Mechanics of the IL2RA Gene Activation Revealed by Modeling and Atomic Force Microscopy

Pascale Milani,Albert Sanchez-Sevilla,José Rocca-Serra,Monique Marilley,Alain Arneodo,Françoise Argoul,Cédric Vaillant,Jean-Marc Egly,Jean Imbert

doi:10.1371/journal.pone.0018811

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.

Highlights

RNA polymerase II-dependent transcription is initiated at a promoter, with a core region able to sustain basal RNA synthesis
According to the importance of these two elements for regulating inducible transcription of the IL2RA gene [39], our aim was to elucidate the possible contribution of supercoiled DNA structures to the intrinsic repression of this gene as well as their actual role in its activation
From the analysis of N = 50 oriented DNA molecules loaded by TBP-TFIIB General Transcription Factors (GTFs), we found that these factors preferentially bind to the putative functional TATA box (Figure 3B, left panel)

Summary

Introduction

RNA polymerase II-dependent transcription is initiated at a promoter, with a core region able to sustain basal RNA synthesis. Recent studies have shown that the DNA sequence codes for high-energy barriers that impair nucleosome formation [14,15,16], and condition the collective assembly of neighboring nucleosomes according to equilibrium statistical ordering principles [16,17,18,19]. These genomic excluding-energybarriers are highly predictive of the nucleosome-free regions (NFRs) observed in vitro and in vivo at the promoter of genes that are constitutively expressed [18,19,20,21]. The relative positioning of these energy barriers with respect to the TSS can condition an inhibitory nucleosomal chromatin environment that represses gene-transcription [19]

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Conclusion