Assembly of specialised chromatin at Fission Yeast Centromeres

Despite the conserved essential function of centromeres, centromeric DNA is not conserved between species. There is strong evidence indicating that centromeres are epigenetically regulated. Although centromeres normally assemble on preferred sequences, these sequences are neither necessary nor sufficient for centromere assembly. For instance, neocentromeres can form upon sequences that previously showed no centromere function. The presence of the histone H3 variant, CENP-A, is thought to be the epigenetic mark that specifies centromere identity. We aim to understand how CENP-A assembly is influenced by sequence and by chromatin context. Schizosaccharomyces pombe centromeres are composed of a central domain which is assembled in CENP-A chromatin and forms the kinetochore, flanked by the heterochromatic (H3K9me2) outer repeat regions. We have previously shown that heterochromatin is required for establishment of CENP-A chromatin, but not for its maintenance. Our analyses suggest that histone acetyltransferases and deacetylases influence CENP-A establishment. In addition, analysis of requirements for CENP-A establishment lead us to propose that a key property of central domain sequences is their ability to direct an environment of low quality pervasive transcription that is permissive for CENP-A chromatin establishment. Genome sequencing of three additional Schizosaccharomyces (S. octosporus, S. japonicus and S. cryophilus) species allowed partial assembly of putative centromere regions. In order to fully assemble the centromeres of these species we are employing PacBio sequencing of long reads in conjunction with analysis of CENP-A and H3K9me2 ChIP-seq to define chromatin domains. Intriguingly, although there is no homology between the centromere sequences of the four Schizosaccharomyces, the organization and architecture are similar. We are investigating the hypothesis that despite the lack of sequence conservation, the Schizosaccharomyces centromeres possess conserved properties that promote assembly of CENP-A chromatin and heterochromatin.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Transforming life. Fragile X syndrome between molecular diagnosis and biosociality

This paper addresses issues of molecular diagnosis as a pivotal life reconfiguration factor, defined both as life forms ("naked life") and as forms of life (Wittgenstein). Drawing on a multi-site ethnographic study carried out from mid-2002 to mid-2003 in the French speaking part of Switzerland as fieldwork for my MA thesis, I describe how these two interrelate in the context of a particular disease, termed "fragile X syndrome", for which a molecular testing routine was proposed since the 90's. It is portrayed in biomedical literature as one of the most common inherited causes of "mental retardation". Its symptomatology, including some physical and behavioral features (e.g. facial dysmorphy), was gradually linked, from the end of 60's to the 90's, to a single genetic causal explanation, first in terms of "chromosome fragility" and further as the fully mutated FMR-1 gene. Molecular diagnosis usually comes after a clinical and family history investigation trajectory. I focus on this "moment" and show that the intervention (engineering) upon "naked life" in the testing lab, its specific products (among others the degree of gene mutation, ie no mutation, pre-mutation or full mutation) and the syndrome inheritance patterns affect the way the syndrome becomes known, experienced and "practiced" by the patients and their families, taking part in their life as "the embodiment of fate, the evil locus" and the "site of hope" (Rabinow, 1999). The biological and the social are re-framed to produce what Rabinow termed "biosociality". In this context, the FMR-1 gene creates not only sociality and inclusion but also new boundaries between "we" and "the others" and new intra-categories in the fragile X communities, (re)shaping identities according to detailed knowledge and articulation between molecular and phenotypic differences

Protein targeting in Schizosaccharomyces pombe

The aim of the work described in this thesis was to develop the fission yeast, Schizosaccharomyces pombe as a model system for studies of the secretory pathway and protein targeting. Two aspects of protein targeting were studied in S. pombe, the characterisation and sorting of the luminal ER protein BiP, and the involvement of ypt/rab proteins in membrane traffic. The homology between BiP proteins from different species was exploited in cloning the gene for S. pombe BiP. The BiP gene was shown to be essential which is consistent with its numerous proposed roles in protein folding and assembly. S. pombe BiP is regulated by a variety of treatments thought to cause an accumulation of misfolded or underglycosylated proteins in the ER, including heat shock. Unusually, S. pombe BiP contains a potential N-linked glycosylation site, which was shown to be partially utilised. The production of antibodies against BiP facilitated an investigation of the structure of the ER in S. pombe. As in mammalian cells the ER of S. pombe has a reticular component. No evidence was obtained for the involvement of microtubules or the actin cytoskeleton in the formation or maintenance of the reticular structure. Amino acids from the C-terminus of S. pombe BiP were attached to the secretory protein acid phosphatase and it was established that ADEL can act as an ER localisation signal on luminal proteins. Signals from other yeast and mammalian species, DDEL, HDEL and KDEL, were also recognised in S. pombe. Members of the ras superfamily isolated in the laboratory using an oligonucleotide representing a conserved region of the GTP-binding domain were characterised. The ypt4 gene represents a novel member of the rab/ypt family. The ypt4 gene is non-essential but in its absence cells contained vacuoles of unusual morphology. An epitope tagged ypt4 protein localised to the surface of vacuoles. These two observations suggest that ypt4 might play some role in vacuole structure or function in S. pombe