Structure and Dynamics of the IL2RA Gene Promoter Figure 4. Tentative model for the activation of IL2RA gene expression. (A) Schematic representation of structural changes induced by the dynamics of a negative supercoil (circled minus) in a region containing the PRRII element (blue bracket) on naked DNA. The supercoil twisting (or DNA untwisting) allows the repositioning of the TSS from a straight portion keeping it closed (red dot), to the curvature apex of the molecule, thus favoring the melting of this region (red star). A red bracket indicates the position of the TATA box. (B) Model of the IL2RA promoter before (resting cell) and after (activated T cells) activation. The upper panel depicts the centrally positioned nucleosome masking part of PRRII (blue bracket), the TATA box and the TSS region, preventing both the PIC formation and Elf-1 binding, and keeping closed the TSS region. After activation, the binding of three HMGA molecules on PRRII induces a negative torsional stress that contributes to the remodeling and/or the ejection of the nucleosome, and allows the binding of the PIC and of Elf-1 on the TATA box (red bracket) and PRRII, respectively (lower panel). The TSS (+1, red star) is at the DNA curvature apex. doi:10.1371/journal.pone.0018811.g004 II entry even in the absence of the TBP [55]. Supercoiled DNA templates were further shown to be required for enhancer- mediated in vitro regulation of the chicken bA-globin and mouse T- cell receptor genes [56]. Negative DNA superhelicity is also involved in the basal level of transcription of the c-myc gene [57]. Very much like the PRRII control region of the IL2RA gene promoter, all these regions with peculiar supercoiled DNA properties appear as ideal target for DNA-binding proteins. In conclusion, by combining DNA modeling and AFM imaging in liquid, we investigated the structure and dynamics of the IL2RA gene promoter and suggested the following model for the activation of gene expression (Figure 4B): a) HMGA1 multiple binding to specific sites in the PRRII region, b) remodeling of the inhibitory nucleosome positioned on the TATA box and TSS via torsional stress propagation, c) timely DNA melting at a specific region via local DNA curvature enhancing and d) recruitment and binding of the PIC. Further studies might be required to determine whether this sequence- specific structural and dynamical activation scenario is peculiar to the human IL2RA gene promoter or can be extended to other inducible RNA Pol II-dependent promoters. Supporting Information Figure S1 Analysis of the complexes of transcription factors and DNA by electrophoretic mobility shift assay. (A) EMSA performed with (from left to right): 563 bp IL2RA naked DNA (lane 1), 563 bp IL2RA DNA and TBP (lane 2), 563 bp IL2RA DNA, TBP and TFIIB (lane 3), 563 bp IL2RA DNA and TFIIB (lane 4) and DNA ladder 100 bp (lane 5). Bands in the gel correspond only to naked DNA and DNA-protein complexes except for the DNA ladder (lane 5). (B) EMSA performed with the 898 bp IL2RA DNA fragment and HMGA for increasing [HMGA1]/[DNA] ratio. The molar ratios of HMGA to DNA were increased from 0 to 3 (0, 1, 2, 3) for lanes 1 to 4. (TIF) Figure S2 AFM imaging of the IL2RA gene promoter region. (A) Large scale (161 mm) AFM imaging in liquid of 1290 bp fragments in aqueous buffer. AFM imaging in liquid of 1290 bp fragments (B, C), 898 bp fragments (D, E) and 563 bp fragments (F, G). (TIF) PLoS ONE | www.plosone.org 8 April 2011 | Volume 6 | Issue 4 | e18811