Structure and Dynamics of the IL2RA Gene Promoter Figure 2. AFM imaging of the IL2RA gene promoter. (A) AFM image of the 1290 bp IL2RA fragment presenting a supercoiled structure. (B) Zoom on the region containing the supercoil, in the white rectangle in panel (A). Grey coding of topographic variations allows the visualization of the 3D shape of the structure. Sections 1 (blue line) and 2 (red line) correspond to the height measurements displayed in panel (D). (C) Schematic drawing of the negative supercoiled structure observed along the red vision axis 2 in panel (b). (D) Height measurements along the red and blue lines in panel (B). The supercoil summits are over 1.5 nm, whereas the double helix height in liquid ranges between 0.8 and 1.2 nm. (E) 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. A loop was formed out of the supercoiled region after 8 min. (F) Corresponding curvature profiles, expressed as Ends ratios (Materials and Methods), along the fragment (window 150 bp, step 1 bp). The position of the TSS (+1) is indicated by a vertical blue line. (G) DG variation profile along the 1290 bp IL2RA fragment. The horizontal line is the mean DG value over the whole gene sequence. doi:10.1371/journal.pone.0018811.g002 Figure S1A). When TBP and TFIIB were incubated with the IL2RA promoter, we detected a (upper) band corresponding to the TBP-TFIIB-IL2RA complex (lane 3). A similar but less intense band was also detected without TFIIB (lane 2) as the signature of the less stable TBP-DNA complex. However, this band disappeared when TBP was not added to the reaction mixture (lane 4), thereby confirming that TFIIB cannot specifically bind to DNA without the presence of TBP. We corroborated the high affinity of IL2RA promoter for GTF binding by visualizing, by AFM, the binding of TBP-TFIIB TFs on the 563 bp IL2RA fragment (Figure 3B, right panels). From the analysis of N = 50 oriented DNA molecules loaded by TBP-TFIIB GTFs, we found that these factors preferentially bind to the putative functional TATA box (Figure 3B, left panel). The fact that the TATA box region corresponds to curved DNA was previously argued as favoring the binding of these TFs as the first step of PIC formation [40]. A similar observation was obtained with the longest 1290 bp IL2RA fragment (data not shown). Nucleosome assembly. We also investigated nucleosome assembly on the 898 bp IL2RA DNA fragment (Materials and Methods) with the specific goal of comparing nucleosome positioning with GTF binding in this constitutively repressed promoter region [19]. From the analysis of N = 100 oriented DNA molecules loaded by a single nucleosome (Figure 3C, right panels), we observed some lack of nucleosome positioning from 2200 bp to 280 bp upstream the TSS (Figure 3C, left panel), exactly where the DNA molecule was predicted to be intrinsically supercoiled both theoretically (Figure 1) and experimentally (Figure 2). This nucleosome free region corresponds to some excluding energy barrier [19] that is coded in the DNA sequence and which further conditions the positioning of a nucleosome at (i) its left foot encompassing the PRRI region and (ii) its right foot on the TATA box and TSS (Figure 3C, left panel). Our AFM study therefore evidenced the presence of an inhibitory nucleosome on the TATA box and TSS, i.e. at the critical location of PIC formation, a point that was observed in vivo in unstimulated T-cells [41]. HMGA1 protein binding. The PRRII region was early on recognized [46] as a target site for HMGA1, a key player in the regulation of inducible transcription of the IL2RA gene. When further investigating the binding of HMGA1 proteins on the 898 bp IL2RA DNA fragment (Materials and Methods and Figure S1B), we observed a preferential binding in the region of intrinsically supercoiled DNA (Figure 3D, right panels) that encompasses PRRII (Figure 3D, left panel) and where PLoS ONE | www.plosone.org 5 April 2011 | Volume 6 | Issue 4 | e18811