Exploiting native forces to capture chromosome conformation in mammalian cell nuclei

Mammalian cells contain the same 2 m-long DNA molecule, but different cell fates imply different three-dimensional genome organization that is tightly linked to gene expression and other cellular functions. This can range from shorter-range chromatin looping between genomic elements such as promoters and enhancers to higher-order folding of whole chromosomes within the confines of cell nuclei. In the past two decades, our understanding of this complex genome architecture and its underlying functions has been advanced through microscopy studies and chromosome conformation capture (3C) techniques. Fixation of the spatial chromatin network relies on cell cross-linking using formaldehyde and is a major experimental component of most molecular biological methods, including 3C-based approaches. However, this step remains a ‘black box’ since it may introduce biases that may skew the resulting data. In fact, such skewing between microscopy and 3C-derived data was recently reported. Here, we address these concerns and provide a novel intrinsic chromosome conformation capture assay (i3C), which allows determination of spatial chromatin interactions without the need for cell cross-linking. We handle mammalian cell nuclei in a close-to-physiological buffer that exploits native forces, such as transcription, to preserve nuclear chromatin folding. We introduce different variations of our intrinsic method that enable us to investigate locus-specific to genome-wide interactions between DNA segments. Using distinct cell lines and chromatin characteristics of the examined loci, we show that most of the native contact signals resemble cross-linked ones and reside within the constraints of topological associated domains (TADs). However, i3C-based tools increase the signal-to-noise ratio and enhance precision of contact determination at regulatory elements and CTCF sites. Moreover, we observed differential contacts by intrinsic techniques and developed an orthogonal validation method called ‘TALE-iD’. Finally, we utilized extracellular signaling by the tumor necrosis factor-α to track pre-established and dynamic chromatin looping. Our findings suggest that cross-linking of chromatin may mask such fine-tuned and temporal-specific dynamics of three-dimensional genome organization. Taken together, intrinsic chromosome conformation capture assays improve our understanding of the true nature of chromatin folding stabilization and shed light on the relevance of native forces on nuclear architecture

Exploiting native forces to capture chromosome conformation in mammalian cell nuclei

Mammalian interphase chromosomes fold into a multitude of loops to fit the confines of cell nuclei, and looping is tightly linked to regulated function. Chromosome conformation capture (3C) technology has significantly advanced our understanding of this structure-to-function relationship. However, all 3C-based methods rely on chemical cross-linking to stabilize spatial interactions. This step remains a “black box” as regards the biases it may introduce, and some discrepancies between microscopy and 3C studies have now been reported. To address these concerns, we developed “i3C”

DNA methylation changes during long-term in vitro cell culture are caused by epigenetic drift

Culture expansion of primary cells evokes highly reproducible DNA methylation (DNAm) changes. We have identified CG dinucleotides (CpGs) that become continuously hyper- or hypomethylated during long-term culture of mesenchymal stem cells (MSCs) and other cell types. Bisulfite barcoded amplicon sequencing (BBA-seq) demonstrated that DNAm patterns of neighboring CpGs become more complex without evidence of continuous pattern development and without association to oligoclonal subpopulations. Circularized chromatin conformation capture (4C) revealed reproducible changes in nuclear organization between early and late passages, while there was no enriched interaction with other genomic regions that also harbor culture-associated DNAm changes. Chromatin immunoprecipitation of CTCF did not show significant differences during long-term culture of MSCs, however culture-associated hypermethylation was enriched at CTCF binding sites and hypomethylated CpGs were devoid of CTCF. Taken together, our results support the notion that DNAm changes during culture-expansion are not directly regulated by a targeted mechanism but rather resemble epigenetic drift

Distinct IL-1α-responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

How cytokine-driven changes in chromatin topology are converted into gene regulatory circuits during inflammation still remains unclear. Here, we show that interleukin (IL)-1α induces acute and widespread changes in chromatin accessibility via the TAK1 kinase and NF-κB at regions that are highly enriched for inflammatory disease-relevant SNPs. Two enhancers in the extended chemokine locus on human chromosome 4 regulate the IL-1α-inducible IL8 and CXCL1-3 genes. Both enhancers engage in dynamic spatial interactions with gene promoters in an IL-1α/TAK1-inducible manner. Microdeletions of p65-binding sites in either of the two enhancers impair NF-κB recruitment, suppress activation and biallelic transcription of the IL8/CXCL2 genes, and reshuffle higher-order chromatin interactions as judged by i4C interactome profiles. Notably, these findings support a dominant role of the IL8 “master” enhancer in the regulation of sustained IL-1α signaling, as well as for IL-8 and IL-6 secretion. CRISPR-guided transactivation of the IL8 locus or cross-TAD regulation by TNFα-responsive enhancers in a different model locus supports the existence of complex enhancer hierarchies in response to cytokine stimulation that prime and orchestrate proinflammatory chromatin responses downstream of NF-κB

Contribution of 3D Chromatin Architecture to the Maintenance of Pluripotency

Maintenance of pluripotency, lineage commitment and differentiation of mammalian embryonic stem cells into all somatic cell types involves differential regulation of different subsets of genes, as does reprogramming of somatic cells back into a pluripotent state. It is now understood that the three-dimensional organization of the human genome asserts a key role in these processes in two ways. First, by providing a largely invariable scaffold onto which dynamic changes in chromatin may manifest; second, by allowing the spatial clustering of genes contributing to the same functional pathways. In this review, we discuss the rapidly growing volume of literature on the structure-to-function relationship of mammalian genomes as regards key developmental transitions of stem cell populations

Binding of nuclear factor kappa B to noncanonical consensus sites reveals its multimodal role during the early inflammatory response

Mammalian cells have developed intricate mechanisms to interpret, integrate, and respond to extracellular stimuli. For example, tumor necrosis factor (TNF) rapidly activates proinflammatory genes, but our understanding of how this occurs against the ongoing transcriptional program of the cell is far from complete. Here, we monitor the early phase of this cascade at high spatiotemporal resolution in TNF-stimulated human endothelial cells. NF-kappa B, the transcription factor complex driving the response, interferes with the regulatory machinery by binding active enhancers already in interaction with gene promoters. Notably, >50% of these enhancers do not encode canonical NF-kappa B binding motifs. Using a combination of genomics tools, we find that binding site selection plays a key role in NF-kappa B-mediated transcriptional activation and repression. We demonstrate the latter by describing the synergy between NF-kappa B and the corepressor JDP2. Finally, detailed analysis of a 2.8-Mbp locus using sub-kbp-resolution targeted chromatin conformation capture and genome editing uncovers how NF-kappa B that has just entered the nucleus exploits pre-existing chromatin looping to exert its multimodal role. This work highlights the involvement of topology in cis-regulatory element function during acute transcriptional responses, where primary DNA sequence and its higher-order structure constitute a regulatory context leading to either gene activation or repression

HMGB2 Loss upon Senescence Entry Disrupts Genomic Organization and Induces CTCF Clustering across Cell Types

Processes like cellular senescence are characterized by complex events giving rise to heterogeneous cell populations. However, the early molecular events driving this cascade remain elusive. We hypothesized that senescence entry is triggered by an early disruption of the cells' three-dimensional (3D) genome organization. To test this, we combined Hi-C, single-cell and population transcriptomics, imaging, and in silico modeling of three distinct cells types entering senescence. Genes involved in DNA conformation maintenance are suppressed upon senescence entry across all cell types. We show that nuclear depletion of the abundant HMGB2 protein occurs early on the path to senescence and coincides with the dramatic spatial clustering of CTCF. Knocking down HMGB2 suffices for senescence-induced CTCF clustering and for loop reshuffling, while ectopically expressing HMGB2 rescues these effects. Our data suggest that HMGB2-mediated genomic reorganization constitutes a primer for the ensuing senescent program

Distinct IL-1 alpha-responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

How cytokine-driven changes in chromatin topology are converted into gene regulatory circuits during inflammation still remains unclear. Here, we show that interleukin (IL)-1 alpha induces acute and widespread changes in chromatin accessibility via the TAK1 kinase and NF-kappa B at regions that are highly enriched for inflammatory disease-relevant SNPs. Two enhancers in the extended chemokine locus on human chromosome 4 regulate the IL-1 alpha-inducible IL8 and CXCL1-3 genes. Both enhancers engage in dynamic spatial interactions with gene promoters in an IL-1 alpha/TAK1-inducible manner. Microdeletions of p65-binding sites in either of the two enhancers impair NF-kappa B recruitment, suppress activation and biallelic transcription of the IL8/CXCL2 genes, and reshuffle higher-order chromatin interactions as judged by i4C interactome profiles. Notably, these findings support a dominant role of the IL8 master enhancer in the regulation of sustained IL-1 alpha signaling, as well as for IL-8 and IL-6 secretion. CRISPR-guided transactivation of the IL8 locus or cross-TAD regulation by TNF alpha-responsive enhancers in a different model locus supports the existence of complex enhancer hierarchies in response to cytokine stimulation that prime and orchestrate proinflammatory chromatin responses downstream of NF-kappa B