Moira Cockell trained as a biologist at the University of Edinburgh and has research and writing experience in the disciplines of biochemistry, genetics, cellular, molecular and structural biology. She obtained a Ph.D in 1990, for studies carried out jointly in Lausanne’s Swiss Institute for Experimental Cancer Research and the Medical Research Council Laboratory of Molecular Biology in Cambridge, England. During more than twenty years at ISREC (1985-2006) Dr Cockell co-authored numerous articles in peer-reviewed journals, gained a strong working knowledge of the biology of cancer and made research contributions in the fields of telomere homeostasis, control of cell division, epigenetics, chromatin structure and transcriptional regulation. In 2001 and 2003 she wrote (successful) requests for external funding of her post as an ISREC research associate. In 2006 Dr Cockell began to pursue freelance writing, editing and scientific-conference-organising activities in parallel with carrying out biomedical research at ISREC (2006-2008) and the Centre Hospitalier Universitaire Vaudois (2006-2007). She is an accredited member of the European Medical Writers Association and completed the EMWA Professional Development Programme (level F) in Medical Writing between 2006 and 2008.
Abstract: Knowledge is a living thing, sustained through dynamic reflexive processes. Whether at the level of cellular signaling pathways, Internet design, or sociocultural interactions, human understanding grows and accrues value through bi-directional transmission across networks of emitters and receptors.
And the cross-fertilization of ideas from different sources is what keeps the process vigorous.
This book represents a coherent milestone in cultivating constructive exchange between experts and specialists from the physical, natural, economic
and human science disciplines. From its sixteen original and highly personal essays portraying multiple facets of the knowledge creation process, emerge a common sense of purpose and a framework of new tools and methodologies for interdisciplinary dialogue.
Abstract: This book unites an international team of leading researchers and educators around the theme of knowledge dialogue. Spanning topics from natural complexity to neuroscience, from education theory to climate change, from immunology to archaeology and human migrations, these renowned multidisciplinarians engage each other, through a series of original essays, in an atmosphere of constructive criticism and with the ambition to build a new foundation for the transdisciplinary approach. It is known that the exact sciences, the social sciences, and the humanities and arts each have their specific tools, methodologies, goals and limitations. This book examines how leading thinkers are addressing the problem of knowledge fragmentation into what C. P. Snow called the âTwo Culturesâ. The authors invite you, the reader, to join their search for reciprocal enlightenment and enrichment as they set out to bridge communication gaps between the traditionally defined disciplines.
Notes: accompanying online material available at www.wkdialogue.ch
Abstract: In Saccharomyces cerevisiae, TBF1, an essential gene, influences telomere function, but also has other roles in the global regulation of transcription. We have identified a new member of the tbf1 gene family in the mammalian pathogen Pneumocystis carinii. We demonstrate by trans-species complementation that its ectopic expression can provide the essential functions of Schizosaccharomyces pombe tbf1 but that there is no rescue between fission and budding yeast orthologues. Our findings indicate that an essential function of this family of proteins has diverged in the budding and fission yeasts and suggest that effects on telomere length or structure are not the primary cause of inviability in S. pombe tbf1 null strains.
Abstract: At yeast telomeres and silent mating-type loci, chromatin assumes a higher-order structure that represses transcription by means of the histone deacetylase Sir2 and structural proteins Sir3 and Sir4. Here, we present a fully reconstituted system to analyze SIR holocomplex binding to nucleosomal arrays. Purified Sir2-3-4 heterotrimers bind chromatin, cooperatively yielding a stable complex of homogeneous molecular weight. Remarkably, Sir2-3-4 also binds naked DNA, reflecting the strong, albeit nonspecific, DNA-binding activity of Sir4. The binding of Sir3 to nucleosomes is sensitive to histone H4 N-terminal tail removal, while that of Sir2-4 is not. Dot1-mediated methylation of histone H3K79 reduces the binding of both Sir3 and Sir2-3-4. Additionally, a byproduct of Sir2-mediated NAD hydrolysis, O-acetyl-ADP-ribose, increases the efficiency with which Sir3 and Sir2-3-4 bind nucleosomes. Thus, in small cumulative steps, each Sir protein, unmodified histone domains, and contacts with DNA contribute to the stability of the silent chromatin complex.
Abstract: Pneumocystis jirovecii is a fungus which causes severe opportunistic infections in immunocompromised humans. The brl1 gene of P. carinii infecting rats was identified and characterized by using bioinformatics in conjunction with functional complementation in Saccharomyces cerevisiae and Schizosaccharomyces pombe. The ectopic expression of this gene rescues null alleles of essential nuclear membrane proteins of the Brr6/Brl1 family in both yeasts.
Abstract: We investigated the role of the evolutionarily conserved protein Lis1 in cell division processes of Caenorhabditis elegans embryos. We identified apparent null alleles of lis-1, which result in defects identical to those observed after inactivation of the dynein heavy chain dhc-1, including defects in centrosome separation and spindle assembly. We raised antibodies against LIS-1 and generated transgenic animals expressing functional GFP-LIS-1. Using indirect immunofluorescence and spinning-disk confocal microscopy, we found that LIS-1 is present throughout the cytoplasm and is enriched in discrete subcellular locations, including the cell cortex, the vicinity of microtubule asters, the nuclear periphery and kinetochores. We established that lis-1 contributes to, but is not essential for, DHC-1 enrichment at specific subcellular locations. Conversely, we found that dhc-1, as well as the dynactin components dnc-1 (p150Glued) and dnc-2 (p50/dynamitin), are essential for LIS-1 targeting to the nuclear periphery, but not to the cell cortex nor to kinetochores. These results suggest that dynein and Lis1, albeit functioning in identical processes, are targeted partially independently of one another.
Abstract: In budding yeast, the silent information regulator Sir2p is a nuclear NAD-dependent deacetylase that is essential for both telomeric and rDNA silencing. All eukaryotic species examined to date have multiple homologues of Sir two (HSTs), which share a highly conserved globular core domain. Here we report that yeast Hst2p and a mammalian Hst2p homologue, hSirT2p, are cytoplasmic in yeast and human cells, in contrast to yHst1p and ySir2p which are exclusively nuclear. Although yHst2p cannot restore silencing in a sir2 deletion, overexpression of yHst2p influences nuclear silencing events in a SIR2 strain, derepressing subtelomeric silencing while increasing repression in the rDNA. In contrast, a form of ySir2p carrying a point mutation in the conserved core domain disrupts both telomeric position effect (TPE) and rDNA repression at low expression levels. This argues that non-nuclear yHst2p can compete for a substrate or ligand specifically required for telomeric, and not rDNA repression.
Abstract: Silent or heritably repressed genes constitute the major fraction of genetic information in higher eukaryotic cells. Budding yeast has very little consecutively repressed DNA, but what exists has served as a paradigm for the molecular analysis of heterochromatin. The major structural constituents of repressed chromatin in yeast are the four core histones and three large chromatin factors called Silent information regulators 2, 3 and 4. How these components assemble DNA into a state that is refractory to transcription remains a mystery. Nonetheless, there have been many recent insights into their molecular structures. This review examines the impact of these results on our understanding of silencing function in budding yeast.
Abstract: Silent information regulator (Sir) 2 is a limiting component of the Sir2/3/4 complex, which represses transcription at subtelomeric and HM loci. Sir2p also acts independently of Sir3p and Sir4p to influence chromatin organization in the rDNA locus. Deleted and mutated forms of Sir2p have been tested for their ability to complement and/or to disrupt silencing. The highly conserved C-terminal domain of Sir2p (aa 199-562) is insufficient to restore repression at either telomeric or rDNA reporters in a sir2Delta background and fails to nucleate silencing when targeted to an appropriate reporter gene. However, its expression in an otherwise wild-type strain disrupts telomeric repression. Similarly, a point mutation (P394L) within this conserved core inactivates the full-length protein but renders it dominant negative for all types of silencing. Deletion of aa 1-198 from Sir2(394L) eliminates its dominant negative effect. Thus we define two distinct functional domains in Sir2p, both essential for telomeric and rDNA repression: the conserved core domain found within aa 199-562 and a second domain that encompasses aa 94-198. Immunolocalization and two-hybrid studies show that aa 94-198 are required for the binding of Sir2p to Sir4p and for the targeting of Sir2p to the nucleolus through another ligand. The globular core domain provides an essential silencing function distinct from that of targeting or Sir complex formation that may reflect its reported mono-ADP-ribosyl transferase activity.
Abstract: Recent studies indicate that the nucleolus is not just a site of ribosome biogenesis. Intriguing links have been found between nucleolar components and the machinery that regulates the cell cycle.
Abstract: Improvements in fluorescence microscopy have allowed us to explore the three-dimensional organization of the nucleus in ways that were impossible ten years ago, revealing subdomains or compartments within the nucleus defined by their enrichments of subsets of factors. Correlations have been drawn between the silencing of a gene and its proximity to a heterochromatic compartment or to the nuclear periphery. The application of genetics and high-resolution microscopy helps examine the creation, maintenance and impact of these compartments on gene expression.
Abstract: In budding yeast genes integrated near telomeres succumb to a variegated pattern of gene repression that requires the silent information regulatory proteins Sir2p, Sir3p and Sir4p, which form a nucleosome-binding complex. Immunolocalization shows that the Sir proteins co-localize with the telomeric repeat binding protein Rap1p and with telomeric DNA in a limited number of foci near the periphery of interphase nuclei. All conditions tested so far that disrupt telomere proximal repression result in a dispersed staining pattern for Sir2p, Sir3p and Sir4p. Although the focal organization is clearly not sufficient for establishing repression, genetic studies suggest that the high local concentration of Sir proteins at telomeric foci facilitates the formation of repressed chromatin. In addition to its telomeric localization, Sir2p is shown by immunostaining and cross-linking to bind a subdomain of the nucleolus. In strains lacking an intact Sir4p, Sir3p also becomes concentrated in the nucleolus by a pathway requiring SIR2 and UTH4. This unexpected localization correlates with observed effects of sir mutations on rDNA stability and longevity, defining a new site of action for silent information regulatory factors. We report a novel WD40 repeat-containing factor, Sif2p, that binds specifically to the Sir4p N-terminus. Like Sir1p and Uth4p, Sif2p antagonizes telomeric silencing by regulating an equilibrium between alternative assembly pathways at different subnuclear loci.
Abstract: Several regions of the Saccharomyces cerevisiae genome are subject to position-dependent transcriptional repression mediated by a multi-component nucleosome-binding complex of silent information regulator proteins (Sir2p, Sir3p and Sir4p). These proteins are present in limiting amounts in the nucleus and are targeted to specific chromosomal regions by interaction with sequence-specific DNA-binding factors. Different sites of repression compete for Sir complexes, although it is not known how Sir distribution is regulated. In a screen for factors that interact with Sir4p amino terminus, we have cloned SIF2, which encodes a WD40-repeat-containing factor that disrupts telomeric silencing when overexpressed. In contrast to deletion of SIR4, SIF2 deletion improved telomeric repression, suggesting that under normal conditions Sif2p antagonizes Sir4p function at telomeres. Sif2p overexpression altered the subnuclear localization of Sir4p, but not its protein expression level, suggesting that Sif2p may recruit Sir4p to nontelomeric sites or repression. The sif2 mutant strains were hypersensitive to a range of stress conditions, but did not have decreased viability and did not alter repression in the rDNA. In conclusion, Sif2p resembles the Sir4p regulatory proteins Sir1p and Uth4p in that it competes for the functional assembly of Sir4p at telomeres, yet unlike Sir1p or Uth4p, it does not target Sir4p to either mating-type or rDNA loci.
Abstract: Transcription in organisms as diverse as yeast and mammals is subject to chromosomal position effects that result in heritable and variegated patterns of gene expression. Two recent studies have employed a reversible protein-DNA crosslinking method to identify the structural components of heterochromatin in budding yeast. The results show that a complex containing the proteins Rap1, Sir2p, Sir3p and Sir4p is physically associated with nucleosomes at telomere proximal regions, but that the repressive chromatin structure extended by Sir3p overexpression has a different composition.
Abstract: The Silent Information Regulatory proteins, Sir3 and Sir4, and the telomeric repeat-binding protein RAP1 are required for the chromatin-mediated gene repression observed at yeast telomeric regions. All three proteins are localized by immunofluorescence staining to foci near the nuclear periphery suggesting a relationship between subnuclear localization and silencing. We present several lines of immunological and biochemical evidence that Sir3, Sir4, and RAP1 interact in intact yeast cells. First, immunolocalization of Sir3 to foci at the yeast nuclear periphery is lost in rap1 mutants carrying deletions for either the terminal 28 or 165 amino acids of RAP1. Second, the perinuclear localization of both Sir3 and RAP1 is disrupted by overproduction of the COOH terminus of Sir4. Third, overproduction of the Sir4 COOH terminus alters the solubility properties of both Sir3 and full-length Sir4. Finally, we demonstrate that RAP1 and Sir4 coprecipitate in immune complexes using either anti-RAP1 or anti-Sir4 antibodies. We propose that the integrity of a tertiary complex between Sir4, Sir3, and RAP1 is involved in both the maintenance of telomeric repression and the clustering of telomeres in foci near the nuclear periphery.
Abstract: Efficient expression of genes under the control of alpha-amylase 2 5'-flanking sequences in exocrine pancreatic cells requires, in addition to the pancreas transcription factor 1 binding site (M. Cockell, B.J. Stevenson, M. Strubin, O. Hagenbüchle, and P. K. Wellauer, Mol. Cell. Biol. 9:2464-2476, 1989), another cis-acting element at positions -60 to -86. This DNA element, which contains an AT-rich core, site for nuclear proteins present not only in the pancreas but also in other tissues and cell lines derived from the endoderm. Purification of binding activities from pancreatic cells by DNA affinity chromatography reveals several distinct proteins ranging in size from 45 to 54 kDa (p45, p47/48, and p54). All of these proteins interact with the specific DNA sequence upon renaturation in vitro. Protein sequencing, electrophoretic mobility shift assay, and immunoblot analyses identify p54 and p47/48 as members of the hepatocyte nuclear factor 3 (HNF3 [forkhead]) family of transcription factors. p54 belongs to the subfamily of HNF3 beta proteins, while p47/48 binding activity includes HNF3 gamma. The cDNAs for two HNF3 beta proteins differing only in N-terminal amino acid sequences were isolated from a pancreatic cDNA library. The mRNAs encoding the two protein species accumulate to different steady-state levels in poly(A)+ RNA of pancreatic cells. Our results support a model by which the pancreas-specific expression of the alpha-amylase gene is mediated by a combination of cell-specific and cell lineage-specific transcription factors.
Abstract: We have characterized binding activities in yeast which recognise the T-rich strand of the yeast ARS consensus element and have purified two of these to homogeneity. One (ACBP-60) is detectable in both nuclear and whole cell extracts, while the other (ACBP-67) is apparent only after fractionation of extracts by heparin-sepharose chromatography. The major binding activity detected in nuclear extracts was purified on a sequence-specific DNA affinity column as a single polypeptide with apparent mobility of 60kDa (ACBP-60). This protein co-fractionates with nuclei, is present at several thousand copies per cell and has a Kd for the T-rich single strand of the ARS consensus between 10(-9) and 10(-10) M. Competition studies with simple nucleic acid polymers show that ACBP-60 has marginally higher affinity for poly dT30 than for a 30 nt oligomer containing the T-rich strand of ARS 307, and approximately 10 fold higher affinity for poly rU. Internal sequence information of purified p60 reveals identity with the open reading frames of genes PUB1 and RNP1 which encode polyuridylate binding protein(s). The second binding activity, ACBP-67, also binds specifically to the T-rich single strand of the ARS consensus, but with considerably lower affinity than ACBP-60. Peptide sequence reveals that the 67kDa protein is identical to the major polyA binding protein in yeast, PAB1.
Abstract: Footprint analysis of the 5'-flanking regions of the alpha-amylase 2, elastase 2, and trypsina genes, which are expressed in the acinar pancreas, showed multiple sites of protein-DNA interaction for each gene. Competition experiments demonstrated that a region from each 5'-flanking region interacted with the same cell-specific DNA-binding activity. We show by in vitro binding assays that this DNA-binding activity also recognizes a sequence within the 5'-flanking regions of elastase 1, chymotrypsinogen B, carboxypeptidase A, and trypsind genes. Methylation interference and protection studies showed that the DNA-binding activity recognized a bipartite motif, the subelements of which were separated by integral helical turns of DNA. The alpha-amylase 2 cognate sequence was found to enhance in vivo transcription of its own promoter in a cell-specific manner, which identified the DNA-binding activity as a transcription factor (PTF 1). The observation that PTF 1 bound to DNA sequences that have been defined as transcriptional enhancers by others suggests that this factor is involved in the coordinate expression of genes transcribed in the acinar pancreas.
Abstract: The positions and relative frequencies of the primary cleavages made by micrococcal nuclease on the DNA of nucleosome core particles have been found by fractionating the double-stranded products of digestion and examining their single-stranded compositions. This approach overcomes the problems caused by secondary events such as the exonucleolytic and pseudo-double-stranded actions of the nuclease and, combined with the use of high resolution gel electrophoresis, enables the cutting site positions to be determined with a higher precision than has been achieved hitherto. The micrococcal nuclease primary cleavage sites lie close (on average, within 0.5 nucleotide) to those previously determined by Lutter (1981) for the nucleases DNase I and DNase II. These similarities show that the accessible regions are the same for all three nucleases, the cleavage sites being dictated by the structure of the nucleosome core. The differences in the final products of the digestion are explained in terms of secondary cleavage events of micrococcal nuclease. While the strongly protected regions of the nucleosome core DNA are common to all three nucleases, there are differences in the relative degrees of cutting at the more exposed sites characteristic of the particular enzyme. In particular, micrococcal nuclease shows a marked polarity in the 3'-5' direction in the cutting rates as plotted along a single strand of the nucleosomal DNA. This is explained in terms of the three-dimensional structure of the nucleosome where, in any accessible region of the double helix, the innermost strand is shielded by the outermost strand on the one side and the histone core on the other. The final part of the paper is concerned with the preference of micrococcal nuclease to cleave at (A,T) sequences in chromatin.
