Showing papers on "Chromosome conformation capture published in 2011"
TL;DR: Using high-resolution chromatin conformation capture methodology, this work examined the spatial configuration of Hox clusters in embryonic mouse tissues where different Hox genes are active and found that spatial compartmentalization may be key to process the colinear activation of these compact gene clusters.
Abstract: The spatial and temporal control of Hox gene transcription is essential for patterning the vertebrate body axis. Although this process involves changes in histone posttranslational modifications, the existence of particular three-dimensional (3D) architectures remained to be assessed in vivo. Using high-resolution chromatin conformation capture methodology, we examined the spatial configuration of Hox clusters in embryonic mouse tissues where different Hox genes are active. When the cluster is transcriptionally inactive, Hox genes associate into a single 3D structure delimited from flanking regions. Once transcription starts, Hox clusters switch to a bimodal 3D organization where newly activated genes progressively cluster into a transcriptionally active compartment. This transition in spatial configurations coincides with the dynamics of chromatin marks, which label the progression of the gene clusters from a negative to a positive transcription status. This spatial compartmentalization may be key to process the colinear activation of these compact gene clusters.
407 citations
TL;DR: NKX2-1, NKX22-2, and MEF2C are proposed as T-ALL oncogenes that are activated by various rearrangements.
327 citations
TL;DR: It is proposed that globule formation represents a higher-order folding state related to clustering of transcribed genes around shared transcription machineries, as previously observed by microscopy.
Abstract: We developed a general approach that combines chromosome conformation capture carbon copy (5C) with the Integrated Modeling Platform (IMP) to generate high-resolution three-dimensional models of chromatin at the megabase scale. We applied this approach to the ENm008 domain on human chromosome 16, containing the α-globin locus, which is expressed in K562 cells and silenced in lymphoblastoid cells (GM12878). The models accurately reproduce the known looping interactions between the α-globin genes and their distal regulatory elements. Further, we find using our approach that the domain folds into a single globular conformation in GM12878 cells, whereas two globules are formed in K562 cells. The central cores of these globules are enriched for transcribed genes, whereas nontranscribed chromatin is more peripheral. We propose that globule formation represents a higher-order folding state related to clustering of transcribed genes around shared transcription machineries, as previously observed by microscopy.
303 citations
TL;DR: It is suggested that the organization of the genome based on 10nm chromatin fibers is sufficient to describe the complexities of nuclear organization and gene regulation.
196 citations
TL;DR: A probabilistic model linking 5C/Hi-C data to physical distances is formulated and a Markov chain Monte Carlo (MCMC) approach called MCMC5C is described to generate a representative sample from the posterior distribution over structures from IF data to reliably interpret the result of their assay and contrast conformations under different conditions.
Abstract: Long-range interactions between regulatory DNA elements such as enhancers, insulators and promoters play an important role in regulating transcription. As chromatin contacts have been found throughout the human genome and in different cell types, spatial transcriptional control is now viewed as a general mechanism of gene expression regulation. Chromosome Conformation Capture Carbon Copy (5C) and its variant Hi-C are techniques used to measure the interaction frequency (IF) between specific regions of the genome. Our goal is to use the IF data generated by these experiments to computationally model and analyze three-dimensional chromatin organization. We formulate a probabilistic model linking 5C/Hi-C data to physical distances and describe a Markov chain Monte Carlo (MCMC) approach called MCMC5C to generate a representative sample from the posterior distribution over structures from IF data. Structures produced from parallel MCMC runs on the same dataset demonstrate that our MCMC method mixes quickly and is able to sample from the posterior distribution of structures and find subclasses of structures. Structural properties (base looping, condensation, and local density) were defined and their distribution measured across the ensembles of structures generated. We applied these methods to a biological model of human myelomonocyte cellular differentiation and identified distinct chromatin conformation signatures (CCSs) corresponding to each of the cellular states. We also demonstrate the ability of our method to run on Hi-C data and produce a model of human chromosome 14 at 1Mb resolution that is consistent with previously observed structural properties as measured by 3D-FISH. We believe that tools like MCMC5C are essential for the reliable analysis of data from the 3C-derived techniques such as 5C and Hi-C. By integrating complex, high-dimensional and noisy datasets into an easy to interpret ensemble of three-dimensional conformations, MCMC5C allows researchers to reliably interpret the result of their assay and contrast conformations under different conditions. http://Dostielab.biochem.mcgill.ca
174 citations
TL;DR: This work adapted the Chromosome Conformation Capture on Chip (4C) assay to systematically map chromosomal interactions in Drosophila melanogaster larval brain tissue and demonstrates that many interactions among PcG target genes exist and that these interactions are guided by overall chromosome architecture.
Abstract: Polycomb group (PcG) proteins bind and regulate hundreds of genes. Previous evidence has suggested that long-range chromatin interactions may contribute to the regulation of PcG target genes. Here, we adapted the Chromosome Conformation Capture on Chip (4C) assay to systematically map chromosomal interactions in Drosophila melanogaster larval brain tissue. Our results demonstrate that PcG target genes interact extensively with each other in nuclear space. These interactions are highly specific for PcG target genes, because non-target genes with either low or high expression show distinct interactions. Notably, interactions are mostly limited to genes on the same chromosome arm, and we demonstrate that a topological rather than a sequence-based mechanism is responsible for this constraint. Our results demonstrate that many interactions among PcG target genes exist and that these interactions are guided by overall chromosome architecture.
171 citations
TL;DR: Findings indicate that the nucleus is pre-organized in a conformation allowing rapid transcriptional reprogramming, and this organization is significantly correlated with cell-type-specific chromatin sites accessible to regulatory factors.
Abstract: The spatial organization of genes in the interphase nucleus plays an important role in establishment and regulation of gene expression. Contradicting results have been reported to date, with little consensus about the dynamics of nuclear organization and the features of the contact loci. In this study, we investigated the properties and dynamics of genomic loci that are in contact with glucocorticoid receptor (GR)-responsive loci. We took a systematic approach, combining genome-wide interaction profiling by the chromosome conformation capture on chip (4C) technology with expression, protein occupancy, and chromatin accessibility profiles. This approach allowed a comprehensive analysis of how distinct features of the linear genome are organized in the three-dimensional nuclear space in the context of rapid gene regulation. We found that the transcriptional response to GR occurs without dramatic nuclear reorganization. Moreover, contrary to the view of transcription-driven organization, even genes with opposite transcriptional responses colocalize. Regions contacting GR-regulated genes are not particularly enriched for GR-regulated loci or for any functional group of genes, suggesting that these subnuclear environments are not organized to respond to a specific factor. The contact regions are, however, highly enriched for DNase I-hypersensitive sites that comprehensively mark cell-type-specific regulatory sites. These findings indicate that the nucleus is pre-organized in a conformation allowing rapid transcriptional reprogramming, and this organization is significantly correlated with cell-type-specific chromatin sites accessible to regulatory factors. Numerous open chromatin loci may be arranged in nuclear domains that are poised to respond to diverse signals in general and to permit efficient gene regulation.
143 citations
TL;DR: In this article, the Drosophila gypsy insulator behaves as a conformational chromatin border that is able to prohibit contacts between a Polycomb response element (PRE) and a distal promoter.
Abstract: Regulation of gene expression involves long-distance communication between regulatory elements and target promoters, but how this is achieved remains unknown. Insulator elements have been proposed to modulate the communication between regulatory elements and promoters due to their ability to insulate genes from regulatory elements or to take part in long-distance interactions. Using a high-resolution chromatin conformation capture (H3C) method, we show that the Drosophila gypsy insulator behaves as a conformational chromatin border that is able to prohibit contacts between a Polycomb response element (PRE) and a distal promoter. On the other hand, two spaced gypsy elements form a chromatin loop that is able to bring an upstream PRE in contact with a downstream gene to mediate its repression. Chromatin immunoprecipitation (ChIP) profiles of the Polycomb protein and its associated H3K27me3 histone mark reflect this insulator-dependent chromatin conformation, suggesting that Polycomb action at a distance can be organized by local chromatin topology.
128 citations
TL;DR: It is proposed that RNAi-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome.
Abstract: RNA silencing pathways are evolutionarily conserved mechanisms that control gene expression via sequence-specific interactions mediated by a small RNA bound to an Argonaute (AGO) effector protein. The paradigm for how RNA silencing controls gene expression at the chromatin level comes from studies in fission yeast, in which the RNAi machinery establishes heterochromatin at the centromere and mating type locus to ensure proper chromosome segregation and to promote stability of repetitive regions. At the centromere, RNAs transcribed from pericentromeric repeats are processed by the Dcr1 endonuclease and Ago1 Argonaute protein, which leads to the recruitment of the histone H3K9 methyltransferase and Swi6/HP1 binding (for review, see Grewal and Elgin 2007).
In Drosophila, it remains unclear whether the RNAi pathway is involved directly in heterochromatin formation. The primary endogenous function of the RNAi/siRNA pathway is to silence the expression of transposable elements (TEs) in the soma (for review, see Okamura and Lai 2008). Silencing is achieved by Dcr-2-mediated cleavage of dsRNAs into 21- to 22-nucleotide (nt) siRNA that are loaded into AGO2, which cleaves the target TE mRNA using its Slicer activity. Less well understood is the function of non-TE endo-siRNAs also produced by Dcr-2 activity and loaded into AGO2, which are generated from hairpin transcripts and regions of 3′ overlap of convergent transcripts (3′ cis-NATs). Two studies implicated AGO2 in heterochromatin formation based on mislocalization of HP1 and desilencing of pericentromeric transcriptional reporters in AGO2 mutants (Deshpande et al. 2005; Fagegaltier et al. 2009). However, direct analysis of HP1 recruitment by chromatin immunoprecipitation (ChIP) and HP1-dependent silencing at small RNA-generating loci led to the suggestion that AGO2 and other Argonaute genes may not be required for heterochromatin formation in the soma (Moshkovich and Lei 2010). Nevertheless, AGO2 or other RNA silencing factors appear to play important roles in chromatin and nuclear organization, such as formation of Polycomb group (PcG) repression bodies (Grimaud et al. 2006) and gypsy chromatin insulator bodies (Lei and Corces 2006).
Chromatin insulators are DNA–protein complexes defined functionally as either barriers that prevent the spread of silent chromatin or enhancer blockers that constrain enhancer–promoter communication. Unlike vertebrates, which possess only one known insulator protein, CTCF (for review, see Phillips and Corces 2009), Drosophila employs at least five different insulator complexes. Two well-characterized insulators are the gypsy [also known as Su(Hw)] insulator and the Fab-8 insulator of the Abd-B locus in the bithorax complex (BX-C) (for review, see Bushey et al. 2008). The gypsy and Fab-8 insulators harbor binding sites for the zinc finger DNA-binding proteins Su(Hw) and CTCF, respectively, and both insulator complexes share a common component: CP190. Despite thousands of distinct DNA-binding sites throughout the genome, insulator proteins concentrate at a small number of nuclear foci, termed insulator bodies, which are dependent on CP190 for their integrity. Highly correlated at least with gypsy insulator function, insulator bodies have been proposed to serve as tethering sites for large chromosomal loops or other higher-order chromatin structures.
It has become increasingly apparent that DNA topology is a critical determinant of gene regulation. While enhancers activate their target promoters over long distances, insulators act to restrict these communications (for review, see Wallace and Felsenfeld 2007). Insulators and other cis-regulatory regions in the Abd-B locus engage in numerous interactions, and the precise topology of the locus has been postulated to be a central mechanism of tissue-specific Abd-B regulation (Cleard et al. 2006; Lanzuolo et al. 2007; Kyrchanova et al. 2008; Bantignies et al. 2011). However, the mechanism by which chromosome looping is achieved at this locus has not been elucidated. Vertebrate CTCF has been demonstrated to mediate chromosomal looping at several developmentally regulated loci in concert with cohesin (for review, see Merkenschlager 2010), but it is not known whether Drosophila CTCF, which only shares homology in the zinc finger DNA-binding domain, retains the capacity to promote looping.
In order to address whether AGO2 functions on chromatin, we performed ChIP-seq analysis of AGO2 in two Drosophila cell lines. Instead of repetitive sequence, AGO2 associates primarily with euchromatic sites, the majority of which correspond to chromatin insulators. Intriguingly, AGO2 chromatin association does not correspond to regions of the genome that produce endo-siRNAs. We demonstrate that AGO2, but not its catalytic activity or other RNAi components, is required for CTCF/CP190-dependent Fab-8 insulator function. Additionally, AGO2 interacts physically with CP190, and depletion of either CP190 or CTCF results in a decrease in AGO2 recruitment throughout the genome. Chromosome conformation capture (3C) experiments demonstrate that CTCF/CP190-dependent looping interactions may regulate AGO2 recruitment to chromatin. Therefore, we propose an RNAi-independent role for AGO2 to promote or stabilize insulator-dependent looping interactions to define transcriptional domains throughout the genome.
121 citations
TL;DR: Three-dimensional conformations of the EBV genome in different latency types indicate that epigenetically stable EBV latency types adopt distinct chromatin architectures that depend on CTCF and mediate alternative promoter targeting by the OriP enhancer.
Abstract: Epstein-Barr Virus (EBV) can establish latent infections with distinct gene expression patterns referred to as latency types. These different latency types are epigenetically stable and correspond to different promoter utilization. Here we explore the three-dimensional conformations of the EBV genome in different latency types. We employed Chromosome Conformation Capture (3C) assay to investigate chromatin loop formation between the OriP enhancer and the promoters that determine type I (Qp) or type III (Cp) gene expression. We show that OriP is in close physical proximity to Qp in type I latency, and to Cp in type III latency. The cellular chromatin insulator and boundary factor CTCF was implicated in EBV chromatin loop formation. Combining 3C and ChIP assays we found that CTCF is physically associated with OriP-Qp loop formation in type I and OriP-Cp loop formation in type III latency. Mutations in the CTCF binding site located at Qp disrupt loop formation between Qp and OriP, and lead to the activation of Cp transcription. Mutation of the CTCF binding site at Cp, as well as siRNA depletion of CTCF eliminates both OriP-associated loops, indicating that CTCF plays an integral role in loop formation. These data indicate that epigenetically stable EBV latency types adopt distinct chromatin architectures that depend on CTCF and mediate alternative promoter targeting by the OriP enhancer.
118 citations
TL;DR: It is concluded that insulator binding proteins rather than PcG complexes are likely to be the major determinants of the long-range higher-order organization of Pcg targets in the nucleus.
Abstract: The genomic binding sites of Polycomb group (PcG) complexes have been found to cluster, forming Polycomb “bodies” or foci in mammalian or fly nuclei. These associations are thought to be driven by interactions between PcG complexes and result in enhanced repression. Here, we show that a Polycomb response element (PRE) with strong PcG binding and repressive activity cannot mediate trans interactions. In the case of the two best-studied interacting PcG targets in Drosophila, the Mcp and the Fab-7 regulatory elements, we find that these associations are not dependent on or caused by the Polycomb response elements they contain. Using functional assays and physical colocalization by in vivo fluorescence imaging or chromosome conformation capture (3C) methods, we show that the interactions between remote copies of Mcp or Fab-7 elements are dependent on the insulator activities present in these elements and not on their PREs. We conclude that insulator binding proteins rather than PcG complexes are likely to be the major determinants of the long-range higher-order organization of PcG targets in the nucleus.
TL;DR: It is demonstrated that loss of CTCF allowed Ig heavy chain recombination, but pre-B cell proliferation and differentiation was severely impaired, and it is a critical factor determining Vκ segment choice for recombination.
TL;DR: The findings indicate that KSHV genomes are organized into chromatin loops mediated by CTCF and cohesin interactions, and that these inter-chromosomal linkages coordinate latent and lytic gene control.
Abstract: Herpesvirus persistence requires a dynamic balance between latent and lytic cycle gene expression, but how this balance is maintained remains enigmatic. We have previously shown that the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) major latency transcripts encoding LANA, vCyclin, vFLIP, v-miRNAs, and Kaposin are regulated, in part, by a chromatin organizing element that binds CTCF and cohesins. Using viral genome-wide chromatin conformation capture (3C) methods, we now show that KSHV latency control region is physically linked to the promoter regulatory region for ORF50, which encodes the KSHV immediate early protein RTA. Other linkages were also observed, including an interaction between the 5′ and 3′ end of the latency transcription cluster. Mutation of the CTCF-cohesin binding site reduced or eliminated the chromatin conformation linkages, and deregulated viral transcription and genome copy number control. siRNA depletion of CTCF or cohesin subunits also disrupted chromosomal linkages and deregulated viral latent and lytic gene transcription. Furthermore, the linkage between the latent and lytic control region was subject to cell cycle fluctuation and disrupted during lytic cycle reactivation, suggesting that these interactions are dynamic and regulatory. Our findings indicate that KSHV genomes are organized into chromatin loops mediated by CTCF and cohesin interactions, and that these inter-chromosomal linkages coordinate latent and lytic gene control.
TL;DR: This Commentary gives an overview of the recent conjunction of ideas regarding chromatin looping in the fields of biology and polymer physics, and explains how specific folding properties emerge upon introducing loops and how this explains a variety of experimental observations.
Abstract: There is rapidly growing evidence that folding of the chromatin fibre inside the interphase nucleus has an important role in the regulation of gene expression. In particular, the formation of loops mediated by the interaction between specific regulatory elements, for instance enhancers and promoters, is crucial in gene control. Biochemical studies that were based on the chromosome conformation capture (3C) technology have confirmed that eukaryotic genomes are highly looped. Insight into the underlying principles comes from polymer models that explore the properties of the chromatin fibre inside the nucleus. Recent models indicate that chromatin looping can explain various properties of interphase chromatin, including chromatin compaction and compartmentalisation of chromosomes. Entropic effects have a key role in these models. In this Commentary, we give an overview of the recent conjunction of ideas regarding chromatin looping in the fields of biology and polymer physics. Starting from simple linear polymer models, we explain how specific folding properties emerge upon introducing loops and how this explains a variety of experimental observations. We also discuss different polymer models that describe chromatin folding and compare them to experimental data. Experimentally testing the predictions of such polymer models and their subsequent improvement on the basis of measurements provides a solid framework to begin to understand how our genome is folded and how folding relates to function.
01 Jul 2011
TL;DR: In this article, the authors describe computational approaches for integrating experimental data with polymer physics, thereby bridging the resolution gap for structural determination of genomes and genomic domains, which has been classically studied using polymer physics and molecular simulations.
Abstract: Over the last decade, and especially after the advent of fluorescent in situ hybridization imaging and chromosome conformation capture methods, the availability of experimental data on genome three-dimensional organization has dramatically increased. We now have access to unprecedented details of how genomes organize within the interphase nucleus. Development of new computational approaches to leverage this data has already resulted in the first three-dimensional structures of genomic domains and genomes. Such approaches expand our knowledge of the chromatin folding principles, which has been classically studied using polymer physics and molecular simulations. Our outlook describes computational approaches for integrating experimental data with polymer physics, thereby bridging the resolution gap for structural determination of genomes and genomic domains.
TL;DR: It is proposed that this unique and evolutionarily conserved genomic architecture of the vertebrate Irx clusters is essential for the coregulation of the first two genes and simultaneously maintains the third gene in a partially independent regulatory landscape.
Abstract: Developmental gene clusters are paradigms for the study of gene regulation; however, the mechanisms that mediate phenomena such as coregulation and enhancer sharing remain largely elusive. Here we address this issue by analysing the vertebrate Irx clusters. We first present a deep enhancer screen of a 2-Mbp span covering the IrxA cluster. Using chromosome conformation capture, we show that enhancer sharing is widespread within the cluster, explaining its evolutionarily conserved organization. We also identify a three-dimensional architecture, probably formed through interactions with CCCTC-binding factor, which is present within both Irx clusters of mouse, Xenopus and zebrafish. This architecture brings the promoters of the first two genes together in the same chromatin landscape. We propose that this unique and evolutionarily conserved genomic architecture of the vertebrate Irx clusters is essential for the coregulation of the first two genes and simultaneously maintains the third gene in a partially independent regulatory landscape.
TL;DR: This outlook describes computational approaches for integrating experimental data with polymer physics, thereby bridging the resolution gap for structural determination of genomes and genomic domains.
Abstract: Over the last decade, and especially after the advent of fluorescent in situ hybridization imaging and chromosome conformation capture methods, the availability of experimental data on genome three-dimensional organization has dramatically increased. We now have access to unprecedented details of how genomes organize within the interphase nucleus. Development of new computational approaches to leverage this data has already resulted in the first three-dimensional structures of genomic domains and genomes. Such approaches expand our knowledge of the chromatin folding principles, which has been classically studied using polymer physics and molecular simulations. Our outlook describes computational approaches for integrating experimental data with polymer physics, thereby bridging the resolution gap for structural determination of genomes and genomic domains.
TL;DR: It is proposed that chromatin globules are commonly formed along chromosomes, in a cell type specific pattern, as a result of frequent long-range interactions among active genes and nearby regulatory elements, consistent with recent observations obtained with genome-wide chromatin interaction mapping.
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TL;DR: In this article, the authors used a maximum likelihood framework to compare SCNA maps and three-dimensional genome architecture as determined by genome-wide chromosome conformation capture (HiC) and described by the proposed fractal-globule (FG) model.
Abstract: The rapid growth of cancer genome structural information provides an opportunity for a better understanding of the mutational mechanisms of genomic alterations in cancer and the forces of selection that act upon them. Here we test the evidence for two major forces, spatial chromosome structure and purifying (or negative) selection, that shape the landscape of somatic copy-number alterations (SCNAs) in cancer1. Using a maximum likelihood framework we compare SCNA maps and three-dimensional genome architecture as determined by genome-wide chromosome conformation capture (HiC) and described by the proposed fractal-globule (FG) model2. This analysis provides evidence that the distribution of chromosomal alterations in cancer is spatially related to three-dimensional genomic architecture and additionally suggests that purifying selection as well as positive selection shapes the landscape of SCNAs during somatic evolution of cancer cells.
TL;DR: Comparative chromatin immunoprecipitation with comprehensive sequencing with epigenetic histone modifications and chromatin conformation capture assays provides new insights into the correlation of endothelial‐expressed GATA2 binding, epigenetic modification, and the determination of endothelium cell specificity.
Abstract: GATA2 is well recognized as a key transcription factor and regulator of cell-type specificity and differentiation. Here, we carried out comparative chromatin immunoprecipitation with comprehensive sequencing (ChIP-seq) to determine genome-wide occupancy of GATA2 in endothelial cells and erythroids, and compared the occupancy to the respective gene expression profile in each cell type. Although GATA2 was commonly expressed in both cell types, different GATA2 bindings and distinct cell-specific gene expressions were observed. By using the ChIP-seq with epigenetic histone modifications and chromatin conformation capture assays; we elucidated the mechanistic regulation of endothelial-specific GATA2-mediated endomucin gene expression, that was regulated by the endothelial-specific chromatin loop with a GATA2-associated distal enhancer and core promoter. Knockdown of endomucin markedly attenuated endothelial cell growth, migration and tube formation. Moreover, abrogation of GATA2 in endothelium demonstrated not only a reduction of endothelial-specific markers, but also induction of mesenchymal transition promoting gene expression. Our findings provide new insights into the correlation of endothelial-expressed GATA2 binding, epigenetic modification, and the determination of endothelial cell specificity.
TL;DR: It is demonstrated that LPS stimulates chromosomal loops in the OPN promoter between NF-κB binding site and AP-1 binding site using chromosome conformation capture technology and identified an essential mechanism to remodel the local chromatin structures and spatial conformations to regulate LPS-induced OPN expression.
Abstract: Osteopontin (OPN) is expressed by various immune cells and modulates both innate and adaptive immune responses. However, the molecular mechanisms that control opn gene expression, especially at the chromatin level, remain largely unknown. We have previously demonstrated many specific cis- and trans-regulatory elements that determine the extent of endotoxin (LPS)-mediated induction of OPN synthesis in murine macrophages. In the present study, we confirm that NF-κB also plays an important role in the setting of LPS-stimulated OPN expression through binding to a distal regulatory element. Importantly, we demonstrate that LPS stimulates chromosomal loops in the OPN promoter between NF-κB binding site and AP-1 binding site using chromosome conformation capture technology. The crucial role of NF-κB and AP-1 in LPS-stimulated DNA looping was confirmed, as small interfering RNA knock-down of NF-κB p65 and AP-1 c-Jun exhibited decreased levels of DNA looping. Furthermore, we demonstrate that p300 can form a complex with NF-κB and AP-1 and is involved in DNA looping and LPS-induced OPN expression. Therefore, we have identified an essential mechanism to remodel the local chromatin structures and spatial conformations to regulate LPS-induced OPN expression.
TL;DR: It is shown that the promoter, upstream region and terminator R3 of active rRNA genes are held together spatially throughout the cell cycle, forming a stable core around which the transcribed region is organized.
Abstract: The Christmas tree view of active ribosomal RNA (rRNA) genes suggests a gene topology in which a large number of nascent rRNA transcripts are prevented from intertwining. The way in which this is achieved has remained unclear. By using a combination of chromatin immunoprecipitation and chromosome conformation capture techniques, we show that the promoter, upstream region and terminator R3 of active rRNA genes are held together spatially throughout the cell cycle, forming a stable core around which the transcribed region is organized. We suggest a new core–helix model for the topology of rRNA genes, that provides a structural basis for the productive synthesis or rRNA.
TL;DR: Data reveal a novel imprinted domain in the Igf2/H19 locus and lead us to propose a model for chromatin folding of this locus on the paternal chromosome.
Abstract: Parental genomic imprinting at the Igf2/H19 locus is controlled by a methylation-sensitive CTCF insulator that prevents the access of downstream enhancers to the Igf2 gene on the maternal chromosome. However, on the paternal chromosome, it remains unclear whether long-range interactions with the enhancers are restricted to the Igf2 promoters or whether they encompass the entire gene body. Here, using the quantitative chromosome conformation capture assay, we show that, in the mouse liver, the endodermal enhancers have low contact frequencies with the Igf2 promoters but display, on the paternal chromosome, strong interactions with the intragenic differentially methylated regions 1 and 2. Interestingly, we found that enhancers also interact with a so-far poorly characterized intergenic region of the locus that produces a novel imprinted long non-coding transcript that we named the paternally expressed Igf2/H19 intergenic transcript (PIHit) RNA. PIHit is expressed exclusively from the paternal chromosome, contains a novel discrete differentially methylated region in a highly conserved sequence and, surprisingly, does not require an intact ICR/H19 gene region for its imprinting. Altogether, our data reveal a novel imprinted domain in the Igf2/H19 locus and lead us to propose a model for chromatin folding of this locus on the paternal chromosome.
TL;DR: A novel mechanism of BCL2 regulation is revealed and SATB1-mediated long-range interaction with the regulation of a gene controlling apoptosis pathway is mechanistically link for the first time.
Abstract: The 279-bp major breakpoint region (mbr) within the 3′-untranslated region (3′-UTR) of the BCL2 gene is a binding site of special AT-rich sequence binding protein 1 (SATB1) that is well known to participate in the long-range regulation of gene transcription. Our previous studies have revealed that the mbr could regulate BCL2 transcription over a 200-kb distance and this regulatory function was closely related to SATB1. This study is to explore the underlying mechanism and its relevance to cellular apoptosis. With chromosome conformation capture (3C) and chromatin immunoprecipitation (ChIP) assays we demonstrated that the mbr could physically interact with BCL2 promoter through SATB1-mediated chromatin looping, which was required for epigenetic modifications of the promoter, CREB accessibility and high expression of the BCL2 gene. During early apoptosis, SATB1 was a key regulator of BCL2 expression. Inhibition of SATB1 cleavage by treatment of cells with a caspase-6 inhibitor or overexpression of mutant SATB1 that was resistant to caspase-6, inhibited disassembly of the SATB1-mediated chromatin loop and restored the BCL2 mRNA level in Jurkat cells. These data revealed a novel mechanism of BCL2 regulation and mechanistically link SATB1-mediated long-range interaction with the regulation of a gene controlling apoptosis pathway for the first time.
TL;DR: The characterization of five novel WREs that localize to a region over 400 kb upstream from MYC suggest that the architecture of the MYC promoter is comprised of distal elements that are juxtaposed through large chromatin loops and that ß-catenin/TCF4 complexes utilize this conformation to activate MYC expression in colon cancer cells.
Abstract: Inappropriate activation of c-Myc (MYC) gene expression by the Wnt/s-catenin signaling pathway is required for colorectal carcinogenesis. The elevated MYC levels in colon cancer cells are attributed in part to s-catenin/TCF4 transcription complexes that are assembled at proximal Wnt/s-catenin responsive enhancers (WREs). Recent studies suggest that additional WREs that control MYC expression reside far upstream of the MYC transcription start site. Here, I report the characterization of five novel WREs that localize to a region over 400 kb upstream from MYC. These WREs harbor nucleosomes with post-translational histone modifications that demarcate enhancer and gene promoter regions. Using quantitative chromatin conformation capture, I show that the distal WREs are aligned with the MYC promoter through large chromatin loops. The chromatin loops are not restricted to colon cancer cells, but are also found in kidney epithelial and lung fibroblast cell lines that lack de-regulated Wnt signaling and nuclear s-catenin/TCF4 complexes. While each chromatin loop is detected in quiescent cells, the positioning of three of the five distal enhancers with the MYC promoter is induced by serum mitogens. These findings suggest that the architecture of the MYC promoter is comprised of distal elements that are juxtaposed through large chromatin loops and that s-catenin/TCF4 complexes utilize this conformation to activate MYC expression in colon cancer cells.
TL;DR: An overview of known lncRNA transcription regulation activities is provided and potential mechanisms by which ncRNAs might exert three-dimensional transcriptional control are discussed and what recent studies have revealed about their role in shaping the authors' genome are discussed.
Abstract: The human genome must be tightly packaged in order to fit inside the nucleus of a cell. Genome organization is functional rather than random, which allows for the proper execution of gene expression programs and other biological processes. Recently, three-dimensional chromatin organization has emerged as an important transcriptional control mechanism. For example, enhancers were shown to regulate target genes by physically interacting with them regardless of their linear distance and even if located on different chromosomes. These chromatin contacts can be measured with the "chromosome conformation capture" (3C) technology and other 3C-related techniques. Given the recent innovation of 3C-derived approaches, it is not surprising that we still know very little about the structure of our genome at high-resolution. Even less well understood is whether there exist distinct types of chromatin contacts and importantly, what regulates them. A new form of regulation involving the expression of long non-coding RNAs (lncRNAs) was recently identified. lncRNAs are a very abundant class of non-coding RNAs that are often expressed in a tissue-specific manner. Although their different subcellular localizations point to their involvement in numerous cellular processes, it is clear that lncRNAs play an important role in regulating gene expression. How they control transcription however is mostly unknown. In this review, we provide an overview of known lncRNA transcription regulation activities. We also discuss potential mechanisms by which ncRNAs might exert three-dimensional transcriptional control and what recent studies have revealed about their role in shaping our genome.
TL;DR: PXR represses human SULT1E1, possibly attenuating the inactivation of estrogen, and removes the ability of HNF4α to loop the promoter and that of PXR to repress the promoter activity.
Abstract: Pregnane X receptor (PXR), acting as a xenobiotic-activated transcription factor, regulates the hepatic metabolism of therapeutics as well as endobiotics such as steroid hormones. Given our finding that PXR activation by rifampicin (RIF) represses the estrogen sulfotransferase (SULT1E1) gene in human primary hepatocytes and hepatocellular carcinoma Huh7 cells, here we have investigated the molecular mechanism of this repression. First the PXR-responsive enhancer was delineated to a 100 bp sequence (-1000/-901), which contains three half sites that constitute the overlapping direct repeat 1 (DR1) and direct repeat 2 (DR2) motifs and two forkhead factor binding sites. siRNA knockdown, chromatin immunoprecipitation and chromatin conformation capture assays were employed to demonstrate that hepatocyte nuclear factor 4α (HNF4α) bound to the PXR-responsive enhancer, and activated the enhancer by looping its position close to the proximal promoter. Upon activation by RIF, PXR indirectly interacted with the enhancer, decreasing the interaction with HNF4α and dissolving the looped SULT1E1 promoter with deacetylation of histone 3. Removal of the DR sites from the enhancer hampers the ability of HNF4α to loop the promoter and that of PXR to repress the promoter activity. Thus, PXR represses human SULT1E1, possibly attenuating the inactivation of estrogen.
TL;DR: It is shown that while loop formation is not in itself sufficient to explain action at a distance, incorporating transient nonspecific and moderate attractive interactions between the chromatin fibers strongly enhances long-distance regulatory interactions and is sufficient to generate a euchromatin-like state.
Abstract: Long-distance regulatory interactions between enhancers and their target genes are commonplace in higher eukaryotes. Interposed boundaries or insulators are able to block these long-distance regulatory interactions. The mechanistic basis for insulator activity and how it relates to enhancer action-at-a-distance remains unclear. Here we explore the idea that topological loops could simultaneously account for regulatory interactions of distal enhancers and the insulating activity of boundary elements. We show that while loop formation is not in itself sufficient to explain action at a distance, incorporating transient nonspecific and moderate attractive interactions between the chromatin fibers strongly enhances long-distance regulatory interactions and is sufficient to generate a euchromatin-like state. Under these same conditions, the subdivision of the loop into two topologically independent loops by insulators inhibits interdomain interactions. The underlying cause of this effect is a suppression of crossings in the contact map at intermediate distances. Thus our model simultaneously accounts for regulatory interactions at a distance and the insulator activity of boundary elements. This unified model of the regulatory roles of chromatin loops makes several testable predictions that could be confronted with in vitro experiments, as well as genomic chromatin conformation capture and fluorescent microscopic approaches.
TL;DR: In vivo data provide novel evidence about the role of RUNX1 in mediating interactions between distal and proximal elements of the HSC gene CD34, and demonstrate by chromosome conformation capture assays in LT‐HSCs that the DRE physically interacts with the human CD34 promoter.
Abstract: The transcription factor RUNX1 is essential to establish the haematopoietic gene expression programme; however, the mechanism of how it activates transcription of haematopoietic stem cell (HSC) genes is still elusive. Here, we obtained novel insights into RUNX1 function by studying regulation of the human CD34 gene, which is expressed in HSCs. Using transgenic mice carrying human CD34 PAC constructs, we identified a novel downstream regulatory element (DRE), which is bound by RUNX1 and is necessary for human CD34 expression in long-term (LT)-HSCs. Conditional deletion of Runx1 in mice harbouring human CD34 promoter–DRE constructs abrogates human CD34 expression. We demonstrate by chromosome conformation capture assays in LT-HSCs that the DRE physically interacts with the human CD34 promoter. Targeted mutagenesis of RUNX binding sites leads to perturbation of this interaction and decreased human CD34 expression in LT-HSCs. Overall, our in vivo data provide novel evidence about the role of RUNX1 in mediating interactions between distal and proximal elements of the HSC gene CD34.
TL;DR: Results are reviewed and some additional insights are provided that elucidate the relationship between structure and function at different hierarchical levels of genome organization.
Abstract: Eukaryotic genomes possess an elaborate and dynamic higher-order structure within the limiting confines of the cell nucleus. Knowledge of the physical principles and the molecular machinery that govern the 3D organization of this structure and its regulation are key to understanding the relationship between genome structure and function. Elegant microscopy and chromosome conformation capture techniques supported by analysis based on polymer models are important steps in this direction. Here, we review results from these efforts and provide some additional insights that elucidate the relationship between structure and function at different hierarchical levels of genome organization.