Finite Element Modeling of Airflow During Phonation

International audienceIn the paper a mathematical model of airflow in human vocal folds is presented. The geometry of the glottal channel is based on measurements of excised human larynges. The airflow is modeled by nonstationary incompressible Navier-Stokes equations in a 2D computational domain, which is deformed in time due to vocal fold vibration. The paper presents numerical results and focuses on flow separation in glottis. Quantitative data from numerical simulations are compared to results of measurements by Particle Image Velocimetry (PIV), performed on a scaled self-oscillating physical model of vocal folds

Condition aux limites transparente pour la propagation acoustique dans un guide recouvert d'un matériau absorbant en présence d'un écoulement uniforme

L'objet de notre travail est de calculer par éléments finis le rayonnement acoustique dans un guide d'onde infini dont une paroi est recouverte d'un matériau absorbant, caractérisé par une impédance Z. Pour tronquer le domaine de calcul, on veut introduire des conditions aux limites transparentes déduites d'une décomposition modale, ce qui nécessite la détermination des modes du guide. En l'absence d'écoulement, la décomposition est aisée pour un guide à parois rigides. En présence d'un matériau absorbant, de nombreuses difficultés apparaissent même en l'absence d'écoulement : l'opérateur n'est plus auto-adjoint et les modes ne sont plus orthogonaux pour le produit scalaire usuel. On montre qu'on peut introduire un « produit scalaire particulier » qui conduit à une « pseudo orthogonalité des modes ». Toutefois, on montre qu'il existe une suite de valeurs « exceptionnelles » de l'impédance Zc pour lesquelles la décomposition modale n'est pas possible. La méthode est étendue au cas d'un écoulement uniforme. L'apparition de dérivées tangentielles de la pression sur la paroi traitée entraîne des difficultés supplémentaires. Un nouveau produit scalaire est introduit, incluant les valeurs de la pression sur la paroi traitée. On montre alors que les modes ne deviennent orthogonaux que si l'un des modes est d'indice élevé et que cette propriété suffit pour définir des conditions transparentes. Dans tous les cas, en dehors des valeurs critiques de l'impédance, on se ramène à la résolution d'une équation de dispersion complexe. La méthode de Newton Raphson est utilisée pour l'identification des modes dans le plan complexe. Nous avons développé une méthode numérique pour calculer les modes ou le rayonnement d'une source, basée sur le couplage entre des éléments finis et les conditions transparentes. Les simulations sont en bon accord, soit avec les résultats analytiques pour les modes, soit avec la pression rayonnée obtenue par une méthode alternative : en entourant le domaine de calcul par des couches absorbantes de type PML (Perfectly Matched Layers)

Changes in chromatin structure during processing of wax-embedded tissue sections

The use of immunofluorescence (IF) and fluorescence in situ hybridisation (FISH) underpins much of our understanding of how chromatin is organised in the nucleus. However, there has only recently been an appreciation that these types of study need to move away from cells grown in culture and towards an investigation of nuclear organisation in cells in situ in their normal tissue architecture. Such analyses, however, especially of archival clinical samples, often requires use of formalin-fixed paraffin wax-embedded tissue sections which need addition steps of processing prior to IF or FISH. Here we quantify the changes in nuclear and chromatin structure that may be caused by these additional processing steps. Treatments, especially the microwaving to reverse fixation, do significantly alter nuclear architecture and chromatin texture, and these must be considered when inferring the original organisation of the nucleus from data collected from wax-embedded tissue sections

Epigenetic control of nuclear architecture

The cell nucleus is a highly structured compartment where nuclear components are thought to localize in non-random positions. Correct positioning of large chromatin domains may have a direct impact on the localization of other nuclear components, and can therefore influence the global functionality of the nuclear compartment. DNA methylation of cytosine residues in CpG dinucleotides is a prominent epigenetic modification of the chromatin fiber. DNA methylation, in conjunction with the biochemical modification pattern of histone tails, is known to lock chromatin in a close and transcriptionally inactive conformation. The relationship between DNA methylation and large-scale organization of nuclear architecture, however, is poorly understood. Here we briefly summarize present concepts of nuclear architecture and current data supporting a link between DNA methylation and the maintenance of large-scale nuclear organization

Poised Transcription Factories Prime Silent uPA Gene Prior to Activation

The association of poised genes with transcription factories may contribute to rapid transcriptional activation in response to stimuli and to silencing when genes are located at the interior of their chromosome territories

Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures

DNA supercoiling is an inherent consequence of twisting DNA and is critical for regulating gene expression and DNA replication. However, DNA supercoiling at a genomic scale in human cells is uncharacterized. To map supercoiling we used biotinylated-trimethylpsoralen as a DNA structure probe to show the genome is organized into supercoiling domains. Domains are formed and remodeled by RNA polymerase and topoisomerase activities and are flanked by GC-AT boundaries and CTCF binding sites. Under-wound domains are transcriptionally active, enriched in topoisomerase I, “open” chromatin fibers and DNaseI sites, but are depleted of topoisomerase II. Furthermore DNA supercoiling impacts on additional levels of chromatin compaction as under-wound domains are cytologically decondensed, topologically constrained, and decompacted by transcription of short RNAs. We suggest that supercoiling domains create a topological environment that facilitates gene activation providing an evolutionary purpose for clustering genes along chromosomes

Nuclear Pore Proteins Nup153 and Megator Define Transcriptionally Active Regions in the Drosophila Genome

Transcriptional regulation is one of the most important processes for modulating gene expression. Though much of this control is attributed to transcription factors, histones, and associated enzymes, it is increasingly apparent that the spatial organization of chromosomes within the nucleus has a profound effect on transcriptional activity. Studies in yeast indicate that the nuclear pore complex might promote transcription by recruiting chromatin to the nuclear periphery. In higher eukaryotes, however, it is not known whether such regulation has global significance. Here we establish nucleoporins as a major class of global regulators for gene expression in Drosophila melanogaster. Using chromatin-immunoprecipitation combined with microarray hybridisation, we show that Nup153 and Megator (Mtor) bind to 25% of the genome in continuous domains extending 10 kb to 500 kb. These Nucleoporin-Associated Regions (NARs) are dominated by markers for active transcription, including high RNA polymerase II occupancy and histone H4K16 acetylation. RNAi–mediated knock-down of Nup153 alters the expression of ∼5,700 genes, with a pronounced down-regulatory effect within NARs. We find that nucleoporins play a central role in coordinating dosage compensation—an organism-wide process involving the doubling of expression of the male X chromosome. NARs are enriched on the male X chromosome and occupy 75% of this chromosome. Furthermore, Nup153-depletion abolishes the normal function of the male-specific dosage compensation complex. Finally, by extensive 3D imaging, we demonstrate that NARs contribute to gene expression control irrespective of their sub-nuclear localization. Therefore, we suggest that NAR–binding is used for chromosomal organization that enables gene expression control

Transcription and Chromatin Organization of a Housekeeping Gene Cluster Containing an Integrated β-Globin Locus Control Region

The activity of locus control regions (LCR) has been correlated with chromatin decondensation, spreading of active chromatin marks, locus repositioning away from its chromosome territory (CT), increased association with transcription factories, and long-range interactions via chromatin looping. To investigate the relative importance of these events in the regulation of gene expression, we targeted the human β-globin LCR in two opposite orientations to a gene-dense region in the mouse genome containing mostly housekeeping genes. We found that each oppositely oriented LCR influenced gene expression on both sides of the integration site and over a maximum distance of 150 kilobases. A subset of genes was transcriptionally enhanced, some of which in an LCR orientation-dependent manner. The locus resides mostly at the edge of its CT and integration of the LCR in either orientation caused a more frequent positioning of the locus away from its CT. Locus association with transcription factories increased moderately, both for loci at the edge and outside of the CT. These results show that nuclear repositioning is not sufficient to increase transcription of any given gene in this region. We identified long-range interactions between the LCR and two upregulated genes and propose that LCR-gene contacts via chromatin looping determine which genes are transcriptionally enhanced

Active and poised promoter states drive folding of the extended HoxB locus in mouse embryonic stem cells

Gene expression states influence the three-dimensional conformation of the genome through poorly understood mechanisms. Here, we investigate the conformation of the murine HoxB locus, a gene-dense genomic region containing closely spaced genes with distinct activation states in mouse embryonic stem (ES) cells. To predict possible folding scenarios, we performed computer simulations of polymer models informed with different chromatin occupancy features, which define promoter activation states or CTCF binding sites. Single cell imaging of the locus folding was performed to test model predictions. While CTCF occupancy alone fails to predict the in vivo folding at genomic length scale of 10 kb, we found that homotypic interactions between active and Polycomb-repressed promoters co-occurring in the same DNA fibre fully explain the HoxB folding patterns imaged in single cells. We identify state-dependent promoter interactions as major drivers of chromatin folding in gene-dense regions

A Gammaherpesviral Internal Repeat Contributes to Latency Amplification

BACKGROUND: Gammaherpesviruses cause important infections of humans, in particular in immunocompromised patients. The genomes of gammaherpesviruses contain variable numbers of internal repeats whose precise role for in vivo pathogenesis is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We used infection of laboratory mice with murine gammaherpesvirus 68 (MHV-68) to explore the biological role of the 40 bp internal repeat of MHV-68. We constructed several mutant viruses partially or completely lacking this repeat. Both in vitro and in vivo, the loss of the repeat did not substantially affect lytic replication of the mutant viruses. However, the extent of splenomegaly, which is associated with the establishment of latency, and the number of ex vivo reactivating and genome positive splenocytes were reduced. Since the 40 bp repeat is part of the hypothetical open reading frame (ORF) M6, it might function as part of M6 or as an independent structure. To differentiate between these two possibilities, we constructed an N-terminal M6STOP mutant, leaving the repeat structure intact but rendering ORF M6 unfunctional. Disruption of ORF M6 did neither affect lytic nor latent infection. In contrast to the situation in lytically infected NIH3T3 cells, the expression of the latency-associated genes K3 and ORF72 was reduced in the latently infected murine B cell line Ag8 in the absence of the 40 bp repeat. CONCLUSIONS/SIGNIFICANCE: These data suggest that the 40 bp repeat contributes to latency amplification and might be involved in the regulation of viral gene expression