Limits to transcriptional silencing in Saccharomyces cerevisiae.

Mating-type switching in the budding yeast Saccharomyces cerevisiae relies on the Sir protein complex to silence HML and HMR, the two loci containing copies of the alleles of the mating type locus, MAT. Sir-based transcriptional silencing has been considered locus-specific, but the recent discovery of rare and transient escapes from silencing at HMLα2 with a sensitive assay called to question if these events extend to the whole locus. Adapting the same assay, we measured that transient silencing failures at HML were more frequent for the α2 gene than α1, similarly to their expression level in unsilenced cells. By coupling a mating assay, at HML we found that one of the two genes at that locus can be transiently expressed while the other gene is maintained silent. Thus, transient silencing loss can be a property of the gene rather than the locus. Cells lacking the SIR1 gene experience epigenetic bistability at HML and HMR. Our previous result led us to ask if HML could allow for two independent epigenetic states within the locus in a sir1Δ mutant. A simple construct using a double fluorescent reporter at HMLα1 and HMLα2 ruled out this possibility. Each HML locus displayed a single epigenetic state. We revisited the question of the correlation between the states of two HML loci in diploid cells, and showed they were independent. Finally, we determined the relative strength of gene repression achieved by Sir-based silencing with that achieved by the a1-α2 repressor.

Single molecule study of DNA collision with elliptical nanoposts conveyed by hydrodynamics.

Periodic arrays of micro- or nanopillars constitute solid-state matrices with excellent properties for DNA size separation. Nanofabrication technologies offer many solutions to tailor the geometry of obstacle arrays, yet most studies have been conducted with cylinders arranged in hexagonal lattices. In this report, we investigate the dynamics of single DNA collision with elliptical nanoposts using hydrodynamic actuation. Our data show that the asymmetry of the obstacles has minor effect on unhooking dynamics, and thus confirm recent predictions obtained by Brownian dynamics simulations. In addition, we show that the disengagement dynamics are correctly predicted by models of electrophoresis, and propose that this consistency is associated to the confinement in slit-like channels. We finally conclude that elliptical posts are expected to marginally improve the performances of separation devices.