Mutations in TUBG1, DYNC1H1, KIF5C and KIF2A cause malformations of cortical development and microcephaly

The genetic causes of malformations of cortical development (MCD) remain largely unknown. Here we report the discovery of multiple pathogenic missense mutations in TUBG1, DYNC1H1 and KIF2A, as well as a single germline mosaic mutation in KIF5C, in subjects with MCD. We found a frequent recurrence of mutations in DYNC1H1, implying that this gene is a major locus for unexplained MCD. We further show that the mutations in KIF5C, KIF2A and DYNC1H1 affect ATP hydrolysis, productive protein folding and microtubule binding, respectively. In addition, we show that suppression of mouse Tubg1 expression in vivo interferes with proper neuronal migration, whereas expression of altered gamma-tubulin proteins in Saccharomyces cerevisiae disrupts normal microtubule behavior. Our data reinforce the importance of centrosomal and microtubule-related proteins in cortical development and strongly suggest that microtubule-dependent mitotic and postmitotic processes are major contributors to the pathogenesis of MCD

Sub-Telomeric core X and Y' Elements in S.cerevisiae Suppress Extreme Variations in Gene Silencing

Telomere Position Effect (TPE) is governed by strong repression signals emitted by telomeres via the Sir2/3/4 Histone Deacetylase complex. These signals are then relayed by weak proto-silencers residing in the subtelomeric core X and Y' elements. Subtelomeres also contain Sub-Telomeric Anti-silencing Regions (STARs). In this study we have prepared telomeres built of different combinations of core X, Y' and STARs and have analyzed them in strains lacking Histone-Acetyltransferase genes as well as in cdc6-1 and Δrif1 strains. We show that core X and Y' dramatically reduce both positive and negative variations in TPE, that are caused by these mutations. We also show that the deletion of Histone-Acetyltransferase genes reduce the silencing activity of an ACS proto-silencer, but also reduce the anti-silencing activity of a STAR. We postulate that core X and Y' act as epigenetic “cushioning” cis-elements

A Single Heterochromatin Boundary Element Imposes Position-Independent Antisilencing Activity in Saccharomyces cerevisiae Minichromosomes

Chromatin boundary elements serve as cis-acting regulatory DNA signals required to protect genes from the effects of the neighboring heterochromatin. In the yeast genome, boundary elements act by establishing barriers for heterochromatin spreading and are sufficient to protect a reporter gene from transcriptional silencing when inserted between the silencer and the reporter gene. Here we dissected functional topography of silencers and boundary elements within circular minichromosomes in Saccharomyces cerevisiae. We found that both HML-E and HML-I silencers can efficiently repress the URA3 reporter on a multi-copy yeast minichromosome and we further showed that two distinct heterochromatin boundary elements STAR and TEF2-UASrpg are able to limit the heterochromatin spreading in circular minichromosomes. In surprising contrast to what had been observed in the yeast genome, we found that in minichromosomes the heterochromatin boundary elements inhibit silencing of the reporter gene even when just one boundary element is positioned at the distal end of the URA3 reporter or upstream of the silencer elements. Thus the STAR and TEF2-UASrpg boundary elements inhibit chromatin silencing through an antisilencing activity independently of their position or orientation in S. cerevisiae minichromosomes rather than by creating a position-specific barrier as seen in the genome. We propose that the circular DNA topology facilitates interactions between the boundary and silencing elements in the minichromosomes

Evidence for silencing compartments within the yeast nucleus: a role for telomere proximity and Sir protein concentration in silencer-mediated repression.

International audienceTranscriptional repression at the silent mating-type loci in yeast requires the targeting of silent information regulator (Sir) proteins through specific interactions formed at cis-acting silencer elements. We show here that a reporter gene flanked by two functional silencers is not repressed when integrated at >200 kb from a telomere. Repression is restored by creation of a new telomere 13 kb from the integrated reporter or by elevated expression of SIRl, SIRS, and/or SIR4. Coupled expression represses in an additive manner, suggesting that all three factors are in limiting concentrations. When overexpressed, Sir3 and Sir4 are dispersed throughout the nucleoplasm, in contrast to wild-type cells where they are clustered in a limited number of foci together with telomeres. Efficient silencer function thus seems to require either proximity to a pool of concentrated Sir proteins, that is, proximity to telomeres, or delocalization of the silencing factors

Cooperation at a distance between silencers and proto-silencers at the yeast HML locus.

Transcriptional repression at the silent yeast mating type loci is achieved through the formation of a particular nucleoprotein complex at specific cis-acting elements called silencers. This complex in turn appears to initiate the spreading of a histone binding protein complex into the surrounding chromatin, which restricts accessibility of the region to the transcription machinery. We have investigated long-range, cooperative effects between silencers by studying the repression of a reporter gene integrated at the HML locus flanked by various combinations of wild-type and mutated silencer sequences. Two silencers can cooperate over >4000 bp to repress transcription efficiently. More importantly, a single binding site for either the repressor activator protein 1 (Rap1), the autonomous replicating sequence (ARS) binding factor 1 (Abf1) or the origin recognition complex (ORC) can enhance the action of a distant silencer without acting as a silencer on its own. Functional cooperativity is demonstrated using a quantitative assay for repression, and varies with the affinity of the binding sites used. Since the repression mechanism is Sir dependent, the Rap1, ORC and/or Abf1 proteins bound to distant DNA elements may interact to create an interface of sufficiently high affinity such that Sir-containing complexes bind, nucleating the silent chromatin state

Efficient generation of narrow-bandwidth picosecond pulses by frequency doubling of femtosecond chirped pulses

International audienceWe demonstrate efficient generation of picosecond narrow-bandwidth pulses by frequency mixing of broadband opposite chirped pulses in a type I doubling crystal. This procedure allows us to produce picosecond pulses that are perfectly synchronized with femtosecond pulses. The experiment shows a decrease of the initial bandwidth by a factor of more than 30, while a high conversion efficiency is maintaine

Dbp5, a DEAD-box protein required for mRNA export, is recruited to the cytoplasmic fibrils of nuclear pore complex via a conserved interaction with CAN/Nup159p.

Dbp5 is a DEAD-box protein essential for mRNA export from the nucleus in yeast. Here we report the isolation of a cDNA encoding human Dbp5 (hDbp5) which is 46% identical to yDbp5p. Like its yeast homologue, hDbp5 is localized within the cytoplasm and at the nuclear rim. By immunoelectron microscopy, the nuclear envelope-bound fraction of Dbp5 has been localized to the cytoplasmic fibrils of the nuclear pore complex (NPC). Consistent with this localization, we show that both the human and yeast proteins directly interact with an N-terminal region of the nucleoporins CAN/Nup159p. In a conditional yeast strain in which Nup159p is degraded when shifted to the nonpermissive temperature, yDbp5p dissociates from the NPC and localizes to the cytoplasm. Thus, Dbp5 is recruited to the NPC via a conserved interaction with CAN/Nup159p. To investigate its function, we generated defective hDbp5 mutants and analysed their effects in RNA export by microinjection in Xenopus oocytes. A mutant protein containing a Glu-->Gln change in the conserved DEAD-box inhibited the nuclear exit of mRNAs. Together, our data indicate that Dbp5 is a conserved RNA-dependent ATPase which is recruited to the cytoplasmic fibrils of the NPC where it participates in the export of mRNAs out of the nucleus

Modeling a disease-correlated tubulin mutation in budding yeast reveals insight into MAP-mediated dynein function

International audienceThe authors of this study employ budding yeast to model a disease-correlated tubulin mutation to determine how it leads to microtubule dysfunction. They find that the mutation leads to alterations in microtubule dynamics, tubulin isotype usage, and a spindle positioning defect that is due to disrupted microtubule-binding by a dynein regulator

Study of plasma heating induced by fast electrons

International audienceWe studied the induced plasma heating in three different kinds of targets: mass limited, foam targets, and large mass targets. The experiment was performed at Ali? Laser Facility of CEA/CESTA. The laser system emitted a ?1 ps pulse with ?10 J energy at a wavelength of ?1 ?m. Mass limited targets had three layers with thicknesses of 10 ?m C8 H 8, 1 ?m C8 H7 Cl, and 10 ?m C8 H8 with size of 100×100 ? m2. Detailed spectroscopic analysis of x rays emitted from the Cl tracer showed that it was possible to heat up the plasma from mass limited targets to a temperature of ?250 eV with density of ? 1021 cm-3. The plasma heating is only produced by fast electron transport in the target, being the 10 ?m C8 H8 overcoating thick enough to prevent any possible direct irradiation of the tracer layer even taking into account mass-ablation due to the prepulse. These results demonstrate that with mass limited targets, it is possible to generate a plasma heated up to several hundreds eV. It is also very important for research concerning high energy density phenomena and for fast ignition (in particular for the study of fast electrons transport and induced heating). © 2009 American Institute of Physics