Figure S3 AFM image treatment and molecule orienta- tion. (A-A’’’) Molecule path skeletonization from an AFM image of the 1290 bp human IL2RA fragment. (B-B’’’) Illustration of the methodology used to orient the IL2RA fragment (19): the pattern of height variation along the axis of the fragment (red) is compared to the calculated pitch variation (black) obtained when using the Bolshoy et al. [44] DNA coding table. The reproducibility in AFM topography measurement and its correlation with sequence- dependent pitch modeling allowed us to orient a significant proportion of the IL2RA molecules imaged by AFM. Indeed there is a significant height difference between the 59 end (low) and 39 end (high), that turn out to be very useful to orient the underlying DNA sequence. The efficiency of this method was confirmed a posteriori by the very accurate positioning of the TBP-TFIIB GTFs at sites corresponding to the TATA sequence (Figure 3B) as expected for a first step of PIC formation. (TIF) Figure S4 AFM imaging of GTF, nucleosome and HMGA1 positioning on the IL2RA gene promoter. (A) AFM imaging of TBP-TFIIB positioning along the 563 bp IL2RA fragment (left panels) and topological profile along the molecule path skeleton (right panels). (B) AFM imaging of mononucleosome positioning along the 898 bp IL2RA fragment (left panels) and topological profile along the molecule path skeleton (right panels) (see Ref. [19]). (C) AFM imaging of HMGA1 positioning along the 898 bp IL2RA fragment (left panels) and topological profile along the molecule path skeleton (right panels). (TIF) Figure S5 Prediction of the DNA structure of a control DNA fragment. (A) AFM images of representative 976 bp pBR control fragments (Materials and Methods) in aqueous buffer. (B) Theoretical analysis of the pBR sequence. The diagrammatic (upper plane) and spectral (lower graph) analyses do not show the presence of supercoiled DNA, as previously revealed for the IL2RA sequence (Figure 1). Window size is 150 bp, step 1 bp, analyzed phasing pitch varies from 5 to 15 bp with step 1 bp. (C) pBR molecule path skeletonization. (D) Height measurement along the red path in (C). (E) Standard deviation of height measurements from 97 pBR molecules (black); for comparison is shown the height standard deviation from 100 IL2RA molecules (blue). (TIF) Figure S6 Time-lapse AFM imaging of the IL2RA gene promoter region. (A) AFM images of a representative 1290 bp IL2RA fragment in aqueous buffer. (B) Time lapse AFM images of this fragment in the enlarged white box in (A) at 0, 4 min, 6 min and 8 min after beginning the observation. The position of the TSS is indicated by a red star. At 4 min, a negative supercoil was observed. In the t = 0 image, the white arrow indicates the position where the supercoiled structure will form. (C) Corresponding curvature profiles, expressed as Ends ratios, along the fragment (window 150 bp, step 1 bp). The position of the TSS is indicated by a vertical blue line. (TIF) References Structure and Dynamics of the IL2RA Gene Promoter Figure S7 Time-lapse AFM imaging of the IL2RA gene promoter region. (A) AFM images of a representative 1290 bp IL2RA fragment in aqueous buffer.(B) Height variation along the DNA molecule path skeleton. (C) Time lapse AFM images of this fragment in the enlarged white box in (A) at 0, 4 min, 6 min and 8 min after beginning the observation. The position of TSS is indicated by a red star. For this molecule the supercoil is already present at t = 0. (D) Corresponding curvature profiles, expressed as Ends ratio, along the fragment (window 150 bp, step 1 bp). The position of the TSS is indicated by a vertical blue line. (TIF) Figure S8 AFM imaging of DNA loop. (A) Enlarged AFM image of the loop formed on the 1290 bp IL2RA fragment (Figure 2). (B) Height variation along the four colored lines in (A); the reference red line is drawn to allow relative positioning of the four topographic curves. (C) Schematic drawing of the corre- sponding loop. (D) Enlarged AFM image of a loop formed on the 900 bp IL2RA fragment. (E) Height variation along the four colored lines in panel (D). (F) Schematic drawing of the corresponding loop. (TIF) Figure S9 AFM image of a DNA loop. (A) Size distribution of DNA loops visualized on 21 images of the 1290 bp and 900 bp IL2RA fragments. (B) AFM images showing a DNA loop on the 1290 bp fragment. (C) AFM images showing a DNA loop on the 898 bp fragment. (TIF) Table S1 List of primers used during the PCR reaction for the amplification of the 1290 bp, 898 bp and 563 bp IL2RA DNA fragments. (DOC) Video S1 Consecutive AFM images of the IL2RA 1290 bp (Figure 2) during continuous scanning in the aqueous buffer at the frequency of 1 frame every 37 seconds. (MOV) Same as Video S1 for the 1290 bp IL2RA (Figure S6). Same as Video S1 for the 1290 bp IL2RA (Figure S7). Video S2 (MOV) Video S3 (MOV) Acknowledgments We are very grateful to A. Benecke, S. Eilebrecht and C. Lavelle for kind gifts of HMGA1 proteins and to A. Travers a careful reading of the manuscript. Author Contributions Conceived and designed the experiments: AA JRS MM. Performed the experiments: PM ASS. Analyzed the data: PM FA MM JRS AA. Contributed reagents/materials/analysis tools: FA MM JME JI. Wrote the paper: AA PM JRS JME JI MM. Contributed to the theoretical modeling: CV MM. 1. Goodrich JA, Cutler G, Tjian R (1996) Contacts in context: promoter specificity and macromolecular interactions in transcription. Cell 84: 825–830. 2. Conaway R, Conaway J (1997) General transcription factors for RNA polymerase II. Prog Nuc Acid Res Mol Biol 56: 327–346. 3. Kornberg RD (2007) The molecular basis of eukaryotic transcription. Proc Natl Acad Sci U S A 104: 12955–12961. 4. Iyer V, Struhl K (1995) Poly(dA:dT), a ubiquitous promoter element that stimulates transcription via its intrinsic DNA structure. EMBO J 14: 2570–2579. 5. Marilley M, Pasero P (1996) Common DNA structural features exhibited by eukaryotic ribosomal gene promoters. Nucleic Acids Res 24: 2204–2211. 6. Schatz T, Langowski J (1997) Curvature and sequence analysis of eukaryotic promoters. J Biomo. Struct Dyn 15: 265–275. 7. Gabrielian AE, Landsman D, Bolshoy A (2000) Curved DNA in promoter sequences. In Silico Biol 1: 183–196. 8. Segal E, Widom J (2009) Poly(dA:dT) tracts: major determinants of nucleosome organization. Curr Opin Struct Biol 19: 65–71. PLoS ONE | www.plosone.org 9 April 2011 | Volume 6 | Issue 4 | e18811