Project description:The chromatin fiber folds into loops, however the mechanisms controlling loop extrusion are still poorly understood. Using super-resolution microscopy, we visualized that loops in intact nuclei are formed by a scaffold of cohesin complexes from which the DNA protrudes. RNA polymerase II decorates the top of the loops and is physically segregated from cohesin. Augmented looping upon increased loading of cohesin on chromosomes causes disruption of Lamin at the nuclear rim and chromatin blending, a homogenous distribution of chromatin within the nucleus. Altering supercoiling via either transcription or topoisomerase inhibition counteracts chromatin blending, increases chromatin condensation, disrupts loop formation and leads to altered cohesin distribution and mobility on chromatin. Overall, negative supercoiling generated by transcription is an important regulator of loop formation in vivo.
Project description:The chromatin fiber folds into loops, however the mechanisms controlling loop extrusion are still poorly understood. Using super-resolution microscopy, we visualized that loops in intact nuclei are formed by a scaffold of cohesin complexes from which the DNA protrudes. RNA polymerase II decorates the top of the loops and is physically segregated from cohesin. Augmented looping upon increased loading of cohesin on chromosomes causes disruption of Lamin at the nuclear rim and chromatin blending, a homogenous distribution of chromatin within the nucleus. Altering supercoiling via either transcription or topoisomerase inhibition counteracts chromatin blending, increases chromatin condensation, disrupts loop formation and leads to altered cohesin distribution and mobility on chromatin. Overall, negative supercoiling generated by transcription is an important regulator of loop formation in vivo.
Project description:DNA supercoiling is essential for life because it controls critical processes, including transcription, replication and recombination. Current methods to measure DNA supercoiling in vivo are laborious and unable to examine single cells. Here we report a method for high-throughput measurement of bacterial DNA supercoiling in vivo. Fluorescent Evaluation of DNA Supercoiling (FEDS) utilizes a plasmid harboring the gene for a green fluorescent protein transcribed by a discovered promoter that responds exclusively to DNA supercoiling, and the gene for a red fluorescent protein transcribed by a constitutive promoter as internal standard. Using FEDS, we uncovered single cell heterogeneity in DNA supercoiling and established that, surprisingly, population-level decreases in DNA supercoiling result from a low mean/high variance DNA supercoiling subpopulation, rather than a homogeneous shift in supercoiling of the whole population. In addition, we identified a regulatory loop in which a gene that decreases DNA supercoiling is transcriptionally repressed when DNA supercoiling increases.
Project description:Determining the conformation of chromatin in cells at the nucleosome level and its relationship to cellular processes has been a central challenge in biology. We show that in quiescent yeast, widespread transcriptional repression coincides with the local compaction of chromatin fibers into structures that are less condensed and more heteromorphic than canonical 30-nanometer forms. Acetylation or substitution of H4 tail residues decompacts fibers and leads to global transcriptional de-repression. Fiber decompaction also increases the rate of loop extrusion by condensin. These findings establish a role for H4 tail-dependent local chromatin fiber folding in regulating transcription and loop extrusion in cells. They also demonstrate the physiological relevance of canonical chromatin fiber folding mechanisms even in the absence of regular 30-nanometer structures.
Project description:Determining the conformation of chromatin in cells at the nucleosome level and its relationship to cellular processes has been a central challenge in biology. We show that in quiescent yeast, widespread transcriptional repression coincides with the local compaction of chromatin fibers into structures that are less condensed and more heteromorphic than canonical 30-nanometer forms. Acetylation or substitution of H4 tail residues decompacts fibers and leads to global transcriptional de-repression. Fiber decompaction also increases the rate of loop extrusion by condensin. These findings establish a role for H4 tail-dependent local chromatin fiber folding in regulating transcription and loop extrusion in cells. They also demonstrate the physiological relevance of canonical chromatin fiber folding mechanisms even in the absence of regular 30-nanometer structures.