control of the immune system response [39]. We observed a very specific DNA structural organization linked to the IL2RA gene promoter sequence [40] that conditions the positioning of a nucleosome on the TATA box and TSS region as previously observed in vivo in unstimulated T-cells [41]. Besides preventing the binding of TBP and TFIIB and in turn PIC formation on the TATA box, this repressive nucleosome positioning likely protects from uncontrolled DNA melting in the TSS region. Time-lapse studies also revealed characteristic conformation changes under local DNA unwinding constraints that eventually facilitate DNA melting at the TSS. We elaborate on a model that incorporates these structural and dynamical DNA features of the IL2RA gene promoter region in its functional chromatin context. In particular we discuss the possible contribution of this constitutive repressive chromatin environment to transcription activation by favoring multiple HMGA1 binding, nucleosome remodeling and DNA unwinding at the TSS. Materials and Methods DNA samples and proteins preparation Three DNA fragments of length 563 bp, 898 bp and 1290 bp containing the 59 proximal promoter region (Figure 1A) of the IL2RA human gene (EMBL:U57613, 12 October 2001) were obtained by PCR amplification of human placenta genomic DNA. Amplifications were performed using the Taq PCR Master Mix Structure and Dynamics of the IL2RA Gene Promoter Kit (Qiagen) (for a list of primers, see Table S1). Mixtures were run on a 1% agarose gel and the isolated DNA fragments were extracted from the corresponding gel slice by using a MinEluteH Gel Extraction Kit (Qiagen). The 976 bp pBR DNA fragment used as a negative control was obtained by EcoRI and NruI digestion of the pBR322 plasmid, separated by 1% agarose gel electrophoresis and purified using a Sephaglass kit (Amersham Pharmacia Biotech). Full-length recombinant histone proteins of Xenopus laevis were over-expressed in bacteria and purified as previously experienced [19]. Full-length recombinant human TBP and TFIIB proteins were produced and purified as previously described [40]. HMGA1a protein (referred to as HMGA1 for simplicity in this report) is a kind gift of A. Benecke (Eilebrecht, S., Brysbaert, G., Wegert, T., Urlaub, H., Benecke, A. & Benecke, B.J. 7SK small nuclear RNA is direct affector of HMGA1 function in transcrip- tion. In preparation). DNA-TPB-TFIIB interaction DNA and protein complexes were assembled using the 563 bp IL2RA DNA fragment in a final volume of 10 ml, with reaction mixture containing 10 mM DNA fragment, 5 ml of binding buffer (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 100 mM NaCl, 10% Glycerol). Human proteins were used in the following amounts: TBP 15 g, TFIIB 125 ng. The reaction mixtures were incubated at 25uC for 30 min. The formation of complexes was controlled Figure 1. Theoretical modeling of the DNA structure of the IL2RA gene promoter. (A) Schematic representation of the 1290 bp human IL2RA gene fragment between nucleotides 2869 bp and +421 bp, including the positive regulatory regions PRRI and PRRII, the TATA box (TACTTAAA) and the TSS (+1). The locations of the 898 bp and 563 bp fragments are also indicated. Positions of major TF binding sites are shown by grey arrows. (B) Theoretical analysis of the 1290 bp IL2RA sequence. Diagrammatic (upper plane) and spectral (lower graph) representation of the curvature as estimated by the Ends ratio at a given position for a given phasing pitch value (Materials and Methods). Red circles correspond to right- handed and left-handed supercoiled DNA. White circles and white line correspond to theoretically predicted and experimentally detected 2D curvatures [40]. (C) 3D modeling of the PRRII region sequence (delimited by the red labels). Left panels show the DNA double helix axis trajectory (thickness = 0.2 nm) from two different orientations. The negative and positive supercoil locations are specified. Right panels display the corresponding DNA molecule at physical scale (thickness = 2 nm). (D) Curvature profiles predicted by modeling the DNA fragment under over- twisting (pitch values 7.5 and 10 bp) or under-twisting (pitch values 11 and 13.5 bp), as compared to the 10.5 bp canonical reference value. The vertical blue line indicates the TSS (+1) position. In (b,d): window size is 150 bp, analyzed phasing pitch varies from 5 to 15 bp with step 1 bp. doi:10.1371/journal.pone.0018811.g001 PLoS ONE | www.plosone.org 2 April 2011 | Volume 6 | Issue 4 | e18811