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SummaryHox genes encode transcription factors that specify segmental identities along the Antero-Posterior body axis. These genes are organized in clusters, where their order corresponds to their activity along the body axis, an evolutionary conserved feature known as collinearity. In Drosophila, the BX-C cluster contains the three most posterior Hox genes, where their collinear activation incorporates progressive replacement of histone modifications, reorganization of 3D chromatin architecture and sequential activation of boundary elements and cis-regulatory regions. To dissect functional hierarchies, we compared chromatin organization in larvae and in cell lines, with a focus on the Abd-B gene. Our work establishes the importance of the Fab-7 boundary element for insulation between 3D domains marked by different histone modifications. Interestingly, we detected a non-canonical inversion of collinear chromatin dynamics at the Abd-B gene, with the active histone domain decreasing in size. This chromatin organization differentially instructed alternative Abd-B promoter use, thereby expanding the possibilities to regulate transcriptional output.

Significance The DNA-binding protein CCCTC-binding factor (CTCF) and the cohesin complex function together to establish chromatin loops and regulate gene expression in mammalian cells. It has been proposed that the cohesin complex moving bidirectionally along DNA extrudes the chromatin fiber and generates chromatin loops when it pauses at CTCF binding sites. To date, the mechanisms by which cohesin localizes at CTCF binding sites remain unclear. In the present study we define two short segments within the CTCF protein that are essential for localization of cohesin complexes at CTCF binding sites. Based on our data, we propose that the N-terminus of CTCF and 3D geometry of the CTCF–DNA complex act as a roadblock constraining cohesin movement and establishing long-range chromatin loops., The DNA-binding protein CCCTC-binding factor (CTCF) and the cohesin complex function together to shape chromatin architecture in mammalian cells, but the molecular details of this process remain unclear. Here, we demonstrate that a 79-aa region within the CTCF N terminus is essential for cohesin positioning at CTCF binding sites and chromatin loop formation. However, the N terminus of CTCF fused to artificial zinc fingers was not sufficient to redirect cohesin to non-CTCF binding sites, indicating a lack of an autonomously functioning domain in CTCF responsible for cohesin positioning. BORIS (CTCFL), a germline-specific paralog of CTCF, was unable to anchor cohesin to CTCF DNA binding sites. Furthermore, CTCF–BORIS chimeric constructs provided evidence that, besides the N terminus of CTCF, the first two CTCF zinc fingers, and likely the 3D geometry of CTCF–DNA complexes, are also involved in cohesin retention. Based on this knowledge, we were able to convert BORIS into CTCF with respect to cohesin positioning, thus providing additional molecular details of the ability of CTCF to retain cohesin. Taken together, our data provide insight into the process by which DNA-bound CTCF constrains cohesin movement to shape spatiotemporal genome organization.

Cell type-specific gene expression is driven through the interplay between lineage-specific transcription factors (TFs) and the chromatin architecture, such as topologically associating domains (TADs), and enhancer-promoter interactions. To elucidate the molecular mechanisms of the cell fate decisions and cell type-specific functions, it is important to understand the interplay between chromatin architectures and TFs. Among enhancers, super-enhancers (SEs) play key roles in establishing cell identity. Adaptive immunity depends on the RAG-mediated assembly of antigen recognition receptors. Hence, regulation of the Rag1 and Rag2 (Rag1/2) genes is a hallmark of adaptive lymphoid lineage commitment. Here, we review the current knowledge of 3D genome organization, SE formation, and Rag1/2 gene regulation during B cell and T cell differentiation.

In early development, the environment triggers mnemonic epigenomic programs resulting in memory and learning experiences to confer cognitive phenotypes into adulthood. To uncover how environmental stimulation impacts the epigenome and genome organization, we used the paradigm of environmental enrichment (EE) in young mice constantly receiving novel stimulation. We profiled epigenome and chromatin architecture in whole cortex and sorted neurons by deep-sequencing techniques. Specifically, we studied chromatin accessibility, gene and protein regulation, and 3D genome conformation, combined with predicted enhancer and chromatin interactions. We identified increased chromatin accessibility, transcription factor binding including CTCF-mediated insulation, differential occupancy of H3K36me3 and H3K79me2, and changes in transcriptional programs required for neuronal development. EE stimuli led to local genome re-organization by inducing increased contacts between chromosomes 7 and 17 (inter-chromosomal). Our findings support the notion that EE-induced learning and memory processes are directly associated with the epigenome and genome organization.

Cell type-specific gene expression is driven through the interplay between lineage-specific transcription factors (TFs) and the chromatin architecture, such as topologically associating domains (TADs), and enhancer-promoter interactions. To elucidate the molecular mechanisms of the cell fate decisions and cell type-specific functions, it is important to understand the interplay between chromatin architectures and TFs. Among enhancers, super-enhancers (SEs) play key roles in establishing cell identity. Adaptive immunity depends on the RAG-mediated assembly of antigen recognition receptors. Hence, regulation of the Rag1 and Rag2 (Rag1/2) genes is a hallmark of adaptive lymphoid lineage commitment. Here, we review the current knowledge of 3D genome organization, SE formation, and Rag1/2 gene regulation during B cell and T cell differentiation.

Chromosomes segregate differentially relative to distinct subnuclear structures, but this genome-wide compartmentalization, pivotal for modulating genome function, remains poorly understood. New genomic mapping methods can reveal chromosome positioning relative to specific nuclear structures. However, computational methods that integrate their results to identify overall intranuclear chromo-some positioning have not yet been developed. We report SPIN, a new method to identify genome-wide nuclear spatial localization patterns. As a proof-of-principle, we use SPIN to integrate nuclear compartment mapping (TSA-seq and DamID) and chromatin interaction data (Hi-C) from K562 cells to identify 10 spatial compartmentalization states genome-wide relative to nuclear speckles, lamina, and nucleoli. These SPIN states show novel patterns of genome spatial organization and their relation to genome function (transcription and replication timing). Comparisons of SPIN states with Hi-C sub-compartments and lamina-associated domains (LADs) from multiple cell types suggest constitutive compartmentalization patterns. By integrating different readouts of higher-order genome organization, SPIN provides critical insights into nuclear spatial and functional compartmentalization.

In early development, the environment triggers mnemonic epigenomic programs resulting in memory and learning experiences to confer cognitive phenotypes into adulthood. To uncover how environmental stimulation impacts the epigenome and genome organization, we used the paradigm of environmental enrichment (EE) in young mice constantly receiving novel stimulation. We profiled epigenome and chromatin architecture in whole cortex and sorted neurons by deep-sequencing techniques. Specifically, we studied chromatin accessibility, gene and protein regulation, and 3D genome conformation, combined with predicted enhancer and chromatin interactions. We identified increased chromatin accessibility, transcription factor binding including CTCF-mediated insulation, differential occupancy of H3K36me3 and H3K79me2, and changes in transcriptional programs required for neuronal development. EE stimuli led to local genome re-organization by inducing increased contacts between chromosomes 7 and 17 (inter-chromosomal). Our findings support the notion that EE-induced learning and memory processes are directly associated with the epigenome and genome organization. We acknowledge support of the Spanish Ministry of Economy and Competitiveness (SAF2011-26216), “Centro de Excelencia Severo Ochoa 2017-2021,” SEV-2016-0571, the CERCA Programme/Generalitat de Catalunya and Jerome Lejeune Foundation, Swiss National Science Foundation Fellowship (PBLAP3_136878) and Co-funded by Marie Curie Actions to CNH. Resources for analyses conducted by SE-G were partially supported by the U.S. National Institutes of Mental Health Funds R01MH104341 and R01MH117790 and by the Social Sciences and Humanities Research Council of Canada (NFRFE-2018-01305). We acknowledge support of the Spanish Ministry of Science and Innovation to the EMBL partnership, Agencia Estatal de Investigaci n (PID2019-110755RB-I00/AEI / 10.13039/501100011033), the European Union’s Horizon 2020 Research and Innovation programme under grant agreement No 848077, Jerôme Lejeune Foundation, NIH (Grant Number: 1R01EB 028159-01), Marató TV3 (#2016/20-30). RP-R resources were supported by R01GM109215. We thank the support of the University of Tübingen for the Open Access Publication Funds contribution.

The discovery of the CRISPR-Cas9 endonuclease has enabled facile genome editing in living cells and organisms. Catalytically inactive Cas9 (dCas9) retains the ability to bind DNA in an RNA-guided fashion, and has additionally been explored as a tool for transcriptional modulation, epigenetic editing, and genomic imaging. This review highlights recent progress and challenges in the development of dCas9 for imaging genomic loci. The emergence and maturation of this technology offers the potential to answer new mechanistic questions about chromosome dynamics and three-dimensional genome organization in vivo.

BackgroundGenomic imprinting is essential for mammalian development and provides a unique paradigm to explore intra-cellular differences in chromatin configuration. So far, the detailed allele-specific chromatin organization of imprinted gene domains has mostly been lacking. Here, we explored the chromatin structure of the two conserved imprinted domains controlled by paternal DNA methylation imprints—theIgf2-H19andDlk1-Dio3domains—and assessed the involvement of the insulator protein CTCF in mouse cells.ResultsBoth imprinted domains are located within overarching topologically associating domains (TADs) that are similar on both parental chromosomes. At each domain, a single differentially methylated region is bound by CTCF on the maternal chromosome only, in addition to multiple instances of bi-allelic CTCF binding. Combinations of allelic 4C-seq and DNA-FISH revealed that bi-allelic CTCF binding alone, on the paternal chromosome, correlates with a first level of sub-TAD structure. On the maternal chromosome, additional CTCF binding at the differentially methylated region adds a further layer of sub-TAD organization, which essentially hijacks the existing paternal-specific sub-TAD organization. Perturbation of maternal-specific CTCF binding site at theDlk1-Dio3locus, using genome editing, results in perturbed sub-TAD organization and bi-allelicDlk1activation during differentiation.ConclusionsMaternal allele-specific CTCF binding at the imprintedIgf2-H19and theDlk1-Dio3domains adds an additional layer of sub-TAD organization, on top of an existing three-dimensional configuration and prior to imprinted activation of protein-coding genes. We speculate that this allele-specific sub-TAD organization provides an instructive or permissive context for imprinted gene activation during development.

Despite recent advances, the underlying functional constraints that shape the three-dimensional organization of eukaryotic genome are not entirely clear. Through comprehensive multivariate analyses of genome-wide datasets, we show that cis and trans interactions in yeast genome have significantly distinct functional associations. In particular, (i) the trans interactions are constrained by coordinated replication and co-varying mutation rates of early replicating domains through interactions among early origins, while cis interactions are constrained by coordination of late replication through interactions among late origins; (ii) cis and trans interactions exhibit differential preference for nucleosome occupancy; (iii) cis interactions are also constrained by the essentiality and co-fitness of interacting genes. Essential gene clusters associate with high average interaction frequency, relatively short-range interactions of low variance, and exhibit less fluctuations in chromatin conformation, marking a physically restrained state of engaged loci that, we suggest, is important to mitigate the epigenetic errors by restricting the spatial mobility of loci. Indeed, the genes with lower expression noise associate with relatively short-range interactions of lower variance and exhibit relatively higher average interaction frequency, a property that is conserved across Escherichia coli, yeast, and mESCs. Altogether, our observations highlight the coordination of replication and the minimization of expression noise, not necessarily co-expression of genes, as potent evolutionary constraints shaping the spatial organization of yeast genome.

Background Our appreciation of the critical role of the genome’s 3D organization in gene regulation is steadily increasing. Recent 3C-based deep sequencing techniques elucidated a hierarchy of structures that underlie the spatial organization of the genome in the nucleus. At the top of this hierarchical organization are chromosomal territories and the megabase-scale A/B compartments that correlate with transcriptional activity within cells. Below them are the relatively cell-type-invariant topologically associated domains (TADs), characterized by high frequency of physical contacts between loci within the same TAD, and are assumed to function as regulatory units. Within TADs, chromatin loops bring enhancers and target promoters to close spatial proximity. Yet, we still have only rudimentary understanding how differences in chromatin organization between different cell types affect cell-type-specific gene expression programs that are executed under basal and challenged conditions. Results Here, we carried out a large-scale meta-analysis that integrated Hi–C data from thirteen different cell lines and dozens of ChIP-seq and RNA-seq datasets measured on these cells, either under basal conditions or after treatment. Pairwise comparisons between cell lines demonstrate a strong association between modulation of A/B compartmentalization, differential gene expression and transcription factor (TF) binding events. Furthermore, integrating the analysis of transcriptomes of different cell lines in response to various challenges, we show that A/B compartmentalization of cells under basal conditions significantly correlates not only with gene expression programs and TF binding profiles that are active under the basal condition but also with those induced in response to treatment. Yet, in pairwise comparisons between different cell lines, we find that a large portion of differential TF binding and gene induction events occur in genomic loci assigned to A compartment in both cell types, underscoring the role of additional critical factors in determining cell-type-specific transcriptional programs. Conclusions Our results further indicate the role of dynamic genome organization in regulation of differential gene expression between different cell types and the impact of intra-TAD enhancer–promoter interactions that are established under basal conditions on both the basal and treatment-induced gene expression programs. Electronic supplementary material The online version of this article (10.1186/s13072-018-0220-2) contains supplementary material, which is available to authorized users.

In mammalian cells, DNA replication timing is controlled at the level of megabase (Mb)-sized chromosomal domains and correlates well with transcription, chromatin structure, and three-dimensional (3D) genome organization. Because of these properties, DNA replication timing is an excellent entry point to explore genome regulation at various levels and a variety of studies have been carried out over the years. However, DNA replication timing studies traditionally required at least tens of thousands of cells, and it was unclear whether the replication domains detected by cell population analyses were preserved at the single-cell level. Recently, single-cell DNA replication profiling methods became available, which revealed that the Mb-sized replication domains detected by cell population analyses were actually well preserved in individual cells. In this article, we provide a brief overview of our current knowledge on DNA replication timing regulation in mammals based on cell population studies, outline the findings from single-cell DNA replication profiling, and discuss future directions and challenges.

Formaldehyde cross-linking is an important component of many technologies, including chromatin immunoprecipita- tion and chromosome conformation capture. The procedure remains empirical and poorly characterized, however, despite a long history of its use in research. Little is known about the specificity of in vivo cross-linking, its efficiency and chemical adducts induced by the procedure. It is time to search this black box. We think it is urgent to draw attention to the un- certainty introduced in results obtained by ChIP and other formaldehyde fixation-based approaches by the fact the cross-linking efficiency of various proteins to DNA and to each other is drastically different and, in the case of in vivo cross-linking, may depend on local conditions within different cellular compartments. Current chromatin research is characterized by the fast accumulation of genome-wide data on the distribution of various regulatory proteins along chromosomes. These data are easily accessible through different databases, and much effort has been made to pour more and more data into the pot. Surprisingly, however, not many scientists are concerned about the validity of the chromatin immunoprecipitation (ChIP) approach. The ChIP procedure was developed >15 years ago (1) and, essentially, the original protocol is still used without paying much attention to its inherent problems, even though it has long been felt that 'the devil is in the ChIP details'. The most problematic step is formal- dehyde fixation. It is commonly believed that for- maldehyde can fix any DNA-protein complex. However, this assumption is far from being

Organizing the DNA to fit inside a spatially constrained nucleus is a challenging problem that has attracted the attention of scientists across all disciplines of science. Increasing evidence has demonstrated the importance of genome geometry in several cellular contexts that affect human health. Among several approaches, the application of sequencing technologies has substantially increased our understanding of this intricate organization, also known as chromatin interactions. These structures are involved in transcriptional control of gene expression by connecting distal regulatory elements with their target genes and regulating co-transcriptional splicing. In addition, chromatin interactions play pivotal roles in the organization of the genome, the formation of structural variants, recombination, DNA replication and cell division. Mutations in factors that regulate chromatin interactions lead to the development of pathological conditions, for example, cancer. In this review, we discuss key findings that have shed light on the importance of these structures in the context of cancers, and highlight the applicability of chromatin interactions as potential biomarkers in molecular medicine as well as therapeutic implications of chromatin interactions.