Loops and the activity of loop extrusion factors constrain chromatin dynamics

Abstract

The chromosomes - DNA polymers and their binding proteins - are compacted into a spatially organized, yet dynamic, three-dimensional structure. Recent genome-wide chromatin conformation capture experiments reveal a hierarchical organization of the DNA structure that is imposed, at least in part, by looping interactions arising from the activity of loop extrusion factors. The dynamics of chromatin reflects the response of the polymer to a combination of thermal fluctuations and active processes. However, how chromosome structure and enzymes acting on chromatin together define its dynamics remains poorly understood. To gain insight into the structure-dynamics relationship of chromatin, we combine high-precision microscopy in living Schizosaccharomyces pombe cells with systematic genetic perturbations and Rouse-model polymer simulations. We first investigated how the activity of two loop extrusion factors, the cohesin and condensin complexes, influences chromatin dynamics. We observed that deactivating cohesin, or to a lesser extent condensin, increased chromatin mobility, suggesting that loop extrusion constrains rather than agitates chromatin motion. Our corresponding simulations reveal that the introduction of loops is sufficient to explain the constraining activity of loop extrusion factors, highlighting that the conformation adopted by the polymer plays a key role in defining its dynamics. Moreover, we find that the number loops or residence times of loop extrusion factors influences the dynamic behavior of the chromatin polymer. Last, we observe that the activity of the INO80 chromatin remodeler, but not the SWI/SNF or RSC complexes, is critical for ATP-dependent chromatin mobility in fission yeast. Taken together we suggest that thermal and INO80-dependent activities exert forces that drive chromatin fluctuations, which are constrained by the organization of the chromosome into loops.

A Scaffolding Element Rewires Local 3D Chromatin Architecture During Differentiation

“…Mechanistically, element A scaffolds the region through its frequent contact with the promoter which constrains the movement of adjacent chromatin regions and confines them to a closer space, thereby favouring the elements in the vicinity of A and the promoter to contact one another more frequently. Upon deletion of element A, chromatin becomes less constrained, in agreement with our cHi-C, 3D-FISH data and with recent simulations 48 . However, despite its enhancer function in cell culture assays, the deletion of element A did not cause significant aberrations to Zfp608 expression.…”

A quantitative ultrastructural timeline of nuclear autophagy reveals a role for dynamin-like protein 1 at the nuclear envelope

“…Cells (PMCPL940, PMCPL857, PMCPL831, PMCPL1319, PMCPL1087) were incubated in sd-N media for three hours before being prepared for imaging on the LLSM. Cells where a Atg39-GFP focus appeared that underwent nucleophagy were selected for further processing using MATLAB scripts to detect Atg39-GFP foci (spots) as described in “Determining Atg39-GFP copy number.” For particle tracking and colocalization analysis the spots were organized into trajectories using previously described MATLAB implementation 58 of the widely-used tracking algorithm ‘track.m’ 59 . Trajectories of a single Atg39-GFP spot that were erroneously split in two (when displacement between two frames exceeded a maximum linking distance) were linked together manually.…”

“…To facilitate monitoring the delivery of Atg39-GFP into vacuoles, we performed these experiments in cells expressing Vph1-mCherry to visualize vacuolar membranes. We measured the intensity of Atg39-GFP foci and used commercial (Imaris) and our own particle tracking algorithms [57][58][59] to quantitatively evaluate their initial appearance at the NE, their rate of growth, and their delivery to the vacuole -all of which occurred at a rate of 1.4 events/cell/hour (Movie 1).…”