DNA supercoiling enhances cooperativity and efficiency of an epigenetic switch

Kamilla Norregaard, Magnus Andersson, Kim Sneppen, Peter Eigil Nielsen, Stanley Brown, and Lene B. Oddershede
PNAS October 22, 2013 110 (43) 17386-17391; https://doi.org/10.1073/pnas.1215907110

  1. Edited by Sankar Adhya, National Institutes of Health, National Cancer Institute, Bethesda, MD, and approved July 8, 2013 (received for review September 12, 2012)

Article Figures & SI

Figures

  • Fig. 1.
    Fig. 1.

    A schematic of CI autoregulation by DNA looping and the experimental assay. Protein complex formation between the λ operator sites displaces the intervening DNA in a loop. The in vivo distance between the operators is ∼2.3 kbp. The bent arrows show the transcription start points of the lytic promoters pL and pR and the lysogenic promoter pRM. The blue rectangles show the adjacent operator subsites OL1, OL2, OL3 and OR1, OR2, OR3. Red X signifies that the promoter is turned off in the shown configuration. CI dimers are shown as green dumbbell structures. (A) Octamer-mediated DNA looping by CI tetramers binding to OL1–OL2 and OR1–OR2. The lytic promoters, pL and pR, are repressed while CI transcription is activated by CI bound to OR2. (B) After octamer-mediated loop formation, an additional CI tetramer can bind cooperatively between OL3 and OR3, thus forming the octamer+tetramer complex. This represses both lytic genes and CI synthesis. (C) A plasmid DNA is tethered to an antidigoxigenin-coated cover slide via a digoxigenin–PNA and a streptavidin-coated μm-sized polystyrene bead via a biotin–PNA. The plasmids contain the entire λ immunity region and the two λ operators, OL and OR, flanked by the PNA-specific sites. CI dimers binding at OL and OR introduce loop formation and cause a detected decrease in the distance between the bead and the surface. (C, Inset) The PNA binds with sequence specificity to a DNA target and forms a triplex invasion complex while displacing the noncomplementary DNA strand in a loop.

  • Fig. 2.
    Fig. 2.

    Juxtaposition kinetics of supercoiled versus relaxed DNA. Histograms of the RMSD for the relaxed plasmid pSB4357-R (A) and for the naturally supercoiled plasmid pSB4357 (B). (C) Portion of chloroquine gel showing distribution of supercoils in the pSB4357 preparation. Entire electrophoretic gel shown is in Fig. S4. (D) Histogram of the relaxation time for all used datasets of pSB4357 (supercoiled plasmid; red) and pSB4357-R (relaxed plasmid; blue).

  • Fig. 3.
    Fig. 3.

    Tethered particle motion time series signal and histograms of the looped and unlooped state for different CI concentrations using the pSB4357 plasmid. (A) Effective length of tether,

    Fig. 3.
    , plotted as a function of time for beads tethered by a supercoiled plasmid. The plotted lines in the trajectory indicate the average excursions for unlooped (dotted line) and looped (dashed line). (B) Histograms of the time series shown in A (same abscissa for all concentrations). (C) Normalized collapsed histograms of looped and unlooped states, represented by the values 0 and 1, respectively, from all obtained datasets (same abscissa for all concentrations). The number of datasets for each concentration is n = 4 for 5 nM CI, n = 7 for 20 nM CI, n = 5 for 40 nM CI, n = 6 for 80 nM CI, and n = 9 for 170 nM CI, and significantly longer time series were used for the analysis than here shown. The looping probability increases as a function of CI concentration.

  • Fig. 4.
    Fig. 4.

    Looping probability as a function of CI concentration for supercoiled DNA and linear relaxed DNA. (A) Data obtained from CI-mediated looping of supercoiled DNA plasmids are shown as solid red circles. Data from CI-mediated looping of linear DNA (solid blue squares) were published by Zurla et al. (14). The hollow black triangles denote data from the relaxed plasmid pSB4357-R, which coincide with the results from linear DNA. Lines show fits of the modified thermodynamic model to data from the supercoiled assay (red) and of the original model to the linear assay (14) (blue), respectively. Error bars represent SD. (B) Illustration of difference between supercoiled and linear assay, on average the effective distance between the two operator sites, OL and OR (marked by blue rectangles), is significantly shorter in the supercoiled configuration (x1) than in the linear configuration (x2). The relaxed pSB4357-R is identical to the structure shown on the left but without supercoils.

Tables

  • Table 1.

    Comparison of free energies

    Reference
    Graphic
    , kcal/mol
    Graphic
    , kcal/mol
    Current study0−1.0
    Zurla et al. (14)1.7−2.4
    Dodd et al. (22)−0.5−3

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DNA supercoiling sharpens an epigenetic switch
Kamilla Norregaard, Magnus Andersson, Kim Sneppen, Peter Eigil Nielsen, Stanley Brown, Lene B. Oddershede
Proceedings of the National Academy of Sciences Oct 2013, 110 (43) 17386-17391; DOI: 10.1073/pnas.1215907110
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