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Articles by Job Dekker in JoVE

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Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

1Program in Gene Function and Expression, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 2Broad Institute of Harvard and Massachusetts Institute of Technology, 3Division of Health Sciences and Technology, Massachusetts Institute of Technology, 4Program for Evolutionary Dynamics, Department of Organismic and Evolutionary Biology, Department of Mathematics, Harvard University, 5Department of Applied Mathematics, Harvard University, 6Department of Physics, Massachusetts Institute of Technology, 7Department of Systems Biology, Harvard Medical School, 8Department of Biology, Massachusetts Institute of Technology


JoVE 1869

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Other articles by Job Dekker on PubMed

Capturing Chromosome Conformation

We describe an approach to detect the frequency of interaction between any two genomic loci. Generation of a matrix of interaction frequencies between sites on the same or different chromosomes reveals their relative spatial disposition and provides information about the physical properties of the chromatin fiber. This methodology can be applied to the spatial organization of entire genomes in organisms from bacteria to human. Using the yeast Saccharomyces cerevisiae, we could confirm known qualitative features of chromosome organization within the nucleus and dynamic changes in that organization during meiosis. We also analyzed yeast chromosome III at the G1 stage of the cell cycle. We found that chromatin is highly flexible throughout. Furthermore, functionally distinct AT- and GC-rich domains were found to exhibit different conformations, and a population-average 3D model of chromosome III could be determined. Chromosome III emerges as a contorted ring.

A Closer Look at Long-range Chromosomal Interactions

Higher-order chromosome organization is emerging as a major determinant of gene regulation. Although the structure of chromatin at the level of individual nucleosomes has been studied in considerable detail, less is known about higher levels of organization. Two new methods have been developed that can be used to obtain detailed information about the higher-order folding of chromatin. Using these methods, long-range looping interactions have been shown to occur upon activation of the murine beta-globin locus, explaining the long-standing question of how gene regulatory elements can act at large genomic distances from their target genes.

A Mechanical Basis for Chromosome Function

We propose that chromosome function is governed by internal mechanical forces generated by programmed tendencies for expansion of the DNA/chromatin fiber against constraining features.

Proximity Among Distant Regulatory Elements at the Beta-globin Locus Requires GATA-1 and FOG-1

Recent evidence suggests that long-range enhancers and gene promoters are in close proximity, which might reflect the formation of chromatin loops. Here, we examined the mechanism for DNA looping at the beta-globin locus. By using chromosome conformation capture (3C), we show that the hematopoietic transcription factor GATA-1 and its cofactor FOG-1 are required for the physical interaction between the beta-globin locus control region (LCR) and the beta-major globin promoter. Kinetic studies reveal that GATA-1-induced loop formation correlates with the onset of beta-globin transcription and occurs independently of new protein synthesis. GATA-1 occupies the beta-major globin promoter normally in fetal liver erythroblasts from mice lacking the LCR, suggesting that GATA-1 binding to the promoter and LCR are independent events that occur prior to loop formation. Together, these data demonstrate that GATA-1 and FOG-1 are essential anchors for a tissue-specific chromatin loop, providing general insights into long-range enhancer function.

Mapping Chromatin Interactions by Chromosome Conformation Capture

Chromosome conformation capture (3C) is one of the only techniques that allows for analysis of an intermediate level of chromosome structure ranging from a few to hundreds of kilobases, a level most relevant for gene regulation. The 3C technique is used to detect physical interactions between sequence elements that are located on the same or on different chromosomes. For instance, physical interactions between distant enhancers and target genes can be measured. The 3C assay uses formaldehyde cross-linking to trap connections between chromatin segments that can, after a number of manipulations, be detected by PCR. This unit describes detailed protocols for performing 3C with yeast Saccharomyces cerevisiae and mammalian cells.

Chromosome Conformation Capture Carbon Copy (5C): a Massively Parallel Solution for Mapping Interactions Between Genomic Elements

Physical interactions between genetic elements located throughout the genome play important roles in gene regulation and can be identified with the Chromosome Conformation Capture (3C) methodology. 3C converts physical chromatin interactions into specific ligation products, which are quantified individually by PCR. Here we present a high-throughput 3C approach, 3C-Carbon Copy (5C), that employs microarrays or quantitative DNA sequencing using 454-technology as detection methods. We applied 5C to analyze a 400-kb region containing the human beta-globin locus and a 100-kb conserved gene desert region. We validated 5C by detection of several previously identified looping interactions in the beta-globin locus. We also identified a new looping interaction in K562 cells between the beta-globin Locus Control Region and the gamma-beta-globin intergenic region. Interestingly, this region has been implicated in the control of developmental globin gene switching. 5C should be widely applicable for large-scale mapping of cis- and trans- interaction networks of genomic elements and for the study of higher-order chromosome structure.

The Active FMR1 Promoter is Associated with a Large Domain of Altered Chromatin Conformation with Embedded Local Histone Modifications

We have analyzed the effects of gene activation on chromatin conformation throughout an approximately 170-kb region comprising the human fragile X locus, which includes a single expressed gene, FMR1 (fragile X mental retardation 1). We have applied three approaches: (i) chromosome conformation capture, which assesses relative interaction frequencies of chromatin segments; (ii) an extension of this approach that identifies domains whose conformation differs from the average, which we developed and named chromosome conformation profiling; and (iii) ChIP analysis of histone modifications. We find that, in normal cells where FMR1 is active, the FMR1 promoter is at the center of a large ( approximately 50 kb) domain of reduced intersegment interactions. In contrast, in fragile X cells where FMR1 is inactive, chromatin conformation is uniform across the entire region. We also find that histone modifications that are characteristic of active genes occur tightly localized around the FMR1 promoter in normal cells and are absent in fragile X cells. Therefore, the expression-correlated change in conformation affects a significantly larger domain than that marked by histone modifications. Domain-wide changes in interaction probability could reflect increased chromatin expansion and may also be related to an altered spatial disposition that results in increased intermingling with unrelated loci. The described approaches are widely applicable to the study of conformational changes of any locus of interest.

The Three 'C' S of Chromosome Conformation Capture: Controls, Controls, Controls

Transcription regulation in higher eukaryotes is controlled by regulatory elements such as enhancers that are recognized by transcription factors. In many cases regulatory elements can be located at distances up to several megabases from their target genes. Recent evidence shows that long-range control of gene expression can be mediated through direct physical interactions between genes and these regulatory elements. Such looping interactions can be detected using the chromosome conformation capture (3C) methodology. Although 3C is experimentally straightforward, to draw meaningful conclusions one must carefully design 3C experiments and implement the conscientious use of controls. The general guidelines presented here should help experimental design and minimize misinterpretation of 3C experiments.

Chromosome Conformation Capture Carbon Copy Technology

Chromosome conformation capture (3C) is used to quantify physical DNA contacts in vivo at high resolution. 3C was first used in yeast to map the spatial chromatin organization of chromosome III, and in higher eukaryotes to demonstrate that genomic DNA elements regulate target genes by physically interacting with them. 3C has been widely adopted for small-scale analysis of functional chromatin interactions along (cis) or between (trans) chromosomes. For larger-scale applications, chromosome conformation capture carbon copy (5C) combines 3C with ligation-mediated amplification (LMA) to simultaneously quantify hundreds of thousands of physical DNA contacts by microarray or ultra-high-throughput DNA sequencing. 5C allows the mapping of extensive networks of physical interactions among large sets of genomic elements throughout the genome. Such networks can provide important biological insights, e.g., by identifying relationships between regulatory elements and their target genes. This unit describes 5C for large-scale analysis of cis- and trans-chromatin interactions in mammalian cells.

Polycomb Response Elements Mediate the Formation of Chromosome Higher-order Structures in the Bithorax Complex

In Drosophila, the function of the Polycomb group genes (PcGs) and their target sequences (Polycomb response elements (PREs)) is to convey mitotic heritability of transcription programmes--in particular, gene silencing. As part of the mechanisms involved, PREs are thought to mediate this transcriptional memory function by building up higher-order structures in the nucleus. To address this question, we analysed in vivo the three-dimensional structure of the homeotic locus bithorax complex (BX-C) by combining chromosome conformation capture (3C) with fluorescent in situ hybridization (FISH) and FISH immunostaining (FISH-I) analysis. We found that, in the repressed state, all major elements that have been shown to bind PcG proteins, including PREs and core promoters, interact at a distance, giving rise to a topologically complex structure. We show that this structure is important for epigenetic silencing of the BX-C, as we find that major changes in higher-order structures must occur to stably maintain alternative transcription states, whereas histone modification and reduced levels of PcG proteins determine an epigenetic switch that is only partially heritable.

Quantitative Analysis of Chromosome Conformation Capture Assays (3C-qPCR)

Chromosome conformation capture (3C) technology is a pioneering methodology that allows in vivo genomic organization to be explored at a scale encompassing a few tens to a few hundred kilobase-pairs. Understanding the folding of the genome at this scale is particularly important in mammals where dispersed regulatory elements, like enhancers or insulators, are involved in gene regulation. 3C technology involves formaldehyde fixation of cells, followed by a polymerase chain reaction (PCR)-based analysis of the frequency with which pairs of selected DNA fragments are crosslinked in the population of cells. Accurate measurements of crosslinking frequencies require the best quantification techniques. We recently adapted the real-time TaqMan PCR technology to the analysis of 3C assays, resulting in a method that more accurately determines crosslinking frequencies than current semiquantitative 3C strategies that rely on measuring the intensity of ethidium bromide-stained PCR products separated by gel electrophoresis. Here, we provide a detailed protocol for this method, which we have named 3C-qPCR. Once preliminary controls and optimizations have been performed, the whole procedure (3C assays and quantitative analyses) can be completed in 7-9 days.

GC- and AT-rich Chromatin Domains Differ in Conformation and Histone Modification Status and Are Differentially Modulated by Rpd3p

Base-composition varies throughout the genome and is related to organization of chromosomes in distinct domains (isochores). Isochore domains differ in gene expression levels, replication timing, levels of meiotic recombination and chromatin structure. The molecular basis for these differences is poorly understood.

Identification and Analysis of Functional Elements in 1% of the Human Genome by the ENCODE Pilot Project

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.

Mapping Networks of Physical Interactions Between Genomic Elements Using 5C Technology

Genomic elements separated by large genomic distances can physically interact to mediate long-range gene regulation and other chromosomal processes. Interactions between genomic elements can be detected using the chromosome conformation capture (3C) technology. We recently developed a high-throughput adaptation of 3C, 3C-carbon copy (5C), that is used to measure networks of millions of chromatin interactions in parallel. As in 3C, cells are treated with formaldehyde to cross-link chromatin interactions. The chromatin is solubilized, digested with a restriction enzyme and ligated at low DNA concentration to promote intra-molecular ligation of cross-linked DNA fragments. Ligation products are subsequently purified to generate a 3C library. The 5C technology then employs highly multiplexed ligation-mediated amplification (LMA) to detect and amplify 3C ligation junctions. The resulting 5C library of ligated primers is analyzed using either microarray detection or ultra-high-throughput DNA sequencing. The 5C protocol described here can be completed in 13 d.

Long-range Chromosomal Interactions and Gene Regulation

Over the last few years important new insights into the process of long-range gene regulation have been obtained. Gene regulatory elements are found to engage in direct physical interactions with distant target genes and with loci on other chromosomes to modulate transcription. An overview of recently discovered long-range chromosomal interactions is presented, and a network approach is proposed to unravel gene-element relationships. Gene expression is controlled by regulatory elements that can be located far away along the chromosome or in some cases even on other chromosomes. Genes and regulatory elements physically associate with each other resulting in complex genome-wide networks of chromosomal interactions. Here we describe several well-characterized cases of long-range interactions involved in the activation and repression of transcription. We speculate on how these interactions may affect gene expression and outline possible mechanisms that may facilitate encounters between distant elements. Finally, we propose that a genome-wide network analysis may provide new insights into the logic of long-range gene regulation.

Mapping in Vivo Chromatin Interactions in Yeast Suggests an Extended Chromatin Fiber with Regional Variation in Compaction

The higher order arrangement of nucleosomes and the level of compaction of the chromatin fiber play important roles in the control of gene expression and other genomic activities. Analysis of chromatin in vitro has suggested that under near physiological conditions chromatin fibers can become highly compact and that the level of compaction can be modulated by histone modifications. However, less is known about the organization of chromatin fibers in living cells. Here, we combine chromosome conformation capture (3C) data with distance measurements and polymer modeling to determine the in vivo mass density of a transcriptionally active 95-kb GC-rich domain on chromosome III of the yeast Saccharomyces cerevisiae. In contrast to previous reports, we find that yeast does not form a compact fiber but that chromatin is extended with a mass per unit length that is consistent with a rather loose arrangement of nucleosomes. Analysis of 3C data from a neighboring AT-rich chromosomal domain indicates that chromatin in this domain is more compact, but that mass density is still well below that of a canonical 30 nm fiber. Our approach should be widely applicable to scale 3C data to real spatial dimensions, which will facilitate the quantification of the effects of chromatin modifications and transcription on chromatin fiber organization.

Gene Regulation in the Third Dimension

Analysis of the spatial organization of chromosomes reveals complex three-dimensional networks of chromosomal interactions. These interactions affect gene expression at multiple levels, including long-range control by distant enhancers and repressors, coordinated expression of genes, and modification of epigenetic states. Major challenges now include deciphering the mechanisms by which loci come together and understanding the functional consequences of these often transient associations.

A Mechanism for Ikaros Regulation of Human Globin Gene Switching

The human beta globin locus consists of an upstream LCR and functional genes arranged sequentially in the order of their expression during development: 5'-HBE1, HBG2, HBG1, HBD, HBB-3'. Haemoglobin switching entails the successive recruitment of these genes into an active chromatin hub (ACH). Here we show that the transcription factor Ikaros plays a major role in the formation of the beta-globin ACH, and in haemoglobin switching. In Plastic mice, where the DNA-binding region of Ikaros is disrupted by a point mutation, there is concomitant marked down-regulation of HBB, and up-regulation of HBG expression. We show for the first time Ikaros and its family member Eos, bind to critical cis elements implicated in haemoglobin switching and deletional hereditary persistence of fetal haemoglobin (HPFH). Chromatin conformation capture (3C) data demonstrated that Ikaros facilitates long-distance DNA looping between the LCR and a region upstream of HBD. This study provides new insights into the mechanism of stage-specific assembly of the beta-globin ACH, and HPFH.

Comprehensive Mapping of Long-range Interactions Reveals Folding Principles of the Human Genome

We describe Hi-C, a method that probes the three-dimensional architecture of whole genomes by coupling proximity-based ligation with massively parallel sequencing. We constructed spatial proximity maps of the human genome with Hi-C at a resolution of 1 megabase. These maps confirm the presence of chromosome territories and the spatial proximity of small, gene-rich chromosomes. We identified an additional level of genome organization that is characterized by the spatial segregation of open and closed chromatin to form two genome-wide compartments. At the megabase scale, the chromatin conformation is consistent with a fractal globule, a knot-free, polymer conformation that enables maximally dense packing while preserving the ability to easily fold and unfold any genomic locus. The fractal globule is distinct from the more commonly used globular equilibrium model. Our results demonstrate the power of Hi-C to map the dynamic conformations of whole genomes.

My5C: Web Tools for Chromosome Conformation Capture Studies

Determining Spatial Chromatin Organization of Large Genomic Regions Using 5C Technology

Spatial organization of chromatin plays an important role at multiple levels of genome regulation. On a global scale, its function is evident in processes like metaphase and chromosome segregation. On a detailed level, long-range interactions between regulatory elements and promoters are essential for proper gene regulation. Microscopic techniques like FISH can detect chromatin contacts, although the resolution is generally low making detection of enhancer-promoter interaction difficult. The 3C methodology allows for high-resolution analysis of chromatin interactions. 3C is now widely used and has revealed that long-range looping interactions between genomic elements are widespread. However, studying chromatin interactions in large genomic regions by 3C is very labor intensive. This limitation is overcome by the 5C technology. 5C is an adaptation of 3C, in which the concurrent use of thousands of primers permits the simultaneous detection of millions of chromatin contacts. The design of the 5C primers is critical because this will determine which and how many chromatin interactions will be examined in the assay. Starting material for 5C is a 3C template. To make a 3C template, chromatin interactions in living cells are cross-linked using formaldehyde. Next, chromatin is digested and subsequently ligated under conditions favoring ligation events between cross-linked fragments. This yields a genome-wide 3C library of ligation products representing all chromatin interactions in vivo. 5C then employs multiplex ligation-mediated amplification to detect, in a single assay, up to millions of unique ligation products present in the 3C library. The resulting 5C library can be analyzed by microarray analysis or deep sequencing. The observed abundance of a 5C product is a measure of the interaction frequency between the two corresponding chromatin fragments. The power of the 5C technique described in this chapter is the high-throughput, high-resolution, and quantitative way in which the spatial organization of chromatin can be examined.

Disease-causing 7.4 Kb Cis-regulatory Deletion Disrupting Conserved Non-coding Sequences and Their Interaction with the FOXL2 Promotor: Implications for Mutation Screening

To date, the contribution of disrupted potentially cis-regulatory conserved non-coding sequences (CNCs) to human disease is most likely underestimated, as no systematic screens for putative deleterious variations in CNCs have been conducted. As a model for monogenic disease we studied the involvement of genetic changes of CNCs in the cis-regulatory domain of FOXL2 in blepharophimosis syndrome (BPES). Fifty-seven molecularly unsolved BPES patients underwent high-resolution copy number screening and targeted sequencing of CNCs. Apart from three larger distant deletions, a de novo deletion as small as 7.4 kb was found at 283 kb 5' to FOXL2. The deletion appeared to be triggered by an H-DNA-induced double-stranded break (DSB). In addition, it disrupts a novel long non-coding RNA (ncRNA) PISRT1 and 8 CNCs. The regulatory potential of the deleted CNCs was substantiated by in vitro luciferase assays. Interestingly, Chromosome Conformation Capture (3C) of a 625 kb region surrounding FOXL2 in expressing cellular systems revealed physical interactions of three upstream fragments and the FOXL2 core promoter. Importantly, one of these contains the 7.4 kb deleted fragment. Overall, this study revealed the smallest distant deletion causing monogenic disease and impacts upon the concept of mutation screening in human disease and developmental disorders in particular.

Gene Dates, Parties and Galas. Symposium on Chromatin Dynamics and Higher Order Organization

Yeast Silent Mating Type Loci Form Heterochromatic Clusters Through Silencer Protein-dependent Long-range Interactions

The organization of eukaryotic genomes is characterized by the presence of distinct euchromatic and heterochromatic sub-nuclear compartments. In Saccharomyces cerevisiae heterochromatic loci, including telomeres and silent mating type loci, form clusters at the nuclear periphery. We have employed live cell 3-D imaging and chromosome conformation capture (3C) to determine the contribution of nuclear positioning and heterochromatic factors in mediating associations of the silent mating type loci. We identify specific long-range interactions between HML and HMR that are dependent upon silencing proteins Sir2p, Sir3p, and Sir4p as well as Sir1p and Esc2p, two proteins involved in establishment of silencing. Although clustering of these loci frequently occurs near the nuclear periphery, colocalization can occur equally at more internal positions and is not affected in strains deleted for membrane anchoring proteins yKu70p and Esc1p. In addition, appropriate nucleosome assembly plays a role, as deletion of ASF1 or combined disruption of the CAF-1 and HIR complexes abolishes the HML-HMR interaction. Further, silencer proteins are required for clustering, but complete loss of clustering in asf1 and esc2 mutants had only minor effects on silencing. Our results indicate that formation of heterochromatic clusters depends on correctly assembled heterochromatin at the silent loci and, in addition, identify an Asf1p-, Esc2p-, and Sir1p-dependent step in heterochromatin formation that is not essential for gene silencing but is required for long-range interactions.

Mechanisms That Regulate Localization of a DNA Double-strand Break to the Nuclear Periphery

DNA double-strand breaks (DSBs) are among the most deleterious forms of DNA lesions in cells. Here we induced site-specific DSBs in yeast cells and monitored chromatin dynamics surrounding the DSB using Chromosome Conformation Capture (3C). We find that formation of a DSB within G1 cells is not sufficient to alter chromosome dynamics. However, DSBs formed within an asynchronous cell population result in large decreases in both intra- and interchromosomal interactions. Using live cell microscopy, we find that changes in chromosome dynamics correlate with relocalization of the DSB to the nuclear periphery. Sequestration to the periphery requires the nuclear envelope protein, Mps3p, and Mps3p-dependent tethering delays recombinational repair of a DSB and enhances gross chromosomal rearrangements. Furthermore, we show that components of the telomerase machinery are recruited to a DSB and that telomerase recruitment is required for its peripheral localization. Based on these findings, we propose that sequestration of unrepaired or slowly repaired DSBs to the nuclear periphery reflects a competition between alternative repair pathways.

Mapping Cis- and Trans- Chromatin Interaction Networks Using Chromosome Conformation Capture (3C)

Expression of genes can be controlled by regulatory elements that are located at large genomic distances from their target genes (in cis), or even on different chromosomes (in trans). Regulatory elements can act at large genomic distances by engaging in direct physical interactions with their target genes resulting in the formation of chromatin loops. Thus, genes and their regulatory elements come in close spatial proximity irrespective of their relative genomic positions. Analysis of interactions between genes and elements will reveal which elements regulate each gene, and will provide fundamental insights into the spatial organization of chromosomes in general. Long-range cis- and trans- interactions can be studied at high resolution using chromosome conformation capture (3C) technology. 3C employs formaldehyde crosslinking to trap physical interactions between loci located throughout the genome. Crosslinked cells are solubilized and chromatin is digested with a restriction enzyme. Chromatin is subsequently ligated under conditions that favor intramolecular ligation. After reversal of the crosslinks, the DNA is purified and interaction frequencies between specific chromosomal loci are determined by quantifying the amounts of corresponding ligation products using polymerase chain reaction (PCR). This chapter describes detailed protocols for 3C analysis of chromatin interactions in the yeast Saccharomyces cerevisiae and in mammalian cells.

Sister Cohesion and Structural Axis Components Mediate Homolog Bias of Meiotic Recombination

Meiotic double-strand break (DSB)-initiated recombination must occur between homologous maternal and paternal chromosomes ("homolog bias"), even though sister chromatids are present. Through physical recombination analyses, we show that sister cohesion, normally mediated by meiotic cohesin Rec8, promotes "sister bias"; that meiosis-specific axis components Red1/Mek1kinase counteract this effect, thereby satisfying an essential precondition for homolog bias; and that other components, probably recombinosome-related, directly ensure homolog partner selection. Later, Rec8 acts positively to ensure maintenance of bias. These complexities mirror opposing dictates for global sister cohesion versus local separation and differentiation of sisters at recombination sites. Our findings support DSB formation within axis-tethered recombinosomes containing both sisters and ensuing programmed sequential release of "first" and "second" DSB ends. First-end release would create a homology-searching "tentacle." Rec8 and Red1/Mek1 also independently license recombinational progression and abundantly localize to different domains. These domains could comprise complementary environments that integrate inputs from DSB repair and mitotic chromosome morphogenesis into the complete meiotic program.

Genomics Tools for Unraveling Chromosome Architecture

The spatial organization of chromosomes inside the cell nucleus is still poorly understood. This organization is guided by intra- and interchromosomal contacts and by interactions of specific chromosomal loci with relatively fixed nuclear 'landmarks' such as the nuclear envelope and the nucleolus. Researchers have begun to use new molecular genome-wide mapping techniques to uncover both types of molecular interactions, providing insights into the fundamental principles of interphase chromosome folding.

Mediator and Cohesin Connect Gene Expression and Chromatin Architecture

Transcription factors control cell-specific gene expression programs through interactions with diverse coactivators and the transcription apparatus. Gene activation may involve DNA loop formation between enhancer-bound transcription factors and the transcription apparatus at the core promoter, but this process is not well understood. Here we report that mediator and cohesin physically and functionally connect the enhancers and core promoters of active genes in murine embryonic stem cells. Mediator, a transcriptional coactivator, forms a complex with cohesin, which can form rings that connect two DNA segments. The cohesin-loading factor Nipbl is associated with mediator-cohesin complexes, providing a means to load cohesin at promoters. DNA looping is observed between the enhancers and promoters occupied by mediator and cohesin. Mediator and cohesin co-occupy different promoters in different cells, thus generating cell-type-specific DNA loops linked to the gene expression program of each cell.

Chemical Genetic Strategy Identifies Histone Deacetylase 1 (HDAC1) and HDAC2 As Therapeutic Targets in Sickle Cell Disease

The worldwide burden of sickle cell disease is enormous, with over 200,000 infants born with the disease each year in Africa alone. Induction of fetal hemoglobin is a validated strategy to improve symptoms and complications of this disease. The development of targeted therapies has been limited by the absence of discrete druggable targets. We developed a unique bead-based strategy for the identification of inducers of fetal hemoglobin transcripts in primary human erythroid cells. A small-molecule screen of bioactive compounds identified remarkable class-associated activity among histone deacetylase (HDAC) inhibitors. Using a chemical genetic strategy combining focused libraries of biased chemical probes and reverse genetics by RNA interference, we have identified HDAC1 and HDAC2 as molecular targets mediating fetal hemoglobin induction. Our findings suggest the potential of isoform-selective inhibitors of HDAC1 and HDAC2 for the treatment of sickle cell disease.

Integrating One-dimensional and Three-dimensional Maps of Genomes

Genomes exist in vivo as complex physical structures, and their functional output (i.e. the gene expression profile of a cell) is related to their spatial organization inside the nucleus as well as to local chromatin status. Chromatin modifications and chromosome conformation are distinct in different tissues and cell types, which corresponds closely with the diversity in gene-expression patterns found in different tissues of the body. The biological processes and mechanisms driving these general correlations are currently the topic of intense study. An emerging theme is that genome compartmentalization - both along the linear length of chromosomes, and in three dimensions by the spatial colocalization of chromatin domains and genomic loci from across the genome - is a crucial parameter in regulating genome expression. In this Commentary, we propose that a full understanding of genome regulation requires integrating three different types of data: first, one-dimensional data regarding the state of local chromatin - such as patterns of protein binding along chromosomes; second, three-dimensional data that describe the population-averaged folding of chromatin inside cells and; third, single-cell observations of three-dimensional spatial colocalization of genetic loci and trans factors that reveal information about their dynamics and frequency of colocalization.

Cell-type-specific Long-range Looping Interactions Identify Distant Regulatory Elements of the CFTR Gene

Identification of regulatory elements and their target genes is complicated by the fact that regulatory elements can act over large genomic distances. Identification of long-range acting elements is particularly important in the case of disease genes as mutations in these elements can result in human disease. It is becoming increasingly clear that long-range control of gene expression is facilitated by chromatin looping interactions. These interactions can be detected by chromosome conformation capture (3C). Here, we employed 3C as a discovery tool for identification of long-range regulatory elements that control the cystic fibrosis transmembrane conductance regulator gene, CFTR. We identified four elements in a 460-kb region around the locus that loop specifically to the CFTR promoter exclusively in CFTR expressing cells. The elements are located 20 and 80 kb upstream; and 109 and 203 kb downstream of the CFTR promoter. These elements contain DNase I hypersensitive sites and histone modification patterns characteristic of enhancers. The elements also interact with each other and the latter two activate the CFTR promoter synergistically in reporter assays. Our results reveal novel long-range acting elements that control expression of CFTR and suggest that 3C-based approaches can be used for discovery of novel regulatory elements.

Enhanced Yeast One-hybrid Assays for High-throughput Gene-centered Regulatory Network Mapping

A major challenge in systems biology is to understand the gene regulatory networks that drive development, physiology and pathology. Interactions between transcription factors and regulatory genomic regions provide the first level of gene control. Gateway-compatible yeast one-hybrid (Y1H) assays present a convenient method to identify and characterize the repertoire of transcription factors that can bind a DNA sequence of interest. To delineate genome-scale regulatory networks, however, large sets of DNA fragments need to be processed at high throughput and high coverage. Here we present enhanced Y1H (eY1H) assays that use a robotic mating platform with a set of improved Y1H reagents and automated readout quantification. We demonstrate that eY1H assays provide excellent coverage and identify interacting transcription factors for multiple DNA fragments in a short time. eY1H assays will be an important tool for mapping gene regulatory networks in Caenorhabditis elegans and other model organisms as well as in humans.

Yeast One-hybrid Assays for Gene-centered Human Gene Regulatory Network Mapping

Gateway-compatible yeast one-hybrid (Y1H) assays provide a convenient gene-centered (DNA to protein) approach to identify transcription factors that can bind a DNA sequence of interest. We present Y1H resources, including clones for 988 of 1,434 (69%) predicted human transcription factors, that can be used to detect both known and new interactions between human DNA regions and transcription factors.

The Three-dimensional Architecture of a Bacterial Genome and Its Alteration by Genetic Perturbation

We have determined the three-dimensional (3D) architecture of the Caulobacter crescentus genome by combining genome-wide chromatin interaction detection, live-cell imaging, and computational modeling. Using chromosome conformation capture carbon copy (5C), we derive ~13 kb resolution 3D models of the Caulobacter genome. The resulting models illustrate that the genome is ellipsoidal with periodically arranged arms. The parS sites, a pair of short contiguous sequence elements known to be involved in chromosome segregation, are positioned at one pole, where they anchor the chromosome to the cell and contribute to the formation of a compact chromatin conformation. Repositioning these elements resulted in rotations of the chromosome that changed the subcellular positions of most genes. Such rotations did not lead to large-scale changes in gene expression, indicating that genome folding does not strongly affect gene regulation. Collectively, our data suggest that genome folding is globally dictated by the parS sites and chromosome segregation.

Translocation Mapping Exposes the Risky Lifestyle of B Cells

Recurrent chromosomal translocations can drive oncogenesis, but how they form has remained elusive. Now, Chiarle et al. (2011) and Klein et al. (2011) characterize the genome-wide spectrum of translocations that form from a single double-stranded break, revealing that specific loci have an intrinsic predisposition for frequent chromosomal rearrangements.

Chromatin Globules: a Common Motif of Higher Order Chromosome Structure?

Recent technological advances in the field of chromosome conformation capture are facilitating tremendous progress in the ability to map the three-dimensional (3D) organization of chromosomes at a resolution of several Kb and at the scale of complete genomes. Here we review progress in analyzing chromosome organization in human cells by building 3D models of chromatin based on comprehensive chromatin interaction datasets. We describe recent experiments that suggest that long-range interactions between active functional elements are sufficient to drive folding of local chromatin domains into compact globular states. We propose 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. Further, we speculate that increasingly longer range interactions can drive aggregation of groups of globular domains. This process would yield a compartmentalized chromosome conformation, consistent with recent observations obtained with genome-wide chromatin interaction mapping.

The Three-dimensional Folding of the α-globin Gene Domain Reveals Formation of Chromatin Globules

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.

Evidence for Transcript Networks Composed of Chimeric RNAs in Human Cells

The classic organization of a gene structure has followed the Jacob and Monod bacterial gene model proposed more than 50 years ago. Since then, empirical determinations of the complexity of the transcriptomes found in yeast to human has blurred the definition and physical boundaries of genes. Using multiple analysis approaches we have characterized individual gene boundaries mapping on human chromosomes 21 and 22. Analyses of the locations of the 5' and 3' transcriptional termini of 492 protein coding genes revealed that for 85% of these genes the boundaries extend beyond the current annotated termini, most often connecting with exons of transcripts from other well annotated genes. The biological and evolutionary importance of these chimeric transcripts is underscored by (1) the non-random interconnections of genes involved, (2) the greater phylogenetic depth of the genes involved in many chimeric interactions, (3) the coordination of the expression of connected genes and (4) the close in vivo and three dimensional proximity of the genomic regions being transcribed and contributing to parts of the chimeric RNAs. The non-random nature of the connection of the genes involved suggest that chimeric transcripts should not be studied in isolation, but together, as an RNA network.

THE STRUCTURE AND FUNCTION OF CHROMATIN AND CHROMOSOMES

No abstract received.

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