An RNA aptamer to HP1/Swi6 facilitates heterochromatin formation at an ectopic locus in S.pombe.

In the fission yeast Schizosaccharomyces pombe (S.pombe), heterochromatin domains are established and maintained by protein complexes that contain numerous RNA binding domains among their components. The fission yeast HP1 protein Swi6 is one such component and contains an unstructured RNA-binding hinge, which is important for the integrity and silencing of heterochromatin. In this study, we have used an RNA aptamer that likely binds to the Swi6 hinge with high affinity, as a tool to perturb the natural interactions mediated by this domain. When the hinge is blocked by the aptamer RNA, Swi6 appears to become less restricted to the pericentromeres and is enriched at specific euchromatic loci. This suggests a role for the Swi6 hinge, along with the chromoshadow domain (previously shown) in controlling the spread of heterochromatin in S.pombe. The study also highlights the potential of using a synthetic aptamer RNA as a tool to perturb nucleic acid - protein interaction in vivo with the objective of understanding the functional relevance of such an interaction.

A Novel Method to Generate MNase Ladders Reveal Rapid Chromatin Remodeling upon Gametogenesis and Mating in Chlamydomonas.

To circumvent nuclei isolation for nucleosomal mapping of wild-type (cell walled) algal cells, we developed a quick and versatile methodology, by abrasion of whole cells (Chlamydomonas, Scenedesmus and yeast), allowing Micrococcal Nuclease (MNase) direct access to nuclear chromatin, in situ. Varying parameters such as bead abrasion, vortex and incubation conditions, we optimized capture of an 'early digest' which may probe chromatin differentially, based on nucleosome accessibility. A comparison of such ladders across vegetative cells, gametes and zygotes revealed an increase in the average nucleosomal repeat length (+17-34nt) upon gametogenesis, indicating a trend of chromatin compaction. Using PCR, we compared promoter enrichment in increasing orders of fractionated nucleosomal repeats (mono-, di-, up to penta-), each differing in cleavability based on chromatin accessibility. Concordant with higher gene expression (mating locus), promoters revealed an enrichment in mono-nucleosomal fractions. Interestingly, the zygote specific gene, MT0828 displayed rapid remodelling from penta-nucleosomal enrichment when completely repressed (vegetative), to intermediate states during gametogenesis (24hrs), which finally shifted to being largely mono-nucleosomal, when induced (1h zygotes). Summarizing three candidate genes from the mating locus, we conclude that the MNase based 'Chromatin Accessibility Assay' can track a range of large-scale rapid chromatin remodelling transitions within the binaries of gene expression.

Predictive Modeling of a Batch Filter Mating Process.

Quantitative characterizations of horizontal gene transfer are needed to accurately describe gene transfer processes in natural and engineered systems. A number of approaches to the quantitative description of plasmid conjugation have appeared in the literature. In this study, we seek to extend that work, motivated by the question of whether a mathematical model can accurately predict growth and conjugation dynamics in a batch process. We used flow cytometry to make time-point observations of a filter-associated mating between two E. coli strains, and fit ordinary differential equation models to the data. A model comparison analysis identified the model formulation that is best supported by the data. Identifiability analysis revealed that the parameters were estimated with acceptable accuracy. The predictive power of the model was assessed by comparison with test data that demanded extrapolation from the training experiments. This study represents the first attempt to assess the quality of model predictions for plasmid conjugation. Our successful application of this approach lays a foundation for predictive modeling that can be used both in the study of natural plasmid transmission and in model-based design of engineering approaches that employ conjugation, such as plasmid-mediated bioaugmentation.