Hopefully, several experimental observations allowed us to reject this objection without denying the influence of the substrate. First, formation of this supercoil was independent of the size of the IL2RA DNA fragment (Figure 2 and Figure S2F and G). Second, for all the IL2RA molecules so far tested (Figure 2, and Figures S6 and S7), the supercoil was found exactly at the same position which coincided with our theoretical modeling prediction (Figure 1). Third, time-lapse studies confirmed that the imaged DNA molecules were sufficiently free to move relative to the substrate. In addition to these thermodynamic and structural features, we observed, consistently with a previous in vivo study [41], that an inhibitory nucleosome was positioned on the PRRII control region, the TATA box and the TSS (Figure 3C). This critically positioned nucleosome, in addition to preventing both TBP and TFIIB binding and in turn PIC formation on the TATA box and Elf-1 binding on PRRII, might also take part in preventing uncontrolled DNA melting by maintaining stably closed the double-helix in the vicinity of the TSS. Furthermore, we showed that another nucleosome was critically positioned on the 59 upstream PRRI control region as an obstacle to the binding of NF- kB, SRF and Sp1 TFs. TSS is poised to open If the increase in thermal stability and the intrinsic supercoiled structure are advantageous to prevent uncontrolled DNA melting in the TSS region (Figure 2G), they should not be obstacles to the opening of the double helix at the time of transcription initiation. Several other structural characteristics of the DNA molecule are known to facilitate base-pair opening including topologically forced DNA curvature that weakens the thermal requirement for strand separation [49]. This is explained by a simultaneous lowering of the stacking energy and by the accumulation of energy within the sugar phosphate backbone which may be further released to open the double helix. In this in vitro study, we showed that the overtwisting of the intrinsic supercoiled structure located in the PRRII region was accompanied by the progressive positioning of the curvature apex at the TSS (Figure 2G and F and Figure S6), with the probable thermodynamic consequence of facilitating a localized melting of the double helix [49,50]. Since this dynamical evolution was observed in the absence of initiation factors, local torsional stress likely induced by the extensive interaction with the charged mica surface, was apparently sufficient to unwind DNA. A sequence-dependent preference to the opening of the double helix at or near the TSS has been reported in an experimental study of different types of promoters [51]. The present AFM study suggests that the intrinsic structure of the IL2RA promoter is a key actor in the activation process of the IL2RA gene (Figure 4A). In vivo probably under the action of architectural TFs, conformational changes induced by DNA unwinding are likely to direct the pre-melting reaction at the prescribed TSS locus. In addition, the modifications detected in DNA flexibility and curvature properties in the TATA box vicinity are likely to facilitate the binding of TBP and TFIIB GTFs at this particular region [40] (Figure 3B) thereby promoting PIC formation. PRRII control region: an adequate substrate for HMGA1 proteins The functional role of PRRII in the regulation of the IL2RA gene expression was first evidenced by a mutation within this region inhibiting its transcription [46]. Binding of HMGA1 (Figure 3D) on an intrinsic supercoiled structure was revealed both by AFM experiment (Figure 2) and theoretical modeling Structure and Dynamics of the IL2RA Gene Promoter (Figure 1). Actually the PRRII sequence is characterized by an anomalous high A+T content (AT-tracts) and the intrinsic supercoil possibly results from the regular spacing of the AT- tracts with a periodicity slightly out of phase with the mean helical pitch. PRRII sequence contains three AT-tracts, separated by 6 and 7 bp, the longest one being 19 bp long, corresponding to three potential binding sites with different affinities for HMGA1. HMGA1 contains AT-hook domains allowing its binding to the minor groove of AT-rich sequences; high affinity actually requires multiple properly phased AT-tracts [52] with spacing ,10 bp. Interestingly, the AT-tracts in PRRII are separated by less that 10 bp which suggests that the observed negative supercoiled DNA structure is the source of specificity for HMGA1 positioning and high affinity binding. Importantly AFM further revealed that the PRRII region is drastically depleted in histone octamer binding [19] (Figure 3C). Because of their relative rigidity, multiple poly(dA:dT) stretches are known to be poorly incorporated into nucleosomes in vivo [4,8,16]. The presence of A-tracts in the PRRII region impairs the structural distortions required for nucleosome formation, leaving some room for HMGA1 binding. A scenario for transcription activation Indeed, HMGA1 binding is likely to be actively involved in the remodeling of the inhibitory nucleosome in response to T-cells activation [53]. Besides HMGA1 binding, additional processes, including histone modifications, must contribute to remodel the repressive nucleosome. Our results suggest that this remodeling might have some dynamical and structural origin. The observed dynamical evolution of the intrinsic left-handed supercoiled structure in the IL2RA promoter region could act as a lever arm helping nucleosome remodeling and/or ejection. This attractive picture is consistent with the experimental observation that when binding to a closed circular DNA at a high protein/ DNA ratio, HMGA1 induces a negative torsional stress which, by accumulation, results in a negative supercoil [53]. In our AFM study, as a substitute to HMGA1 action, the electrostatic interaction of free DNA with a positively charged mica surface could induce some dynamical overtwisting of the supercoil that ultimately might destabilize the nucleosome positioned on the TATA box and TSS. We propose the following scenario for IL2RA transcription activation. In a physiological context, resting T-cells contain low but detectable amounts of HMGA1, insufficient to induce nucleosome ejection. Following activation, the increase in concentration of this protein and its binding on its three specific sites (AT-tracts) could act as a switch by giving the torsional stress necessary to promote remodeling of the inhibitory nucleosome, and in turn Elf-1 fixation. In addition, the propagation of this torsional stress (DNA unwinding), accompa- nied by a shift of the DNA curvature apex towards the TSS, would also facilitate DNA melting and the recruitment of the RNA Pol II at the TATA box. The importance of supercoiled structures in the regulation of transcription process has been the subject of increasing interest. For example, in prokaryotes, the ilvPG gene of E. coli requires an intrinsically supercoiled DNA template located at the promoter to allow the binding at position 292 bp of the integration host factor (IHF). This peculiar structure was shown to mediate the translocation of superhelical energy, to facilitate stabilization in the 210 bp promoter region, and to activate transcription by increasing the rate of open complex formation [54]. These mechanisms are likely to be important for the regulation of transcription in eukaryotes as well. In S.cerevisiae CUP1 gene, negatively supercoiled DNA templates apparently allow RNA Pol PLoS ONE | www.plosone.org 7 April 2011 | Volume 6 | Issue 4 | e18811