It is becoming increasingly clear that the shape of the genome importantly influences transcription regulation. Pluripotent stem cells such as embryonic stem cells were recently shown to organize ...their chromosomes into topological domains that are largely invariant between cell types. Here we combine chromatin conformation capture technologies with chromatin factor binding data to demonstrate that inactive chromatin is unusually disorganized in pluripotent stem-cell nuclei. We show that gene promoters engage in contacts between topological domains in a largely tissue-independent manner, whereas enhancers have a more tissue-restricted interaction profile. Notably, genomic clusters of pluripotency factor binding sites find each other very efficiently, in a manner that is strictly pluripotent-stem-cell-specific, dependent on the presence of Oct4 and Nanog protein and inducible after artificial recruitment of Nanog to a selected chromosomal site. We conclude that pluripotent stem cells have a unique higher-order genome structure shaped by pluripotency factors. We speculate that this interactome enhances the robustness of the pluripotent state.
The spatial organization of chromosomes influences many nuclear processes including gene expression. The cohesin complex shapes the 3D genome by looping together CTCF sites along chromosomes. We show ...here that chromatin loop size can be increased and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. Cohesin’s DNA release factor WAPL restricts this loop extension and also prevents looping between incorrectly oriented CTCF sites. We reveal that the SCC2/SCC4 complex promotes the extension of chromatin loops and the formation of topologically associated domains (TADs). Our data support the model that cohesin structures chromosomes through the processive enlargement of loops and that TADs reflect polyclonal collections of loops in the making. Finally, we find that whereas cohesin promotes chromosomal looping, it rather limits nuclear compartmentalization. We conclude that the balanced activity of SCC2/SCC4 and WAPL enables cohesin to correctly structure chromosomes.
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•Hi-C analysis demonstrates that chromatin loop size can be increased genome-wide•The duration with which cohesin embraces DNA determines the length of chromatin loops•Haploid genetics reveals that the SCC2/SCC4 complex promotes loop extension•Cohesin limits the compartmentalization of chromatin within the nucleus
Cohesin's dynamic association with DNA determines the length of chromatin loops and allows this complex to correctly structure chromosomes.
Specialized adult somatic cells, such as cardiomyocytes (CMs), are highly differentiated with poor renewal capacity, an integral reason underlying organ failure in disease and aging. Among the least ...renewable cells in the human body, CMs renew approximately 1% annually. Consistent with poor CM turnover, heart failure is the leading cause of death. Here, we show that an active version of the Hippo pathway effector YAP, termed YAP5SA, partially reprograms adult mouse CMs to a more fetal and proliferative state. One week after induction, 19% of CMs that enter S-phase do so twice, CM number increases by 40%, and YAP5SA lineage CMs couple to pre-existing CMs. Genomic studies showed that YAP5SA increases chromatin accessibility and expression of fetal genes, partially reprogramming long-lived somatic cells in vivo to a primitive, fetal-like, and proliferative state.
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•Creation of a mouse conditionally expressing active YAP called YAP5SA•YAP5SA in adult cardiomyocytes (CMs) induces a more primitive transcriptional state•YAP5SA activates developmental enhancers•YAP5SA expression in CMs causes CM hyperplasia and overall heart hypercellularity
As highly differentiated cells, cardiomyocytes have poor renewal capacity, a contributing factor to heart failure in aging and disease. Monroe et al. created a mouse conditionally overexpressing active YAP (YAP5SA) and show that YAP5SA expression induces adult cardiomyocytes to adopt a more proliferative state with fetal-like chromatin and transcriptional landscapes.
CCCTC-binding factor (CTCF) is an architectural protein involved in the three-dimensional (3D) organization of chromatin. In this study, we assayed the 3D genomic contact profiles of a large number ...of CTCF binding sites with high-resolution 4C-seq. As recently reported, our data also suggest that chromatin loops preferentially form between CTCF binding sites oriented in a convergent manner. To directly test this, we used CRISPR/Cas9 genome editing to delete core CTCF binding sites in three loci, including the CTCF site in the Sox2 super-enhancer. In all instances, CTCF and cohesin recruitment were lost, and chromatin loops with distal, convergent CTCF sites were disrupted or destabilized. Re-insertion of oppositely oriented CTCF recognition sequences restored CTCF and cohesin recruitment, but did not re-establish chromatin loops. We conclude that CTCF binding polarity plays a functional role in the formation of higher-order chromatin structure.
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•CTCF binding polarity determines chromatin looping•Inverted or disengaged CTCF sites do not necessarily form new chromatin loops•Cohesin recruitment to CTCF sites is independent of loop formation•Phenocopied linear but altered 3D chromatin landscape can affect gene expression
CCCTC-binding factor (CTCF) shapes the three-dimensional genome. Here, de Wit et al. provide direct evidence for CTCF binding polarity playing an underlying role in chromatin looping. Cohesin association persists, but inverted CTCF sites fail to loop, which can sometimes lead to long-range changes in gene expression.
•4C-seq identifies chromatin loops, contact domains and inter-domain contacts.•A detailed protocol for preparation of 4C-seq template is provided.•A pipeline is provided which processes multiplexed ...4C-seq reads from FASTQ files.
Chromosome conformation capture (3C) methods measure DNA contact frequencies based on nuclear proximity ligation, to uncover in vivo genomic folding patterns. 4C–seq is a derivative 3C method, designed to search the genome for sequences contacting a selected genomic site of interest. 4C-seq employs inverse PCR and next generation sequencing to amplify, identify and quantify its proximity ligated DNA fragments. It generates high-resolution contact profiles for selected genomic sites based on limited amounts of sequencing reads. 4C-seq can be used to study multiple aspects of genome organization. It primarily serves to identify specific long-range DNA contacts between individual regulatory DNA modules, forming for example regulatory chromatin loops between enhancers and promoters, or architectural chromatin loops between cohesin- and CTCF- associated domain boundaries. Additionally, 4C-seq contact profiles can reveal the contours of contact domains and can identify the structural domains that co-occupy the same nuclear compartment. Here, we present an improved step-by-step protocol for sample preparation and the generation of 4C-seq sequencing libraries, including an optimized PCR and 4C template purification strategy. In addition, a data processing pipeline is provided which processes multiplexed 4C-seq reads directly from FASTQ files and generates files compatible with standard genome browsers for visualization and further statistical analysis of the data such as peak calling using peakC. The protocols and the pipeline presented should readily allow anyone to generate, visualize and interpret their own high resolution 4C contact datasets.
We present the experimental protocol and data analysis toolbox for multi-contact 4C (MC-4C), a new proximity ligation method tailored to study the higher-order chromatin contact patterns of selected ...genomic sites. Conventional chromatin conformation capture (3C) methods fragment proximity ligation products for efficient analysis of pairwise DNA contacts. By contrast, MC-4C is designed to preserve and collect large concatemers of proximity ligated fragments for long-molecule sequencing on an Oxford Nanopore or Pacific Biosciences platform. Each concatemer of proximity ligation products represents a snapshot topology of a different individual allele, revealing its multi-way chromatin interactions. By inverse PCR with primers specific for a fragment of interest (the viewpoint) and DNA size selection, sequencing is selectively targeted to thousands of different complex interactions containing this viewpoint. A tailored statistical analysis toolbox is able to generate background models and three-way interaction profiles from the same dataset. These profiles can be used to distinguish whether contacts between more than two regulatory sequences are mutually exclusive or, conversely, simultaneously occurring at chromatin hubs. The entire procedure can be completed in 2 w, and requires standard molecular biology and data analysis skills and equipment, plus access to a third-generation sequencing platform.
Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism in which specialized low-fidelity DNA polymerases bypass replication-blocking lesions, and it is usually associated with ...mutagenesis. In Saccharomyces cerevisiae a key event in TLS is the monoubiquitination of PCNA, which enables recruitment of the specialized polymerases to the damaged site through their ubiquitin-binding domain. In mammals, however, there is a debate on the requirement for ubiquitinated PCNA (PCNA-Ub) in TLS. We show that UV-induced Rpa foci, indicative of single-stranded DNA (ssDNA) regions caused by UV, accumulate faster and disappear more slowly in Pcna(K164R/K164R) cells, which are resistant to PCNA ubiquitination, compared to Pcna(+/+) cells, consistent with a TLS defect. Direct analysis of TLS in these cells, using gapped plasmids with site-specific lesions, showed that TLS is strongly reduced across UV lesions and the cisplatin-induced intrastrand GG crosslink. A similar effect was obtained in cells lacking Rad18, the E3 ubiquitin ligase which monoubiquitinates PCNA. Consistently, cells lacking Usp1, the enzyme that de-ubiquitinates PCNA exhibited increased TLS across a UV lesion and the cisplatin adduct. In contrast, cells lacking the Rad5-homologs Shprh and Hltf, which polyubiquitinate PCNA, exhibited normal TLS. Knocking down the expression of the TLS genes Rev3L, PolH, or Rev1 in Pcna(K164R/K164R) mouse embryo fibroblasts caused each an increased sensitivity to UV radiation, indicating the existence of TLS pathways that are independent of PCNA-Ub. Taken together these results indicate that PCNA-Ub is required for maximal TLS. However, TLS polymerases can be recruited to damaged DNA also in the absence of PCNA-Ub, and perform TLS, albeit at a significantly lower efficiency and altered mutagenic specificity.
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 of stimulating transcription initiation and elongation from target gene promoters and a role of recruiting cohesin for the creation of chromosomal domains, the engagement of CTCF sites in chromatin looping and the activation of distal target genes.
Detailed genomic contact maps have revealed that chromosomes are structurally organized in megabase-sized topologically associated domains (TADs) that encompass smaller subTADs. These domains ...segregate in the nuclear space to form active and inactive nuclear compartments, but cause and consequence of compartmentalization are largely unknown. Here, we combined lacO/lacR binding platforms with allele-specific 4C technologies to track their precise position in the three-dimensional genome upon recruitment of NANOG, SUV39H1, or EZH2. We observed locked genomic loci resistant to spatial repositioning and unlocked loci that could be repositioned to different nuclear subcompartments with distinct chromatin signatures. Focal protein recruitment caused the entire subTAD, but not surrounding regions, to engage in new genomic contacts. Compartment switching was found uncoupled from transcription changes, and the enzymatic modification of histones per se was insufficient for repositioning. Collectively, this suggests that trans-associated factors influence three-dimensional compartmentalization independent of their cis effect on local chromatin composition and activity.
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•4C technology reveals nuclear repositioning of genomic loci upon protein recruitment•Given subTAD can adopt multiple nuclear positions depending on the recruited factor•Switching between nuclear compartments can be uncoupled from transcriptional changes•Histone-modifying SUV39H1 relies on its chromodomain for locus repositioning
Wijchers et al. combine 4C technology with lacO/lacR binding platforms to show that a given genomic locus can adopt multiple nuclear positions depending on the recruited protein. Focal protein recruitment can move an entire subTAD, but repositioning appeared largely unrelated to changes in its chromatin composition and activity.
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