Kerklingh, E. | bioRxiv Science

bioRxiv2021

Dynamic epistasis analysis reveals how chromatin remodeling regulates transcriptional bursting

Transcriptional bursting has been linked to the stochastic positioning of nucleosomes. However, how bursting is regulated by remodeling of promoter nucleosomes is unknown. Here, we use single-molecule live-cell imaging of GAL10 transcription in budding yeast to measure how transcriptional bursting changes upon single and double perturbations of chromatin remodeling factors, the transcription factor Gal4 and preinitiation complex (PIC) components. Using dynamic epistasis analysis, we reveal how remodeling of different nucleosomes regulates individual transcriptional bursting parameters. At the nucleosome covering the Gal4 binding sites, RSC acts synergistically with Gal4 binding to facilitate each burst. Conversely, nucleosome remodeling at the TATA box controls only the first burst upon galactose induction. In the absence of remodelers, nucleosomes at canonical TATA boxes are displaced by TBP binding to allow for transcription activation. Overall, our results reveal how promoter nucleosome remodeling, together with transcription factor and PIC binding regulates the kinetics of transcriptional bursting.

molecular biology↗

bioRxiv2019

Synaptotagmin-1 and Doc2b exhibit distinct membrane remodeling mechanisms

While the role of Synaptotagmin-1 in living cells has been described in detail, it remains a challenge to dissect the contribution of membrane remodelling by its two cytoplasmic C2 domains (C2AB) to the Ca2+-secretion coupling mechanism. Here, we study membrane remodeling using pairs of optically-trapped beads coated with SNARE-free synthetic membranes. We find that the soluble C2AB domain of Syt1 strongly affects the probability and strength of membrane-membrane interactions in a strictly Ca2+- and protein-dependent manner. A lipid mixing assay with confocal imaging reveals that at low Syt1 concentrations, no hemifusion is observed. Notably, for similar low concentrations of Doc2b hemifusion does occur. Consistently, both C2AB fragments cause a reduction in the membrane bending modulus, as measured by an AFM-based method. This lowering of the energy required for membrane deformation likely contributes to the overall Ca2+-secretion triggering mechanism by calcium sensor proteins. When comparing symmetrical (both sides) and asymmetrical (one side) presence of protein on the membranes, Syt1 favors an asymmetrical but Doc2b a symmetrical configuration, as inferred from higher tether probabilities and break forces. This provides support for the direct bridging hypothesis for Syt-1, while hinting to possible preference for protein-protein (and not protein-membrane) interactions for Doc2b. Overall, our study sheds new light on the mechanism of Ca2+ induced fusion triggering, which is essential for fundamental understanding of secretion of neurotransmitters and endocrine substances.

biophysics↗