Composition and functional specificity of SWI2/SNF2 class chromatin remodeling complexes

By regulating the structure of chromatin, ATP-dependent chromatin remodeling complexes (remodelers) perform critical functions in the maintenance, transmission and expression of the eukaryotic genome. Although all known chromatin-remodeling complexes contain an ATPase as a central motor subunit, a number of distinct classes have been recognized. Recent studies have emphasized a more extensive functional diversification among closely related chromatin remodeling complexes than previously anticipated. Here, we discuss recent insights in the functional differences between two evolutionary conserved subclasses of SWI/SNF-related chromatin remodeling factors. One subfamily comprises yeast SWI/SNF, fly BAP and mammalian BAF, whereas the other subfamily includes yeast RSC, fly PBAP and mammalian PBAF. We review the subunit composition, conserved protein modules and biological functions of each of these subclasses of SWI/SNF remodelers. In particular, we will focus on the roles of specific subunits in developmental gene control and human diseases. Recent findings suggest that functional diversification among SWI/SNF complexes allows the eukaryotic cell to fine-tune and integrate the execution of diverse biological programs involving the expression, maintenance and duplication of its genome.     

Sensitivity of NS3 serine proteases from hepatitis C virus genotypes 2 and 3 to the inhibitor BILN 2061

Hepatitis C virus (HCV) displays a high degree of genetic variability. Six genotypes and more than 50 subtypes have been identified to date. In this report, kinetic profiles were determined for NS3 proteases of genotypes 1a, 1b, 2ac, 2b, and 3a, revealing no major differences in activity. In vitro sensitivity studies with BILN 2061 showed a decrease in affinity for proteases of genotypes 2 and 3 (K(i), 80 to 90 nM) compared to genotype 1 enzymes (K(i), 1.5 nM). To understand the reduced sensitivity of genotypes 2 and 3 to BILN 2061, active-site residues in the proximity of the inhibitor binding site were replaced in the genotype-1b enzyme with the corresponding genotype-2b or -3a residues. The replacement of five residues at positions 78, 79, 80, 122, and 132 accounted for most of the reduced sensitivity of genotype 2b, while replacement of residue 168 alone could account for the reduced sensitivity of genotype 3a. BILN 2061 remains a potent inhibitor of these non-genotype-1 NS3-NS4A proteins, with K(i) values below 100 nM. This in vitro potency, in conjunction with the good pharmacokinetic data reported for humans, suggests that there is potential for BILN 2061 as an antiviral agent for individuals infected with non-genotype-1 HCV.     

In vitro measurement of cell death with the annexin A5 affinity assay

One of the hallmarks of cell death is the cell surface-expression of phosphatidylserine. Expression of phosphatidylserine at the cell surface can be measured in vitro with the phosphatidylserine-binding protein annexin A5 conjugated to fluorochromes. This measurement can be made by flow cytometry or by confocal scanning-laser microscopy. The annexin A5 affinity assay comprises the incubation of cells stimulated to execute cell death with fluorescence-labeled annexin A5 and propidium iodide. Living cells are annexin A5-negative and propidium iodide negative, cells in the early phases of cell death are annexin A5 positive-and propidium iodide-negative, and secondary necrotic cells are annexin A5-positive and propidium iodide-positive. The entire procedure takes about 30 minutes for flow cytometry and 45 minutes for confocal scanning-laser microscopy. Various precautions and considerations are discussed further in the protocol described here.     

Smad5 determines murine amnion fate through the control of bone morphogenetic protein expression and signalling levels

Smad5 is an intracellular mediator of bone morphogenetic protein (Bmp) signalling. It is essential for primordial germ cell (PGC) development, for the development of the allantois and for amnion closure, as demonstrated by loss of Bmp signalling. By contrast, the appearance of ectopic PGC-like cells and regionalized ectopic vasculogenesis and haematopoiesis in thickened Smad5(m1/m1) amnion are amnion defects that have not been associated with loss of Bmp signalling components. We show that defects in amnion and allantois can already be detected at embryonic day (E) 7.5 in Smad5 mutant mice. However, ectopic Oct4-positive (Oct4(+)) and alkaline phosphatase-positive (AP(+)) cells appear suddenly in thickened amnion at E8.5, and at a remote distance from the allantois and posterior primitive streak, suggesting a change of fate in situ. These ectopic Oct4(+), AP(+) cells appear to be Stella negative and hence cannot be called bona fide PGCs. We demonstrate a robust upregulation of Bmp2 and Bmp4 expression, as well as of Erk and Smad activity, in the Smad5 mutant amnion. The ectopic expression of several Bmp target genes in different domains and the regionalized presence of cells of several Bmp-sensitive lineages in the mutant amnion suggest that different levels of Bmp signalling may determine cell fate. Injection of rBMP4 in the exocoelom of wild-type embryos can induce thickening of amnion, mimicking the early amnion phenotype in Smad5 mutants. These results support a model in which loss of Smad5 results paradoxically in gain of Bmp function defects in the amnion.     

GENOTYPE‐BY‐TEMPERATURE INTERACTIONS MAY HELP TO MAINTAIN CLONAL DIVERSITY IN ASTERIONELLA FORMOSA (BACILLARIOPHYCEAE)

Marine and freshwater phytoplankton populations often show large clonal diversity, which is in disagreement with clonal selection of the most vigorous genotype(s). Temporal fluctuation in selection pressures in variable environments is a leading explanation for maintenance of such genetic diversity. To test the influence of temperature as a selection force in continually (seasonally) changing aquatic systems we carried out reaction norms experiments on co‐occurring clonal genotypes of a ubiquitous diatom species, Asterionella formosa Hassall, across an environmentally relevant range of temperatures. We report within population genetic diversity and extensive diversity in genotype‐specific reaction norms in growth rates and cell size traits. Our results showed genotype by environment interactions, indicating that no genotype could outgrow all others across all temperature environments. Subsequently, we constructed a model to simulate the relative proportion of each genotype in a hypothetical population based on genotype and temperature‐specific population growth rates. This model was run with different seasonal temperature patterns. Our modeling exercise showed a succession of two to several genotypes becoming numerically dominant depending on the underlying temperature pattern. The results suggest that (temperature) context dependent fitness may contribute to the maintenance of genetic diversity in isolated populations of clonally reproducing microorganisms in temporally variable environments.     

VEGF-induced vascular permeability is mediated by FAK

Endothelial cells (ECs) form cell-cell adhesive junctional structures maintaining vascular integrity. This barrier is dynamically regulated by vascular endothelial growth factor (VEGF) receptor signaling. We created an inducible knockin mouse model to study the contribution of the integrin-associated focal adhesion tyrosine kinase (FAK) signaling on vascular function. Here we show that genetic or pharmacological FAK inhibition in ECs prevents VEGF-stimulated permeability downstream of VEGF receptor or Src tyrosine kinase activation in vivo. VEGF promotes tension-independent FAK activation, rapid FAK localization to cell-cell junctions, binding of the FAK FERM domain to the vascular endothelial cadherin (VE-cadherin) cytoplasmic tail, and direct FAK phosphorylation of β-catenin at tyrosine-142 (Y142) facilitating VE-cadherin-β-catenin dissociation and EC junctional breakdown. Kinase inhibited FAK is in a closed conformation that prevents VE-cadherin association and limits VEGF-stimulated β-catenin Y142 phosphorylation. Our studies establish a role for FAK as an essential signaling switch within ECs regulating adherens junction dynamics.     

Pongamia pinnata: An Untapped Resource for the Biofuels Industry of the Future

Pongamia pinnata (L.) Pierre is a fast-growing leguminous tree with the potential for high oil seed production and the added benefit of the ability to grow on marginal land. These properties support the suitability of this plant for large-scale vegetable oil production required by a sustainable biodiesel industry. The future success of P. pinnata as a sustainable source of feedstock for the biofuels industry is dependent on an extensive knowledge of the genetics, physiology and propagation of this legume. In particular, research should be targeted to maximizing plant growth as it relates to oil biosynthesis. This review assesses and integrates the biological, chemical and genetic attributes of the plant, providing the basis for future research into Pongamia’s role in an emerging industry.