not in the context of gene-environment interaction studies. The aim of this study was to examine the interaction of genes and family dysfunction in contributing to conduct problems in young people. Parents of 278 monozygotic and 378 dizygotic twin pairs, aged 5-18, from the CaStANET birth cohort twin register were questioned about zygosity, conduct problems and family environment. Using structural equation modeling we tested for main and interactive effects of genes and family dysfunction modelled as an environmental 'moderator variable'. Both main and gene-environment interaction effects were highly significant. It was concluded that a risk genotype conferring susceptibility to family dysfunction is responsible for most of the variance in antisocial symptoms in childhood and adolescence.Full Text.
Cashikar, A. G., Duennwald, M. L. and Lindquist, S. L. (2005) A chaperone pathway in protein disaggregation: Hsp26 alters the nature of protein aggregates to facilitate reactivation by Hsp104. J Biol Chem. Apr 20; [Epub ahead of print] Cellular protein folding is challenged by environmental stress and aging which lead to aberrant protein conformations and aggregation. One way to antagonize the detrimental consequences of protein misfolding is to reactivate vital proteins from aggregates. In the yeast Saccharomyces cerevisiae, Hsp104 facilitates disaggregation and reactivates aggregated proteins with assistance from Hsp70 (Ssa1) and Hsp40 (Ydj1). The small heat shock proteins (sHsps), Hsp26 and Hsp42, also function in the recovery of misfolded proteins and prevent aggregation in vitro, but their in vivo roles in protein homeostasis remain elusive. We observed that after a sub-lethal heat shock, a majority of Hsp26 becomes insoluble. Its return to the soluble state during recovery depends on the presence of Hsp104. Further, cells lacking Hsp26 are impaired in the disaggregation of an easily assayed heat-aggregated reporter protein, luciferase. In vitro, Hsp104, Ssa1 and Ydj1 reactivate luciferase:Hsp26 co-aggregates 20-fold more efficiently than luciferase aggregates alone. sHsps also facilitate the Hsp104-mediated solubilization of polyglutamine in yeast. Thus, Hsp26 renders aggregates more accessible to Hsp104/Ssa1/Ydj1. sHsps partially suppress toxicity, even in the absence of Hsp104, potentially by sequestering polyQ from toxic interactions with other proteins. Hence, Hsp26 plays an important role in pathways that defend cells against environmental stress and the types of protein misfolding seen in neurodegenerative disease.PDF
Cerimele, F., Battle, T., Lynch, R., Frank, D. A., Murad, E., Cohen, C., Macaron, N., Sixbey, J., Smith, K., Watnick, R. S., Eliopoulos, A., Shehata, B. and Arbiser, J. L. (2005) Reactive oxygen signaling and MAPK activation distinguish Epstein-Barr virus (EBV)-positive versus EBV-negative Burkitt's lymphoma. Proceedings of the National Academy of Sciences of the United States of America. 102 175-179. Burkitt's lymphoma (BL) is an aggressive B cell neoplasm, which is one of the most common neoplasms of childhood. It is highly widespread in East Africa, where it appears in endemic form associated with Epstein-Barr virus (EBV) infection, and around the world in a sporadic form in which EBV infection is much less common. In addition to being the first human neoplasm to be associated with EBV, BL is associated with a characteristic translocation, in which the Ig promoter is translocated to constitutively activate the c-myc oncogene. Although many BLs respond well to chemotherapy, a significant fraction fails to respond to therapy, leading to death. In this article, we demonstrate that EBV-positive BL expresses high levels of activated mitogen-activated protein kinase and reactive oxygen species (ROS), and that ROS directly regulate NF-kappaB activation. EBV-negative BLs exhibit activation of phosphoinositol 3-kinase, but do not have elevated levels of ROS. Elevated reactive oxygen may play a role in diverse forms of viral carcinogenesis in humans, including cancers caused by EBV, hepatitis B, C, and human T cell lymphotropic virus. Our findings imply that inhibition of ROS may be useful in the treatment of EBV-induced neoplasia.Full Text
Chen, C. Z. and Lodish, H. F. (2005) MicroRNAs as regulators of mammalian hematopoiesis. Seminars in Immunology. 17 155-165. MicroRNAs (miRNAs) are an abundant class of similar to 22 nucleotide non-coding RNAs and play important regulatory roles in animal and plant development at the post-transcriptional level. Many miRNAs cloned from mouse bone marrow cells are differentially regulated in various hematopoietic lineages, suggesting that they might influence hematopoietic lineage differentiation. miR-181, a miRNA specifically expressed in B cells within mouse bone marrow, promotes B-cell differentiation when expressed in hematopoietic stem/progenitor cells. Some human miRNAs are linked to leukemias: the miR-15a/miR-16 locus is frequently deleted or down-regulated in patients with B-cell chronic lymphocytic leukemia and miR-142 is at a translocation site found in a case of aggressive B-cell leukemia. Collectively, these results indicate that miRNAs may be important regulators of mammalian hematopoiesis. Here, we provide background on the biogenesis and function of miRNAs and discuss how miRNA-mediated post-transcriptional regulation may influence the development and function of blood cells. Full Text
Clarke , A. S., Tang, T. T., Ooi, D. L. and Orr-Weaver, T. L. (2005) POLO Kinase Regulates the Drosophila Centromere Cohesion Protein MEI-S332. Dev Cell. 8 53-64. Accurate segregation of chromosomes is critical to ensure that each daughter cell receives the full genetic complement. Maintenance of cohesion between sister chromatids, especially at centromeres, is required to segregate chromosomes precisely during mitosis and meiosis. The Drosophila protein MEI-S332, the founding member of a conserved protein family, is essential in meiosis for maintaining cohesion at centromeres until sister chromatids separate at the metaphase II/anaphase II transition. MEI-S332 localizes onto centromeres in prometaphase of mitosis or meiosis I, remaining until sister chromatids segregate. We elucidated a mechanism for controlling release of MEI-S332 from centromeres via phosphorylation by POLO kinase. We demonstrate that POLO antagonizes MEI-S332 cohesive function and that full POLO activity is needed to remove MEI-S332 from centromeres, yet this delocalization is not required for sister chromatid separation. POLO phosphorylates MEI-S332 in vitro, POLO and MEI-S332 bind each other, and mutation of POLO binding sites prevents MEI-S332 dissociation from centromeres. Full Text
Claycomb, J. M. and Orr-Weaver, T. L. (2005) Developmental gene amplification: insights into DNA replication and gene expression. Trends Genet. 21 149-62. In the formation of a complex organism and the differentiation of specific cell types, there are often demands for high levels of particular gene products. These demands can be met by increasing transcription or translation, or by decreasing the rate of mRNA or protein turnover. Although these are the most common means to increase expression levels, there is another mechanism: gene amplification. Developmental gene amplification is a DNA replication-based process whereby specific genes are replicated above the copy number of surrounding sequences, resulting in an increase in the template available for transcription. Recent microarray studies in Drosophila melanogaster have identified two additional amplicons, suggesting that developmental gene amplification might be more widely used than was previously thought. Furthermore, work in both Drosophila and the related fly, Sciara coprophila, has yielded insights into the mechanisms, regulatory sequences and proteins controlling DNA replication during gene amplification, including a link between transcription factors and origin usage. Full Text.
Curran, S., Purcell, S., Craig, I., Asherson, P. and Sham, P. (2005) The serotonin transporter gene as a QTL for ADHD. American Journal of Medical Genetics Part B-Neuropsychiatric Genetics. 134B 42-47. Molecular studies of attention deficit hyperactivity disorder (ADHD) have identified susceptibility genes for the categorically diagnosed disorder using operational diagnostic criteria. Here, we take a QTL approach to mapping genes for ADHD using a composite continuous index of ADHD behavior in a large epidemiological sample. Previous studies of clinical ADHD suggest that two functional polymorphisms in the serotonin transporter gene (SLC6A4), one in the 5'-regulatory region of the gene (5-HTTLPR) and the other a VNTR (5-HTTVNTR) in the second intron, as well as a single nucleotide polyrnorphism in the 3'-untranslated region (3'-UTR SNP), may be associated with the disorder. Here, we investigate these polymorphisms as well as an additional ten SNPs spread across the gene. We found significant association with the long (L) allele of the 5-HTTLPR; P = 0.019, but neither the 5-HTTVNTR nor the T-UTR SNP were significantly associated. Significant associations (P < 0.05) were found for a further 5 the 10 other markers tested. We found evidence for two haplotype blocks spanning the region. We found strong evidence for association with the first haplotype block (comprised of four markers), with the significance of a combined primary and secondary test of association reaching an empirical P value = 0.0054 for the global test and an empirical P value = 0.00081 for the largest local test. Thus, we show here that SLC6A4, which has a major influence on brain serotonin availability, may be a QTL for ADHD .Full Text
Daly, M. J. and Altshuler, D. (2005) Partners in crime. Nat Genet. 37 337-8. The genetic culprits that contribute to common diseases remain at large, despite dedicated sleuthing by many laboratories. A new study evaluates the power of genome-wide searches for variants acting in combination, with results that are both unexpected and encouraging. Full Text.
Dinulescu, D. M., Ince, T. A., Quade, B. J., Shafer, S. A., Crowley, D. and Jacks, T. (2005) Role of K-ras and Pten in the development of mouse models of endometriosis and endometrioid ovarian cancer. Nature Medicine. 11 63-70. Epithelial ovarian tumors present a complex clinical, diagnostic and therapeutic challenge because of the difficulty of early detection, lack of known precursor lesions and high mortality rates. Endometrioid ovarian carcinomas are frequently associated with endometriosis, but the mechanism for this association remains unknown. Here we present the first genetic models of peritoneal endometriosis and endometrioid ovarian adenocarcinoma in mice, both based on the activation of an oncogenic K-ras allele. In addition, we find that expression of oncogenic K-ras or conditional Pten deletion within the ovarian surface epithelium gives rise to preneoplastic ovarian lesions with an endometrioid glandular morphology. Furthermore, the combination of the two mutations in the ovary leads to the induction of invasive and widely metastatic endometrioid ovarian adenocarcinomas with complete penetrance and a disease latency of only 7 weeks. The ovarian cancer model described in this study recapitulates the specific tumor histomorphology and metastatic potential of the human disease. Full Text.
Dobbin, K. K., Beer, D. G., Meyerson, M., Yeatman, T. J., Gerald, W. L., Jacobson, J. W., Conley, B., Buetow, K. H., Heiskanen, M., Simon, R. N., Minna, J. D., Girard, L., Misek, D. E., Taylor, J. M. G., Hanash, S., Naoki, K., Hayes, D. N., Ladd-Acosta, C., Enkemann, S. A., Viale, A. and Giordano, T. J. (2005) Interlaboratory comparability study of cancer gene expression analysis using oligonucleotide microarrays. Clinical Cancer Research. 11 565-572. A key step in bringing gene expression data into clinical practice is the conduct of large studies to confirm preliminary models. The performance of such confirmatory studies and the transition to clinical practice requires that microarray data from different laboratories are comparable and reproducible. We designed a study to assess the comparability of data front four laboratories that will conduct a larger microarray profiling confirmation project in lung adenocarcinomas. To test the feasibility of combining data across laboratories, frozen tumor tissues, cell line pellets, and purified RNA samples were analyzed at each of the four laboratories. Samples of each type and several subsamples from each tumor and each cell line were blinded before being distributed. The laboratories followed a common protocol for all steps of tissue processing, RNA extraction. and microarrav analysis using Affymetrix Human Genome U133A arrays. High within-laboratory and between-laboratory correlations were observed on the purified RNA samples. the cell tines. and the frozen tumor tissues. Intraclass correlation within laboratories was only slightly stronger than between laboratories, and the intraclass correlation tended to be weakest for genes expressed at low levels and showing small variation. Finally, hierarchical cluster analysis revealed that the repeated samples clustered to-ether regardless of the laboratory in which the experiments were done. The findings indicate that under properly controlled conditions it is feasible to perform complete tumor microarray analysis, from tissue processing to hybridization and scanning, at multiple independent laboratories for a single study.Full Text.
Endres, M., Ahmadi, M., Kruman, I., Biniszkiewicz, D., Meisel, A. and Gertz, K. (2005) Folate deficiency increases postischemic brain injury. Stroke. 36 321-325. Background and Purpose - Folate deficiency and resultant hyperhomocysteinemia impair vascular function and increase stroke risk. We tested the hypothesis that folate deficiency and high homocysteine levels promote DNA damage and increase brain injury after cerebral ischemia/reperfusion. Methods - 129/Sv mice, uracil-DNA glycosylase - deficient (Ung(-/-)) mice, and Ung(+/+\) littermate mice were exposed to a folate-deficient diet for 3 months and then subjected to 30-minute middle cerebral artery (MCA) occlusion and reperfusion. Plasma homocysteine levels and physiological parameters were measured in selected animals. Outcome measures were neurological sensorimotor deficits, infarct size measured by computer-assisted volumetry, and oxidative DNA damage measured by a colorimetric assay. Results - Exposure to a folate-deficient diet for 3 months conferred approximate to6- to 10-fold higher plasma homocysteine levels than those associated with a normal diet. Cerebral lesion volumes and neurological deficits after MCA occlusion and 72-hour reperfusion were significantly 2.1-fold increased in folate-deficient 129/SV wild-type mice compared with those associated with a normal diet, which could not be explained by obvious differences in physiological parameters. Abasic sites, hallmarks of oxidative DNA damage, were significantly increased in DNA from the ischemic brain of folate-deficient animals at early time points after MCA occlusion. Folate deficiency further increased brain lesion size in animals lacking uracil-DNA glycosylase compared with wild-type littermate mice. Conclusions - Folate deficiency and resultant hyperhomocysteinemia are not only associated with increased stroke risk but increase oxidative DNA damage and ischemic lesion size after MCA occlusion/reperfusion.Full Text.
Fink, G. R. (2005) A transforming principle. Cell. 120 153-4. Full Text.
Gimelbrant, A. A., Ensminger, A. W., Qi, P. M., Zucker, J. and Chess, A. (2005) Monoallelic expression and asynchronous replication of p120 catenin in mouse and human cells. Journal of Biological Chemistry. 280 1354-1359. The number of autosomal mammalian genes subject to random monoallelic expression has been limited to genes highly specific to the function of chemosensory neurons or lymphocytes, making this phenomenon difficult to address systematically. Here we demonstrate that asynchronous DNA replication can be used as a marker for the identification of novel genes with monoallelic expression and identify p120 catenin, a gene involved in cell adhesion, as belonging to this class. p120 is widely expressed; its presence in available cell lines allowed us to address quantitative aspects of monoallelic expression. We show that the epigenetic choice of active allele is clonally stable and that biallelic clones express p120 at twice the level of monoallelic clones. Unlike previous reports about genes of this type, we found that expression of p120 can be monoallelic in one cell type and strictly biallelic in another. We show that in human lymphoblasts, the silencing of one allele is incomplete. These unexpected properties are likely to be widespread, as we show that the Tlr4 gene shares them. Identification of monoallelic expression of a nearly ubiquitous gene indicates that this type of gene regulation is more common than previously thought. This has important implications for carcinogenesis and definition of cell identity.Full Text.
Giraldez, A. J., Cinalli, R. M., Glasner, M. E., Enright, A. J., Thomson, J. M., Baskerville, S., Hammond, S. M., Bartel, D. P. and Schier, A. F. (2005) MicroRNAs regulate brain morphogenesis in zebrafish. Science. 308 833-838. MicroRNAs (miRNAs) are small RNAs that regulate gene expression posttranscriptionally. To block all miRNA formation in zebrafish, we generated maternal-zygotic dicer (MZdicer) mutants that disrupt the Dicer ribonuclease III and double-stranded RNA-binding domains. Mutant embryos do not process precursor miRNAs into mature miRNAs, but injection of preprocessed miRNAs restores gene silencing, indicating that the disrupted domains are dispensable for later steps in silencing. MZdicer mutants undergo axis formation and differentiate multiple cell types but display abnormal morphogenesis during gastrulation, brain formation, somitogenesis, and heart development. Injection of miR-430 miRNAs rescues the brain defects in MZdicer mutants, revealing essential roles for miRNAs during morphogenesis. Full Text.
Gordon, D. B., Nekludova, L., McCallum, S. and Fraenkel, E. (2005) TAMO: a flexible, object-oriented framework for analyzing transcriptional regulation using DNA-sequence motifs. Bioinformatics.May19 [Epub ahead of print] SUMMARY: TAMO (Tools for Analysis of MOtifs) is an object-oriented computational framework for interpreting transcriptional regulation using DNA-sequence motifs. To simplify the application of multiple motif discovery programs to genome-wide data, TAMO provides a sophisticated motif object with interfaces to several popular programs. In addition, TAMO provides modules for integrating motif analysis with diverse data sources including genomic sequences, microarrays, and various databases. Finally, TAMO includes tools for sequence analysis, algorithms for scoring, comparing and clustering motifs, and several useful statistical tests. Recently, we have applied these tools to analyze tens of thousands of motifs derived from hundreds of microarray experiments (Harbison et al., 2004). AVAILABILITY: TAMO is a Python/C++ package and requires Python 2.3 or higher. Source code and documentation are available at http://web.wi.mit.edu/fraenkel/TAMO/. PDF.
Gribnau, J., Luikenhuis, S., Hochedlinger, K., Monkhorst, K. and Jaenisch, R. (2005) X chromosome choice occurs independently of asynchronous replication timing. J Cell Biol. 168 365-73. In mammals, dosage compensation is achieved by X chromosome inactivation in female cells. Xist is required and sufficient for X inactivation, and Xist gene deletions result in completely skewed X inactivation. In this work, we analyzed skewing of X inactivation in mice with an Xist deletion encompassing sequence 5 KB upstream of the promoter through exon 3. We found that this mutation results in primary nonrandom X inactivation in which the wild-type X chromosome is always chosen for inactivation. To understand the molecular mechanisms that affect choice, we analyzed the role of replication timing in X inactivation choice. We found that the two Xist alleles and all regions tested on the X chromosome replicate asynchronously before the start of X inactivation. However, analysis of replication timing in cell lines with skewed X inactivation showed no preference for one of the two Xist alleles to replicate early in S-phase before the onset of X inactivation, indicating that asynchronous replication timing does not play a role in skewing of X inactivation. Full Text
Hirschhorn, J. N. and Daly, M. J. (2005) Genome-wide association studies for common diseases and complex traits. Nature Reviews Genetics. 6 95-108. Genetic factors strongly affect susceptibility to common diseases and also influence disease-related quantitative traits. Identifying the relevant genes has been difficult, in part because each causal gene only makes a small contribution to overall heritability. Genetic association studies offer a potentially powerful approach for mapping causal genes with modest effects, but are limited because only a small number of genes can be studied at a time. Genome-wide association studies will soon become possible, and could open new frontiers in our understanding and treatment of disease. However, the execution and analysis of such studies will require great care.Full Text
Hochedlinger, K., Yamada, Y., Beard, C. and Jaenisch, R. (2005) Ectopic expression of oct-4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues. Cell. 121 465-77. The POU-domain transcription factor Oct-4 is normally expressed in pluripotent cells of the mammalian embryo. In addition, germ-cell tumors and a few somatic tumors show detectable expression of Oct-4. While Oct-4's role during preimplantation development is to maintain embryonic cells in a pluripotent state, little is known about its potential oncogenic properties. Here we investigate the effect of ectopic Oct-4 expression on somatic tissues of adult mice using a doxycycline-dependent expression system. Activation of Oct-4 results in dysplastic growths in epithelial tissues that are dependent on continuous Oct-4 expression. Dysplastic lesions show an expansion of progenitor cells and increased beta-catenin transcriptional activity. In the intestine, Oct-4 expression causes dysplasia by inhibiting cellular differentiation in a manner similar to that in embryonic cells. These data show that certain adult progenitors remain competent to interpret key embryonic signals and support the notion that progenitor cells are a driving force in tumorigenesis. Full Text
Hug, C. and Lodish, H. F. (2005) Visfatin: A new adipokine. Science. 307 366-367. Full Text.
Hug, C. and Lodish, H. F. (2005) The role of the adipocyte hormone adiponectin in cardiovascular disease. Curr Opin Pharmacol. 5 129-34. Adiponectin, a novel hormone made by fat tissue, regulates energy metabolism and endothelial activation. Serum levels of adiponectin are reduced in conditions that are associated with an increased risk of cardiovascular disease, such as diabetes and the metabolic syndrome. Adiponectin trimers assemble into higher-order oligomers, which have different signaling properties. Adiponectin trimers and a C-terminal globular domain activate AMP-activated protein kinase, whereas hexamer and high-molecular weight isoforms activate nuclear factor-kappaB signaling pathways. Exogenous adiponectin corrects metabolic defects that are associated with insulin resistance, and might protect the endothelium from the progression of cardiovascular disease. Receptors for adiponectin have been described and might provide future therapeutic targets for the treatment of cardiovascular disease.Full Text.
Jenner, R. G. and Young, R. A. (2005) Insights into host responses against pathogens from transcriptional profiling. Nat Rev Microbiol. 3 281-94. DNA microarrays have allowed us to monitor the effects of pathogens on host-cell gene expression programmes in great depth and on a broad scale. The comparison of results that have been generated by these studies is complex, and such a comparison has not previously been attempted in a systematic manner. In this review, we have collated and compared published transcriptional-profiling data from 32 studies that involved 77 different host-pathogen interactions, and have defined a common host-transcriptional-response. We outline gene expression patterns in the context of Toll-like receptor and pathogen-mediated signalling pathways, and summarize the contributions that transcriptional-profiling studies have made to our understanding of the infectious disease process.Full Text.
Kim, S. W., Kim, S., Tracy, J. B., Jasanoff, A. and Bawendi, M. G. (2005) Phosphine oxide polymer for water-soluble nanoparticles. J Am Chem Soc. 127 4556-7. A phosphine oxide polymer was developed using bis(dichlorophosphino)ethane and poly(ethylene glycol). This polymer system was used to transfer various nanoparticles from organic solvents to water, retaining their physical properties and reactivities.Full Text.
Koralov, S. B., Novobrantseva, K. I., Hochedlinger, K., Jaenisch, R. and Rajewsky, K. (2005) Direct in vivo V-H to J(H) rearrangement violating the 12/23 rule. Journal of Experimental Medicine. 201 341-348. V(D)J recombination at the immunoglobulin heavy chain (IgH) locus follows the 12/23 rule to ensure the correct assembly of the variable region gene segments. Here, we report characterization of an in vivo model that allowed us to study recombination violating the 12/23 rule, namely a mouse strain lacking canonical D elements in its IgH locus. We demonstrate that V-H to J(H) joining can support the generation of all B cell subsets. However, the process is inefficient in that B cells and antibodies derived from the D-H-less allelle are not detectable if the latter is combined with a wild-type IgH allele. There is no preferential usage of any particular V-H gene family or J(H) element in V(H)J(H) junctions, indicating that 23/23-guided recombination is possible, but is a low frequency event at the IgH locus in vivo.Full Text.
Lee, L. A., Lee, E., Anderson, M. A., Vardy, L., Tahinci, E., Ali, S. M., Kashevsky, H., Benasutti, M., Kirschner, M. W. and Orr-Weaver, T. L. (2005) Drosophila genome-scale screen for PAN GU kinase substrates identifies Mat89Bb as a cell cycle regulator. Dev Cell. 8 435-42. Although traditional organism-based mutational analysis is powerful in identifying genes involved in specific biological processes, limitations include incomplete coverage and time required for gene identification. Biochemical screens using cell transfection or yeast two-hybrid methods are rapid, but they are limited by cDNA library quality. The recent establishment of "uni-gene sets" has made it feasible to biochemically screen an organism's entire genome. Radiolabeled protein pools prepared from the Drosophila Gene Collection were used in a Drosophila in vitro expression cloning ("DIVEC") screen for substrates of PAN GU kinase, which is crucial for S-M embryonic cell cycles. Ablation of one identified substrate, Mat89Bb, by RNAi produces a polyploid phenotype similar to that of pan gu mutants. Xenopus embryos injected with Mat89Bb morpholinos arrest with polyploid nuclei, and Mat89Bb RNAi in HeLa cells gives rise to multinucleated cells. Thus, Mat89Bb plays an evolutionarily conserved role as a crucial regulator of both cell cycle and development. Full Text.
Lewis, B. P., Burge, C. B. and Bartel, D. P. (2005) Conserved Seed Pairing, Often Flanked by Adenosines, Indicates that Thousands of Human Genes are MicroRNA Targets. Cell. 120 15-20. We predict regulatory targets of vertebrate microRNAs (miRNAs) by identifying mRNAs with conserved complementarity to the seed (nucleotides 2-7) of the miRNA. An overrepresentation of conserved adenosines flanking the seed complementary sites in mRNAs indicates that primary sequence determinants can supplement base pairing to specify miRNA target recognition. In a four-genome analysis of 3' UTRs, approximately 13,000 regulatory relationships were detected above the estimate of false-positive predictions, thereby implicating as miRNA targets more than 5300 human genes, which represented 30% of our gene set. Targeting was also detected in open reading frames. In sum, well over one third of human genes appear to be conserved miRNA targets. Full Text.
Lim, L. P., Lau, N. C., Garrett-Engele, P., Grimson, A., Schelter, J. M., Castle, J., Bartel, D. P., Linsley, P. S. and Johnson, J. M. (2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature. 433 769-773. MicroRNAs ( miRNAs) are a class of noncoding RNAs that post-transcriptionally regulate gene expression in plants and animals(1,2). To investigate the influence of miRNAs on transcript levels, we transfected miRNAs into human cells and used microarrays to examine changes in the messenger RNA profile. Here we show that delivering miR-124 causes the expression profile to shift towards that of brain, the organ in which miR-124 is preferentially expressed, whereas delivering miR-1 shifts the profile towards that of muscle, where miR-1 is preferentially expressed. In each case, about 100 messages were downregulated after 12 h. The 30 untranslated regions of these messages had a significant propensity to pair to the 50 region of the miRNA, as expected if many of these messages are the direct targets of the miRNAs(3). Our results suggest that metazoan miRNAs can reduce the levels of many of their target transcripts, not just the amount of protein deriving from these transcripts. Moreover, miR-1 and miR-124, and presumably other tissue-specific miRNAs, seem to downregulate a far greater number of targets than previously appreciated, thereby helping to define tissue-specific gene expression in humans. Full Text
Lowery, L. A. and Sive, H. (2005) Initial formation of zebrafish brain ventricles occurs independently of circulation and requires the nagie oko and snakehead/atp1a1a.1 gene products. Development. 132 2057-67.Epub 2005 Mar 23 The mechanisms by which the vertebrate brain develops its characteristic three-dimensional structure are poorly understood. The brain ventricles are a highly conserved system of cavities that form very early during brain morphogenesis and that are required for normal brain function. We have initiated a study of zebrafish brain ventricle development and show here that the neural tube expands into primary forebrain, midbrain and hindbrain ventricles rapidly, over a 4-hour window during mid-somitogenesis. Circulation is not required for initial ventricle formation, only for later expansion. Cell division rates in the neural tube surrounding the ventricles are higher than between ventricles and, consistently, cell division is required for normal ventricle development. Two zebrafish mutants that do not develop brain ventricles are snakehead and nagie oko. We show that snakehead is allelic to small heart, which has a mutation in the Na(+)K(+) ATPase gene atp1a1a.1. The snakehead neural tube undergoes normal ventricle morphogenesis; however, the ventricles do not inflate, probably owing to impaired ion transport. By contrast, mutants in nagie oko, which was previously shown to encode a MAGUK family protein, fail to undergo ventricle morphogenesis. This correlates with an abnormal brain neuroepithelium, with no clear midline and disrupted junctional protein expression. This study defines three steps that are required for brain ventricle development and that occur independently of circulation: (1) morphogenesis of the neural tube, requiring nok function; (2) lumen inflation requiring atp1a1a.1 function; and (3) localized cell proliferation. We suggest that mechanisms of brain ventricle development are conserved throughout the vertebrates.Full Text
Lyckman, A. W., Fan, G. P., Rios, M., Jaenisch, R. and Sur, M. (2005) Normal eye-specific patterning of retinal inputs to mutine subcortical visual nuclei in the absence of brain-derived neurotrophic factor. Visual Neuroscience. 22 27-36. Brain-derived neurotrophic factor (BDNF) is a preferred ligand for a member of the tropomyosin-related receptor family, trkB. Activation of trkB is implicated in various activity-independent as well as activity-dependent growth processes in many developing and mature neural systems. In the subcortical visual system, where electrical activity has been implicated in normal development, both differential survival, as well as remodeling of axonal arbors, have been suggested to contribute to eye-specific segregation of retinal ganglion cell inputs. Here, we tested whether BDNF is required for eye-specific segregation of visual inputs to the lateral geniculate nucleus and the superior colliculus, and two other major subcortical target fields in mice. We report that eye-specific patterning is normal in two mutants that lack BDNF expression during the segregation period: a germ-line knockout for BDNF, and a conditional mutant in which BDNF expression is absent or greatly reduced in the central nervous system. We conclude that the availability of BDNF is not necessary for eye-specific segregation in subcortical visual nuclei. Full Text.
Mallory, A. C., Bartel, D. P. and Bartel, B. (2005) microRNA-Directed Regulation of Arabidopsis AUXIN RESPONSE FACTOR17 Is Essential for Proper Development and Modulates Expression of Early Auxin Response Genes. Plant Cell. 17 1360-1375. The phytohormone auxin plays critical roles during plant growth, many of which are mediated by the auxin response transcription factor (ARF) family. microRNAs (miRNAs), endogenous 21-nucleotide riboregulators, target several mRNAs implicated in auxin responses. miR160 targets ARF10, ARF16, and ARF17, three of the 23 Arabidopsis thaliana ARF genes. Here, we describe roles of miR160-directed ARF17 posttranscriptional regulation. Plants expressing a miRNA-resistant version of ARF17 have increased ARF17 mRNA levels and altered accumulation of auxin-inducible GH3-like mRNAs, YDK1/GH3.2, GH3.3, GH3.5, and DFL1/GH3.6, which encode auxin-conjugating proteins. These expression changes correlate with dramatic developmental defects, including embryo and emerging leaf symmetry anomalies, leaf shape defects, premature inflorescence development, altered phyllotaxy along the stem, reduced petal size, abnormal stamens, sterility, and root growth defects. These defects demonstrate the importance of miR160-directed ARF17 regulation and implicate ARF17 as a regulator of GH3-like early auxin response genes. Many of these defects resemble phenotypes previously observed in plants expressing viral suppressors of RNA silencing and plants with mutations in genes important for miRNA biogenesis or function, providing a molecular rationale for phenotypes previously associated with more general disruptions of miRNA function..Full Text
Marszalek, J. R., Kitidis, C., Dirusso, C. C. and Lodish, H. F. (2005) Long-chain Acyl CoA synthetase 6 preferentially promotes DHA metabolism.J Biol Chem. Jan 17; [Epub ahead of print] Previously we demonstrated that supplementation with the polyunsaturated fatty acids (PUFA) arachidonic acid (AA) or docosahexaenoic acid (DHA) increased neurite outgrowth of PC12 cells during differentiation, and that over-expression of rat acyl-CoA synthetase long-chain family member 6 (Acsl6 - formerly ACS2) further increased PUFA-enhanced neurite outgrowth. However, whether Acsl6 over-expression enhanced the amount of PUFA accumulated in the cells or altered the partitioning of any FA into phospholipids (PLs) or triglycerides (TAGs) was unknown. Here we show that Acsl6 over-expression specifically promotes DHA internalization, activation to DHA-CoA, and accumulation in differentiating PC12 cells. In contrast, OA and AA internalization and activation to OA-CoA and AA-CoA were increased only marginally by Acsl6 over-expression. Additionally, the level of total cellular PLs was increased in Acsl6 over-expressing cells when the medium was supplemented with AA and DHA, but not with OA. Acsl6 over-expression increased the incorporation of [(14)C]-labeled OA, AA or DHA into PLs and TAGs. These results do not support a role for Acsl6 in the specific targeting of FAs into PLs or TAGs. Rather, our data support the hypothesis that Acsl6 functions primarily in DHA metabolism, and that its over-expression increases DHA and AA internalization primarily during the first 24 hours of neuronal differentiation to stimulate PL synthesis and enhance neurite outgrowth. PDF
Mutsuddi, M., Chaffee, B., Cassidy, J., Silver, S. J., Yootle, T. L. and Rebay, I. (2005) Using Drosophila to decipher how mutations associated with human branchio-oto-renal syndrome and optical defects compromise the protein tyrosine phosphatase and transcriptional functions of Eyes absent. Genetics.2005 Mar 31; [Epub ahead of print] Eyes absent (EYA) proteins are defined by a conserved C-terminal EYA domain (ED) that both contributes to its function as a transcriptional coactivator by mediating protein-protein interactions and possesses intrinsic protein tyrosine phosphatase activity. Mutations in human EYA1 result in an autosomal dominant disorder called branchio-oto-renal (BOR) syndrome as well as congenital cataracts and ocular defects (OD). Both BOR and OD associated missense mutations alter residues in the conserved ED as do three missense mutations identified from Drosophila eya alleles. To investigate the molecular mechanisms whereby these mutations disrupt EYA function, we tested their activity in a series of assays that measured in vivo function, phosphatase activity, transcriptional capability, and protein-protein interactions. We find that the OD associated mutations retain significant in vivo activity whereas those derived from BOR patients show a striking decrease or loss of in vivo functionality. Protein-protein interactions, either with its partner transcription factor Sine oculis or with EYA itself, were not significantly compromised. Finally, the results of the biochemical assays suggest that both loss of protein tyrosine phosphatase activity and reduced transcriptional capability contributes to the impaired EYA function associated with BOR/OD syndrome, thus shedding new light into the molecular mechanisms underlying this disease.PDF
Orimo, A., Gupta, P. B., Sgroi, D. C., Arenzana-Seisdedos, F., Delaunay, T., Naeem, R., Carey, V. J., Richardson, A. L. and Weinberg, R. A. (2005) Stromal Fibroblasts Present in Invasive Human Breast Carcinomas Promote Tumor Growth and Angiogenesis through Elevated SDF-1/CXCL12 Secretion. Cell. 121 335-48. Fibroblasts often constitute the majority of the stromal cells within a breast carcinoma, yet the functional contributions of these cells to tumorigenesis are poorly understood. Using a coimplantation tumor xenograft model, we demonstrate that carcinoma-associated fibroblasts (CAFs) extracted from human breast carcinomas promote the growth of admixed breast carcinoma cells significantly more than do normal mammary fibroblasts derived from the same patients. The CAFs, which exhibit the traits of myofibroblasts, play a central role in promoting the growth of tumor cells through their ability to secrete stromal cell-derived factor 1 (SDF-1); CAFs promote angiogenesis by recruiting endothelial progenitor cells (EPCs) into carcinomas, an effect mediated in part by SDF-1. CAF-secreted SDF-1 also stimulates tumor growth directly, acting through the cognate receptor, CXCR4, which is expressed by carcinoma cells. Our findings indicate that fibroblasts within invasive breast carcinomas contribute to tumor promotion in large part through the secretion of SDF-1.Full Text
Pan, H., Yan, B. S., Rojas, M., Shebzukhov, Y. V., Zhou, H. W., Kobzik, L., Higgins, D. E., Daly, M. J., Bloom, B. R. and Kramnik, I. (2005) Ipr1 gene mediates innate immunity to tuberculosis. Nature. 434 767-772. An estimated eight million people are infected each year with the pathogen Mycobacterium tuberculosis, and more than two million die annually(1). Yet only about 10% of those infected develop tuberculosis. Genetic variation within host populations is known to be significant in humans and animals(2,3), but the nature of genetic control of host resistance to tuberculosis remains poorly understood. Previously we mapped a new genetic locus on mouse chromosome 1, designated sst1 (for supersusceptibility to tuberculosis 1)(4). Here we show that this locus mediates innate immunity in sst1 congenic mouse strains and identify a candidate gene, Intracellular pathogen resistance 1 (Ipr1), within the sst1 locus. The Ipr1 gene is upregulated in the sst1 resistant macrophages after activation and infection, but it is not expressed in the sst1 susceptible macrophages. Expression of the Ipr1 transgene in the sst1 susceptible macrophages limits the multiplication not only of M. tuberculosis but also of Listeria monocytogenes and switches a cell death pathway of the infected macrophages from necrosis to apoptosis. Our data indicate that the Ipr1 gene product might have a previously undocumented function in integrating signals generated by intracellular pathogens with mechanisms controlling innate immunity, cell death and pathogenesis.Full Text
Petryshen, T. L., Middleton, F. A., Kirby, A., Aldinger, K. A., Purcell, S., Tahl, A. R., Morley, C. P., McGann, L., Gentile, K. L., Rockwell, G. N., Medeiros, H. M., Carvalho, C., Macedo, A., Dourado, A., Valente, J., Ferreira, C. P., Patterson, N. J., Azevedo, M. H., Daly, M. J., Pato, C. N., Pato, M. T. and Sklar, P. (2005) Support for involvement of neuregulin 1 in schizophrenia pathophysiology. Molecular Psychiatry. 10 366-374. Schizophrenia is a common, multigenic psychiatric disorder. Linkage studies, including a recent meta-analysis of genome scans, have repeatedly implicated chromosome 8p12-p23.1 in schizophrenia susceptibility. More recently, significant association with a candidate gene on 8p12, neuregulin 1 (NRG1), has been reported in several European and Chinese samples. We investigated NRG1 for association in schizophrenia patients of Portuguese descent to determine whether this gene is a risk factor in this population. We tested NRG1 markers and haplotypes for association in 111 parent-proband trios, 321 unrelated cases, and 242 control individuals. Associations were found with a haplotype that overlaps the risk haplotype originally reported in the Icelandic population ('Hap(ICE)'), and two haplotypes located in the 30 end of NRG1 ( all P<0.05). However, association was not detected with Hap(ICE) itself. Comparison of NRG1 transcript expression in peripheral leukocytes from schizophrenia patients and unaffected siblings identified 3.8-fold higher levels of the SMDF variant in patients ( P = 0.039). Significant positive correlations ( P<0.001) were found between SMDF and HRG-beta 2 expression and between HRG-gamma and ndf43 expression, suggesting common transcriptional regulation of NRG1 variants. In summary, our results suggest that haplotypes across NRG1 and multiple NRG1 variants are involved in schizophrenia.Full Text.
Purcell, S., Sham, P. and Daly, M. J. (2005) Parental phenotypes in family-based association analysis. American Journal of Human Genetics. 76 249-259. Family-based association designs are popular, because they offer inherent control of population stratification based on age, sex, ethnicity, and environmental exposure. However, the efficiency of these designs is hampered by current analytic strategies that consider only offspring phenotypes. Here, we describe the incorporation of parental phenotypes and, specifically, the inclusion of parental genotype-phenotype correlation terms in association tests, providing a series of tests that effectively span an efficiency-robustness spectrum. The model is based on the between-within-sibship association model presented in 1999 by Fulker and colleagues for quantitative traits and extended here to nuclear families. By use of a liability-threshold-model approach, standard dichotomous and/or qualitative disease phenotypes can be analyzed (and can include appropriate corrections for phenotypically ascertained samples), which allows for the application of this model to analysis of the commonly used affected-proband trio design. We show that the incorporation of parental phenotypes can considerably increase power, as compared with the standard transmission/disequilibrium test and equivalent quantitative tests, while providing both significant protection against stratification and a means of evaluating the contribution of stratification to positive results. This methodology enables the extraction of more information from existing family-based collections that are currently being genotyped and analyzed by use of standard approaches.Full Text.
Quinzii, C. M., Kattah, A. G., Naini, A., Akman, H. O., Mootha, V. K., DiMauro, S. and Hirano, M. (2005) Coenzyme Q deficiency and cerebellar ataxia associated with an aprataxin mutation. Neurology. 64 539-541. Primary muscle coenzyme Q10 (CoQ10) deficiency is an apparently autosomal recessive condition with heterogeneous clinical presentations. Patients with these disorders improve with CoQ10 supplementation. In a family with ataxia and CoQ10 deficiency, analysis of genome-wide microsatellite markers suggested linkage of the disease to chromosome 9p13 and led to identification of an aprataxin gene ( APTX) mutation that causes ataxia oculomotor apraxia (AOA1 [MIM606350]). The authors' observations indicate that CoQ10 deficiency may contribute to the pathogenesis of AOA1.
Rebay, I., Silver, S. J. and Tootle, T. L. (2005) New vision from Eyes absent: transcription factors as enzymes. Trends Genet. 21 163-171. Post-translational modifications such as phosphorylation provide versatile context-specific strategies for modulating transcription factor activity. In the prevailing view of this process, modifying enzymes indirectly influence gene expression by shuttling to and from the nucleus where they alter the activity of their target transcription factors. However, a new paradigm has recently been suggested from studies of Eyes absent (EYA), a member of a conserved network of transcriptional regulators implicated in the development of numerous tissues and organs including the eye, ear, muscle and kidney. These findings indicate that EYA operates both as a transcriptional coactivator and as the prototype of a novel class of protein tyrosine phosphatases. The regulatory potential of such a bifunctional transcription factor is enormous and suggests a new layer of dynamic regulation in which transcription factors themselves might provide intrinsic enzymatic activities to fine-tune nuclear output. Full Text
Sagerstrom, C. G., Gammill, L. S., Veale, R. and Sive, H. (2005) Specification of the enveloping layer and lack of autoneuralization in zebrafish embryonic explants. Developmental Dynamics. 232 85-97. We have analyzed the roles of cell contact during determination of the outermost enveloping layer (EVL) and deeper neurectoderm in zebrafish embryos. Outer cells, but not deeper cells, are specified to express the EVL-specific marker, cyt1 by late blastula. EVL specification requires cell contact or close cell proximity, because cyt1 is not expressed after explant dissociation. The EVL may be homologous to the Xenopus epithelial layer, including the ventral larval epidermis. While Xenopus epidermal cytokeratin gene expression is activated by bone morphogenetic protein (BMP) signaling, zebrafish cyt1 is not responsive to BMPs. Zebrafish early gastrula ectodermal explants are specified to express the neural markers opl (zic1) and otx2, and this expression is prevented by BMP4. Dissociation of zebrafish explants prevents otx2 and opl expression, suggesting that neural specification in zebrafish requires cell contact or close cell proximity. This finding is in contrast to the case in Xenopus, where ectodermal dissociation leads to activation of neural gene expression, or autoneuralization. Our data suggest that distinct mechanisms direct development of homologous lineages in different vertebrates.Full Text.
Sangster, T. A. and Queitsch, C. (2005) The HSP90 chaperone complex, an emerging force in plant development and phenotypic plasticity. Curr Opin Plant Biol. 8 86-92. The essential cellular functions of the molecular chaperone HSP90 have been intensively investigated in fungal and mammalian model systems. Several recent publications have highlighted the importance of this chaperone complex in plant development and responsiveness to external stimuli. In particular, HSP90 is crucial for R-protein-mediated defense against pathogens. Other facets of HSP90 function in plants include its involvement in phenotypic plasticity, developmental stability, and buffering of genetic variation. Plants have emerged as powerful tools that complement other model systems in attempts to extend our knowledge of the myriad impacts of protein folding and chaperone function. Full Text
Sarbassov Dos D. , David A. Guertin, Siraj M. Ali, David M. Sabatini (2005) Phosphorylation and Regulation of Akt/PKB by the Rictor-mTOR Complex Science, Vol 307, Issue 5712, 1098-1101. Deregulation of Akt/protein kinase B (PKB) is implicated in the pathogenesis of cancer and diabetes. Akt/PKB activation requires the phosphorylation of Thr308 in the activation loop by the phosphoinositide-dependent kinase 1 (PDK1) and Ser473 within the carboxyl-terminal hydrophobic motif by an unknown kinase. We show that in Drosophila and human cells the target of rapamycin (TOR) kinase and its associated protein rictor are necessary for Ser473 phosphorylation and that a reduction in rictor or mammalian TOR (mTOR) expression inhibited an Akt/PKB effector. The rictor-mTOR complex directly phosphorylated Akt/PKB on Ser473 in vitro and facilitated Thr308 phosphorylation by PDK1. Rictor-mTOR may serve as a drug target in tumors that have lost the expression of PTEN, a tumor suppressor that opposes Akt/PKB activation. Full Text.
Shorter, J. and Lindquist, S. (2005) Navigating the ClpB channel to solution. Nat Struct Mol Biol. 12 4-6. New work places the Hsp70/DnaK system in an opening role in protein disaggregation, facilitating the extraction of individual polypeptides from the aggregate surface to the axial channel of Hsp104/ClpB. It also reinforces a long-standing belief that reactivation rather than simple clearance of aggregated protein is necessary for cell stress tolerance. Full Text.
Silver, S. J. and Rebay, I. (2005) Signaling circuitries in development: insights from the retinal determination gene network. Development. 132 3-13. Context-specific integration of information received from the Notch, Transforming growth factor beta, Wingless/Wnt, Hedgehog and Epidermal growth factor receptor signaling pathways sets the stage for deployment of the retinal determination gene network (RDGN), a group of transcription factors that collectively directs the formation of the eye and other tissues. Recent investigations have revealed how these transcription factors are regulated by their interactions with each other and with effectors of the above signaling pathways. Further study of the RDGN may provide insights into how common cues can generate context-specific responses, a key aspect of developmental regulation that remains poorly understood. Full Text
Singer, J. B., Hill, A. E., Nadeau, J. H. and Lander, E. S. (2005) Mapping quantitative trait loci for anxiety in chromosome substitutions strains of mice. Genetics. 169 855-862. Anxious behavior in the mouse is a complex quantitative phenotype that varies widely among inbred mouse strains. We examined a panel of chromosome substitution strains bearing individual A/J chromosomes in an otherwise C57BL/6J background in open-field and light-dark transition tests. Our results confirmed previous reports of quantitative trait loci (QTL) on chromosomes 1, 4, and 15 and identified novel loci on chromosomes 6 and 17. The studies were replicated in two separate laboratories. Systematic differences in the overall activity level were found between the two facilities, but the presence of the QTL was confirmed in both laboratories. We also identified specific effects on open-field defecation and center avoidance and distinguished them from overall open-field activity .Full Text.
Srivastava, A., Metaxas, A. C., So, P., Matsudaira, P., Ehrlich, D. and Georghiou, G. E. (2005) Numerical simulation of DNA sample preconcentration in microdevice electrophoresis. Electrophoresis. 26 1130-1143. A numerical model is presented for the accurate and efficient prediction of preconcentration and transport of DNA during sample introduction and injection in microcapillary electrophoresis. The model incorporates conservation laws for the different buffer ions, salt ions, and DNA sample, coupled through a Gaussian electric field to account for the field modifications that cause electromigration. The accuracy and efficiency required to capture the physics associated with such a complex transient problem are realized by the use of the finite element-flux corrected transport (FE-FCT) algorithm in two dimensions. The model has been employed for the prediction of DNA sample preconcentration and transport during electrophoresis in a double-T injector microdevice. To test its validity, the numerical results have been compared with the corresponding experimental data under similar conditions, and excellent agreement has been found. Finally, detailed results from a simulation of DNA sample preconcentration in electrophoretic microdevices are presented using as parameters the electric field strength and the other species concentrations. The effect of the Tris concentration on sample stacking is also investigated. These results demonstrate the great potential offered by the model for future optimization of such microchip devices with respect to significantly enhanced speed and resolution of sample separation.PDF
Tachibana, C., Yoo, J. Y., Tagne, J. B., Kacherovsky, N., Lee, T. I. and Young, E. T. (2005) Combined global localization analysis and transcriptome data identify genes that are directly coregulated by Adr1 and Cat8. Molecular and Cellular Biology. 25 2138-2146. In Saccharomyces cerevisiae, glucose depletion causes a profound alteration in metabolism, mediated in part by global transcriptional changes. Many of the transcription factors that regulate these changes act combinatorially. We have analyzed combinatorial regulation by Adr1 and Cat8, two transcription factors that act during glucose depletion, by combining genome-wide expression and genome-wide binding data. We identified 32 genes that are directly activated by Adr1, 28 genes that are directly activated by Cat8, and 14 genes that are directly regulated by both. Our analysis also uncovered promoters that Adr1 binds but does not regulate and promoters that are indirectly regulated by Cat8, stressing the advantage of combining global expression and global localization analysis to find directly regulated targets. At most of the coregulated promoters, the in vivo binding of one factor is independent of the other, but Adr1 is required for optimal Cat8 binding at two promoters with a poor match to the Cat8 binding consensus. In addition, Cat8 is required for Adr1 binding at promoters where Adr1 is not required for transcription. These data provide a comprehensive analysis of the direct, indirect, and combinatorial requirements for these two global transcription factors. Full Text.
Thomas, M., Lu, J. J., Ge, Q., Zhang, C., Chen, J. and Klibanov, A. M. (2005) Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung. Proc Natl Acad Sci U S A. 102 5679-5684. High-molecular-mass polyethylenimines (PEIs) are widely used vectors for nucleic acid delivery. We found that removal of the residual N-acyl moieties from commercial linear 25-kDa PEI enhanced its plasmid DNA delivery efficiency 21 times in vitro, as well as 10,000 times in mice with a concomitant 1,500-fold enhancement in lung specificity. Several additional linear PEIs were synthesized by acid-catalyzed hydrolysis of poly(2-ethyl-2-oxazoline), yielding the pure polycations. PEI87 and PEI217 exhibited the highest efficiency in vitro: 115-fold and 6-fold above those of the commercial and deacylated PEI25s, respectively; moreover, PEI87 delivered DNA to mouse lung as efficiently as the pure PEI25 but at a lower concentration and with a 200-fold lung specificity. These improvements stem from an increase in the number of protonatable nitrogens, which presumably results in a tighter condensation of plasmid DNA and a better endosomal escape of the PEI/DNA complexes. As a validation of the potential of such linear, fully deacylated PEIs in gene therapy for lung diseases, systemic delivery in mice of the complexes of a short interfering RNA (siRNA) against a model gene, firefly luciferase, and PEI25 or PEI87 afforded a 77% and 93% suppression of the gene expression in the lungs, respectively. Furthermore, a polyplex of a siRNA against the influenza viral nucleocapsid protein gene and PEI87 resulted in a 94% drop of virus titers in the lungs of influenza-infected animals. Full Text.
Tong, W., Zhang, J. and Lodish, H. F. (2005) Lnk inhibits erythropoiesis and Epo-dependent JAK2 activation and downstream signaling pathways. Blood. Feb 10; [Epub ahead of print]. Erythropoietin (Epo), along with its receptor EpoR, is the principal regulator of red cell development. Upon Epo addition, the EpoR signaling the JAK2 tyrosine kinase, activates multiple pathways including Stat5, PI-3K/Akt, and p42/44MAPK. The adaptor protein Lnk is implicated in cytokine receptor signaling. Here, we showed that Lnk-deficient mice have elevated numbers of erythroid progenitors, and that splenic CFU-e progenitors are hypersensitive to Epo. Lnk-/- mice also exhibit superior recovery after erythropoietic stress. In addition, Lnk deficiency resulted in enhanced Epo-induced signaling pathways in splenic erythroid progenitors. Conversely, Lnk overexpression inhibits Epo-induced cell growth in 32D/EpoR cells. In primary culture of fetal liver cells, Lnk overexpression inhibits Epo-dependent erythroblast differentiation and induces apoptosis. Lnk blocks three major signaling pathways, Stat5, Akt, and MAPK, induced by Epo in primary erythroblasts. In addition, the Lnk SH2 domain is essential for its inhibitory function, whereas the conserved tyrosine near the C-terminus and the PH domain of Lnk are not critical. Furthermore, wild type Lnk, but not the Lnk SH2 mutant, becomes tyrosine-phosphorylated following Epo administration and inhibits EpoR phosphorylation and JAK2 activation. Hence, Lnk, through its SH2 domain, negatively modulates EpoR signaling by attenuating JAK2 activation, and regulates Epo-mediated erythropoiesis.PDF
Tootle, T. L. and Rebay, I. (2005) Post-translational modifications influence transcription factor activity: A view from the ETS superfamily. Bioessays. 27 285-298. Transcription factors provide nodes of information integration by serving as nuclear effectors of multiple signaling cascades, and thus elaborate layers of regulation, often involving post-translational modifications, modulating and coordinate activities. Such modifications can rapidly and reversibly regulate virtually all transcription factor functions, including subcellular localization, stability, interactions with cofactors, other post-translational modifications and transcriptional activities. Aside from analyses of the effects of serine/threonine phosphorylation, studies on post-translational modifications of transcription factors are only in the initial stages. In particular, the regulatory possibilities afforded by combinatorial usage of and competition between distinct modifications on an individual protein are immense, and with respect to large families of closely related transcription factors, offer the potential of conferring critical specificity. Here we will review the post-translational modifications known to regulate ETS transcriptional effectors and will discuss specific examples of how such modifications influence their activities to highlight emerging paradigms in transcriptional regulation.Full Text.
Xie, X. H., Lu, J., Kulbokas, E. J., Golub, T. R., Mootha, V., Lindblad-Toh, K., Lander, E. S. and Kellis, M. (2005) Systematic discovery of regulatory motifs in human promoters and 3 ' UTRs by comparison of several mammals. Nature. 434 338-345. Comprehensive identification of all functional elements encoded in the human genome is a fundamental need in biomedical research. Here, we present a comparative analysis of the human, mouse, rat and dog genomes to create a systematic catalogue of common regulatory motifs in promoters and 30 untranslated regions ( 3 0 UTRs). The promoter analysis yields 174 candidate motifs, including most previously known transcription-factor binding sites and 105 new motifs. The 3'-UTR analysis yields 106 motifs likely to be involved in post-transcriptional regulation. Nearly one-half are associated with microRNAs ( miRNAs), leading to the discovery of many new miRNA genes and their likely target genes. Our results suggest that previous estimates of the number of human miRNA genes were low, and that miRNAs regulate at least 20% of human genes. The overall results provide a systematic view of gene regulation in the human, which will be refined as additional mammalian genomes become available.Full Text
Zaman, M. H., Kamm, R. D., Matsudaira, P. T. and Lauffenburger, D. A. (2005) Computational Model for Cell Migration in Three- Dimensional Matrices. Biophysical Journal. BioFast Published May 20. While computational models for cell migration on two- dimensional substrata [DiMilla et al., Biophysical Journal (1991)] have described how various molecular and cellular properties and physiochemical processes are integrated to accomplish cell locomotion, the same issues, along with certain new ones, might contribute differently to a model for migration within three-dimensional matrices. To address this more complicated situation, we have developed a computational model for cell migration in three-dimensional matrices using a force-based dynamics approach. This model determines an overall locomotion velocity vector, comprising speed and direction, for individual cells based on internally-generated forces transmitted into external traction forces and considering a time-scale during which multiple attachment and detachment events are integrated. Key parameters characterize cell and matrix properties, including cell/matrix adhesion and mechanical and steric properties of the matrix; critical underlying molecular properties are incorporated explicitly or implicitly. Model predictions agree well with experimental results for the limiting case of migration on two-dimensional substrata as well as recent experiments in three-dimensional natural tissues and synthetic gels. Certain predicted features such as biphasic behavior of speed with density of matrix ligands for three-dimensional migration are qualitatively similar to their two-dimensional counterparts, but new effects generally absent in two-dimensional systems such as effects due to matrix sterics and mechanics are now predicted to arise in many three-dimensional situations. As one particular example manifestation of these effects, the optimal levels of cell receptor expression and matrix ligand density yielding maximal migration are dependent on matrix mechanical compliance .PDF
Zhang X., Odom, D. T., Koo, S. H., Conkright, M. D., Canettieri, G., Best, J., Chen, H., Jenner, R., Herbolsheimer, E., Jacobsen, E., Kadam, S., Ecker, J. R., Emerson, B., Hogenesch, J. B., Unterman, T., Young, R. A. and Montminy, M. (2005) Genome-wide analysis of cAMP-response element binding protein occupancy, phosphorylation, and target gene activation in human tissues. Proc Natl Acad Sci U S A Mar 7; [Epub ahead of print]. Hormones and nutrients often induce genetic programs via signaling pathways that interface with gene-specific activators. Activation of the cAMP pathway, for example, stimulates cellular gene expression by means of the PKA-mediated phosphorylation of cAMP-response element binding protein (CREB) at Ser-133. Here, we use genome-wide approaches to characterize target genes that are regulated by CREB in different cellular contexts. CREB was found to occupy approximately 4,000 promoter sites in vivo, depending on the presence and methylation state of consensus cAMP response elements near the promoter. The profiles for CREB occupancy were very similar in different human tissues, and exposure to a cAMP agonist stimulated CREB phosphorylation over a majority of these sites. Only a small proportion of CREB target genes was induced by cAMP in any cell type, however, due in part to the preferential recruitment of the coactivator CREB-binding protein to those promoters. These results indicate that CREB phosphorylation alone is not a reliable predictor of target gene activation and that additional CREB regulatory partners are required for recruitment of the transcriptional apparatus to the promoter PDF
Zhang, C. C. and Lodish, H. F. (2005) Murine hematopoietic stem cells change their surface phenotype during ex vivo expansion. Blood. Feb 8; [Epub ahead of print] Ex vivo expansion of hematopoietic stem cells (HSCs) is important for many clinical applications and knowledge of the surface phenotype of ex vivo expanded HSCs will be critical to their purification and analysis. Here we developed a simple culture system for bone marrow (BM) HSCs using low levels of SCF, TPO, IGF-2, and FGF-1 in serum-free medium. As measured by competitive repopulation analyses, there was a greater than 20-fold increase in numbers of long-term (LT) HSCs after a 10-day culture of total BM cells. Culture of BM "side population" (SP) cells, a highly-enriched stem cell population, for 10 days resulted in an ~8 fold expansion of repopulating HSCs. Similar to freshly isolated HSCs, repopulating HSCs after culture were positive for the stem cell markers Sca-1, Kit, and CD31 and receptors for IGF-2. Surprisingly, prion protein and Tie-2, that are present on freshly isolated HSCs, were not on cultured HSCs. Two other HSC markers, Endoglin and Mpl, were expressed only on a portion of cultured HSCs. Therefore the surface phenotype of ex vivo expanded HSCs is different from that of freshly isolated HSCs, but this plasticity of surface phenotype does not significantly alter their repopulation capability.PDF.
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