encompasses three binding sites for the architectural factor HMGA1 and one for Elf-1, a lymphoid-specific protein that belongs to the ETS TF family. According to the importance of these two elements for regulating inducible transcription of the IL2RA gene [39], our aim was to elucidate the possible contribution of supercoiled DNA structures to the intrinsic repression of this gene as well as their actual role in its activation. As noticed earlier [9], the periodic distribution of A/T stretches participates in determining the shape of bent DNA. If the periodicity is equal to the DNA canonical pitch ,10.5 bp, DNA forms an easily detectable planar curve. However, if this periodicity is significantly different, then DNA forms either a right- or a left-handed 3D superhelix which turns out to be difficult to detect in practice. To unravel the presence of such supercoiled structures, we used a method based on computer modeling of the DNA trajectory using the 16 dinucleotide steps [44,45]. Indeed, by artificially ‘‘twisting’’ (i.e. decreasing the phasing pitch) or ‘‘untwisting’’ (i.e. increasing the phasing pitch) the molecule, it is possible to detect a left- or a right-handed supercoil respectively by making them appear as a 2D bent curve. When reconstructing the curvature landscape (Figure 1B) from the successive Ends ratio profiles (Materials and Methods) obtained along the IL2RA promoter for increasing pitch values from 5 bp to 15 bp (Figure 1D), we indentified two new important conformational characteristics of the promoter. Besides the detected 2D curvatures (white circles on the grey-coded curvature map, Figure 1B, upper plane) identified on the Ends ratio profile for the canonical 10.5 bp pitch value, two Ends ratio peaks were obtained for pitch values ,7.5 bp and 13.5 bp as the signature of negative and positive supercoiled DNA respectively (red circles on the grey-coded curvature map, Figure 1B, upper plane). Importantly, both these negative and positive supercoils co-map with PRRII [46]. These results were substantiated when using the MAPall software [5] to represent the 3D path of the DNA molecule in the vicinity of the PRRII region (Figure 1C). Two different supercoils coexist: a large amplitude, negative one which encloses a smaller positive one. This second one is hardly visible when the diameter of the double- helix is represented at the physical scale (Figure 1C, right panels); it becomes clear when focusing on the double-helix axis only (Figure 1C, left panels). AFM imaging of PRRII DNA supercoiling AFM imaging in liquid (Materials and Methods) allowed us to validate the theoretical prediction of the presence of an intrinsic supercoiled structure in the IL2RA promoter region (Figure 2A and Figure S2) and of its handedness as left-handed, i.e. negative (Figure 2C) as spectacularly revealed when zooming on the corresponding region (Figure 2B). From a quantitative point of view, the height profiles obtained along two different vision axis exhibit maxima as high as 1.4–1.6 nm along the supercoil that significantly exceed the characteristic ,0.9–1.1 nm height of the double-helix lying on the mica surface (Figure 2D). Note that the standard deviation of height measurements from 100 molecules is fairly constant (,0.3 nm) along the entire molecule trajectory (Figure S5E). After orientating the DNA molecule (Material and Methods and Figure S3), the supercoil was found to be located between positions 2160620 bp and 270620 bp from the TSS, confirming its co-localization with the PRRII element. Impor- tantly, this supercoil was robustly observed on the 1290 bp DNA fragment but also on the shortest fragment of length 563 bp (figure S2F and G). Furthermore, when using the 976 bp pBR DNA fragment as a control (Materials and Methods), no evidence of 3D supercoiled structure was ever obtained (Figure S5A, C-E), in agreement with theoretical modeling (Figure S5B). This experi- Structure and Dynamics of the IL2RA Gene Promoter mental AFM study thus demonstrated the existence of a left- handed supercoiled structure in the PRRII region. However, to detect the presence of a small right-handed supercoiled structure, as also predicted by theoretical modeling (Figure 1), a higher resolution of the AFM tip needs to be achieved. Time-lapse AFM analysis of DNA structural changes AFM observations made in liquid allow the tracking of DNA conformational changes in time, provided conditions for low adhesion to the support are satisfied. In such ionic conditions, the molecules explored different conformations although interactions with the mica surface remained sufficient to maintain their local 2D confinement necessary for AFM imaging [19,40]. In addition, the scanning frequency was increased so that we could register one image of the same molecule every 37 seconds. We produced 3 movies that are available online (Videos S1, S2 and S3). As seen in selected frames (Figure 2E, and Figures S6 and S7), all these molecules exhibit a left-handed supercoiled structure at the same location as theoretically predicted (Figure 1). To visualize structural modifications occurring at the IL2RA promoter, we zoomed on images taken at the beginning of the observation (T0) and successively 4, 6 and 8 minutes later (T4, T6 and T8 respectively). The supercoil encompassing the PRRII region definitely changed geometry (Figure 2E and Figure S6). Some negative overwinding of the supercoiled region progressively invaded the structure, eventually leading to the formation of a DNA loop (Figure 2E, bottom panel). Successive topographical profiles registered on the loop unambiguously demonstrated the left-handedness of this structure (Figure S8A–C). The analysis of as many as 21 of such left-handed DNA loops (Figure S9) assesses its positioning in the region extending from PRRII to the TATA box and TSS (Figure 3A). Despite some variability in size, these loops have a characteristic length ,260 bp (,94 nm) which is about twice the persistence length of B-DNA (,50 nm) [25,26]. To further analyze the consequences of these morphological changes on the curvature properties of these IL2RA molecules, we compared the Ends ratio profiles (Materials and Methods) obtained at different times (Figure 2F, Figures S6C and S7C). At T0, the DNA molecule had a curved region slightly upstream the TSS, in good agreement with the theoretical curvature profile predicted for the canonical pitch value 10.5 bp (Figure 1D). When time proceeded, the position of the curvature apex shifted towards the TSS. When comparing the curvature profiles obtained at later time T8 (Figure 2F and Figure S6C), with the theoretical profiles predicted when changing the pitch value (Figure 1D), we showed that the observed shift in the curvature apex towards the TSS could be mimicked by artificially increasing locally the pitch to values ,11–11.5 bp. Thus, the observed dynamical evolution of the curvature properties of the DNA molecule in the TATA box and TSS regions would be the signature of some local unwinding of the DNA double helix, likely weakening the thermal requirement for strand separation (i.e. lowering DG at the TSS, see Figure 2G) and ultimately leading to base-pair opening at the required position as suggested by sharp decrease observed on the height profile in the TSS region (see Figure S7B). Mapping of functional and architectural proteins along the IL2RA promoter by AFM Having examined the intrinsic structural properties of the IL2RA promoter, we next investigated the role of these properties in the formation of DNA-protein complexes. TBP-TFIIB binding. Electrophoretic mobility shift assays (EMSAs) were performed on the IL2RA promoter with the presence of TBP and TFIIB GTFs (Materials and Methods and PLoS ONE | www.plosone.org 4 April 2011 | Volume 6 | Issue 4 | e18811