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HICOMB 2018: Computing Chromosome Conformation

HICOMB 2018: Computing Chromosome Conformation

Keynote for HICOMB (17th IEEE International Workshop on High Performance Computational Biology) 2018.

James Taylor

May 21, 2018
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  1. @jxtx
    https://speakerdeck.com/jxtx

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  2. Acknowledgements
    Chromatin analysis and methods developed by Michael E. G. Sauria.
    Chromosome conformation paints: Teresa Luperchio and Karen Reddy
    HiFive available from github.com/bxlab/hifive, or .
    Our lab: Enis Afgan, Dannon Baker, Boris Brenerman, Min Hyung Cho,
    Dave Clements, Peter DeFord, Sam Guerler, Nathan Roach, Michael E. G.
    Sauria, German Uritskiy
    Other collaborators: Anton Nekrutenko and the group,

    Craig Stewart and the group

    Ross Hardison and the VISION group

    Jennifer Phillips-Cremins and Victor Corces (sub-TADS and HiFive)

    Johnston, Kim, Hilser, and DiRuggiero labs (JHU Biology)

    Battle, Langmead, Leek, Schatz, Timp lab (JHU Genomics Collective)
    NHGRI (HG005133, HG004909, HG005542, HG005573, HG006620)

    NIDDK (DK065806) and NSF (DBI 0543285, DBI 0850103)
    funded by the National Science Foundation
    Award #ACI-1445604
    install with bioconda

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  3. Gene Regulation:
    ■ How is control of gene expression encoded in the genome?
    ■ How can we detect the elements involved?
    ■ How do they act in a coordinated way in the cell?
    ■ How does their epigenomic modification contribute to development?
    Evolution:
    ■ What is the relationship between evolutionary constraint and function?
    ■ What mechanisms and patterns influence mutagenesis?
    Data intensive science:
    ■ How can we support increasingly data intensive and
    quantitatively complex science?
    ■ How can we improve the efficiency of scientific discovery?
    ■ How can we improve the quality the resulting science?
    ■ How can we support more reproducible research?

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  4. Gene control at a distance

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  5. (Adapted from Hardison and Taylor 2012)
    General TFs
    RNA Pol II
    RNA
    P300
    Transcription Factors
    Promoter
    cis-regulatory Module
    ~100s of Kilobases
    apart in genomic
    distance
    H3K4me3
    H3K4me1
    H4Ac

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  6. Evidence for 3D interactions in gene
    regulation

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  7. Insulator proteins

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  8. yellow
    wing blade
    body cuticle
    mouth parts
    denticle belts
    bristles, denticle belts,
    aristae
    tarsal claws
    The yellow locus in Drosophila
    (Geyer and Corces, Genes and Development, 1992, etc)

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  9. yellow
    gypsy retrotransposon (at -700bp)
    wing blade
    body cuticle
    mouth parts
    denticle belts
    bristles, denticle belts,
    aristae
    tarsal claws
    The yellow locus in Drosophila
    (Geyer and Corces, Genes and Development, 1992, etc)

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  10. yellow
    gypsy retrotransposon (at -700bp)
    su(Hw) binding site cluster
    wing blade
    body cuticle
    mouth parts
    denticle belts
    bristles, denticle belts,
    aristae
    tarsal claws
    The yellow locus in Drosophila
    (Geyer and Corces, Genes and Development, 1992, etc)

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  11. yellow
    -1868 -+660 +1310 +2940
    -700
    su(Hw) binding site cluster
    wing blade
    body cuticle
    mouth parts
    denticle belts
    bristles, denticle belts,
    aristae
    tarsal claws
    The yellow locus in Drosophila
    (Geyer and Corces, Genes and Development, 1992, etc)

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  12. Repression of transcription by su(Hw)
    - 1868
    -800R
    -800
    -700R
    +660R
    +660
    +1310
    +1310R
    +2490R
    body mouth
    wing cuticle hooks TATA
    ~ ' + +
    t
    + i
    " + i
    + i
    + + +
    + + +
    I
    !
    =
    + + + i
    i
    + + + ,
    t
    J
    + + + i
    bristles
    tarsal
    claws
    Figure 2. Summary of y phenotypes in trans-
    formed lines. (Top) The relative location with
    respect to the TATA box of different tissue-
    specific enhancers responsible for the expres-
    sion of the y gene in various tissues. Numbers
    at left indicate the location of the insertion
    site of the su(Hw)-binding region into the y
    gene in the various plasmids used for germ-
    line transformation. Each lane summarizes
    information on transformed lines obtained
    with each plasmid. The position of the in-
    serted sequences relative to various y enhanc-
    ers is indicated diagrammatically by a triangle
    that represents the su(Hw)-binding region;
    the solid circles represent the su(Hw) protein;
    the arrow indicates the orientation of the in-
    serted sequences relative to the y gene. The
    coloration of each tissue is indicated by +
    (wild type) or - (mutant) signs.
    required to produce the observed phenotype in these
    transformants, we analyzed the effect of mutations in
    The alleles used in this experiment were su(Hw) v, a null
    allele caused by a deletion of most of the su(Hw) gene,
    Cold Spring Harbor Laboratory Press
    on November 1, 2015 - Published by
    genesdev.cshlp.org
    Downloaded from
    yellow
    -1868 -+660 +1310 +2940
    -700
    Blocks tissue specific
    enhancer activity
    regardless of location
    relative to TSS or
    insert orientation
    (Geyer and Corces, Genes and Development, 1992, etc)

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  13. Insulator proteins
    (Yang and Corces, Current Opinion in Genetics & Development, 2012)

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  14. Insulator proteins
    (Yang and Corces, Current Opinion in Genetics & Development, 2012)

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  15. tissue-specific enhancers to target promoters by
    forming alternative chromatin loop domains. It
    is conceivable that these domains not only
    block inappropriate enhancers but also facilitate
    interaction between distant enhancers and the
    target promoter.
    References and Notes
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    and M. Levine, V. Pirrotta, and G. Felsenfeld for
    discussion, communication of unpublished results,
    and reading of the manuscript. Supported by NIH
    grant 5RO158458-02 (H.N.C.).
    4 October 2000; accepted 15 December 2000
    Loss of Insulator Activity by
    Paired Su(Hw) Chromatin
    Insulators
    Ekaterina Muravyova,1 Anton Golovnin,1,2,3 Elena Gracheva,1
    Aleksander Parshikov,1 Tatiana Belenkaya,1 Vincenzo Pirrotta,3*
    Pavel Georgiev1
    Chromatin insulators are regulatory elements that block the action of tran-
    scriptional enhancers when interposed between enhancer and promoter. The
    Drosophila Suppressor of Hairy wing [Su(Hw)] protein binds the Su(Hw) insu-
    lator and prevents enhancer-promoter interaction by a mechanism that is not
    understood. We show that when two copies of the Su(Hw) insulator element,
    instead of a single one, are inserted between enhancer and promoter, insulator
    activity is neutralized and the enhancer-promoter interaction may instead be
    facilitated. This paradoxical phenomenon could be explained by interactions
    between protein complexes bound at the insulators.
    The Drosophila gypsy retrotransposon con-
    tains a chromatin insulator that consists of
    cluster of 12 binding sites for the Su(Hw)
    zinc-finger protein (1–6). In the presence of
    Su(Hw) protein binding, the insulator blocks
    the activity of an enhancer separated from the
    promoter by an Su(Hw) binding region.
    However, this insulator action fails in certain
    genetic rearrangements that introduce more
    than one gypsy retrotransposon in the region
    of the yellow gene (7). The loss of insulator
    activity might result from intrachromosomal
    pairing between the two gypsy retrotrans-
    posons, causing chromatin to fold and allow-
    ing the enhancer to contact the promoter.
    Alternatively, interaction between the pro-
    Fig. 4. Insulator-mediated loop formation. (A)
    A suHw insulator (S) may interact with other
    nuclear sites/insulators (I), separating the en-
    hancer (E) and the promoter (P) into distinct
    domains and blocking their interaction. (B) In-
    teractions between two tandem suHw insula-
    tors fail to sequester the enhancer and may
    even facilitate enhancer-promoter interaction
    by “looping out” the intervening DNA. (C) En-
    hancer blocking may be strengthened by the
    preferred interactions between two suHw insu-
    lators flanking the enhancer.
    www.sciencemag.org SCIENCE VOL 291 19 JANUARY 2001 49
    245, R339 (1983).
    14. F. K. Stephan, G. Becker, Physiol. Behav. 46, 731 (1989).
    15. K.-A. Stokkan, S. Yamazaki, H. Tei, Y. Sakaki, M.
    Menaker, unpublished data.
    16. Serum concentrations of corticosterone were measured
    with a commercial radioimmunoassay kit (Coat-A-
    Count, Diagnostic Products, Los Angeles). One rat
    showed 207.9 and 41.0 ng/ml and another showed
    105.8 and 68.9 ng/ml at 3 hours after lights were turned
    on (“prefeeding”) and 9.5 hours after lights were turned
    on (“basal”), respectively. The difference between our
    results and those reported in (13) may be due to the
    fact that our animals were just weaned and growing
    rapidly, so that any restrictions in food access may be
    stressful. Aging markedly reduces the prefeeding corti-
    costerone secretion in rats exposed to RF [S. Honma et
    al., Am. J. Physiol. 271, R1514 (1996)].
    as intraperitoneal injections for 7 days. Control ani-
    mals received 0.2 ml of DMSO.
    18. On the seventh day of treatment, the serum level of
    corticosterone, 30 min after injection, was 581 Ϯ
    174 (SEM) ng/ml (n ϭ 6) and 39 Ϯ 17 ng/ml (n ϭ 6)
    in animals receiving corticosterone and DMSO injec-
    tions, respectively.
    19. A. Balsalobre et al., Science 289, 2344 (2000).
    20. Both ad lib feeding and food access restricted to the
    light period are probably highly abnormal for rats in
    the field.
    21. S.-I. Inouye, H. Kawamura, Proc. Natl. Acad. Sci.
    U.S.A. 76, 5962 (1979).
    22. S. Yamazaki, M. C. Kerbeshian, C. G. Hocker, G. D.
    Block, M. Menaker, J. Neurosci. 18, 10709 (1998).
    23. R. Y. Moore, D. C. Klein, Brain Res. 71, 17 (1974).
    25. J. D. Plautz, M. Kaneko, J. C. Hall, S. A. Kay, Science
    278, 1632 (1997).
    26. D. Whitmore, N. S. Foulkes, P. Sassone-Corsi, Nature
    404, 87 (2000).
    27. F. Damiola et al., Genes Dev. 14, 2950 (2000).
    28. We thank M. Quigg for measuring corticosterone
    concentrations and K. M. Greene and S. C. Miller for
    technical assistance. This work was supported in part
    by the NSF Center for Biological Timing, NIH grant
    MH 56647 (to M.M.); by travel grant 130173/410
    from the Norwegian Research Council (to K.-A.S.);
    and by a research grant from the Japanese Ministry of
    Education, Science, Sports and Culture and the Japa-
    nese Ministry of Health and Welfare (to H.T.).
    26 September 2000; accepted 13 December 2000
    Effects of cis Arrangement of
    Chromatin Insulators on
    Enhancer-Blocking Activity
    Haini N. Cai* and Ping Shen
    Chromatin boundary elements or insulators are believed to regulate gene
    activity in complex genetic loci by organizing specialized chromatin structures.
    Here, we report that the enhancer-blocking activity of the Drosophila suHw
    insulator is sensitive to insulator copy number and position. Two tandem copies
    of suHw were ineffective in blocking various enhancers from a downstream
    promoter. Moreover, an enhancer was blocked more effectively from a pro-
    moter by two flanking suHw insulators than by a single intervening one. Thus,
    insulators may modulate enhancer-promoter interactions by interacting with
    each other and facilitating the formation of chromatin loop domains.
    Insulators regulate gene activity in diverse or-
    ganisms (1–8). The defining feature of insula-
    tors as a class of regulatory elements is their
    ability to block enhancer-promoter interactions
    when positioned interveningly. One of the best
    characterized insulators is suHw, a 340–base
    pair (bp) element from the Drosophila gypsy
    retrotransposon. It protects transgenes from
    chromosomal position effects and blocks vari-
    ous enhancer-promoter interactions (9–13).
    SUHW, a zinc-finger DNA binding protein,
    and MOD(MDG4), a BTB domain protein, are
    essential for suHw function (13–16). Using
    divergently transcribed reporter genes in trans-
    genic Drosophila embryos, we have shown that
    skipped stripe 2 enhancer, directs reporter ex-
    pression in a composite pattern of broad dorsal
    activation and dominant ventral repression of
    the E2 stripe (Fig. 1, A and D) (13, 17, 18). A
    single 340-bp suHw insulator element in the
    VS2 transgene partially blocked the upstream
    VRE enhancer (Fig. 1, B and D). Two tandem
    suHw elements (arranged as direct repeats)
    were inserted between VRE and E2, resulting in
    VSS2. Instead of enhanced blockage, VSS2 em-
    bryos exhibited a loss of suHw insulator activ-
    ity (Fig. 1, C and D). This was observed in most
    VSS2 embryos (Fig. 1D) and in all 10 indepen-
    dent VSS2 lines, indicating that it is unlikely to
    be caused by chromosomal position effects.
    (Fig. 2, B and H), whereas two tandem suHw
    elements (NSSH) did not block the NEE en-
    hancer (Fig. 2, C and H). A second group of
    transgenes uses a twist mesoderm enhancer
    (PE) and an evenskipped stripe 3 enhancer (E3)
    (13). Both enhancers are active when separated
    by the L spacer (PL3) (Fig. 2, D and H).
    Insertion of a suHw element in the PS3 trans-
    gene blocked the upstream PE enhancer (Fig. 2,
    E and H), whereas two tandem suHw elements
    (PSS3) did not block the PE enhancer (Fig. 2, F
    and H). Replacing one of the two suHw ele-
    ments in PSS3 with a spacer of comparable size
    (A) restored the enhancer-blocking activity of
    the remaining suHw in PSA3 embryos (Fig.
    2G), indicating that loss of insulator activity
    with two suHw elements is not due to the
    spacing change but to the presence of the addi-
    tional insulator. Genomic PCR with individual
    NSH, NSSH, PS3, and PSS3 lines indicated that
    the transgenes were structurally intact (Fig. 2I).
    These results suggest that the loss of insulator
    activity with tandemly arranged suHw is inde-
    pendent of the enhancer tested.
    The enhancer-blocking activity of suHw
    may require its interaction with other sites
    (or insulators) within the nucleus. A second
    suHw nearby may compete dominantly for the
    existing suHw and affect the neighboring en-
    hancer-promoter interactions, depending on the
    cis arrangement of these elements. To test this
    hypothesis, we constructed the SVS2 transgene
    in which the VRE enhancer is flanked by two
    suHw elements. In contrast to the loss of insu-
    on October 24, 2016
    http://science.sciencemag.org/
    Downloaded from
    of suHw were ineffective in blocking various enhancers from a downstream
    promoter. Moreover, an enhancer was blocked more effectively from a pro-
    moter by two flanking suHw insulators than by a single intervening one. Thus,
    insulators may modulate enhancer-promoter interactions by interacting with
    each other and facilitating the formation of chromatin loop domains.
    Insulators regulate gene activity in diverse or-
    ganisms (1–8). The defining feature of insula-
    tors as a class of regulatory elements is their
    ability to block enhancer-promoter interactions
    when positioned interveningly. One of the best
    characterized insulators is suHw, a 340–base
    pair (bp) element from the Drosophila gypsy
    retrotransposon. It protects transgenes from
    chromosomal position effects and blocks vari-
    ous enhancer-promoter interactions (9–13).
    SUHW, a zinc-finger DNA binding protein,
    and MOD(MDG4), a BTB domain protein, are
    essential for suHw function (13–16). Using
    divergently transcribed reporter genes in trans-
    genic Drosophila embryos, we have shown that
    an enhancer blocked from the downstream pro-
    moter by suHw is fully competent to activate an
    upstream promoter (12).
    To probe the insulator mechanism, we test-
    ed the effect of suHw copy number on its
    insulator strength in Drosophila embryos. The
    zerknullt enhancer VRE (ventral repression el-
    ement) has been shown to be partially blocked
    by suHw (12). In blastoderm embryos, the V2
    transgene containing VRE and E2, an even-
    skipped stripe 2 enhancer, directs reporter ex-
    pression in a composite pattern of broad dorsal
    activation and dominant ventral repression of
    the E2 stripe (Fig. 1, A and D) (13, 17, 18). A
    single 340-bp suHw insulator element in the
    VS2 transgene partially blocked the upstream
    VRE enhancer (Fig. 1, B and D). Two tandem
    suHw elements (arranged as direct repeats)
    were inserted between VRE and E2, resulting in
    VSS2. Instead of enhanced blockage, VSS2 em-
    bryos exhibited a loss of suHw insulator activ-
    ity (Fig. 1, C and D). This was observed in most
    VSS2 embryos (Fig. 1D) and in all 10 indepen-
    dent VSS2 lines, indicating that it is unlikely to
    be caused by chromosomal position effects.
    Genomic polymerase chain reaction (PCR)
    analysis of independent VS2 and VSS2 lines
    further verified the structural integrity of the
    transgenes in vivo (Fig. 1E) (19).
    To determine whether the loss of insulator
    function in VSS2 embryos is enhancer-specific,
    we constructed transgenes using a rhomboid
    neuroectodermal enhancer (NEE) and a hairy
    stripe 1 enhancer (H1) (13). The NLH embryos
    containing NEE and H1 enhancers separated by
    a 1.4-kb neutral spacer (L) exhibited a compos-
    ite lacZ pattern directed by both enhancers (Fig.
    2, A and H). A single suHw element in the NSH
    transgene blocked the upstream NEE enhancer
    (A) restored the enhancer-blocking activity of
    the remaining suHw in PSA3 embryos (Fig.
    2G), indicating that loss of insulator activity
    with two suHw elements is not due to the
    spacing change but to the presence of the addi-
    tional insulator. Genomic PCR with individual
    NSH, NSSH, PS3, and PSS3 lines indicated that
    the transgenes were structurally intact (Fig. 2I).
    These results suggest that the loss of insulator
    activity with tandemly arranged suHw is inde-
    pendent of the enhancer tested.
    The enhancer-blocking activity of suHw
    may require its interaction with other sites
    (or insulators) within the nucleus. A second
    suHw nearby may compete dominantly for the
    existing suHw and affect the neighboring en-
    hancer-promoter interactions, depending on the
    cis arrangement of these elements. To test this
    hypothesis, we constructed the SVS2 transgene
    in which the VRE enhancer is flanked by two
    suHw elements. In contrast to the loss of insu-
    lator function seen in VSS2 embryos, the VRE
    enhancer is more effectively blocked in SVS2
    embryos than in VS2 embryos (Fig. 3, A, B, and
    D). Thus, it is the tandem arrangement rather
    than physical proximity that causes the loss of
    insulator activity. VRE-mediated dorsal activa-
    tion of the divergently transcribed miniwhite is
    also diminished in SVS2 embryos (19), indicat-
    ing that VRE is blocked from promoters on
    either side. suHw-mediated blockage of VRE is
    significantly reduced in SVS2/mod(mdg4)u1
    embryos (Fig. 3C), indicating that a MOD-
    (MDG4)-mediated complex is required for the
    enhanced insulator activity (13, 16, 20). VSS2,
    Department of Cellular Biology, University of Georgia,
    Athens, GA 30602, USA.
    *To whom correspondence should be addressed.
    www.sciencemag.org SCIENCE VOL 291 19 JANUARY 2001 493

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  16. pression was studied in a su(Hw)– back-
    ground. In five lines, the absence of Su(Hw)
    protein reduced white expression, implying
    blocked. Howe
    flanked by two
    ed at position
    transcription s
    the yellow gen
    and wings. In
    expression dec
    not change in
    activation of t
    yellow enhanc
    posed insulato
    yellow, the ins
    tors between
    promoters may
    stead of block
    lator between
    removed, yield
    expression in
    pressed, showi
    enhancers in the majority of the lines.
    Fig. 3. Model of the double insulator bypass.
    (A) A single insulator blocks enhancer-promot-
    er interaction. (B) Two insulators may interact
    with one another through the protein complex-
    es bound to them, forming a loop and bringing
    the enhancers closer to the promoter.
    (Muravyova et al, Science, 2001)

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  17. Capturing Chromosome Conformation

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  18. antibody to chicken C␮ (M1) (Southern Biotechnology
    Associates, Birmingham, AL) and then with polyclonal
    fluorescein isothiocyanate–conjugated goat antibodies
    to mouse IgG (Fab)
    2
    (Sigma). Predominantly sIgM(ϩ)
    subclones were excluded from the analysis, because
    they most likely originated from cells that were already
    sIgM(ϩ) at the time of subcloning.
    23. For Ig light chain sequencing, PCR amplification
    and sequencing of the rearranged light chain V
    segments were performed as previously described
    (19), except that high-fidelity PfuTurbo polymer-
    ase (Stratagene) was used with primer pair V␭1/
    V␭2 for PCR, and primer V␭3 was used for se-
    quencing (17). Only one nucleotide change, which
    most likely reflects a PCR-introduced artifact, was
    noticed in the V-J-3Ј intron region in a total of 80
    0.5-kb-long sequences from AIDϪ/ϪE cells.
    24. We thank M. Reth and T. Brummer for kindly provid-
    ing the MerCreMer plasmid vector; P. Carninci and Y.
    Hayashizaki for construction of the riken1 bursal
    cDNA library; A. Peters and K. Jablonski for excellent
    technical help; and C. Stocking and J. Lo
    ¨hrer for
    carefully reading the manuscript. Supported by grant
    Bu 631/2-1 from the Deutsche Forschungsgemein-
    shaft, by the European Union Framework V programs
    “Chicken Image” and “Genetics in a Cell Line,” and by
    Japan Society for the Promotion of Science Postdoc-
    toral Fellowships for Research Abroad.
    22 October 2001; accepted 18 December 2001
    Capturing Chromosome
    Conformation
    Job Dekker,1* Karsten Rippe,2 Martijn Dekker,3 Nancy Kleckner1
    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 G
    1
    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 chro-
    mosome III could be determined. Chromosome III emerges as a contorted ring.
    Important chromosomal activities have been
    linked with both structural properties and
    spatial conformations of chromosomes. Local
    properties of the chromatin fiber influence
    gene expression, origin firing, and DNA re-
    pair [e.g., (1, 2)]. Higher order structural
    features—such as formation of the 30-nm
    fiber, chromatin loops and axes, and inter-
    chromosomal connections—are important for
    chromosome morphogenesis and also have
    roles in gene expression and recombination.
    Activities such as transcription and timing of
    replication have been related to overall spa-
    affords a resolution of 100 to 200 nm at best,
    which is insufficient to define chromosome
    conformation. DNA binding proteins fused to
    green fluorescent protein permit visualization
    of individual loci, but only a few positions
    can be examined simultaneously. Multiple
    loci can be visualized with fluorescence in
    situ hybridization (FISH), but this requires
    severe treatment that may affect chromosome
    organization.
    We developed a high-throughput method-
    ology, Chromosome Conformation Capture
    (3C), which can be used to analyze the over-
    of purified nuclei is largely intact, as shown
    below.
    For quantification of cross-linking fre-
    quencies, cross-linked DNA is digested with
    a restriction enzyme and then subjected to
    ligation at very low DNA concentration. Un-
    der such conditions, ligation of cross-linked
    fragments, which is intramolecular, is strong-
    ly favored over ligation of random fragments,
    which is intermolecular. Cross-linking is then
    reversed and individual ligation products are
    detected and quantified by the polymerase
    chain reaction (PCR) using locus-specific
    primers. Control template is generated in
    which all possible ligation products are
    present in equal abundance (7). The cross-
    linking frequency (X) of two specific loci is
    determined by quantitative PCR reactions us-
    ing control and cross-linked templates, and X
    is expressed as the ratio of the amount of
    product obtained using the cross-linked tem-
    plate to the amount of product obtained with
    the control template (Fig. 1B). X should be
    directly proportional to the frequency with
    which the two corresponding genomic sites
    interact (10).
    Control experiments show that formation
    of ligation products is strictly dependent on
    both ligation and cross-linking (Fig. 1C). In
    general, X decreases with increasing separa-
    tion distance in kb along chromosome III
    (“genomic site separation”). Cross-linking
    frequencies for both the left telomere and the
    centromere of chromosome III with each of
    R E P O R T S
    on April 19, 2012
    www.sciencemag.org
    Downloaded from
    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 G
    1
    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 chro-
    mosome III could be determined. Chromosome III emerges as a contorted ring.
    Important chromosomal activities have been
    linked with both structural properties and
    spatial conformations of chromosomes. Local
    properties of the chromatin fiber influence
    gene expression, origin firing, and DNA re-
    pair [e.g., (1, 2)]. Higher order structural
    features—such as formation of the 30-nm
    fiber, chromatin loops and axes, and inter-
    chromosomal connections—are important for
    chromosome morphogenesis and also have
    roles in gene expression and recombination.
    Activities such as transcription and timing of
    replication have been related to overall spa-
    tial nuclear disposition of different regions
    and their relationships to the nuclear enve-
    lope [e.g., (3–6)]. At each of these levels,
    chromosome organization is highly dynamic,
    varying both during the cell cycle and among
    different cell types.
    Analysis of chromosome conformation is
    complicated by technical limitations. Elec-
    tron microscopy, while affording high reso-
    lution, is laborious and not easily applicable
    to studies of specific loci. Light microscopy
    affords a resolution of 100 to 200 nm at best,
    which is insufficient to define chromosome
    conformation. DNA binding proteins fused to
    green fluorescent protein permit visualization
    of individual loci, but only a few positions
    can be examined simultaneously. Multiple
    loci can be visualized with fluorescence in
    situ hybridization (FISH), but this requires
    severe treatment that may affect chromosome
    organization.
    We developed a high-throughput method-
    ology, Chromosome Conformation Capture
    (3C), which can be used to analyze the over-
    all spatial organization of chromosomes and
    to investigate their physical properties at high
    resolution. The principle of our approach is
    outlined in Fig. 1A (7). Intact nuclei are
    isolated (8) and subjected to formaldehyde
    fixation, which cross-links proteins to other
    proteins and to DNA. The overall result is
    cross-linking of physically touching seg-
    ments throughout the genome via contacts
    between their DNA-bound proteins. The rel-
    ative frequencies with which different sites
    have become cross-linked are then deter-
    mined. Analysis of genome-wide interaction
    frequencies provides information about gen-
    eral nuclear organization as well as physical
    properties and conformations of chromo-
    somes. We have used intact yeast nuclei for
    all experiments. Although the method can be
    performed using intact cells, the signals are
    considerably lower, making quantification
    difficult (9). The general nuclear organization
    which is intermolecular. Cross-linking is then
    reversed and individual ligation products are
    detected and quantified by the polymerase
    chain reaction (PCR) using locus-specific
    primers. Control template is generated in
    which all possible ligation products are
    present in equal abundance (7). The cross-
    linking frequency (X) of two specific loci is
    determined by quantitative PCR reactions us-
    ing control and cross-linked templates, and X
    is expressed as the ratio of the amount of
    product obtained using the cross-linked tem-
    plate to the amount of product obtained with
    the control template (Fig. 1B). X should be
    directly proportional to the frequency with
    which the two corresponding genomic sites
    interact (10).
    Control experiments show that formation
    of ligation products is strictly dependent on
    both ligation and cross-linking (Fig. 1C). In
    general, X decreases with increasing separa-
    tion distance in kb along chromosome III
    (“genomic site separation”). Cross-linking
    frequencies for both the left telomere and the
    centromere of chromosome III with each of
    12 other positions along that same chromo-
    some (Fig. 1, C and D) were determined
    using nuclei isolated from exponentially
    growing haploid cells. Interestingly, the two
    telomeres of chromosome III interact more
    frequently than predicted from their genomic
    site separation, which suggests that the chro-
    mosome ends are in close spatial proximity.
    This is expected because yeast telomeres are
    known to occur in clusters (11, 12).
    We next applied our method to an analysis
    of centromeres and of homologous chromo-
    somes (“homologs”) during meiosis in yeast
    (7). In mitotic and premeiotic cells, centro-
    meres are clustered near the spindle pole
    body (13, 14) and homologous chromosomes
    are loosely associated (15–17). These fea-
    tures change markedly when cells enter mei-
    osis (13). The centromere cluster is rapidly
    lost and is not restored until just before the
    first meiotic division. Loose interactions be-
    1Department of Molecular and Cellular Biology, Har-
    vard University, Cambridge, MA 02138, USA. 2Mole-
    kulare Genetik (H0700), Deutsches Krebsforschungs-
    zentrum, Im Neuenheimer Feld 280, and Kirchhoff-
    Institut fu
    ¨r Physik, Physik Molekularbiologischer Pro-
    zesse, Universita
    ¨t Heidelberg, Schro
    ¨derstrasse 90,
    D-69120 Heidelberg, Germany. 32e Oosterparklaan
    272, 3544 AX Utrecht, Netherlands.
    *To whom correspondence should be addressed. E-
    mail: [email protected]
    15 FEBRUARY 2002 VOL 295 SCIENCE www.sciencemag.org
    1306
    on April 19, 201
    www.sciencemag.org
    Downloaded from

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  19. Protein/DNA interaction complex

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  20. 1. Cross link to capture close interactions

    View full-size slide

  21. 2. Restriction Enzyme Digestion

    View full-size slide

  22. 3. Ligation of DNA within complex

    View full-size slide

  23. 4. Purify DNA and measure interactions

    View full-size slide

  24. 1. Crosslink
    Protein/DNA
    complex
    2. Restriction
    Enzyme
    Digest
    3. Biotin fill
    and Ligate
    4. Pull down
    Junctions
    4. Sequence
    Hi-C for measuring chromatin interactions
    some were consistent across chromosomes: th
    A B
    E F
    Count matrix, for pairs of restriction
    fragments or larger bins
    (Lieberman-Aiden et al. 2009)

    View full-size slide

  25. Supplemental Figure 1.Proximity-mediated ligation assays.
    Cross-link
    Cells
    Restriction Enzyme
    Digestion
    Ligation
    Reaction
    Reverse
    Cross-linking
    Quantitative PCR
    Ligation
    Reaction
    Reverse
    Cross-linking
    Cloning and Sequencing
    or
    PCR and Microarray
    Biotin
    Labeling
    and Reaction
    Reaction
    Reverse
    Cross-linking
    Sonication
    Fragmentation
    and Streptavidin
    Precipitation
    Addition of
    Sequencing
    Linkers
    High-throughput
    Sequencing
    Sonication
    Fragmentation
    and Antibody
    Precipitation
    Addition of
    RE Linkers
    Ligation
    Reaction
    Restriction
    Enzyme Digest
    and Sequencing
    Linker Addition
    High-throughput
    Sequencing
    Reverse
    Cross-linking
    and Primer
    Annealing
    Nick Repair and PCR
    Enrichment
    Addition of
    Sequencing
    Linkers
    High-throughput
    Sequencing
    ChIA-PET
    many to many
    HiC
    all to all
    4C
    one to all
    5C
    many to many
    3C
    one to one
    RE Fragments
    DNA Binding Protein
    Cross-linked Protein
    Sequencing Linker
    MmeI-containing Linker
    Antibody
    Streptavidin
    Biotin
    RE Site
    Sonication Break Site
    Ligation Event
    Targeted Primers
    Universal Primers
    (Sauria et al. Genome Biology 2015)

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  26. Ѱ-C Analysis
    Raw Count Matrix
    ora
    hillips Nora
    ty
    KR

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  27. Ѱ-C Analysis
    Raw Count Matrix
    ora
    hillips Nora
    ty
    KR
    hillips Nora
    Normalization
    &JK
    % J K
    GJ
    GK
    DJK
    ɿ ' GJ
    GK
    EJK

    FH
    ɿ 1PJTTPO GJ GK % EJK



    &JK &YQFDUFE DPVOU CFUXFFO CJOT J BOE K
    % J K
    %JTUBODF DPNQPOFOU FJUIFS FYQMJDJU PS FNQJSJDBMMZ FT
    GJ
    GK #JO TQFDJöD DPSSFDUJPO GBDUPS EJSFDUMZ MFBSOFE PS QBSBN
    ċ

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