The human genome folds to create thousands of intervals, called "contact domains," that exhibit enhanced contact frequency within themselves. "Loop domains" form because of tethering between two loci-almost always bound by CTCF and cohesin-lying on the same chromosome. "Compartment domains" form when genomic intervals with similar histone marks co-segregate. Here, we explore the effects of degrading cohesin. All loop domains are eliminated, but neither compartment domains nor histone marks are affected. Loss of loop domains does not lead to widespread ectopic gene activation but does affect a significant minority of active genes. In particular, cohesin loss causes superenhancers to co-localize, forming hundreds of links within and across chromosomes and affecting the regulation of nearby genes. We then restore cohesin and monitor the re-formation of each loop. Although re-formation rates vary greatly, many megabase-sized loops recovered in under an hour, consistent with a model where loop extrusion is rapid.

Cohesin Loss Eliminates All Loop Domains

The three-dimensional structure of chromosomes plays an important role in gene expression regulation and also influences the repair of radiation-induced DNA damage. Genomic aberrations that disrupt chromosome spatial domains can lead to diseases including cancer, but how the 3D genome structure responds to DNA damage is poorly understood. Here, we investigate the impact of DNA damage response and repair on 3D genome folding using Hi-C experiments on wild type cells and ataxia telangiectasia mutated (ATM) patient cells. We irradiate fibroblasts, lymphoblasts, and ATM-deficient fibroblasts with 5 Gy X-rays and perform Hi-C at 30 minutes, 24 hours, or 5 days after irradiation. We observe that 3D genome changes after irradiation are cell type-specific, with lymphoblastoid cells generally showing more contact changes than irradiated fibroblasts. However, all tested repair-proficient cell types exhibit an increased segregation of topologically associating domains (TADs). This TAD boundary strengthening after irradiation is not observed in ATM deficient fibroblasts and may indicate the presence of a mechanism to protect 3D genome structure integrity during DNA damage repair.

/pdf/radiation-induced-dna-damage-and-repair-effects-on-3d-genome-1szkpne2wd.pdf

Radiation-induced DNA damage and repair effects on 3D genome organization.

Summary The spatial organization of chromosomes by structural maintenance of chromosomes (SMC) complexes is vital to organisms from bacteria to humans 1,2. SMC complexes were recently found to be motors that extrude DNA loops 3–11. It remains unclear, however, what happens when multiple SMC complexes encounter one another in vivo on the same DNA, how encounters are resolved, or how interactions help organize an active genome 12. Here, we set up a “crash-course track” system to study what happens when SMC complexes encounter one another. Using the parS/ParB system, which loads SMC complexes in a targeted manner13–17, we engineered the Bacillus subtilis chromosome to have multiple SMC loading sites. Chromosome conformation capture (Hi-C) analyses of over 20 engineered strains show an amazing variety of never-before-seen chromosome folding patterns. Polymer simulations indicate these patterns require SMC complexes to traverse past each other in vivo, contrary to the common assumption that SMC complexes mutually block each other’s extrusion activity 18. Our quantitative model of bypassing predicted that increasing the numbers of SMCs on the chromosome could overwhelm the bypassing mechanism, create SMC traffic jams, and lead to major chromosome reorganization. We validated these predictions experimentally. We posit that SMC complexes traversing one another is part of a larger phenomenon of bypassing large steric barriers which enables these loop extruders to spatially organize a functional and busy genome.

/pdf/dna-loop-extruding-smc-complexes-can-traverse-one-another-in-256yvtvth5.pdf

DNA-loop extruding SMC complexes can traverse one another in vivo

One of the hallmarks of DNA damage is the rapid spreading of phosphorylated histone H2A (γ-H2AX) around a DNA double-strand break (DSB) In the budding yeast Saccharomyces cerevisiae, nearly all H2A isoforms can be phosphorylated, either by Mec1ATR or Tel1ATM checkpoint kinases We induced a site-specific DSB with HO endonuclease at the MAT locus on chromosome III and monitored the formation of γ-H2AX by chromatin immunoprecipitation (ChIP)-qPCR in order to uncover the mechanisms by which Mec1ATR and Tel1ATM propagate histone modifications across chromatin With either kinase, γ-H2AX spreads as far as ∼50 kb on both sides of the lesion within 1 h; but the kinetics and distribution of modification around the DSB are significantly different The total accumulation of phosphorylation is reduced by about half when either of the two H2A genes is mutated to the nonphosphorylatable S129A allele Mec1 activity is limited by the abundance of its ATRIP partner, Ddc2 Moreover, Mec1 is more efficient than Tel1 at phosphorylating chromatin in trans-at distant undamaged sites that are brought into physical proximity to the DSB We compared experimental data to mathematical models of spreading mechanisms to determine whether the kinases search for target nucleosomes by primarily moving in three dimensions through the nucleoplasm or in one dimension along the chromatin Bayesian model selection indicates that Mec1 primarily uses a three-dimensional diffusive mechanism, whereas Tel1 undergoes directed motion along the chromatin

https://www.pnas.org/content/pnas/early/2020/08/14/2002126117.full.pdf

Yeast ATM and ATR kinases use different mechanisms to spread histone H2A phosphorylation around a DNA double-strand break.

Chromatin integrity is key for cell homeostasis and for preventing pathological development. Alterations in core chromatin components, histone proteins, recently came into the spotlight through the discovery of their driving role in cancer. Building on these findings, in this review, we discuss how histone variants and their associated chaperones safeguard genome stability and protect against tumorigenesis. Accumulating evidence supports the contribution of histone variants and their chaperones to the maintenance of chromosomal integrity and to various steps of the DNA damage response, including damaged chromatin dynamics, DNA damage repair, and damage-dependent transcription regulation. We present our current knowledge on these topics and review recent advances in deciphering how alterations in histone variant sequence, expression, and deposition into chromatin fuel oncogenic transformation by impacting cell proliferation and cell fate transitions. We also highlight open questions and upcoming challenges in this rapidly growing field.

https://www.mdpi.com/2073-4409/9/11/2424/pdf

Histone Variants: Guardians of Genome Integrity.

DNA Double-Strand Breaks (DSBs) repair is essential to safeguard genome integrity. Upon DSBs, the ATM PI3K kinase rapidly triggers the establishment of megabase-sized, γH2AX-decorated chromatin domains which further act as seeds for the formation of DNA Damage Response (DDR) foci1. How these foci are rapidly assembled in order to establish a “repair-prone” environment within the nucleus is yet unclear. Topologically Associating Domains (TADs) are a key feature of 3D genome organization that regulate transcription and replication, but little is known about their contribution to DNA repair processes2,3. Here we found that TADs are functional units of the DDR, instrumental for the correct establishment of γH2AX/53BP1 chromatin domains in a manner that involves one-sided cohesin-mediated loop extrusion on both sides of the DSB. We propose a model whereby H2AX-containing nucleosomes are rapidly phosphorylated as they actively pass by DSB-anchored cohesin. Our work highlights the critical impact of chromosome conformation in the maintenance of genome integrity and provides the first example of a chromatin modification established by loop extrusion.

/pdf/loop-extrusion-as-a-mechanism-for-dna-double-strand-breaks-4eolfcth2d.pdf

Loop extrusion as a mechanism for DNA Double-Strand Breaks repair foci formation

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Loop extrusion as a mechanism for DNA Double-Strand Breaks repair foci formation