by gel electrophoresis (1.5% agarose) (Figure S1A) and AFM imaging. Nucleosome reconstitution Nucleosome reconstitution was performed by the salt dialysis procedure. The reaction was stopped in TE buffer (10 mM Tris- HCl, pH 7.4, 1 mM EDTA) and 10 mM NaCl. Nucleosomes were reconstituted with a histone/DNA ratio of 1/0.8. The formation of NCP was controlled by gel electrophoresis (1.5% agarose) (data not shown) and AFM imaging [19]. HMGA1-DNA complexes The isolated 898 bp IL2RA fragment (1.5 pmol) was incubated with increasing amounts of HMGA1 in 18 ml of binding buffer (17 mM Tris, pH 7.8, 41 mM NaCl, and 1.7 mM EDTA). After incubation for 30 min room temperature, the formation of complexes was controlled by gel electrophoresis (6.5% polyacryl- amide in 0.25X TBE) (Figure S1B). For AFM imaging we used a molar ratio of HMGA to DNA of 3:1. AFM imaging AFM imaging was performed in solution by using a Nanoscope IIIa microscope (Veeco/Digital Instruments, Santa Barbara, CA) equipped with a type-E scanner and in tapping mode [40]. Freshly cleaved mica (muscovite mica, grade V-I, SP1) served as a support for sample adsorption. DNA preparations, nucleosome reconsti- tution solution and DNA-TBP-TFIIB reaction mixture were diluted in different buffers according to different experimental protocols. In the presence of doubly charged counter ions (Ni2+) in the solution, DNA molecules are able to equilibrate on the surface before being captured in a given conformation [22,42]. For scanning in solution, we used the following buffer solutions: N Buffer A: 10 mM Tris-HCl, 0.1 mM NiCl2, pH 7.9 N Buffer B: 10 mM Tris-HCl, 1 mM NiCl2, pH 7.9 For DNA imaging (Figure S2), DNA preparations were diluted (0.3 ng/ml) in buffer A. A 10 ml droplet of the solution was deposited onto freshly cleaved mica and incubated for two minutes. A 100 ml volume of buffer A was added in the liquid cell prior to imaging. For time-lapse studies, images were registered and the direction of scanning was from top to bottom. The size of the window was 160.25 mm and the scanning frequency 3.38 Hz, which gave one image every 37 seconds. For DNA-TBP-TFIIB complexes, nucleosomes, and HMGA1- DNA complexes imaging, reaction mixtures were diluted ,4-fold in the buffer B. A 10 ml droplet of the solution was deposited onto freshly cleaved mica and incubated for two minutes. A 100 ml volume of buffer B was added in the liquid cell prior to imaging. AFM images were recorded from 161 mm or 262 mm frames, at a line scan rate of 2.18 Hz and a resolution of 5126512 pixels. For all scans, commercial silicon nitride probes (type NP-S, Veeco Instruments) were used at a drive frequency of 8–9.5 kHz at ambient temperature. Image analysis Image treatment and analysis were performed with the ‘‘Scanning Adventure’’ software [27]. Briefly, line-by-line sec- ond-order flattening was first applied, followed by thresholding and filtering. Zooms on individual DNA fragments, nucleosomes or DNA-TBP-TFIIB complexes were achieved and the DNA molecule path was skeletonized; then topographical analysis and length measurements were carried out (Figure S3). The results enabled a measurement of (i) the height variation along the DNA Structure and Dynamics of the IL2RA Gene Promoter molecules which allowed us to orient the DNA fragment [19] (Figure S3), (ii) the DNA-protein complexes height (Figure S4) and (iii) the mapping of the nucleosome and TBP-TFIIB positions along the DNA fragments. From the measurement of the length of free DNA outside the nucleosome, namely L+ (resp. L2) for the longest (resp. shortest) arm, and in turn of the DNA complexed into nucleosome Lc = L - (L + + L 2), where L is the total length of the considered DNA fragment, we positioned the dyad on this fragment by adding Lc/2 to either L+ or L2 as previously described [19]. The position of the TBP-TFIIB-DNA complex was determined as corresponding to the local height maximum along the DNA skeleton. The same procedure was applied for the HMGA1-DNA complex. Positions and lengths were measured in nm and converted in bp with the approximate rise of 0.36 nm per bp [27]. For time-lapse images analysis, DNA conformational changes were followed on successive scans of the same molecule by calculating the Ends ratio along the molecule for each scan, with a 150 bp window and a 1 bp step. The Ends ratio is a measurement of the DNA curvature as given by the ratio: (length along the molecule)/(distance between the two ends of the DNA segment) in a selected window. These changes were registered in series of about 10 images on different molecules. DNA modeling DNA duplex stability. The thermodynamic library [43] characterizing all 10 Watson-Crick nearest-neighbor interactions in DNA was used to calculate DNA duplex stability DG. These thermodynamic data provide an experimental basis for predicting the stability of any DNA duplex region by inspection of its primary sequence. Each calculated value takes into account the contribution of the surrounding nucleotides. We used the MAPall software [5] (previously PACS DNA), with a 150 bp window and a 1 bp step. All calculations were carried out with parameters and conditions (1 M NaCl, 25uC and pH 7) previously used by SantaLucia et al [43]. Curvature analysis and superhelix detection. The DNA path was calculated as the result of one translation and three rotations (roll, tilt and twist) at each nucleotide step using Bolshoy et al. values [44]. This DNA coding table was previously shown to provide very good fit of experimental DNA data from AFM analysis in liquid [40]. For superhelix detection, we used a method based on computer modeling allowing the detection of a left- handed (negative) as well as a right-handed (positive) superhelical organization of DNA encoded by the nucleotide sequence [40,45]. The superhelical structure was made apparent by calculating the 3D DNA trajectory at different pitch values. When properly phased, the intrinsically curved elements make the DNA double helix path curved in a 2D plane. The coordinates calculated from the 3D trajectory of the helix axis were used to compute the Ends ratio in a 2D-curvature map. The phasing pitch value was expressed as a number of base pairs per turn. Results Theoretical modeling: prediction of the DNA structure of the IL2RA promoter We investigated the 1290 bp 59 proximal promoter region of the IL2RA human gene [39] (Figure 1A), that includes: (i) the TSS, (ii) the core promoter with the TATA box (TACTTAAA), (iii) two positive regulatory regions, PRRI and PRRII, lying respectively at position 2289/2216 bp and 2137/264 bp upstream of the major TSS. PRRI contains binding sites for sequence-specific TFs, NF-kB, SRF and Sp1, whereas PRRII PLoS ONE | www.plosone.org 3 April 2011 | Volume 6 | Issue 4 | e18811