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Lickwar C.R.,Carolina Center for the Genome science | Mueller F.,Institute Of Biologie Of Lecole Normale Superieure | Mueller F.,Institute Pasteur Paris | Lieb J.D.,Carolina Center for the Genome science
Nature Protocols | Year: 2013

Competition chromatin immunoprecipitation (competition ChIP) enables experimenters to measure protein-DNA dynamics at a single locus or across the entire genome, depending on the detection method. Competition ChIP relies on a cell containing two copies of a single DNA-associated factor, with each copy of the factor differentially epitope tagged. One of the copies is expressed constitutively and the second is induced as a competitor. The ratio of isoforms associated with discrete genomic locations is detected by ChIP-on-chip (ChIP-chip) or ChIP-sequencing (ChIP-seq). The rate at which the resident isoform of the protein is replaced by the competitor at each binding location enables the calculation of residence time for that factor at each site of interaction genome wide. Here we provide a detailed protocol for designing and performing competition ChIP experiments in Saccharomyces cerevisiae, which takes ∼5 d to complete (not including strain production and characterizations, which may take as long as 6 months). Included in this protocol are guidelines for downstream bioinformatic analysis to extract residence times throughout the genome. © 2013 Nature America, Inc. All rights reserved.

Berger N.,French National Institute for Agricultural Research | Dubreucq B.,French National Institute for Agricultural Research | Roudier F.,Institute Of Biologie Of Lecole Normale Superieure | Dubos C.,French National Institute for Agricultural Research | Lepiniec L.,French National Institute for Agricultural Research
Plant Cell | Year: 2011

LEAFY COTYLEDON2 (LEC2) is a master regulator of seed development in Arabidopsis thaliana. In vegetative organs, LEC2 expression is negatively regulated by Polycomb Repressive Complex2 (PRC2) that catalyzes histone H3 Lys 27 trimethylation (H3K27me3) and plays a crucial role in developmental phase transitions. To characterize the cis-regulatory elements involved in the transcriptional regulation of LEC2, molecular dissections and functional analyses of the promoter region were performed in vitro, both in yeast and in planta. Two cis-activating elements and a cis-repressing element (RLE) that is required for H3K27me3 marking were characterized. Remarkably, insertion of the RLE cis-element into pF3H, an unrelated promoter, is sufficient for repressing its transcriptional activity in different tissues. Besides improving our understanding of LEC2 regulation, this study provides important new insights into the mechanisms underlying H3K27me3 deposition and PRC2 recruitment at a specific locus in plants. © American Society of Plant Biologists. All rights reserved.

Ten Hoopen R.,University of Cambridge | Cepeda-Garcia C.,University of Cambridge | Cepeda-Garcia C.,Centro Andaluz Of Biologia Molecular Y Medicina Regenerativa Cabimer | Fernandez-Arruti R.,University of Cambridge | And 5 more authors.
Current Biology | Year: 2012

Background: Budding yeast is a unique model to dissect spindle orientation in a cell dividing asymmetrically. In yeast, this process begins with the capture of pole-derived astral microtubules (MTs) by the polarity determinant Bud6p at the cortex of the bud in G 1. Bud6p couples MT growth and shrinkage with spindle pole movement relative to the contact site. This activity resides in N-terminal sequences away from a domain linked to actin organization. Kip3p (kinesin-8), a MT depolymerase, may be implicated, but other molecular details are essentially unknown. Results: We show that Bud6p and Kip3p play antagonistic roles in controlling the length of MTs contacting the bud. The stabilizing role of Bud6p required the plus-end-tracking protein Bim1p (yeast EB1). Bim1p bound Bud6p N terminus, an interaction that proved essential for cortical capture of MTs in vivo. Moreover, Bud6p influenced Kip3p dynamic distribution through its effect on MT stability during cortical contacts via Bim1p. Coupling between Kip3p-driven depolymerization and shrinkage at the cell cortex required Bud6p, Bim1p, and dynein, a minus-end-directed motor helping tether the receding plus ends to the cell cortex. Validating these findings, live imaging of the interplay between dynein and Kip3p demonstrated that both motors decorated single astral MTs with dynein persisting at the plus end in association with the site of cortical contact during shrinkage at the cell cortex. Conclusions: Astral MT shrinkage linked to Bud6p involves its direct interaction with Bim1p and the concerted action of two MT motors - Kip3p and dynein. © 2012 Elsevier Ltd All rights reserved.

Boulin T.,Institute Of Biologie Of Lecole Normale Superieure | Boulin T.,French Institute of Health and Medical Research | Boulin T.,French National Center for Scientific Research | Hobert O.,Columbia University
Wiley Interdisciplinary Reviews: Developmental Biology | Year: 2012

This review aims to provide an overview of the technologies which make the nematode Caenorhabditis elegans an attractive genetic model system. We describe transgenesis techniques and forward and reverse genetic approaches to isolate mutants and clone genes. In addition, we discuss the new possibilities offered by genome engineering strategies and next-generation genome analysis tools. © 2011 Wiley Periodicals, Inc.

Delgehyr N.,University of Cambridge | Delgehyr N.,Institute Of Biologie Of Lecole Normale Superieure | Rangone H.,University of Cambridge | Fu J.,University of Cambridge | And 5 more authors.
Current Biology | Year: 2012

Klp10A is a kinesin-13 of Drosophila melanogaster that depolymerizes cytoplasmic microtubules [1]. In interphase, it promotes microtubule catastrophe [2-4]; in mitosis, it contributes to anaphase chromosome movement by enabling tubulin flux [1, 5]. Here we show that Klp10A also acts as a microtubule depolymerase on centriolar microtubules to regulate centriole length. Thus, in both cultured cell lines and the testes, absence of Klp10A leads to longer centrioles that show incomplete 9-fold symmetry at their ends. These structures and associated pericentriolar material undergo fragmentation. We also show that in contrast to mammalian cells where depletion of CP110 leads to centriole elongation [6], in Drosophila cells it results in centriole length diminution that is overcome by codepletion of Klp10A to give longer centrioles than usual. We discuss how loss of centriole capping by CP110 might have different consequences for centriole length in mammalian [6-8] and insect cells and also relate these findings to the functional interactions between mammalian CP110 and another kinesin-13, Kif24, that in mammalian cells regulates cilium formation. © 2012 Elsevier Ltd.

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