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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.Julia Franzen et al. investigate if changes in DNA methylation at specific genetic loci during cell culture expansion are due to a specific mechanism or gradual deregulation of an epigenetic state. Their results suggest that changes in CpG methylation are due to indirect epigenetic drift, rather than a consequence of targeting by DNA methyltransferases.

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”, a novel approach for capturing spatial interactions without a need for cross‐linking. We apply i3C to intact nuclei of living cells and exploit native forces that stabilize chromatin folding. Using different cell types and loci, computational modeling, and a methylation‐based orthogonal validation method, “TALE‐iD”, we show that native interactions resemble cross‐linked ones, but display improved signal‐to‐noise ratios and are more focal on regulatory elements and CTCF sites, while strictly abiding to topologically associating domain restrictions. Synopsis i3C captures chromatin folding in intact nuclei without a need for cross‐linking and reveals that native interactions resemble cross‐linked ones, yet they display improved signal‐to‐noise ratios and highlight how cis ‐elements and TADs contribute to 3D genomic organization. i3C addresses the issue of potential biases introduced in 3C‐based studies by formaldehyde cross‐linking and harsh treatments. This protocol is robust, sensitive, and significantly faster than the conventional approach, and relies on native forces to preserve spatial interactions in uncross‐linked nuclei. Overall, i3C interaction profiles resemble conventional ones and highlight the contribution of both active and inactive regulatory regions in chromatin looping, as well as that of the restrictions imposed by topologically associating domain boundaries. i3C complements the existing toolkit and can prove especially useful for analyzing dense interaction matrices at high resolution. Graphical Abstract i3C captures chromatin folding in intact nuclei without a need for cross‐linking and reveals that native interactions resemble cross‐linked ones, yet they display improved signal‐to‐noise ratios and highlight how cis ‐elements and TADs contribute to 3D genomic organization.

Transcription factories contain all three mammalian RNA polymerases, each actively transcribing a different subset of genes. This protocol describes how to isolate large factory fragments for the analysis of associated protein and RNA content. Mammalian cell nuclei contain three RNA polymerases (RNAP I, RNAP II and RNAP III), which transcribe different gene subsets, and whose active forms are contained in supramolecular complexes known as 'transcription factories.' These complexes are difficult to isolate because they are embedded in the 3D structure of the nucleus. Factories exchange components with the soluble nucleoplasmic pool over time as gene expression programs change during development or disease. Analysis of their content can provide information on the nascent transcriptome and its regulators. Here we describe a protocol for the isolation of large factory fragments under isotonic salt concentrations in <72 h. It relies on DNase I–mediated detachment of chromatin from the nuclear substructure of freshly isolated, unfixed cells, followed by caspase treatment to release multi-megadalton factory complexes. These complexes retain transcriptional activity, and isolation of their contents is compatible with downstream analyses by mass spectrometry (MS) or RNA-sequencing (RNA-seq) to catalog the proteins and RNA associated with sites of active transcription.

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.

Abstract The advent of the chromosome conformation capture (3C) and related technologies has profoundly renewed our understaning of three-dimensional chromatin organization in mammalian nuclei. Alongside these experimental approaches, numerous computational tools for handling, normalizing, visualizing, and ultimately detecting interactions in 3C-type datasets are being developed. Here, we present Bloom, a comprehensive method for the analysis of 3C-type data matrices on the basis of Dirichlet process mixture models that addresses two important open issues. First, it retrieves occult interaction patterns from sparse data, like those derived from single-cell Hi-C experiments; thus, bloomed sparse data can now be used to study interaction landscapes at sub-kbp resolution. Second, it detects enhancer-promoter interactions with high sensitivity and inherently assigns an interaction frequency score (IFS) to each contact. Using enhancer perturbation data of different throughput, we show that IFS accurately quantifies the regulatory influence of each enhancer on its target promoter. As a result, Bloom allows decoding of complex regulatory landscapes by generating functionally-relevant enhancer atlases solely on the basis of 3C-type of data. Competing Interest Statement The authors have declared no competing interest.

Abstract Culture expansion of primary cells evokes highly reproducible DNA methylation (DNAm) changes at specific sites in the genome. These changes might be due to an directly regulated epigenetic process, or to gradual deregulation of the epigenetic state, which is often referred to as “epigenetic drift”. We have identified CG dinucleotides (CpGs) that become continuously hyper- or hypomethylated in the course of culture expansion of mesenchymal stem cells (MSCs) and other cell types. During reprogramming into induced pluripotent stem cells (iPSCs) particularly the culture-associated hypomethylation is reversed simultaneously with age-associated and pluripotency-associated DNAm changes. Bisulfite barcoded amplicon sequencing (BBA-seq) demonstrated that upon passaging the DNAm patterns of neighboring CpGs become more complex without evidence of continuous pattern development and without association to oligoclonal subpolulations of MSCs at later passages. Circularized chromatin conformation capture (4C) revealed reproducible changes in nuclear organization between early and late passages, while there was no preferential 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 indicate that DNAm changes during culture-expansion resembles epigenetic drift, which seems to occur in relation to chromatin conformation. Competing Interest Statement WW is cofounder of Cygenia GmbH (www.cygenia.com), which can provide service for epigenetic analysis to other scientists. JF contributes to this company, too. All other authors do not have a conflict of interest to declare. Footnotes * We noticed a mistake in the script for the hemimethylation analysis. In fact, hemimethylation at the culture-associated CpGs was much less pronounced. We have therefore tempered this aspect in a revised manuscript. The title and discussion was changed accordingly. We dearly apologize for this confusion.

Ageing-relevant processes, like cellular senescence, are characterized by complex, often stochastic, events giving rise to heterogeneous cell populations. We hypothesized that entry into senescence of different primary human cells can be triggered by one early molecular event affecting the spatial organization of chromosomes. To test this, we combined whole-genome chromosome conformation capture, population and single-cell transcriptomics, super-resolution imaging, and functional analyses applied on proliferating and replicatively-senescent populations from three distinct human cell types. We found a number of genes involved in DNA conformation maintenance being suppressed upon senescence across cell types. Of these, the abundant high mobility group (HMG) B1 and B2 nuclear factors are quantitatively removed from cell nuclei before typical senescence markers appear, and mark a subset of topologically-associating domain (TAD) boundaries. Their loss coincides with obvious reorganization of chromatin interactions via the dramatic spatial clustering of CTCF foci. HMGB2 knock-down recapitulates this senescence-induced CTCF clustering, while also affecting insulation at TAD boundaries. We accordingly propose that HMGB-mediated deregulation of chromosome conformation constitutes a primer for the ensuing senescent program across cell types.