Project description:Mammalian chromosomes are partitioned into topologically associating domains (TADs) by the loop-extrusion activity of cohesin that is blocked at specific DNA sites bound by CTCF. Chromosome structure inside TADs is highly variable in single cells, yet little is known about its temporal dynamics, how it influences the rates and durations of chromosomal contacts, and how it depends on CTCF and cohesin. To address these questions we combine two quantitative live-cell imaging strategies that minimize locus-specific confounding effects. We show that loop extrusion by cohesin globally reduces the mobility of the chromatin fiber in living cells, while also increasing the rates of formation and durations of contacts between sequences inside the same TAD. Quantitative analysis of high-resolution microscopy data reveals that contacts assemble and disassemble frequently in the course of the cell cycle, and become substantially more frequent and longer in the presence of convergent CTCF sites. Comparison with polymer modeling additionally reveals that cohesin-mediated CTCF loops last around 10 minutes on average. Our data support the notion that chromosome structure within TADs is highly dynamic and provide a quantitative framework for understanding the principles that link chromosome structure to biological function.
Project description:Mammalian chromosomes are partitioned into topologically associating domains (TADs) by the loop-extrusion activity of cohesin that is blocked at specific DNA sites bound by CTCF. Chromosome structure inside TADs is highly variable in single cells, yet little is known about its temporal dynamics, how it influences the rates and durations of chromosomal contacts, and how it depends on CTCF and cohesin. To address these questions we combine two quantitative live-cell imaging strategies that minimize locus-specific confounding effects. We show that loop extrusion by cohesin globally reduces the mobility of the chromatin fiber in living cells, while also increasing the rates of formation and durations of contacts between sequences inside the same TAD. Quantitative analysis of high-resolution microscopy data reveals that contacts assemble and disassemble frequently in the course of the cell cycle, and become substantially more frequent and longer in the presence of convergent CTCF sites. Comparison with polymer modeling additionally reveals that cohesin-mediated CTCF loops last around 10 minutes on average. Our data support the notion that chromosome structure within TADs is highly dynamic and provide a quantitative framework for understanding the principles that link chromosome structure to biological function.
Project description:Mammalian chromosomes are partitioned into topologically associating domains (TADs) by the loop-extrusion activity of cohesin that is blocked at specific DNA sites bound by CTCF. Chromosome structure inside TADs is highly variable in single cells, yet little is known about its temporal dynamics, how it influences the rates and durations of chromosomal contacts, and how it depends on CTCF and cohesin. To address these questions we combine two quantitative live-cell imaging strategies that minimize locus-specific confounding effects. We show that loop extrusion by cohesin globally reduces the mobility of the chromatin fiber in living cells, while also increasing the rates of formation and durations of contacts between sequences inside the same TAD. Quantitative analysis of high-resolution microscopy data reveals that contacts assemble and disassemble frequently in the course of the cell cycle, and become substantially more frequent and longer in the presence of convergent CTCF sites. Comparison with polymer modeling additionally reveals that cohesin-mediated CTCF loops last around 10 minutes on average. Our data support the notion that chromosome structure within TADs is highly dynamic and provide a quantitative framework for understanding the principles that link chromosome structure to biological function.
Project description:Mammalian chromosomes are partitioned into topologically associating domains (TADs) by the loop-extrusion activity of cohesin that is blocked at specific DNA sites bound by CTCF. Chromosome structure inside TADs is highly variable in single cells, yet little is known about its temporal dynamics, how it influences the rates and durations of chromosomal contacts, and how it depends on CTCF and cohesin. To address these questions we combine two quantitative live-cell imaging strategies that minimize locus-specific confounding effects. We show that loop extrusion by cohesin globally reduces the mobility of the chromatin fiber in living cells, while also increasing the rates of formation and durations of contacts between sequences inside the same TAD. Quantitative analysis of high-resolution microscopy data reveals that contacts assemble and disassemble frequently in the course of the cell cycle, and become substantially more frequent and longer in the presence of convergent CTCF sites. Comparison with polymer modeling additionally reveals that cohesin-mediated CTCF loops last around 10 minutes on average. Our data support the notion that chromosome structure within TADs is highly dynamic and provide a quantitative framework for understanding the principles that link chromosome structure to biological function.
Project description:Recent studies of genome-wide chromatin interactions have revealed that the human genome is partitioned into many self-associating topological domains. The boundary sequences are enriched for binding sites of CTCF and the cohesin complex, implicating these two factors in the establishment or maintenance of topological domains. To determine the role of cohesin and CTCF in higher order chromatin architecture in human cells, we proteolytically cleaved the cohesin complex from interphase chromatin and examined changes in chromosomal organization as well as transcriptome. We observed a general loss of local chromosomal interactions upon disruption of cohesin complex, but the topological domains remain intact. However, we found that depletion of CTCF by RNA interference in these cells not only reduced intra-domain interactions but also increased inter-domain interactions. Further more, distinct groups of genes become mis-regulated upon depletion of cohesin and CTCF. Taken together, these observations suggest that CTCF and cohesin contribute in different ways to chromatin organization and gene regulation. Hi-C and mRNA-seq experiments in Cohesin and CTCF depleted HEK293 cells
Project description:Developmental gene expression is often controlled by distal regulatory DNA elements called enhancers. Distant enhancer action is restricted to structural chromosomal domains that are flanked by CTCF-associated boundaries and formed through cohesin chromatin loop extrusion. To better understand how enhancers, genes and CTCF boundaries together form structural domains and control expression, we used a bottom-up approach, building series of active regulatory landscapes in inactive chromatin. We demonstrate here that gene transcription levels and activity over time reduce with increased enhancer distance. The enhancer recruits cohesin to stimulate domain formation and engage flanking CTCF sites in loop formation. It requires cohesin exclusively for the activation of distant genes, not of proximal genes, with nearby CTCF boundaries supporting efficient long-range enhancer action. Our work supports a dual activity model for enhancers: its classic role to stimulate transcription initiation and elongation from target gene promoters and a role to recruit cohesin for the creation of chromosomal domains, the engagement of CTCF sites in chromatin looping, and the activation of distal target genes.