Mechanics of the IL2RA Gene Activation Revealed by Modeling and Atomic Force Microscopy Pascale Milani1,2,3, Monique Marilley1,2,3,5, Albert Sanchez-Sevilla1, Jean Imbert4,5, Ce´ dric Vaillant2,3, Fran c¸oise Argoul2,3, Jean-Marc Egly6, Jose´ Rocca-Serra1,5, Alain Arneodo2,3* 1 RGFCP EA 3290, Faculte´ de Me´decine, Marseille, France, 2 Universite´ de Lyon, Lyon, France, 3 Laboratoire Joliot-Curie and Laboratoire de Physique, CNRS, Ecole Normale Supe´rieure de Lyon, Lyon, France, 4 U928 Inserm, TAGC, Marseille, France, 5 Universite´ de la Me´diterrane´e, Marseille, France, 6 Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, CNRS/INSERM/Universite´ de Strasbourg, Illkirch, France Abstract Transcription implies recruitment of RNA polymerase II and transcription factors (TFs) by DNA melting near transcription start site (TSS). Combining atomic force microscopy and computer modeling, we investigate the structural and dynamical properties of the IL2RA promoter and identify an intrinsically negative supercoil in the PRRII region (containing Elf-1 and HMGA1 binding sites), located upstream of a curved DNA region encompassing TSS. Conformational changes, evidenced by time-lapse studies, result in the progressive positioning of curvature apex towards the TSS, likely facilitating local DNA melting. In vitro assays confirm specific binding of the General Transcription Factors (GTFs) TBP and TFIIB over TATA-TSS position, where an inhibitory nucleosome prevented preinitiation complex (PIC) formation and uncontrolled DNA melting. These findings represent a substantial advance showing, first, that the structural properties of the IL2RA promoter are encoded in the DNA sequence and second, that during the initiation process DNA conformation is dynamic and not static. Citation: Milani P, Marilley M, Sanchez-Sevilla A, Imbert J, Vaillant C, et al. (2011) Mechanics of the IL2RA Gene Activation Revealed by Modeling and Atomic Force Microscopy. PLoS ONE 6(4): e18811. doi:10.1371/journal.pone.0018811 Editor: Axel Imhof, Ludwig-Maximilians-Universita¨t M u¨nchen, Germany Received November 15, 2010; Accepted March 16, 2011; Published April 13, 2011 Copyright: 2011 Milani et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Conseil Re´gional PACA, the Conseil Ge´ne´ral des Bouches du Rhoˆ ne, the Conseil Re´gional Rhoˆ ne-Alpes (Emergence 2005) and the Agence Nationale de la Recherche under project DNAnucl (ANR-06-PCVI-0026). P. Milani was supported by the "Collectivite´ Territoriale de Corse". The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: alain.arneodo@ens-lyon.fr Introduction RNA polymerase II-dependent transcription is initiated at a promoter, with a core region able to sustain basal RNA synthesis. Initiating transcription implies the binding of GTFs and RNA polymerase II on the promoter as well as the localized melting of DNA in the TSS region [1–3]. Initiation thus requires that the DNA structure facilitates the binding of these factors as well as the appropriate DNA melting. Special attention has been devoted to the DNA structure in the promoter region as a plausible physical support for promoter activation [4–8]. Indeed, since the discovery of intrinsically curved DNA [9], many experiments have established that the local sequence-dependent physical (structural, mechanical) properties of the DNA double helix are one of the major ingredients in DNA-protein interactions [10,11]. In particular, the energy cost for nucleosome formation presents a sequence-dependent contribution which may influence the positioning of nucleosomes, and in turn the access of TFs to their target sites thereby contributing to gene expression regulation [12,13]. Recent studies have shown that the DNA sequence actually codes for high-energy barriers that impair nucleosome formation [14–16], and condition the collective assembly of neighboring nucleosomes according to equilibrium statistical ordering principles [16–19]. These genomic excluding-energy- barriers are highly predictive of the nucleosome-free regions (NFRs) observed in vitro and in vivo at the promoter of genes that are constitutively expressed [18–21]. Alternatively, the relative positioning of these energy barriers with respect to the TSS can also condition an inhibitory nucleosomal chromatin environment that represses gene-transcription [19]. Among the various experimental techniques that have been used to investigate DNA binding and flexibility, atomic force microscopy (AFM) has proved to be very efficient. It can be used to estimate the persistence length of DNA via end-to-end distance measurements [22–26], but more interestingly it can give access, via thermodynamical averaging over a population of conforma- tions, to both the intrinsic DNA curvature and bending flexibility with a surprisingly good resolution of 2–3 double-helix pitches [23,24,27]. AFM imaging has also been applied to characterize the chromatin structure at the nanoscale level [28]. Nucleosome assembly has been visualized, mainly in air, and in the specific context of positioning sequences (e.g. Xenopus 5S rDNA and 601 DNA sequences) and arrays of concatenated repetitions of these sequences [28–31]. Only recently, AFM imaging in liquid has provided new insight into the role of the DNA sequence on the collective organization of nucleosomes along the eukaryotic chromatin fiber [19,32]. Single-molecule time-lapse AFM ap- proach has also proved to be capable of detecting the dynamics of DNA conformational changes [33,34], of telomeric nucleosomes [35], of unwrapping of DNA from the histone core in the absence of ATP-dependent chromatin remodelers [36] and of molecular systems involved in transcription [37] and replication [38]. Here we combined AFM imaging in liquid and physical DNA modeling to investigate the structural and dynamical DNA properties, nucleosome assembly and TF binding in the promoter region of the human IL2RA gene which plays a major role in the PLoS ONE | www.plosone.org 1 April 2011 | Volume 6 | Issue 4 | e18811