Variant Histone H2afv reprograms DNA methylation during early zebrafish development.

The DNA methylome is re-patterned during discrete phases of vertebrate development. In zebrafish, there are 2 waves of global DNA demethylation and re-methylation: the first occurs before gastrulation when the parental methylome is changed to the zygotic pattern and the second occurs after formation of the embryonic body axis, during organ specification. The occupancy of the histone variant H2A.Z and regions of DNA methylation are generally anti-correlated, and it has been proposed that H2A.Z restricts the boundaries of highly methylated regions. While many studies have described the dynamics of methylome changes during early zebrafish development, the factors involved in establishing the DNA methylation landscape in zebrafish embryos have not been identified. We test the hypothesis that the zebrafish ortholog of H2A.Z (H2afv) restricts DNA methylation during development. We find that, in control embryos, bulk genome methylation decreases after gastrulation, with a nadir at the bud stage, and peaks during mid-somitogenesis; by 24 hours post -fertilization, total DNA methylation levels return to those detected in gastrula. Early zebrafish embryos depleted of H2afv have significantly more bulk DNA methylation during somitogenesis, suggesting that H2afv limits methylation during this stage of development. H2afv deficient embryos are small, with multisystemic abnormalities. Genetic interaction experiments demonstrate that these phenotypes are suppressed by depletion of DNA methyltransferase 1 (Dnmt1). This work demonstrates that H2afv is essential for global DNA methylation reprogramming during early vertebrate development and that embryonic development requires crosstalk between H2afv and Dnmt1.

Non-Canonical Wnt Predominates in Activated Rat Hepatic Stellate Cells, Influencing HSC Survival and Paracrine Stimulation of Kupffer Cells.

The Wnt system is highly complex and is comprised of canonical and non-canonical pathways leading to the activation of gene expression. Our aim was to examine changes in the expression of Wnt ligands and regulators during hepatic stellate cell (HSC) transdifferentiation and assess the relative contributions of the canonical and non-canonical Wnt pathways in fibrogenic activated HSC. The expression profile of Wnt ligands and regulators in HSC was not supportive for a major role for ß-catenin-dependent canonical Wnt signalling, this verified by inability to induce Topflash reporter activity in HSC even when expressing a constitutive active ß-catenin. We detected expression of Wnt5a in activated HSC which can signal via non-canonical mechanisms and showed evidence for non-canonical signalling in these cells involving phosphorylation of Dvl2 and pJNK. Stimulation of HSC or Kupffer cells with Wnt5a regulated HSC apoptosis and expression of TGF-ß1 and MCP1 respectively. We were unable to confirm a role for ß-catenin-dependent canonical Wnt in HSC and instead propose autocrine and paracrine functions for Wnts expressed by activated HSC via non-canonical pathways. The data warrant detailed investigation of Wnt5a in liver fibrosis.

Diffraction-based automated crystal centering.

A fully automated procedure for detecting and centering protein crystals in the X-ray beam of a macromolecular crystallography beamline has been developed. A cryo-loop centering routine that analyzes video images with an edge detection algorithm is first used to determine the dimensions of the loop holding the sample; then low-dose X-rays are used to record diffraction images in a grid over the edge and face plane of the loop. A three-dimensional profile of the crystal based on the number of diffraction spots in each image is constructed. The derived center of mass is then used to align the crystal to the X-ray beam. Typical samples can be accurately aligned in approximately 2-3 min. Because the procedure is based on the number of ;good' spots as determined by the program Spotfinder, the best diffracting part of the crystal is aligned to the X-ray beam.