Mechanics of the IL2RA gene activation revealed by modeling and atomic force microscopy.

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.

Nucleosome positioning by genomic excluding-energy barriers.

Recent genome-wide nucleosome mappings along with bioinformatics studies have confirmed that the DNA sequence plays a more important role in the collective organization of nucleosomes in vivo than previously thought. Yet in living cells, this organization also results from the action of various external factors like DNA-binding proteins and chromatin remodelers. To decipher the code for intrinsic chromatin organization, there is thus a need for in vitro experiments to bridge the gap between computational models of nucleosome sequence preferences and in vivo nucleosome occupancy data. Here we combine atomic force microscopy in liquid and theoretical modeling to demonstrate that a major sequence signaling in vivo are high-energy barriers that locally inhibit nucleosome formation rather than favorable positioning motifs. We show that these genomic excluding-energy barriers condition the collective assembly of neighboring nucleosomes consistently with equilibrium statistical ordering principles. The analysis of two gene promoter regions in Saccharomyces cerevisiae and the human genome indicates that these genomic barriers direct the intrinsic nucleosome occupancy of regulatory sites, thereby contributing to gene expression regulation.

Atomic force microscopy of DNA in solution and DNA modelling show that structural properties specify the eukaryotic replication initiation site.

The replication origins (ORIs) of Schizosaccharomyces pombe, like those in most eukaryotes, are long chromosomal regions localized within A+T-rich domains. Although there is no consensus sequence, the interacting proteins are strongly conserved, suggesting that DNA structure is important for ORI function. We used atomic force microscopy in solution and DNA modelling to study the structural properties of the Spars1 origin. We show that this segment is the least stable of the surrounding DNA (9 kb), and contains regions of intrinsically bent elements (strongly curved and inherently supercoiled DNAs). The pORC-binding site co-maps with a superhelical DNA region, where the spatial arrangement of adenine/thymine stretches may provide the binding substrate. The replication initiation site (RIP) is located within a strongly curved DNA region. On pORC unwinding, this site shifts towards the apex of the curvature, thus potentiating DNA melting there. Our model is entirely consistent with the sequence variability, large size and A+T-richness of ORIs, and also accounts for the multistep nature of the initiation process, the specificity of pORC-binding site(s), and the specific location of RIP. We show that the particular DNA features and dynamic properties identified in Spars1 are present in other eukaryotic origins.

Gradual melting of a replication origin (Schizosaccharomyces pombe ars1): in situ atomic force microscopy (AFM) analysis.

Local DNA melting is integral to fundamental processes such as replication or transcription. In vivo, these two processes do not occur on molecules free in solution but, instead, involve DNA molecules which are organized into DNA/proteins complexes. Atomic force microscopy imaging offers a possibility to look at individual molecules. It allowed us to follow the progress of local denaturation in liquid, but with the added constraints of DNA lying on a surface. We present a kinetic analysis of the mapping of the temperature-driven melting seen at a replication origin (Schizosaccharomyces pombe ars1). The results indicate an expected base composition dependency, but also a strong extremity effect. Noteworthy, a "structural" effect is clearly occurring - which is shown by the greater susceptibility of the strongly curved region present in the sequence to unwind. DNA melting, at this place, is seen to occur after an increase in the curvature amplitude and a simultaneous shift of the nucleotide sequence positioned at the apex. Because this may determine the position of the Replication Initiation (R.I.) site, the result suggests that eukaryotic replication origins, although described as possessing no consensus sequences, may well have their mechanics sustained by the properties of common structural features. Our analysis may, therefore, provide new information that will give genuine insights on how DNA molecules behave when organized into primosomes, replisomes, promoter initiation complexes, etc. and thus, be essential to better understanding the way genes function.

TBP binding capacity of the TATA box is associated with specific structural properties: AFM study of the IL-2R alpha gene promoter.

DNA is not only a nucleotide sequence which allows the binding of regulators but its intrinsic structural properties such as curvature and flexibility are also viewed as playing an active role in the regulation of transcription. Our combination of computer modelling and AFM imaging allow direct access to DNA curvature and flexibility. We have searched for these DNA structural features involved in transcription regulation within the IL-2Ralpha gene promoter. Investigation of these structural characteristics shows concordant results. First, in the core promoter, the region containing the functional TATA box shows intrinsic curvature associated with a peculiar distribution of flexibility. Both these inherent properties are characteristic of this region as compared with the other parts of the promoter. Second, the proximal promoter exhibits two important regions: a first one flexible and curved, followed by a segment of rigid linear DNA, each localised within one of the two Positive Regulatory Regions PRRI and PRRII respectively. Based on these observations, we propose different roles for DNA curvature and/or flexibility in promoter sequences.

Fine mapping of inherent flexibility variation along DNA molecules: validation by atomic force microscopy (AFM) in buffer.

Curvature and flexibility are structural properties of central importance to genome function. However, due to the difficulties in finding suitable experimental conditions, methods for studying one without the interference of the other have proven to be difficult. We propose a new approach that provides a measure of inherent flexibility of DNA by taking advantage of two powerful techniques, X-ray crystallography and nuclear magnetic resonance. Both techniques are able to detect local curvature on DNA fragments but, while the first analyzes DNA in the solid state, the second works on DNA in solution. Comparison of the two data sets allowed us to calculate the relative contribution to flexibility of the three rotations and three translations, which relate successive base pair planes for the ten different dinucleotide steps. These values were then used to compute the variation of flexibility along a given nucleotide sequence. This allowed us to validate the method experimentally through comparisons with maps of local fluctuations in DNA molecule trajectory constructed from atomic force microscopy imaging in solution. We conclude that the six dinucleotide-step parameters defined here provide a powerful tool for the exploration of DNA structure and, consequently will make an important contribution to our understanding of DNA-sequence-dependent biological processes.

Accuracy of AFM measurements of the contour length of DNA fragments adsorbed on mica in air and in aqueous buffer.

The measurement by atomic force microscope of the contour length of DNA fragments adsorbed on mica has been made as accurate as possible by revisiting the different steps of image acquisition and processing. In air, the DNA helical rise was estimated at 2.97 +/- 0.15 A per base pair (bp) (mean +/- standard deviation) by imaging a 648-bp DNA fragment and 2.95 +/- 0.14 A per bp for a 115-bp fragment. This confirms earlier observations suggesting that drying DNA fragments on mica in the presence of nickel induces limited conformational changes. At this point the exact nature of these conformational changes remains unknown. Simple hypotheses are the transconformation of stretches of the DNA molecules to the A-form of the double helix or alteration of the helix structure at the points of contact between DNA and mica. By contrast, in aqueous buffer, the measured helical rise was 3.14 +/- 0.15 A per bp for the 648-bp fragment and 3.17 +/- 0.13 A per bp for the 1115-bp fragment. Thus, measured helical rises do not depend on the fragment length and are significantly shorter than the 3.38 A per bp measured by crystallography, but close to the 3.18 A per bp found in NMR studies. These findings are discussed with respect to discrepancies in earlier results published in the literature.