Chromatin-chromatin Interactions Research Articles

Revealing the Biophysics of Lamina-Associated Domain Formation by Integrating Theoretical Modeling and High-Resolution Imaging.

The interactions between chromatin and the nuclear lamina orchestrate cell type-specific gene activity by forming lamina-associated domains (LADs) which preserve cellular characteristics through gene repression. However, unlike the interactions between chromatin segments, the strength of chromatin-lamina interactions and their dependence on cellular environment are not well understood. Here, we develop a theory to predict the size and shape of peripheral heterochromatin domains by considering the energetics of chromatin-chromatin interactions, the affinity between chromatin and the nuclear lamina and the kinetics of methylation and acetylation9in human mesenchymal stem cells (hMSCs). Through the analysis of super-resolution images of peripheral heterochromatin domains using this theoretical framework, we determine the nuclear lamina-wide distribution of chromatin-lamina affinities. We find that the extracted affinity is highly spatially heterogeneous and shows a bimodal distribution, indicating regions along the lamina with strong chromatin binding and those exhibiting vanishing chromatin affinity interspersed with some regions exhibiting a relatively diminished chromatin interactions, in line with the presence of structures such as nuclear pores. Exploring the role of environmental cues on peripheral chromatin, we find that LAD thickness increases when hMSCs are cultured on a softer substrate, in correlation with contractility-dependent translocation of histone deacetylase 3 (HDAC3) from the cytosol to the nucleus. In soft microenvironments, chromatin becomes sequestered at the nuclear lamina, likely due to the interactions of HDAC3 with the chromatin anchoring protein LAP2 β ,increasing chromatin-lamina affinity, as well as elevated levels of the intranuclear histone methylation. Our findings are further corroborated by pharmacological interventions that inhibit contractility, as well as by manipulating methylation levels using epigenetic drugs. Notably, in the context of tendinosis, a chronic condition characterized by collagen degeneration, we observed a similar increase in the thickness of peripheral chromatin akin to that of cells cultured on soft substrates consistent with theoretical predictions. Our findings underscore the pivotal role of the microenvironment in shaping genome organization and highlight its relevance in pathological conditions.

Active transcription and epigenetic reactions synergistically regulate meso-scale genomic organization

In interphase nuclei, chromatin forms dense domains of characteristic sizes, but the influence of transcription and histone modifications on domain size is not understood. We present a theoretical model exploring this relationship, considering chromatin-chromatin interactions, histone modifications, and chromatin extrusion. We predict that the size of heterochromatic domains is governed by a balance among the diffusive flux of methylated histones sustaining them and the acetylation reactions in the domains and the process of loop extrusion via supercoiling by RNAPII at their periphery, which contributes to size reduction. Super-resolution and nano-imaging of five distinct cell lines confirm the predictions indicating that the absence of transcription leads to larger heterochromatin domains. Furthermore, the model accurately reproduces the findings regarding how transcription-mediated supercoiling loss can mitigate the impacts of excessive cohesin loading. Our findings shed light on the role of transcription in genome organization, offering insights into chromatin dynamics and potential therapeutic targets.

Regulatory mechanisms and context-dependent roles of TAL1 in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy derived from thymic T-cell precursors. Approximately 40-60% of T-ALL cases exhibit aberrant overexpression of the TAL1 oncogenic transcription factor. Here, we provide a comprehensive view of the TAL1-induced transcriptional program in human T-ALL cells using a rapid protein degradation system coupled with integrative approaches. Our study demonstrates that TAL1 targets can be classified into several groups, each of which exhibits unique gene expression kinetics, chromatin features, and regulatory mechanisms. Group A genes are highly dependent on TAL1, many of which are not expressed in normal T cells or TAL1-negative T-ALL cells, representing an oncogenic TAL1 signature. The TAL1 complex predominantly activates group A genes. TAL1's effect is not replaceable with its regulatory partners GATA3 or RUNX1. In contrast, group B genes, many of which are generally expressed across different T-ALL subgroups, exhibit densely-connected chromatin-chromatin interactions and demonstrate the collaborative roles played by TAL1 with other transcription factors. Interestingly, TAL1 cooperates with NOTCH1 to regulate gene expression in TAL1-positive T-ALL cells, whereas it potentially antagonizes the NOTCH1-MYC pathway and leads to lethality in TAL1-negative/ TLX3-positive cells, demonstrating the context-dependent roles of TAL1.

Variants in the DDX6-CXCR5 autoimmune disease risk locus influence the regulatory network in immune cells and salivary gland.

Fine mapping and bioinformatic analysis of the DDX6-CXCR5 genetic risk association in Sjögren's Disease (SjD) and Systemic Lupus Erythematosus (SLE) identified five common SNPs with functional evidence in immune cell types: rs4938573, rs57494551, rs4938572, rs4936443, rs7117261. Functional interrogation of nuclear protein binding affinity, enhancer/promoter regulatory activity, and chromatin-chromatin interactions in immune, salivary gland epithelial, and kidney epithelial cells revealed cell type-specific allelic effects for all five SNPs that expanded regulation beyond effects on DDX6 and CXCR5 expression. Mapping the local chromatin regulatory network revealed several additional genes of interest, including lnc-PHLDB1-1. Collectively, functional characterization implicated the risk alleles of these SNPs as modulators of promoter and/or enhancer activities that regulate cell type-specific expression of DDX6, CXCR5, and lnc-PHLDB1-1, among others. Further, these findings emphasize the importance of exploring the functional significance of SNPs in the context of complex chromatin architecture in disease-relevant cell types and tissues.

Effects of chromatin-nuclear envelope and chromatin-chromatin interactions on chromatin spatial accessibility from ensembles of single-cell 3D chromatin models at whole diploid genome scale.

Chromatin-chromatin and chromatin-nuclear envelope interactions play important roles in determining 3D genome organization. However, how chromatin spatial accessibilities in 3D space at single-cell level are dictated by these interactions is not known beyond Mb-scale A/B compartments. Here we study the effects of chromatin-nuclear envelope and chromatin-chromatin interactions on the 3D accessibility of chromatins in single cells. We obtained mapping of the LAD-regions on chromosomes based on the DamID assay, which are locations where chromatin interacts with lamin proteins from the nuclear envelope. Furthermore, we identify the most relevant chromatin-chromatin interactions from Hi-C measurements. This is achieved by removing random chromatin-chromatin polymer interactions due to collision effects arising from nuclear confinement. We have successfully constructed large ensembles of single-cell 3D polymer models of the whole diploid genome of mouse Embryonic Stem Cells (mESC) at 200 kb resolution. With four ensembles of chromatin conformations at different conditions, each with 2.0×104 single-cell 3D diploid whole genomes, we analyze the effects of these interactions on chromatin accessibility in single cells. Our results show there are quantifiable effects of chromatin-chromatin interactions and LAD-lamin interactions on 3D chromatin accessibilities.

Phase separation of AB compartments are modulated by lamina-chromatin interaction.

Recent experiments reveal that chromatin is able to bind to nuclear lamina proteins such as lamin A/C and lamin B1. Loci with high binding affinity to the lamina are called Lamin Associated Domains (LADs), primarily made up of inactive chromatin. This connection between heterochromatin and the nuclear envelope points to the possibility of further chromatin organization based on nucleus placement. Here we explore such a phenomenon by simulating five different human chromosomes in close proximity to a spherical confinement, which is attractive only to loci marked as LADs. Utilizing the chromatin types taken from the cell line GM12878, the chromosomes were modeled using the Minimal Chromatin Model (MiChroM) at 50 Kbp resolution. This model ensures that the main fold of the chromatin is due to compartmentalization, while the lamina interaction falls into the perturbative regime. According to the simulations, introducing lamina interaction alone can drive the chromatin conformation from the inverted nucleus to the conventional nucleus, and vice versa. Also, the presence of lamina interaction enhances compartmentalization at the intrachromosomal level. Further, depleted lamina simulations show that only chromatin-chromatin interactions are not enough to reproduce the experimental interchromosomal Hi-C maps. In contrast, the addition of nuclear lamina-chromatin interaction increases the frequency of interchromosomal euchromatin contact. This indicates that the nuclear lamina plays an important role in chromatin folding and dynamics.

Interplay of epigenetic regulation and chromatin-lamina interaction modulates the morphology of lamina-associated domains.

In eukaryotic nuclei, a significant portion of transcriptionally repressed chromatin is anchored to the nuclear lamina (NL), leading to the formation of lamina-associated domains (LADs). LADs are marked by increased histone methylation levels and are physically or chemically tethered to NL via proteins such as lamina-associated polypeptide 2β (LAP2β). The spatial organization of LADs exerts stimulating effects on gene repression and cell function. Here, we propose a phase-field model of chromatin organization to address the mechanisms underlying the morphological regulation of LADs. The model includes chromatin-chromatin interactions, chromatin-lamina interactions, and epigenetic kinetics of acetylation and methylation. We find that the strength of chromatin-lamina interaction drives the peripheral localization of heterochromatin, and its competition with the chromatin-chromatin interactions plays a role in determining the LAD shape. We derive a relation for the LAD thickness in terms of the methylation rates and chromatin-lamina affinity, which we validate using whole-genome numerical simulations. Comparing simulations with super-resolution images of in-vitro nuclei under histone deacetylase (HDAC) inhibiting conditions quantitatively validates the model, presenting novel means of inferring physical parameters such as epigenetic rates and chromatin-lamina affinity from nuclei super-resolution images. Our findings suggest that the association of HDAC3 and LAP2β modulates the LAD organization, serving as a potential mechanism to respond to the microenvironmental stimuli. We numerically predict the whole-genome chromatin organization in nuclei under varying chemo-mechanical environments such as epigenetic inhibition and substrate stiffnesses. Our experimental results further validate our model while quantitatively estimating the histone methylation rate and chromatin-lamina affinity in each condition. Our work has significant relevance in understanding the roles of microenvironmental factors in regulating chromatin morphology, which provides insights into the cell response to vital biological processes, including development, cancer metastasis, and degenerative diseases.

Co-Immunoprecipitation (Co-Ip) in Mammalian Cells.

The cell is a fantastic place where molecules dynamically move through the various cellular structures and compartments and meet each other, either transiently or in more stable complexes. These complexes have always a specific biological function; thus, it is important to identify and characterize the interaction between molecules, either DNA/RNA, DNA/DNA, protein/DNA, protein/protein, and so on. polycomb group proteins (PcG proteins) are epigenetic repressors involved in important physiologic processes as development and differentiation. They act on the chromatin through the formation of a repressive environment involving histone modification, recruitment of co-repressors, and chromatin-chromatin interactions. PcG form multiprotein complexes, whose characterization required several approaches. In this chapter, I will describe the co-immunoprecipitation (Co-IP) protocol, an easy method used to identify and analyze multiprotein complexes. In Co-IP, an antibody is used to isolate its target antigen, along with its binding partners, from a mixed sample. The binding partners purified with the immunoprecipitated protein can be identified by Western blot or by mass spectrometry.

Mechanisms of chromate carcinogenesis by chromatin alterations.

In a dynamic environment, organisms must constantly mount an adaptive response to new environmental conditions in order to survive. Novel patterns of gene expression, driven by attendant changes in chromatin architecture, aid in adaptation and survival. Critical mechanisms in the control of gene transcription govern new spatiotemporal chromatin-chromatin interactions that make regulatory DNA elements accessible to the transcription factors that control the response. Consequently, agents that disrupt chromatin structure are likely to have a direct impact on the transcriptional programs of cells and organisms and to drive alterations in fundamental physiological processes. In this regard, hexavalent chromium (Cr(VI)) is of special interest because it interacts directly with cellular proteins, DNA, and other macromolecules, and is likely to upset cell functions that may cause generalized damage to the organism. Here, we will highlight chromium-mediated mechanisms that disrupt chromatin architecture and discuss how these mechanisms are integral to its carcinogenic properties. Emerging evidence indicates that Cr(VI) targets euchromatin, particularly in genomic locations flanking the binding sites of the essential transcription factors CTCF and AP1, and, in so doing, they disrupt nucleosomal architecture. Ultimately, the ensuing changes, if occurring in critical regulatory domains, may establish a new chromatin state, either toxic or adaptive, that will be governed by the corresponding gene transcription changes in key biological processes associated with that state.

Nuclear position modulates long-range chromatin interactions.

The human genome is non-randomly organized within the cell nucleus. Spatial mapping of genome folding by biochemical methods and imaging has revealed extensive variation in locus interaction frequencies between cells in a population and between homologs within an individual cell. Commonly used mapping approaches typically examine either the relative position of genomic sites to each other or the position of individual loci relative to nuclear landmarks. Whether the frequency of specific chromatin-chromatin interactions is affected by where in the nuclear space a locus is located is unknown. Here, we have simultaneously mapped at the single cell level the interaction frequencies and radial position of more than a hundred locus pairs using high-throughput imaging to ask whether the location within the nucleus affects interaction frequency. We find strong enrichment of many interactions at specific radial positions. Position-dependency of interactions was cell-type specific, correlated with local chromatin type, and cell-type-specific enriched associations were marked by increased variability, sometimes without a significant decrease in mean spatial distance. These observations demonstrate that the folding of the chromatin fiber, which brings genomically distant loci into proximity, and the position of that chromatin fiber relative to nuclear landmarks, are closely linked.

Enhancer-promoter interaction maps provide insights into skeletal muscle-related traits in pig genome

BackgroundGene expression programs are intimately linked to the interplay of active cis regulatory elements mediated by chromatin contacts and associated RNAs. Genome-wide association studies (GWAS) have identified many variants in these regulatory elements that can contribute to phenotypic diversity. However, the functional interpretation of these variants remains nontrivial due to the lack of chromatin contact information or limited contact resolution. Furthermore, the distribution and role of chromatin-associated RNAs in gene expression and chromatin conformation remain poorly understood. To address this, we first present a comprehensive interaction map of nuclear dynamics of 3D chromatin-chromatin interactions (H3K27ac BL-HiChIP) and RNA-chromatin interactions (GRID-seq) to reveal genomic variants that contribute to complex skeletal muscle traits.ResultsIn a genome-wide scan, we provide systematic fine mapping and gene prioritization from GWAS leading signals that underlie phenotypic variability of growth rate, meat quality, and carcass performance. A set of candidate functional variants and 54 target genes previously not detected were identified, with 71% of these candidate functional variants choosing to skip over their nearest gene to regulate the target gene in a long-range manner. The effects of three functional variants regulating KLF6 (related to days to 100 kg), MXRA8 (related to lean meat percentage), and TAF11 (related to loin muscle depth) were observed in two pig populations. Moreover, we find that this multi-omics interaction map consists of functional communities that are enriched in specific biological functions, and GWAS target genes can serve as core genes for exploring peripheral trait-relevant genes.ConclusionsOur results provide a valuable resource of candidate functional variants for complex skeletal muscle-related traits and establish an integrated approach to complement existing 3D genomics by exploiting RNA-chromatin and chromatin-chromatin interactions for future association studies.

Hexavalent chromium disrupts chromatin architecture.

Accessibility of DNA elements and the orchestration of spatiotemporal chromatin-chromatin interactions are critical mechanisms in the regulation of gene transcription. Thus, in an ever-changing milieu, cells mount an adaptive response to environmental stimuli by modulating gene expression that is orchestrated by coordinated changes in chromatin architecture. Correspondingly, agents that alter chromatin structure directly impact transcriptional programs in cells. Heavy metals, including hexavalent chromium (Cr(VI)), are of special interest because of their ability to interact directly with cellular protein, DNA and other macromolecules, resulting in general damage or altered function. In this review we highlight the chromium-mediated mechanisms that promote disruption of chromatin architecture and how these processes are integral to its carcinogenic properties. Emerging evidence shows that Cr(VI) targets nucleosomal architecture in euchromatin, particularly in genomic locations flanking binding sites of the essential transcription factors CTCF and AP1. Ultimately, these changes contribute to an altered chromatin state in critical gene regulatory regions, which disrupts gene transcription in functionally relevant biological processes.

ENEMAL, an enhancer RNA transcribed from a distal MALAT1 enhancer, promotes NEAT1 long isoform expression.

Emerging evidence has shown that active enhancers are abundantly transcribed, generating long non-coding RNAs, called enhancer RNAs (eRNAs). While putative eRNAs are often observed from RNA sequencing, the roles of most eRNAs remain largely unknown. Previously, we identified putative enhancer regions at the MALAT1 locus that form chromatin-chromatin interactions under hypoxia, and one of these enhancers is located about 30 kb downstream of the NEAT1 gene and -20 kb upstream of the MALAT1 gene (MALAT1-20 kb enhancer). Here, we report that a novel eRNA, named eRNA of the NEAT1-MALAT1-Locus (eNEMAL), is transcribed from the MALAT1-20 kb enhancer and conserved in primates. We found that eNEMAL is upregulated in response to hypoxia in multiple breast cancer cell lines, but not in non-tumorigenic MCF10A cells. Overexpression and knockdown of eNEMAL revealed that alteration of eNEMAL level does not affect MALAT1 expression. Instead, we found that eNEMAL upregulates the long isoform of NEAT1 (NEAT1_2) without increasing the total NEAT1 transcript level in MCF7 breast cancer cells, suggesting that eNEMAL has a repressive effect on the 3'-end polyadenylation process required for generating the short isoform of NEAT1 (NEAT1_1). Altogether, we demonstrated that an eRNA transcribed from a MALAT1 enhancer regulates NEAT1 isoform expression, implicating the MALAT1-20 kb enhancer and its transcript eNEMAL in co-regulation of MALAT1 and NEAT1 in response to hypoxia in breast cancer cells.

Epstein-Barr virus inactivates the transcriptome and disrupts the chromatin architecture of its host cell in the first phase of lytic reactivation.

Epstein-Barr virus (EBV), a herpes virus also termed HHV 4 and the first identified human tumor virus, establishes a stable, long-term latent infection in human B cells, its preferred host. Upon induction of EBV’s lytic phase, the latently infected cells turn into a virus factory, a process that is governed by EBV. In the lytic, productive phase, all herpes viruses ensure the efficient induction of all lytic viral genes to produce progeny, but certain of these genes also repress the ensuing antiviral responses of the virally infected host cells, regulate their apoptotic death or control the cellular transcriptome. We now find that EBV causes previously unknown massive and global alterations in the chromatin of its host cell upon induction of the viral lytic phase and prior to the onset of viral DNA replication. The viral initiator protein of the lytic cycle, BZLF1, binds to >105 binding sites with different sequence motifs in cellular chromatin in a concentration dependent manner implementing a binary molar switch probably to prevent noise-induced erroneous induction of EBV’s lytic phase. Concomitant with DNA binding of BZLF1, silent chromatin opens locally as shown by ATAC-seq experiments, while previously wide-open cellular chromatin becomes inaccessible on a global scale within hours. While viral transcripts increase drastically, the induction of the lytic phase results in a massive reduction of cellular transcripts and a loss of chromatin-chromatin interactions of cellular promoters with their distal regulatory elements as shown in Capture-C experiments. Our data document that EBV’s lytic cycle induces discrete early processes that disrupt the architecture of host cellular chromatin and repress the cellular epigenome and transcriptome likely supporting the efficient de novo synthesis of this herpes virus.

Optogenetics and CRISPR: A New Relationship Built to Last.

Since the breakthrough discoveries that CRISPR-Cas9 nucleases can be easily programmed and employed to induce targeted double-strand breaks in mammalian cells, the gene editing field has grown exponentially. Today, CRISPR technologies based on engineered class II CRISPR effectors facilitate targeted modification of genes and RNA transcripts. Moreover, catalytically impaired CRISPR-Cas variants can be employed as programmable DNA binding domains and used to recruit effector proteins, such as transcriptional regulators, epigenetic modifiers or base-modifying enzymes, to selected genomic loci. The juxtaposition of CRISPR and optogenetics enables spatiotemporally confined and highly dynamic genome perturbations in living cells and animals and holds unprecedented potential for biology and biomedicine.Here, we provide an overview of the state-of-the-art methods for light-control of CRISPR effectors. We will detail the plethora of exciting applications enabled by these systems, including spatially confined genome editing, timed activation of endogenous genes, as well as remote control of chromatin-chromatin interactions. Finally, we will discuss limitations of current optogenetic CRISPR tools and point out routes for future innovation in this emerging field.

Spatial organization impacts function

Genome Organization Genomes are organized into topologically associating domains (TADs), which concentrate chromatin-chromatin interactions and modulate gene expression. Using the mouse X chromosome–inactivation center (XIC) as a genetic model, van Bemmel et al. quantified the extent to which TADs regulate gene transcription. Xist and Tsix are two noncoding RNAs that overlap in the same genetic locus but are transcribed from opposite directions and thus are sense:antisense. By swapping promoters into each other's TAD and measuring the resulting expression kinetics of Xist and Tsix , the authors showed that spatial partitioning of promoters was essential to ensure the correct developmental timing of X inactivation by XIC. Nat. Genet. 51 , 1024 (2019).

Hypoxia induces cancer cell-specific chromatin interactions and increases MALAT1 expression in breast cancer cells

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long noncoding RNA overexpressed in various cancers that promotes cell growth and metastasis. Although hypoxia has been shown to up-regulate MALAT1, only hypoxia-inducible factors (HIFs) have been implicated in activation of the MALAT1 promoter in specific cell types and other molecular mechanisms associated with hypoxia-mediated MALAT1 up-regulation remain largely unknown. Here, we demonstrate that hypoxia induces cancer cell-specific chromatin-chromatin interactions between newly identified enhancer-like cis-regulatory elements present at the MALAT1 locus. We show that hypoxia-mediated up-regulation of MALAT1 as well as its antisense strand TALAM1 occurs in breast cancer cells, but not in nontumorigenic mammary epithelial cells. Our analyses on the MALAT1 genomic locus discovered three novel putative enhancers that are located upstream and downstream of the MALAT1 gene body. We found that parts of these putative enhancers are epigenetically modified to a more open chromatin state under hypoxia in breast cancer cells. Furthermore, our chromosome conformation capture experiment demonstrated that noncancerous cells and breast cancer cells exhibit different interaction profiles under both normoxia and hypoxia, and only breast cancer cells gain specific chromatin interactions under hypoxia. Although the HIF-2α protein can enhance the interaction between the promoter and the putative 3' enhancer, the gain of chromatin interactions associated with other upstream elements, such as putative -7 and -20 kb enhancers, were HIF-independent events. Collectively, our study demonstrates that cancer cell-specific chromatin-chromatin interactions are formed at the MALAT1 locus under hypoxia, implicating a novel mechanism of MALAT1 regulation in cancer.

Characteristic arrangement of nucleosomes is predictive of chromatin interactions at kilobase resolution.

High-throughput chromosome conformation capture (3C) technologies, such as Hi-C, have made it possible to survey 3D genome structure. However, obtaining 3D profiles at kilobase resolution at low cost remains a major challenge. Therefore, we herein present an algorithm for precise identification of chromatin interaction sites at kilobase resolution from MNase-seq data, termed chromatin interaction site detector (CISD), and a CISD-based chromatin loop predictor (CISD_loop) that predicts chromatin–chromatin interactions (CCIs) from low-resolution Hi-C data. We show that the predictions of CISD and CISD_loop overlap closely with chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) anchors and loops, respectively. The validity of CISD/CISD_loop was further supported by a 3C assay at about 5 kb resolution. Finally, we demonstrate that only modest amounts of MNase-seq and Hi-C data are sufficient to achieve ultrahigh resolution CCI maps. Our results suggest that CCIs may result in characteristic nucleosomes arrangement patterns flanking the interaction sites, and our algorithms may facilitate precise and systematic investigations of CCIs on a larger scale than hitherto have been possible.

IRegNet3D: three-dimensional integrated regulatory network for the genomic analysis of coding and non-coding disease mutations

The mechanistic details of most disease-causing mutations remain poorly explored within the context of regulatory networks. We present a high-resolution three-dimensional integrated regulatory network (iRegNet3D) in the form of a web tool, where we resolve the interfaces of all known transcription factor (TF)-TF, TF-DNA and chromatin-chromatin interactions for the analysis of both coding and non-coding disease-associated mutations to obtain mechanistic insights into their functional impact. Using iRegNet3D, we find that disease-associated mutations may perturb the regulatory network through diverse mechanisms including chromatin looping. iRegNet3D promises to be an indispensable tool in large-scale sequencing and disease association studies.

Capture-C reveals preformed chromatin interactions between HIF-binding sites and distant promoters.

Hypoxia‐inducible factor (HIF) directs an extensive transcriptional cascade that transduces numerous adaptive responses to hypoxia. Pan‐genomic analyses, using chromatin immunoprecipitation and transcript profiling, have revealed large numbers of HIF‐binding sites that are generally associated with hypoxia‐inducible transcripts, even over long chromosomal distances. However, these studies do not define the specific targets of HIF‐binding sites and do not reveal how induction of HIF affects chromatin conformation over distantly connected functional elements. To address these questions, we deployed a recently developed chromosome conformation assay that enables simultaneous high‐resolution analyses from multiple viewpoints. These assays defined specific long‐range interactions between intergenic HIF‐binding regions and one or more promoters of hypoxia‐inducible genes, revealing the existence of multiple enhancer–promoter, promoter–enhancer, and enhancer–enhancer interactions. However, neither short‐term activation of HIF by hypoxia, nor long‐term stabilization of HIF in von Hippel–Lindau (VHL)‐defective cells greatly alters these interactions, indicating that at least under these conditions, HIF can operate on preexisting patterns of chromatin–chromatin interactions that define potential transcriptional targets and permit rapid gene activation by hypoxic stress.