Biomedical subjects

Nathalie Aulner

Publications and source records attributed to Nathalie Aulner.

5 recordsLinked to original sources

PubMed2006

Cell-based assays using primary endothelial cells to study multiple steps in inflammation.

Cell-based assays are powerful tools for drug discovery and provide insight into complex signal transduction pathways in higher eukaryotic cells. Information gleaned from assays that monitor a cellular phenotype can be used to elucidate the details of a single pathway and to establish patterns of cross talk between pathways. By selecting the appropriate cell model, cell-based assays can be used to understand the function of a specific cell type in a complex disease process such as inflammation. We have used human umbilical vein endothelial cells to establish three cell-based, phenotypic assays that query different stages of a major signaling pathway activated in inflammation. One assay analyzes the tumor necrosis factor alpha (TNFalpha)-induced translocation of the transcription factor NF-kappaB from the cytoplasm into the nucleus 20 min after stimulation with TNFalpha. Two more assays monitor the expression of E-selectin and VCAM-1, 4 and 24 h after stimulation with TNFalpha. Indirect immunofluorescence and high-throughput automated microscopy were used to analyze cells. Imaging was performed with the IN Cell Analyzer 3000. All assays proved to be highly robust. Z' values between 0.7 and 0.8 make each of the three assays well suited for use in high-throughput screening for drug or probe discovery.

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PubMed2005

Sequence characteristics of functional siRNAs.

RNA interference in mammalian cells is actively used to conduct genetic screens, to identify and to validate targets, and to elucidate regulators and modifiers of cellular pathways. To ensure the specificity and efficacy of the active 21mer siRNA molecules, it is pertinent to develop a strategy for their rational design. Here we show that most functional siRNAs have characteristic sequence features. We tested 601 siRNAs targeting one exogenous and three endogenous genes. The efficacy of the siRNAs was determined at the protein level. Using a decision tree algorithm in combination with information analysis, our analyses revealed four sets of rules with a mean knockdown efficacy ranging from 60% to 73%. (To distinguish between percentages used to describe the quality of an siRNA and the percentages used to describe parts of data sets we underlined the former throughout this paper.) The best rule comprises an A/U at positions 10 and 19, a G/C at position 1, and more than three A/Us between positions 13 and 19, in the sense strand of the siRNA sequence. Using these rules, there is a 99.9% chance of designing an effective siRNA in a set of three with more than 50% knockdown efficiency in a biological readout.

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PubMed2004

Drosophila nipped-B protein supports sister chromatid cohesion and opposes the stromalin/Scc3 cohesion factor to facilitate long-range activation of the cut gene.

The Drosophila melanogaster Nipped-B protein facilitates transcriptional activation of the cut and Ultrabithorax genes by remote enhancers. Sequence homologues of Nipped-B, Scc2 of Saccharomyces cerevisiae, and Mis4 of Schizosaccharomyces pombe are required for sister chromatid cohesion during mitosis. The evolutionarily conserved Cohesin protein complex mediates sister chromatid cohesion, and Scc2 and Mis4 are needed for Cohesin to associate with chromosomes. Here, we show that Nipped-B is also required for sister chromatid cohesion but that, opposite to the effect of Nipped-B, the stromalin/Scc3 component of Cohesin inhibits long-range activation of cut. To explain these findings, we propose a model based on the chromatin domain boundary activities of Cohesin in which Nipped-B facilitates cut activation by alleviating Cohesin-mediated blocking of enhancer-promoter communication.

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PubMed2002

Modification of position-effect variegation by competition for binding to Drosophila satellites.

White-mottled (w(m4)) position-effect variegation (PEV) arises by translocation of the white gene near the pericentric AT-rich 1.688 g/cm3 satellite III (SATIII) repeats of the X chromosome of Drosophila. The natural and artificial A*T-hook proteins D1 and MATH20 modify w(m4) PEV in opposite ways. D1 binds SATIII repeats and enhances PEV, presumably via a recruitment of protein partners, whereas MATH20 suppresses it. We show that D1 and MATH20 compete for binding to identical sites of SATIII repeats in vitro and that conditional MATH20 expression results in a displacement of D1 from pericentric heterochromatin in vivo. In the presence of intermediate levels of MATH20, we show that this displacement becomes selective for SATIII repeats. These results strongly suggest that the suppression of w(m4) PEV by MATH20 is due to a displacement of D1 from its preferred binding sites and provide additional support for a direct role of D1 in the assembly of AT-rich heterochromatin.

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PubMed2002

The AT-hook protein D1 is essential for Drosophila melanogaster development and is implicated in position-effect variegation.

We have analyzed the expression pattern of the D1 gene and the localization of its product, the AT hook-bearing nonhistone chromosomal protein D1, during Drosophila melanogaster development. D1 mRNAs and protein are maternally contributed, and the protein localizes to discrete foci on the chromosomes of early embryos. These foci correspond to 1.672- and 1.688-g/cm(3) AT-rich satellite repeats found in the centromeric heterochromatin of the X and Y chromosomes and on chromosomes 3 and 4. D1 mRNA levels subsequently decrease throughout later development, followed by the accumulation of the D1 protein in adult gonads, where two distributions of D1 can be correlated to different states of gene activity. We show that the EP473 mutation, a P-element insertion upstream of D1 coding sequences, affects the expression of the D1 gene and results in an embryonic homozygous lethal phenotype correlated with the depletion of D1 protein during embryogenesis. Remarkably, decreased levels of D1 mRNA and protein in heterozygous flies lead to the suppression of position-effect variegation (PEV) of the white gene in the white-mottled (w(m4h)) X-chromosome inversion. Our results identify D1 as a DNA-binding protein of known sequence specificity implicated in PEV. D1 is the primary factor that binds the centromeric 1.688-g/cm(3) satellite repeats which are likely involved in white-mottled variegation. We propose that the AT-hook D1 protein nucleates heterochromatin assembly by recruiting specialized transcriptional repressors and/or proteins involved in chromosome condensation.

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