Dissecting the transcriptional regulatory network of embryonic stem cells

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2008.Includes bibliographical references.The process by which a single fertilized egg develops into a human being with over 200 cell types, each with a distinct gene expression pattern controlling its cellular state, is poorly understood. An understanding of the transcriptional regulatory networks that establish and maintain gene expression programs in mammalian cells is fundamental to understand development and should provide the foundation for improved diagnosis and treatment of disease. Although it is not yet feasible to map the entirety of these networks in vertebrate cells, recent work in embryonic stem (ES) cells has demonstrated that core features of the network can be discovered by focusing on key transcriptional regulators and their target genes. Here, I describe important insights that have emerged from such studies and highlight how similar approaches can be used to discover the core networks of other vertebrate cell types. Knowledge of the regulatory networks controlling gene expression programs and cell states can guide efforts to reprogram cell states and holds great promise for both disease therapeutics and regenerative medicine.by Megan F. Cole.Ph.D

Interchanging Functionality Among Homologous Elongation Factors Using Signatures of Heterotachy

Numerous models of molecular evolution have been formulated to describe the forces that shape sequence divergence among homologous proteins. These models have greatly enhanced our understanding of evolutionary processes. Rarely are such models empirically tested in the laboratory, and even more rare, are such models exploited to generate novel molecules useful for synthetic biology. Here, we experimentally demonstrate that the heterotachy model of evolution captures signatures of functional divergence among homologous elongation factors (EFs) between bacterial EF-Tu and eukaryotic eEF1A. These EFs are GTPases that participate in protein translation by presenting aminoacylated-tRNAs to the ribosome. Upon release from the ribosome, the EFs are recharged by nucleotide exchange factors EF-Ts in bacteria or eEF1B in eukaryotes. The two nucleotide exchange factors perform analogous functions despite not being homologous proteins. The heterotachy model was used to identify a set of sites in eEF1A/EF-Tu associated with eEF1B binding in eukaryotes and another reciprocal set associated with EF-Ts binding in bacteria. Introduction of bacterial EF-Tu residues at these sites into eEF1A protein efficiently disrupted binding of cognate eEF1B as well as endowed eEF1A with the novel ability to bind bacterial EF-Ts. We further demonstrate that eEF1A variants, unlike yeast wild-type, can function in a reconstituted in vitro bacterial translation system. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00239-013-9540-9) contains supplementary material, which is available to authorized users

Evolutionary Toxicology: Population-Level Effects of Chronic Contaminant Exposure on the Marsh Frogs (Rana ridibunda) of Azerbaijan

We used molecular methods and population genetic analyses to study the effects of chronic contaminant exposure in marsh frogs from Sumgayit, Azerbaijan. Marsh frogs inhabiting wetlands in Sumgayit are exposed to complex mixtures of chemical contaminants, including petroleum products, pesticides, heavy metals, and many other industrial chemicals. Previous results documented elevated estimates of genetic damage in marsh frogs from the two most heavily contaminated sites. Based on mitochondrial DNA (mtDNA) control region sequence data, the Sumgayit region has reduced levels of genetic diversity, likely due to environmental degradation. The Sumgayit region also acts as an ecological sink, with levels of gene flow into the region exceeding gene flow out of the region. Additionally, localized mtDNA heteroplasmy and diversity patterns suggest that one of the most severely contaminated sites in Sumgayit is acting as a source of new mutations resulting from an increased mutation rate. This study provides an integrated method for assessing the cumulative population impacts of chronic contaminant exposure by studying both population genetic and evolutionary effects

Genome-Wide ENU Mutagenesis in Combination with High Density SNP Analysis and Exome Sequencing Provides Rapid Identification of Novel Mouse Models of Developmental Disease

BACKGROUND Mice harbouring gene mutations that cause phenotypic abnormalities during organogenesis are invaluable tools for linking gene function to normal development and human disorders. To generate mouse models harbouring novel alleles that are involved in organogenesis we conducted a phenotype-driven, genome-wide mutagenesis screen in mice using the mutagen N-ethyl-N-nitrosourea (ENU). METHODOLOGY/PRINCIPAL FINDINGS ENU was injected into male C57BL/6 mice and the mutations transmitted through the germ-line. ENU-induced mutations were bred to homozygosity and G3 embryos screened at embryonic day (E) 13.5 and E18.5 for abnormalities in limb and craniofacial structures, skin, blood, vasculature, lungs, gut, kidneys, ureters and gonads. From 52 pedigrees screened 15 were detected with anomalies in one or more of the structures/organs screened. Using single nucleotide polymorphism (SNP)-based linkage analysis in conjunction with candidate gene or next-generation sequencing (NGS) we identified novel recessive alleles for Fras1, Ift140 and Lig1. CONCLUSIONS/SIGNIFICANCE In this study we have generated mouse models in which the anomalies closely mimic those seen in human disorders. The association between novel mutant alleles and phenotypes will lead to a better understanding of gene function in normal development and establish how their dysfunction causes human anomalies and disease.This work was enabled by the Australian Phenomics Network and partly supported by funding from the Australian Government’s National Collaborative Research Infrastructure Strategy, a Strategic Grant from the Faculty of Medicine, Nursing and Health Sciences at Monash University, and the Victorian Government’s Operational Infrastructure Support Program. IS acknowledges support through the NH&MRC R. Douglas Wright and ARC Future Fellowship schemes. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Influence of Grain Boundary Character on Creep Void Formation in Alloy 617

Alloy 617, a high temperature creep-resistant, nickel-based alloy, is being considered for the primary heat exchanger for the Next Generation Nuclear Plant (NGNP) which will operate at temperatures exceeding 760oC. Orientation imaging microscopy (OIM) is used to characterize the grain boundaries in the vicinity of creep voids that develop during high temperature creep tests (800-1000oC at creep stresses ranging from 20-85 MPa) terminated at creep strains ranging from 5-40%. Observations using optical microscopy indicate creep rate does not significantly influence the creep void fraction at a given creep strain. Preliminary analysis of the OIM data indicates voids tend to form on grain boundaries parallel, perpendicular or 45o to the tensile axis, while few voids are found at intermediate inclinations to the tensile axis. Random grain boundaries intersect most voids while CSL-related grain boundaries did not appear to be consistently associated with void development

Spatial variability in sustainable development trajectories in South Africa:provincial level safe and just operating spaces

The Sustainable Development Goals (SDGs) represents the first globally agreed framework to address human development and environmental stewardship in an integrated way. One approach to summarising national SDG status is our “barometer for inclusive sustainable development in South Africa”. The barometer downscales global social and planetary boundaries to provide status and trends for 20 critical indicators of environmental stress and social deprivation. In this paper, we explore the sub-national heterogeneity in sustainable development indicators by creating barometers defining the ‘safe and just operating space’ for South Africa’s nine provinces. Our results show that environmental stress varies significantly and provinces need to focus on quite different issues. Although generally environmental stress is increasing, there are areas where it is decreasing, most notably, marine harvesting. Social deprivation results show more of a pattern with high levels of deprivation in employment, income and safety across the provinces, and historically disadvantaged provinces showing the most deprivation overall. Although deprivation is generally decreasing, there are notable exceptions such as food security in six provinces. Our provincial barometers and trend plots are novel in that they present comparable environmental and social data on key indicators over time for all South Africa’s provinces. They are visual tools that communicate the range of key challenges and risks that provincial governments face, and are non-specialist and accessible to a range of audiences. In addition, the paper provides a critical case study of spatial disaggregation of national data that is required for the SDGs implementation

Transcriptional role of cyclin D1 in development revealed by a “genetic-proteomic” screen

Author manuscript: 2010 September 22.Cyclin D1 belongs to the core cell cycle machinery, and it is frequently overexpressed in human cancers[superscript 1, 2]. The full repertoire of cyclin D1 functions in normal development and oncogenesis is unclear at present. Here we developed Flag- and haemagglutinin-tagged cyclin D1 knock-in mouse strains that allowed a high-throughput mass spectrometry approach to search for cyclin D1-binding proteins in different mouse organs. In addition to cell cycle partners, we observed several proteins involved in transcription. Genome-wide location analyses (chromatin immunoprecipitation coupled to DNA microarray; ChIP-chip) showed that during mouse development cyclin D1 occupies promoters of abundantly expressed genes. In particular, we found that in developing mouse retinas—an organ that critically requires cyclin D1 function[superscript 3, 4]—cyclin D1 binds the upstream regulatory region of the Notch1 gene, where it serves to recruit CREB binding protein (CBP) histone acetyltransferase. Genetic ablation of cyclin D1 resulted in decreased CBP recruitment, decreased histone acetylation of the Notch1 promoter region, and led to decreased levels of the Notch1 transcript and protein in cyclin D1-null (Ccnd1-/-) retinas. Transduction of an activated allele of Notch1 into Ccnd1-/- retinas increased proliferation of retinal progenitor cells, indicating that upregulation of Notch1 signalling alleviates the phenotype of cyclin D1-deficiency. These studies show that in addition to its well-established cell cycle roles, cyclin D1 has an in vivo transcriptional function in mouse development. Our approach, which we term ‘genetic–proteomic’, can be used to study the in vivo function of essentially any protein

Detection of mammaglobin mRNA in peripheral blood is associated with high grade breast cancer: Interim results of a prospective cohort study

<p>Abstract</p> <p>Background</p> <p>We sought to examine the detection rate of cancer cells in peripheral blood (PBL) and in bone marrow (BM) using an established 7-gene marker panel and evaluated whether there were any definable associations of any individual gene with traditional predictors of prognosis.</p> <p>Methods</p> <p>Patients with T1-T3 primary breast cancer were enrolled into a prospective, multi-institutional cohort study. In this interim analysis 215 PBL and 177 BM samples were analyzed by multimarker, real-time RT-PCR analysis designed to detect circulating and disseminated breast cancer cells.</p> <p>Results</p> <p>At a threshold of three standard deviations from the mean expression level of normal controls, 63% (136/215) of PBL and 11% (19/177) of BM samples were positive for at least one cancer-associated marker. Marker positivity in PBL demonstrated a statistically significant association with grade II-III (vs. grade I; p = 0.0083). Overexpression of the mammaglobin (<it>mam</it>) gene alone had a statistically significant association with high tumor grade (p = 0.0315), and showed a trend towards ER-negative tumors and a high risk category. There was no association between marker positivity in PBL and the pathologic (H&E) and/or molecular (RT-PCR) status of the axillary lymph nodes (ALN).</p> <p>Conclusion</p> <p>This study suggests that molecular detection of circulating cancer cells in PBL detected by RT-PCR is associated with high tumor grade and specifically that overexpression of the <it>mam </it>gene in PBL may be a poor prognostic indicator. There was no statistically significant association between overexpression of cancer-associated genes in PBL and ALN status, supporting the concept of two potentially separate metastatic pathways.</p

The genetic landscape of high-risk neuroblastoma

Neuroblastoma is a malignancy of the developing sympathetic nervous system that often presents with widespread metastatic disease, resulting in survival rates of less than 50%1. To determine the spectrum of somatic mutation in high-risk neuroblastoma, we studied 240 cases using a combination of whole exome, genome and transcriptome sequencing as part of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. Here we report a low median exonic mutation frequency of 0.60 per megabase (0.48 non-silent), and remarkably few recurrently mutated genes in these tumors. Genes with significant somatic mutation frequencies included ALK (9.2% of cases), PTPN11 (2.9%), ATRX (2.5%, an additional 7.1% had focal deletions), MYCN (1.7%, a recurrent p.Pro44Leu alteration), and NRAS (0.83%). Rare, potentially pathogenic germline variants were significantly enriched in ALK, CHEK2, PINK1, and BARD1. The relative paucity of recurrent somatic mutations in neuroblastoma challenges current therapeutic strategies reliant upon frequently altered oncogenic drivers

A protein methylation pathway in Chlamydomonas flagella is active during flagellar resorption

Author Posting. © American Society for Cell Biology, 2008. This article is posted here by permission of American Society for Cell Biology for personal use, not for redistribution. The definitive version was published in Molecular Biology of the Cell 19 (2008): 4319-4327, doi:10.1091/mbc.E08-05-0470.During intraflagellar transport (IFT), the regulation of motor proteins, the loading and unloading of cargo and the turnover of flagellar proteins all occur at the flagellar tip. To begin an analysis of the protein composition of the flagellar tip, we used difference gel electrophoresis to compare long versus short (i.e., regenerating) flagella. The concentration of tip proteins should be higher relative to that of tubulin (which is constant per unit length of the flagellum) in short compared with long flagella. One protein we have identified is the cobalamin-independent form of methionine synthase (MetE). Antibodies to MetE label flagella in a punctate pattern reminiscent of IFT particle staining, and immunoblot analysis reveals that the amount of MetE in flagella is low in full-length flagella, increased in regenerating flagella, and highest in resorbing flagella. Four methylated proteins have been identified in resorbing flagella, using antibodies specific for asymmetrically dimethylated arginine residues. These proteins are found almost exclusively in the axonemal fraction, and the methylated forms of these proteins are essentially absent in full-length and regenerating flagella. Because most cells resorb cilia/flagella before cell division, these data indicate a link between flagellar protein methylation and progression through the cell cycle.This work was supported by National Institutes of Health Grant DK071720 (R.D.S.) and National Science Foundation Grant MCB 0418877 (R.D.S.)