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WNT signaling activates MYC expression in cancer cells. Here we report that this involves an oncogenic super-enhancer-mediated tethering of active MYC alleles to nuclear pores to increase transcript export rates. As the decay of MYC transcripts is more rapid in the nucleus than in the cytoplasm, the oncogenic super-enhancer-facilitated export of nuclear MYC transcripts expedites their escape from the nuclear degradation system in colon cancer cells. The net sum of this process, as supported by computer modeling, is greater cytoplasmic MYC messenger RNA levels in colon cancer cells than in wild type cells. The cancer-cell-specific gating of MYC is regulated by AHCTF1 (also known as ELYS), which connects nucleoporins to the oncogenic super-enhancer via β-catenin. We conclude that WNT signaling collaborates with chromatin architecture to post-transcriptionally dysregulate the expression of a canonical cancer driver. Oncogenic MYC expression involves super-enhancer-mediated tethering of MYC alleles to nuclear pores, thus increasing messenger RNA export. This is regulated by AHCTF1 and β-catenin.

WNT signaling enhances MYC expression in cancer cells to increase the rate of cell proliferation. We have recently found that this principle involves the gating of MYC to nuclear pores mediated by an oncogenic super-enhancer in a ß-catenin-dependent manner in colon cancer cells. This phenomenon, which is absent in normal cells, leads to pathological levels of MYC expression.

The chromosome conformation capture method and its derivatives, such as circularized chromosome conformation capture, carbon copy chromosome conformation capture, high-throughput chromosome conformation capture and capture high-throughput chromosome conformation capture, have pioneered our understanding of the principles of chromosome folding in the nucleus. These technical advances, however, cannot precisely quantitate interaction frequency in very small input samples. Here we describe a protocol for the Nodewalk assay, which is based on converting chromosome conformation capture DNA samples to RNA and subsequently to cDNA using strategically placed primers. This pipeline enables the quantitative analyses of chromatin fiber interactions without compromising its sensitivity down to300 cells, making it suitable for MiSeq analyses of higher-order chromatin structures in biopsies, circulating tumor cells and transitional cell states, for example. Importantly, the quality of the Nodewalk sample can be assessed before sequencing to avoid unnecessary costs. Moreover, it enables analyses from hundreds of different restriction enzyme fragment viewpoints within the same initial small input sample to uncover complex, genome-wide networks. Following optimization of the different steps, the entire protocol can be completed within 2 weeks.

The relationship between stochastic transcriptional bursts and dynamic 3D chromatin states is not well understood due to poor sensitivity and/or resolution of current chromatin structure-based assays. Consequently, it is not well established if enhancers operate individually and/or in clusters to coordinate gene transcription. In the current study, we introduce Nodewalk, which uniquely combines high sensitivity with high resolution to enable the analysis of chromatin networks in minute input material. The >10,000-fold increase in sensitivity over other many-to-all competing methods uncovered that active chromatin hubs identified in large input material, corresponding to 10 000 cells, flanking theMYClocus are primarily virtual. Thus, the close agreement between chromatin interactomes generated from aliquots corresponding to less than 10 cells with randomly re-sampled interactomes, we find that numerous distal enhancers positioned within flanking topologically associating domains (TADs) converge onMYCin largely mutually exclusive manners. Moreover, when comparing with several enhancer baits, the assignment of theMYClocus as the node with the highest dynamic importance index, indicates that it isMYCtargeting its enhancers, rather thanvice versa.Dynamic changes in the configuration of the boundary between TADs flankingMYCunderlie numerous stochastic encounters with a diverse set of enhancers to depict the plasticity of its transcriptional regulation. Such an arrangement might increase the fitness of the cancer cell by increasing the probability ofMYCtranscription in response to a wide range of environmental cues encountered by the cell during the neoplastic process.

WNT signaling activates MYC expression in cancer cells. Here we report that this involves an oncogenic super-enhancer-mediated tethering of active MYC alleles to nuclear pores to increase transcript export rates. As the decay of MYC transcripts is more rapid in the nucleus than in the cytoplasm, the oncogenic super-enhancer-facilitated export of nuclear MYC transcripts expedites their escape from the nuclear degradation system in colon cancer cells. The net sum of this process, as supported by computer modeling, is greater cytoplasmic MYC messenger RNA levels in colon cancer cells than in wild type cells. The cancer-cell-specific gating of MYC is regulated by AHCTF1 (also known as ELYS), which connects nucleoporins to the oncogenic super-enhancer via β-catenin. We conclude that WNT signaling collaborates with chromatin architecture to post-transcriptionally dysregulate the expression of a canonical cancer driver.

Transcriptionally active and inactive chromatin domains tend to segregate into separate sub-nuclear compartments to maintain stable expression patterns. However, here we uncovered an inter-chromosomal network connecting active loci enriched in circadian genes to repressed lamina-associated domains (LADs). The interactome is regulated by PARP1 and its co-factor CTCF. They not only mediate chromatin fiber interactions but also promote the recruitment of circadian genes to the lamina. Synchronization of the circadian rhythm by serum shock induces oscillations in PARP1-CTCF interactions, which is accompanied by oscillating recruitment of circadian loci to the lamina, followed by the acquisition of repressive H3K9me2 marks and transcriptional attenuation. Furthermore, depletion of H3K9me2/3, inhibition of PARP activity by olaparib, or downregulation of PARP1 or CTCF expression counteracts both recruitment to the envelope and circadian transcription. PARP1- and CTCF-regulated contacts between circadian loci and the repressive chromatin environment at the lamina therefore mediate circadian transcriptional plasticity.

Heritable and reversible chromatin modifications govern phenotypic plasticity and stability by enabling the organism to alter or maintain its transcriptome in response to environmental cues. Separation of active and inactive chromatin domains in the three-dimensional (3D) space of the nucleus is considered to play an essential role in the maintenance of epigenetic memories, and provides an additional layer of transcriptional regulation. Heterochromatin compartments are largely absent form pluripotent cells and form during differentiation at the nuclear periphery and around the nucleolus, where multiple layers of heritable epigenetic modifications coordinate the repression of gene-poor regions and developmentally repressed genes. In the interior of the nucleus, enhancer elements establish cell type–specific expression patterns via regulated physical contacts with their target genes. In this chapter we discuss the basic principles and factors that regulate 3D genome organization and enhancer action at a distance, and describe how these features regulate phenotypic plasticity during development, ageing and senescence. Finally, we will discuss recent models that describe cancer development as an extreme form of pathologically increased epigenetic plasticity, which drives tumor evolution under constantly changing selective pressure.

Mammalian physiology exhibits 24-hour cyclicity due to circadian rhythms of gene expression controlled by transcription factors (TF) that comprise molecular clocks. Core clock TFs bind to the genome at enhancer sequences to regulate circadian gene expression, but not all binding sites are equally functional. Here we demonstrate that circadian gene expression in mouse liver is controlled by rhythmic chromatin interactions between enhancers and promoters. Rev-erbα, a core repressive TF of the clock, opposes functional loop formation between Rev-erbα-regulated enhancers and circadian target gene promoters by recruitment of the NCoR-HDAC3 corepressor complex, histone deacetylation, and eviction of the elongation factor BRD4 and the looping factor MED1. Thus, a repressive arm of the molecular clock operates by rhythmically modulating chromatin loops to control circadian gene transcription.

This year is the tenth anniversary of the publication in this journal of a model suggesting the existence of 'tumour progenitor genes'. These genes are epigenetically disrupted at the earliest stages of malignancies, even before mutations, and thus cause altered differentiation throughout tumour evolution. The past decade of discovery in cancer epigenetics has revealed a number of similarities between cancer genes and stem cell reprogramming genes, widespread mutations in epigenetic regulators, and the part played by chromatin structure in cellular plasticity in both development and cancer. In the light of these discoveries, we suggest here a framework for cancer epigenetics involving three types of genes: 'epigenetic mediators', corresponding to the tumour progenitor genes suggested earlier; 'epigenetic modifiers' of the mediators, which are frequently mutated in cancer; and 'epigenetic modulators' upstream of the modifiers, which are responsive to changes in the cellular environment and often linked to the nuclear architecture. We suggest that this classification is helpful in framing new diagnostic and therapeutic approaches to cancer.

The fetal transcription factor PLAG1 is found to be overexpressed in cancers, and has been suggested to bind the insulin like growth factor 2 (IGF2) P3 promoter, and to activate the IGF2 gene. The expression of IGF2 has partly been linked to loss of CTCF-dependent chromatin insulator function at the H19 imprinting control region (ICR). We investigated the role of PLAG1 for IGF2 regulation in Hep3B and JEG-3 cell lines. Chromatin immunoprecipitation revealed cell type-specific binding of PLAG1 to the IGF2 P3 promoter, which was substantially insensitive to recombinant PLAG1 overexpression in the endogenous context. We hypothesized that the H19 chromatin insulator may be involved in the cell type-specific PLAG1 response. By using a GFP reporter gene/insulator assay plasmid construct with and without the H19 ICR and/or an SV40 enhancer, we confirm that the effect of the insulator is specifically associated with the activity of the IGF2 P3 promoter in the GFP reporter system, and furthermore, that the reporter insulator is functional in JEG-3 but not in Hep3B cells. FACS analysis was used to assess the function of PLAG1 in low endogenously expressing, but Zn-inducible stable PLAG1 expressing JEG-3 cell clones. Considerable increase in IGF2 expression upon PLAG1 induction with a partial insulator overriding activity was found using the reporter constructs. This is in contrast to the effect of the endogenous IGF2 gene which was insensitive to PLAG1 expression in JEG-3, while modestly induced the already highly expressed IGF2 gene in Hep3B cells. We suggest that the PLAG1 binding to the IGF2 P3 promoter and IGF2 expression is cell type-specific, and that the PLAG1 transcription factor acts as a transcriptional facilitator that partially overrides the insulation by the H19 ICR.

Uncoupling Proteins (UCPs) are integral ion channels residing in the inner mitochondrial membrane. UCP2 is ubiquitously expressed, while UCP3 is found primarily in muscles and adipose tissue. Although the exact molecular mechanism of action is controversial, it is generally agreed that both homologues function to facilitate mitochondrial fatty acid oxidation. UCP2 and -3 expression is activated by the peroxisome proliferator-activated receptors (PPARs), but so far no PPAR response element has been reported in the vicinity of the Ucp2 and Ucp3 genes. Using genome-wide profiling of PPARgamma occupancy in 3T3-L1 adipocytes we demonstrate that PPARgamma associates with three chromosomal regions in the vicinity of the Ucp3 locus and weakly with a site in intron 1 of the Ucp2 gene. These sites are isolated from the nearest neighboring sites by >900 kb. The most prominent PPARgamma binding site in the Ucp2 and Ucp3 loci is located in intron 1 of the Ucp3 gene and is the only site that facilitates PPARgamma transactivation of a heterologous promoter. This site furthermore transactivates the endogenous Ucp3 promoter, and using chromatin conformation capture we show that it loops out to specifically interact with the Ucp2 promoter and intron 1. Our data indicate that PPARgamma transactivation of both UCP2 and -3 is mediated through this novel enhancer in Ucp3 intron 1.

Recent observations highlight that the mammalian genome extensively communicates with itself via long-range chromatin interactions. The causal link between such chromatin cross-talk and epigenetic states is, however, poorly understood. We identify here a network of physically juxtaposed regions from the entire genome with the common denominator of being genomically imprinted. Moreover, CTCF-binding sites within the H19 imprinting control region (ICR) not only determine the physical proximity among imprinted domains, but also transvect allele-specific epigenetic states, identified by replication timing patterns, to interacting, nonallelic imprinted regions during germline development. We conclude that one locus can directly or indirectly pleiotropically influence epigenetic states of multiple regions on other chromosomes with which it interacts.

An overall link between the potential for gene transcription and the timing of replication in S phase is now well established in metazoans. Here we discuss emerging evidence that highlights the possibility that replication timing is causally linked with epigenetic reprogramming. In particular, we bring together conclusions from a range of studies to propose a model in which reprogramming factors determine the timing of replication and the implementation of reprogramming events requires passage through S phase. These considerations have implications for our understanding of development, evolution and diseases such as cancer.

The pioneering chromosome conformation capture (3C) method provides the opportunity to study chromosomal folding in the nucleus. It is based on formaldehyde cross-linking of living cells followed by enzyme digestion, intramolecular ligation and quantitative (Q)-PCR analysis. However, 3C requires prior knowledge of the bait and interacting sequence (termed interactor) rendering it less useful for genome-wide studies. As several recent reports document, this limitation has been overcome by exploiting a circular intermediate in a variant of the 3C method, termed 4C (for circular 3C). The strategic positioning of primers within the bait enables the identification of unknown interacting sequences, which form part of the circular DNA. Here, we describe a protocol for our 4C method, which produces a high-resolution interaction map potentially suitable for the analysis of cis-regulatory elements and for comparison with chromatin marks obtained by chromatin immunoprecipitation (ChIP) on chip at the sites of interaction. Following optimization of enzyme digestions and amplification conditions, the protocol can be completed in 2-3 weeks.

Despite considerable efforts, the spatial link between the nuclear architecture and the genome remains enigmatic. The 4C method, independently innovated in four different laboratories, might in combination with other methods change that. As this method is based on the unbiased identification of sequences interacting with specific baits, there are unique opportunities for unravelling the secrets of how the genome functions in 3D.

Accumulating evidence converges on the possibility that chromosomes interact with each other to regulate transcription in trans. To systematically explore the epigenetic dimension of such interactions, we devised a strategy termed circular chromosome conformation capture (4C). This approach involves a circularization step that enables high-throughput screening of physical interactions between chromosomes without a preconceived idea of the interacting partners. Here we identify 114 unique sequences from all autosomes, several of which interact primarily with the maternally inherited H19 imprinting control region. Imprinted domains were strongly overrepresented in the library of 4C sequences, further highlighting the epigenetic nature of these interactions. Moreover, we found that the direct interaction between differentially methylated regions was linked to epigenetic regulation of transcription in trans. Finally, the patterns of interactions specific to the maternal H19 imprinting control region underwent reprogramming during in vitro maturation of embryonic stem cells. These observations shed new light on development, cancer epigenetics and the evolution of imprinting.

Despite considerable efforts, our understanding of the organization of higher order chromatin conformations in single cells and how these relate to chromatin marks remains poor. We have earlier invented the Chromatin In Situ Proximity (ChrISP) technique to determine proximities between chromatin fibers within a single chromosome. Here we used ChrISP to identify chromosome 11-specific hubs that are enriched in the H3K9me2 mark and that project towards the nuclear membrane in finger-like structures. Conversely, chromosome 11-specfic chromatin hubs, visualized by the presence of either H3K9me1 or H3K9me3 marks, are chromosome-wide and largely absent at the nuclear periphery. As the nuclear periphery-specific chromatin hubs were lost in the induced reduction of H3K9me2 levels, they likely represent Large Organization Chromatin in Lysine Methylation (LOCK) domains, previously identified by ChIP-seq analysis. Strikingly, the downregulation of the H3K9me2/3 marks also led to the chromosome-wide compaction of chromosome 11, suggesting a pleiotropic function of these features not recognized before. The ChrISP-mediated visualization of dynamic chromatin states in single cells thus provides an analysis of chromatin structures with a resolution far exceeding that of any other light microscopic technique.

Current techniques for analyzing chromatin structures are hampered by either poor resolution at the individual cell level or the need for a large number of cells to obtain higher resolution. This is a major problem as it hampers our understanding of chromatin conformation in single cells and how these respond to environmental cues. Here we describe a new method, chromatin in situ proximity (ChrISP), which reproducibly scores for proximities between two different chromatin fibers in 3-D with a resolution of ∼170Å in single cells. The technique is based on the in situ proximity ligation assay (ISPLA), but ChrISP omits the rolling circle amplification step (RCA). Instead, the proximities between chromatin fibers are visualized by a fluorescent connector oligonucleotide DNA, here termed splinter, forming a circular DNA with another circle-forming oligonucleotide, here termed backbone, upon ligation. In contrast to the regular ISPLA technique, our modification enables detection of chromatin fiber proximities independent of steric hindrances from nuclear structures. We use this method to identify higher order structures of individual chromosomes in relation to structural hallmarks of interphase nuclei and beyond the resolution of the light microscope.

The genome is dynamically organized in the nuclear space in a manner that reflects and influences nuclear functions. Developmental processes that govern the formation and maintenance of epigenetic memories are also tightly linked to adaptive changes in the physical and functional landscape of the nuclear architecture. Biological and biophysical principles governing the three-dimensional folding of chromatin are therefore central to our understanding of epigenetic regulation during adaptive responses and in complex diseases, such as cancer. Accumulating evidence points to the direction that global alterations in nuclear architecture and chromatin folding conspire with unstable epigenetic states of the primary chromatin fiber to drive the phenotypic plasticity of cancer cells.

It is generally assumed that CTCF-binding sites are synonymous with the demarcation of expression domains by promoting the formation of chromatin loops. We have proposed earlier, however, that such features may be context-dependent. In support of this notion, we show here that chromatin loop structures, impinging on CTCF-binding sites 1/2 and 3/4 at the 5' and 3'-ends, respectively, within the maternal allele of the H19 imprinting control region (ICR), differ significantly. Although abrogation of CTCF binding to the maternal H19 ICR allele results in loss of chromatin loops in the 3'-region, there is a dramatic gain of long-range chromatin loops impinging on the 5'-region. As the degree of occupancy of its four CTCF-binding sites discriminates between the chromatin insulator and replication timing functions, we submit that the CTCF-binding sites within the H19 ICR are functionally diverse and organize context-dependent higher order chromatin conformations.

DNA is folded into increasingly complex yet highly mobile structures to organize the chromosomes. In the interphase nucleus, chromosomes or part of the chromosomes encounter one another preferentially at the boundaries between chromosomal territories. Although this situation implies that the preferred chromosomal neighborhood is a key determinant of interactions between chromosomes, what this means in functional terms is currently not well understood. Using the H19 imprinting control region as a window, it has been demonstrated that epigenetic information of the primary chromatin fiber has dual functions. Thus, epigenetic marks not only influence the proximity between chromatin fibers but also transfer epigenetic states between chromatin fibers both in cis and in trans. High-throughput sequence and DNA fluorescence it situ hybridization (FISH) analyses reveal that these features require chromatin movements that are restricted in space and time. The mechanisms involved in the establishment of chromosome interactomes may provide insight of fundamental importance into pivotal regulatory processes in the nucleus, such as the coordination of transcriptional programs and replication timing.

The genome forms extensive and dynamic physical interactions with itself in the form of chromosome loops and bridges, thus exploring the three-dimensional space of the nucleus. It is now possible to examine these interactions at the molecular level, and we have gained glimpses of their functional implications. Chromosomal interactions can contribute to the silencing and activation of genes within the three-dimensional context of the nuclear architecture. Technical advances in detecting these interactions contribute to our understanding of the functional organization of the genome, as well as its adaptive plasticity in response to environmental changes during development and disease.

Underlies many diseases

The Kcnq1 imprinting control region (ICR) located in intron 10 of the Kcnq1 gene is unmethylated on the paternal chromosome and methylated on the maternal chromosome and has been implicated in the manifestation of parent-of-origin-specific expression of six neighboring genes. The unmethylated Kcnq1 ICR harbors bidirectional silencer activity and drives expression of an antisense RNA, Kcnq1ot1, which overlaps the Kcnq1 coding region. To elucidate whether the Kcnq1ot1 RNA plays a role in the bidirectional silencing activity of the Kcnq1 ICR, we have characterized factor binding sites by genomic footprinting and tested the functional consequence of various deletions of these binding sites in an episome-based system. Deletion of the elements necessary for Kcnq1ot1 promoter function resulted in the loss of silencing activity. Furthermore, interruption of Kcnq1ot1 RNA production by the insertion of a polyadenylation sequence downstream of the promoter also caused a loss of both silencing activity and methylation spreading. Thus, the antisense RNA plays a key role in the silencing function of the ICR. Double-stranded RNA (dsRNA)-mediated RNA interference is unlikely to be involved, as the ICR is active irrespective of the simultaneous production of dsRNA from the genes it silences.

A new study shows that during transcription, the TH2 interleukin gene cluster is organized into several small chromatin loops connected at their base by the protein SATB1. This first detailed glimpse of chromatin folding provides a new perspective on the coordination of cell type–specific gene expression.

Chromatin modifications influence all the nuclear functions that are templated by the DNA, and regulate when and how the genome should be expressed and copied. As chromatin states are reversible in nature, elaborate mechanisms have evolved to ensure the stable inheritance and maintenance of chromatin states that define differentiation stage- and cell type–specific gene expression patterns. Importantly, these heritable marks, termed epigenetic modifications, can also be reprogrammed by environmental signals. The heritability and reversibility of chromatin modifications thus regulate developmental processes in multicellular organisms by balancing between the robust maintenance of the phenotype against environmental perturbations and phenptypic plasticity during adaptive responses. In this process chromatin has been envisaged to function as a platform for both buffering against and integrating environmental cues, enabling the propagation of transient signals over time. In this chapter we will discuss the brief history and recent advances of chromatin-based processes to provide an overview about the role of chromatin in development and the deregulation of these processes in diseases.