Job Dekker

University of Massachusetts Medical School, Worcester, Massachusetts, United States

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Publications (91)1455.53 Total impact

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    ABSTRACT: SMC condensin complexes play a central role in compacting and resolving replicated chromosomes in virtually all organisms, yet how they accomplish this remains elusive. In Bacillus subtilis, condensin is loaded at centromeric parS sites, where it encircles DNA and individualizes newly replicated origins. Using chromosome conformation capture and cytological assays, we show that condensin recruitment to origin-proximal parS sites is required for the juxtaposition of the two chromosome arms. Recruitment to ectopic parS sites promotes alignment of large tracks of DNA flanking these sites. Importantly, insertion of parS sites on opposing arms indicates that these "zip-up" interactions only occur between adjacent DNA segments. Collectively, our data suggest that condensin resolves replicated origins by promoting the juxtaposition of DNA flanking parS sites, drawing sister origins in on themselves and away from each other. These results are consistent with a model in which condensin encircles the DNA flanking its loading site and then slides down, tethering the two arms together. Lengthwise condensation via loop extrusion could provide a generalizable mechanism by which condensin complexes act dynamically to individualize origins in B. subtilis and, when loaded along eukaryotic chromosomes, resolve them during mitosis. © 2015 Wang et al.; Published by Cold Spring Harbor Laboratory Press.
    Genes & development 08/2015; 29(15):1661-75. DOI:10.1101/gad.265876.115 · 12.64 Impact Factor
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    ABSTRACT: We have examined the three-dimensional organization of the yeast genome during quiescence by a chromosome capture technique as a means of understanding how genome organization changes during development. For exponentially growing cells we observe high levels of inter-centromeric interaction but otherwise a predominance of intrachromosomal interactions over interchromosomal interactions, consistent with aggregation of centromeres at the spindle pole body and compartmentalization of individual chromosomes within the nucleoplasm. Three major changes occur in the organization of the quiescent cell genome. First, intrachromosomal associations increase at longer distances in quiescence as compared to growing cells. This suggests that chromosomes undergo condensation in quiescence, which we confirmed by microscopy by measurement of the intrachromosomal distances between two sites on one chromosome. This compaction in quiescence requires the condensin complex. Second, inter-centromeric interactions decrease, consistent with prior data indicating that centromeres disperse along an array of microtubules during quiescence. Third, inter-telomeric interactions significantly increase in quiescence, an observation also confirmed by direct measurement. Thus, survival during quiescence is associated with substantial topological reorganization of the genome. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
    Nucleic Acids Research 07/2015; DOI:10.1093/nar/gkv723 · 9.11 Impact Factor
  • Jon-Matthew Belton · Job Dekker
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    ABSTRACT: Hi-C enables simultaneous detection of interaction frequencies between all possible pairs of restriction fragments in the genome. The Hi-C method is based on chromosome conformation capture (3C), which uses formaldehyde cross-linking to fix chromatin regions that interact in three-dimensional space, irrespective of their genomic locations. In the Hi-C protocol described here, cross-linked chromatin is digested with HindIII and the ends are filled in with a nucleotide mix containing biotinylated dCTP. These fragments are ligated together, and the resulting chimeric molecules are purified and sheared to reduce length. Finally, biotinylated ligation junctions are pulled down with streptavidin-coated beads, linked to high-throughput sequencing adaptors, and amplified via polymerase chain reaction (PCR). The resolution of the Hi-C data set will depend on the depth of sequencing and choice of restriction enzyme. When sufficient sequence reads are obtained, information on chromatin interactions and chromosome conformation can be derived at single restriction fragment resolution for complete genomes. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 07/2015; 2015(7):pdb.prot085209. DOI:10.1101/pdb.prot085209 · 4.63 Impact Factor
  • Jon-Matthew Belton · Job Dekker
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    ABSTRACT: Chromosome conformation capture (3C) has revolutionized the ways in which the conformation of chromatin and its relationship to other molecular functions can be studied. 3C-based techniques are used to determine the spatial arrangement of chromosomes in organisms ranging from bacteria to humans. In particular, they can be applied to the study of chromosome folding and organization in model organisms with small genomes and for which powerful genetic tools exist, such as budding yeast. Studies in yeast allow the mechanisms that establish or maintain chromatin structure to be analyzed at very high resolution with relatively low cost, and further our understanding of these fundamental processes in higher eukaryotes as well. Here we provide an overview of chromatin structure and introduce methods for performing 3C, with a focus on studies in budding yeast. Variations of the basic 3C approach (e.g., 3C-PCR, 5C, and Hi-C) can be used according to the scope and goals of a given experiment. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 07/2015; 2015(7):pdb.top077552. DOI:10.1101/pdb.top077552 · 4.63 Impact Factor
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    ABSTRACT: We describe a Hi-C-based method, Micro-C, in which micrococcal nuclease is used instead of restriction enzymes to fragment chromatin, enabling nucleosome resolution chromosome folding maps. Analysis of Micro-C maps for budding yeast reveals abundant self-associating domains similar to those reported in other species, but not previously observed in yeast. These structures, far shorter than topologically associating domains in mammals, typically encompass one to five genes in yeast. Strong boundaries between self-associating domains occur at promoters of highly transcribed genes and regions of rapid histone turnover that are typically bound by the RSC chromatin-remodeling complex. Investigation of chromosome folding in mutants confirms roles for RSC, "gene looping" factor Ssu72, Mediator, H3K56 acetyltransferase Rtt109, and the N-terminal tail of H4 in folding of the yeast genome. This approach provides detailed structural maps of a eukaryotic genome, and our findings provide insights into the machinery underlying chromosome compaction. Copyright © 2015 Elsevier Inc. All rights reserved.
    Cell 06/2015; 162(1). DOI:10.1016/j.cell.2015.05.048 · 33.12 Impact Factor
  • Jon-Matthew Belton · Job Dekker
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    ABSTRACT: Chromosome conformation capture carbon copy (5C) is a high-throughput method for detecting ligation products of interest in a chromosome conformation capture (3C) library. 5C uses ligation-mediated amplification (LMA) to generate carbon copies of 3C ligation product junctions using single-stranded oligonucleotide probes. This procedure produces a 5C library of short DNA molecules which represent the interactions between the corresponding restriction fragments. The 5C library can be amplified using universal primers containing the Illumina paired-end adaptor sequences for subsequent high-throughput sequencing. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 06/2015; 2015(6):pdb.prot085191. DOI:10.1101/pdb.prot085191 · 4.63 Impact Factor
  • Jon-Matthew Belton · Job Dekker
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    ABSTRACT: Chromosome conformation capture (3C) is a method for studying chromosomal organization that takes advantage of formaldehyde cross-linking to measure the spatial association of two pieces of chromatin. The 3C method begins with whole-cell formaldehyde fixation of chromatin. After cell lysis, solubilized chromatin is digested with a type II restriction endonuclease, and cross-linked DNA fragments are ligated together. Cross-links are reversed by degradation with proteinase K, and chimeric DNA molecules are purified by standard phenol:chloroform extraction. The resulting 3C library represents chromatin fragments that may be separated by large genomic distances or located on different chromosomes, but are close enough in three-dimensional space for cross-linking. Locus-specific oligonucleotide primers are used to detect interactions of interest in the 3C library using end-point polymerase chain reaction (PCR). © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 06/2015; 2015(6):pdb.prot085175. DOI:10.1101/pdb.prot085175 · 4.63 Impact Factor
  • Jon-Matthew Belton · Job Dekker
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    ABSTRACT: In experiments using chromosome conformation capture followed by PCR (3C-PCR) or chromosome conformation capture carbon copy (5C), it is critical to control for intrinsic biases in the restriction fragments of interest and the probes or primers used for detection. Characteristics such as GC%, annealing temperature, efficiency of 3C primers or 5C probes, and length of restriction fragment can cause variations in primer or probe performance and fragment ligation efficiency. Bias can be measured empirically by production of a random control library, as described here, to be used with the 3C library of interest. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 06/2015; 2015(6):pdb.prot085183. DOI:10.1101/pdb.prot085183 · 4.63 Impact Factor
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    ABSTRACT: The three-dimensional organization of a genome plays a critical role in regulating gene expression, yet little is known about the machinery and mechanisms that determine higher-order chromosome structure. Here we perform genome-wide chromosome conformation capture analysis, fluorescent in situ hybridization (FISH), and RNA-seq to obtain comprehensive three-dimensional (3D) maps of the Caenorhabditis elegans genome and to dissect X chromosome dosage compensation, which balances gene expression between XX hermaphrodites and XO males. The dosage compensation complex (DCC), a condensin complex, binds to both hermaphrodite X chromosomes via sequence-specific recruitment elements on X (rex sites) to reduce chromosome-wide gene expression by half. Most DCC condensin subunits also act in other condensin complexes to control the compaction and resolution of all mitotic and meiotic chromosomes. By comparing chromosome structure in wild-type and DCC-defective embryos, we show that the DCC remodels hermaphrodite X chromosomes into a sex-specific spatial conformation distinct from autosomes. Dosage-compensated X chromosomes consist of self-interacting domains (∼1 Mb) resembling mammalian topologically associating domains (TADs). TADs on X chromosomes have stronger boundaries and more regular spacing than on autosomes. Many TAD boundaries on X chromosomes coincide with the highest-affinity rex sites and become diminished or lost in DCC-defective mutants, thereby converting the topology of X to a conformation resembling autosomes. rex sites engage in DCC-dependent long-range interactions, with the most frequent interactions occurring between rex sites at DCC-dependent TAD boundaries. These results imply that the DCC reshapes the topology of X chromosomes by forming new TAD boundaries and reinforcing weak boundaries through interactions between its highest-affinity binding sites. As this model predicts, deletion of an endogenous rex site at a DCC-dependent TAD boundary using CRISPR/Cas9 greatly diminished the boundary. Thus, the DCC imposes a distinct higher-order structure onto X chromosomes while regulating gene expression chromosome-wide.
    Nature 06/2015; DOI:10.1038/nature14450 · 42.35 Impact Factor
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    ABSTRACT: In addition to mediating sister chromatid cohesion during the cell cycle, the cohesin complex associates with CTCF and with active gene regulatory elements to form long-range interactions between its binding sites. Genome-wide chromosome conformation capture had shown that cohesin's main role in interphase genome organization is in mediating interactions within architectural chromosome compartments, rather than specifying compartments per se. However, it remained unclear how cohesin-mediated interactions contribute to the regulation of gene expression. We have found that the binding of CTCF and cohesin is highly enriched at enhancers and in particular at enhancer arrays or 'super-enhancers' in mouse thymocytes. Using local and global chromosome conformation capture we demonstrate that enhancer elements associate not just in linear sequence, but also in 3-D, and that spatial enhancer clustering is facilitated by cohesin. The conditional deletion of cohesin from non-cycling thymocytes preserved enhancer position, H3K27ac, H4K4me1 and enhancer transcription, but weakened interactions between enhancers. Interestingly, ~50% of deregulated genes reside in the vicinity of enhancer elements, suggesting that cohesin regulates gene expression through spatial clustering of enhancer elements. We propose a model for cohesin-dependent gene regulation where spatial clustering of enhancer elements acts as a unified mechanism for both, enhancer-promoter 'connections' and 'insulation'. Published by Cold Spring Harbor Laboratory Press.
    Genome Research 02/2015; 25(4). DOI:10.1101/gr.184986.114 · 13.85 Impact Factor
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    Job Dekker
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    ABSTRACT: Genetic and epigenetic inheritance through mitosis is critical for dividing cells to maintain their state. This process occurs in the context of large-scale re-organization of chromosome conformation during prophase leading to the formation of mitotic chromosomes, and during the reformation of the interphase nucleus during telophase and early G1. This review highlights how recent studies over the last 5 years employing chromosome conformation capture combined with classical models of chromosome organization based on decades of microscopic observations, are providing new insights into the three-dimensional organization of chromatin inside the interphase nucleus and within mitotic chromosomes. One striking observation is that interphase genome organization displays cell type-specific features that are related to cell type-specific gene expression, whereas mitotic chromosome folding appears universal and tissue invariant. This raises the question of whether or not there is a need for an epigenetic memory for genome folding. Herein, the two different folding states of mammalian genomes are reviewed and then models are discussed wherein instructions for cell type-specific genome folding are locally encoded in the linear genome and transmitted through mitosis, e.g., as open chromatin sites with or without continuous binding of transcription factors. In the next cell cycle these instructions are used to re-assemble protein complexes on regulatory elements which then drive three-dimensional folding of the genome from the bottom up through local action and self-assembly into higher order levels of cell type-specific organization. In this model, no explicit epigenetic memory for cell type-specific chromosome folding is required.
    Epigenetics & Chromatin 11/2014; 7(1):25. DOI:10.1186/1756-8935-7-25 · 4.46 Impact Factor
  • Bryan R. Lajoie · Job Dekker · Noam Kaplan
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    ABSTRACT: Over the last decade, development and application of a set of molecular genomic approaches based on the chromosome conformation capture method (3C), combined with increasingly powerful imaging approaches, have enabled high resolution and genome-wide analysis of the spatial organization of chromosomes. The aim of this paper is to provide guidelines for analyzing and interpreting data obtained with genome-wide 3C methods such as Hi-C and 3C-seq that rely on deep sequencing to detect and quantify pairwise chromatin interactions genome-wide.
    Methods 11/2014; 72. DOI:10.1016/j.ymeth.2014.10.031 · 3.22 Impact Factor
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    ABSTRACT: Eukaryotic genomes are folded into three-dimensional structures, such as self-associating topological domains, the borders of which are enriched in cohesin and CCCTC-binding factor (CTCF) required for long-range interactions. How local chromatin interactions govern higher-order folding of chromatin fibres and the function of cohesin in this process remain poorly understood. Here we perform genome-wide chromatin conformation capture (Hi-C) analysis to explore the high-resolution organization of the Schizosaccharomyces pombe genome, which despite its small size exhibits fundamental features found in other eukaryotes. Our analyses of wild-type and mutant strains reveal key elements of chromosome architecture and genome organization. On chromosome arms, small regions of chromatin locally interact to form 'globules'. This feature requires a function of cohesin distinct from its role in sister chromatid cohesion. Cohesin is enriched at globule boundaries and its loss causes disruption of local globule structures and global chromosome territories. By contrast, heterochromatin, which loads cohesin at specific sites including pericentromeric and subtelomeric domains, is dispensable for globule formation but nevertheless affects genome organization. We show that heterochromatin mediates chromatin fibre compaction at centromeres and promotes prominent inter-arm interactions within centromere-proximal regions, providing structural constraints crucial for proper genome organization. Loss of heterochromatin relaxes constraints on chromosomes, causing an increase in intra- and inter-chromosomal interactions. Together, our analyses uncover fundamental genome folding principles that drive higher-order chromosome organization crucial for coordinating nuclear functions.
    Nature 10/2014; 516(7531). DOI:10.1038/nature13833 · 42.35 Impact Factor
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    Proceedings of the National Academy of Sciences 08/2014; 111(33-33):E3366. DOI:10.1073/pnas.1410434111 · 9.81 Impact Factor
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    ABSTRACT: A new level of chromosome organization, topologically associating domains (TADs), was recently uncovered by chromosome conformation capture (3C) techniques. To explore TAD structure and function, we developed a polymer model that can extract the full repertoire of chromatin conformations within TADs from population-based 3C data. This model predicts actual physical distances and to what extent chromosomal contacts vary between cells. It also identifies interactions within single TADs that stabilize boundaries between TADs and allows us to identify and genetically validate key structural elements within TADs. Combining the model's predictions with high-resolution DNA FISH and quantitative RNA FISH for TADs within the X-inactivation center (Xic), we dissect the relationship between transcription and spatial proximity to cis-regulatory elements. We demonstrate that contacts between potential regulatory elements occur in the context of fluctuating structures rather than stable loops and propose that such fluctuations may contribute to asymmetric expression in the Xic during X inactivation.
    Cell 05/2014; 157(4):950-63. DOI:10.1016/j.cell.2014.03.025 · 33.12 Impact Factor
  • Journal of Investigative Dermatology 05/2014; 134(1}, Meeting Abstract = {444):S77. · 6.37 Impact Factor
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    ABSTRACT: With the completion of the human genome sequence, attention turned to identifying and annotating its functional DNA elements. As a complement to genetic and comparative genomics approaches, the Encyclopedia of DNA Elements Project was launched to contribute maps of RNA transcripts, transcriptional regulator binding sites, and chromatin states in many cell types. The resulting genome-wide data reveal sites of biochemical activity with high positional resolution and cell type specificity that facilitate studies of gene regulation and interpretation of noncoding variants associated with human disease. However, the biochemically active regions cover a much larger fraction of the genome than do evolutionarily conserved regions, raising the question of whether nonconserved but biochemically active regions are truly functional. Here, we review the strengths and limitations of biochemical, evolutionary, and genetic approaches for defining functional DNA segments, potential sources for the observed differences in estimated genomic coverage, and the biological implications of these discrepancies. We also analyze the relationship between signal intensity, genomic coverage, and evolutionary conservation. Our results reinforce the principle that each approach provides complementary information and that we need to use combinations of all three to elucidate genome function in human biology and disease.
    Proceedings of the National Academy of Sciences 04/2014; 111(17). DOI:10.1073/pnas.1318948111 · 9.81 Impact Factor
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    Noam Kaplan · Job Dekker
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    ABSTRACT: Despite advances in DNA sequencing technology, assembly of complex genomes remains a major challenge, particularly for genomes sequenced using short reads, which yield highly fragmented assemblies. Here we show that genome-wide in vivo chromatin interaction frequency data, which are measurable with chromosome conformation capture-based experiments, can be used as genomic distance proxies to accurately position individual contigs without requiring any sequence overlap. We also use these data to construct approximate genome scaffolds de novo. Applying our approach to incomplete regions of the human genome, we predict the positions of 65 previously unplaced contigs, in agreement with alternative methods in 26/31 cases attempted in common. Our approach can theoretically bridge any gap size and should be applicable to any species for which global chromatin interaction data can be generated.
    Nature Biotechnology 11/2013; 31(12). DOI:10.1038/nbt.2768 · 39.08 Impact Factor
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    ABSTRACT: V(D)J joining is mediated by RAG recombinase during early B-lymphocyte development in the bone marrow (BM). Activation-induced deaminase initiates isotype switching in mature B cells of secondary lymphoid structures. Previous studies questioned the strict ontological partitioning of these processes. We show that pro-B cells undergo robust switching to a subset of immunoglobulin H (IgH) isotypes. Chromatin studies reveal that in pro-B cells, the spatial organization of the Igh locus may restrict switching to this subset of isotypes. We demonstrate that in the BM, V(D)J joining and switching are interchangeably inducible, providing an explanation for the hyper-IgE phenotype of Omenn syndrome.
    Genes & development 11/2013; 27(22):2439-44. DOI:10.1101/gad.227165.113 · 12.64 Impact Factor
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    ABSTRACT: Mitotic chromosomes are among the most recognizable structures in the cell, yet for over a century their internal organization remains largely unsolved. We applied chromosome conformation capture methods, 5C and Hi-C, across the cell cycle and revealed two alternative three-dimensional folding states of the human genome. We show that the highly compartmentalized and cell-type-specific organization described previously for non-synchronous cells is restricted to interphase. In metaphase, we identify a homogenous folding state, which is locus-independent, common to all chromosomes, and consistent among cell types, suggesting a general principle of metaphase chromosome organization. Using polymer simulations, we find that metaphase Hi-C data are inconsistent with classic hierarchical models, and is instead best described by a linearly-organized longitudinally compressed array of consecutive chromatin loops.
    Science 11/2013; 342(6161). DOI:10.1126/science.1236083 · 31.48 Impact Factor

Publication Stats

12k Citations
1,455.53 Total Impact Points

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Institutions

  • 2003–2015
    • University of Massachusetts Medical School
      • Department of Biochemistry and Molecular Pharmacology
      Worcester, Massachusetts, United States
  • 2012–2013
    • University of Massachusetts Amherst
      • Department of Biochemistry and Molecular Biology
      Amherst Center, Massachusetts, United States