Research

Understanding the impact of environmental effects on the mechanical and physical properties of DNA stay a decisive challenging to well understanding the live of cells. As everybody knows, DNA is an essential component of the cellular machinery. It adopted a lot of conformation from packaging chromosome to the commonly-know double helix and it play a crucial role in lot biological processes such as transcription or replication.

Indeed, it was experimentally found that DNA is a polymer which can stiffen, abruptly bend or be partially denatured in function of its sequence. This local events radically alterne the physicals properties of the DNA molecular in terme of flexibility and elasticity.

So, is crucial to understanding the physics of this molecule. In this framework, we develop tools to quantify, both experimentally and theoretically the impact of physical and chemical changes on the mechanics of a DNA molecule.

Present Project

Do Lamina-associated domains act as tuning actors configuring the mechanical constraints of the chromatin domain at nuclear periphery ?

Advents in cell imaging and sequencing technologies enhance our understanding of genome conformation and gene regulation, and spark advancements in modeling these processes in three dimensions (3D). Many properties of chromatin that underlie its physical and biological behavior however remain poorly understood.

Paradoxically, while genes, topologically-associated domains (TADs), sub-TAD chromosomal contacts or lamina-associated domains (LADs) can easily be mapped linearly, mechanisms regulating their physical properties, formation, maintenance or rearrangement remain elusive.

We examine here how LADs impose mechanical constraints on physical properties of inter-LAD regions at the nuclear periphery (NP). Based on polymer physics, we model the chromatin fiber as a coarse-grained semi-flexible chain of connected beads anchored to the NP at each end, mimicking two LADs that restrict the 3D accessible space in-between.

Schematic representation of the in vitro Local3D model. The chromatin fiber is anchored by 2 point(LADs), at the nuclear pheriphery

Dynamic Monte Carlo simulations (DMC) are used to statistically describe and generate a representative ensemble of configurations accessible for the sub-chromatin chain in this specific geometry, restricted by LADs and the NP. By performing DMC simulations, we examine the out-of-equilibrium dynamics of the system to determine whether LADs are able to configure two distinct metastable long-lived chromatin states. These states are (i) the average position of inter-LAD regions modeled to fluctuate around a location close to the NP and (ii) the average position of inter-LAD regions stay away from the NP. Long-lifed refers to a residence time larger than the exploration time defined when the system continuously oscillates through all conformation states accessible for the chromatin which is constrain inbetween the modeled LADs.

Parameters in our modeling include genomic length of inter-LAD domains, LAD length, chromatin persistence length and Euclidean distance between two LADs along the NP. Our study aims to establish whether lamin B1 LADs act as preferential actors that modulate mechanical constraints and physical properties of the inter-LAD chromatin fiber, and thereby dynamically contribute to regulating gene expression in these regions.

Thesis Project

Experimental and numerical simulation

My project constists in both using the Thethered Particle Motion (or TPM) methode and modeling the DNA behaviour by numerical simulations.

Experiment approach: TPM is a recent experimental single molecule technique which consists in grafting one end of the DNA molecular (length LDNA) on a substrat and a nanometric particule (with raduis Rpar) at the other end. The brownianne motion of the tethered nanoparticle is followed. The amplitude of the motion RExp|| of this complex DNA/particle that directly depends on the apparent length of the DNA molecule, so of its conformational changes of DNA molecules.

Schematic representation of a TPM Bio-chip experiment

The TPM technique is well adapted to address questions concerning conformational changes of the DNA molecule such as curvatures, or denaturation bubble. Since conformational degrees of freedom are not hampered by any external constraints with TPM methode. Moreover, the IPBS group, which has a recognized expertise in TPM techniques, has developed a biochip that enables the high parallelization of TPM and the analysis enought molecules simultaneously [1]. Thanks to the ensuing high-throughput data acquisition, we obtain a large accumulation of individual statistics that permits us to get results with a good accuracy. In that way, the 2D projection of the bead displacement relative to the anchoring point of the DNA molecule gives access to its root-mean-squared end-to-end distance projected on the grafting surface, noted Rexp||.

1) 2D projection of the nanoparticle displacement 2) Root-mean-squared end-to-end distance projected R exp||, usually called Amplitude of the motion 3) Probability distribution of Rexp||

[1] Plénat, T., Tardin, C., Rousseau, P., Salomé, L., 2012. High-throughput single-molecule analysis of DNA-protein interactions by tethered particle motion. Nucleic Acids Research. 40 (12) : e89.

Numerical simulation approach: I performed Kinetic Monte Carlo simulations on the particle-DNA complex to predict the particle to anchor 2D-distance. The current simulation model is based on statistical model of polymer described at the mesoscopic scale [2].

To simulate the TPM geometry I model the DNA-particle complex as a chain of N(:25 or 50) connected small spheres of radius a and a larger final particle of radius Rpar. Numerical displacement of the labeled particle is obtained by performing a Kinetic Monte Carlo Simulation with the followings constraints: hard-wall condition, freely rotative joint and hard sphere.

Schematic representation of numerical simulation

The 2D-vector of the particle position is measured throughout simulations and utilized to estimate the amplitude of motion and average being taken along the trajectory. We obtain RSimu|| which would be compare at the TPM experiment measurement.

[2] M. Manghi, C. Tardin, J. Baglio, P. Rousseau, L. Salomé, N. Destainville, 2010. Probing DNA conformational changes with high temporal resolution by tethered particle motion, Phys. Biol. 7, 046003.

Theoretical aspect: Obtaining most degree of pertinant physical parameter of the DNA requires few step of analysis procedure. The calculation of the root-mean-square end-to-end distance of the DNA molecule RDNA from the RExp|| or RSimu|| requires an appropriate theoretical model and correcting for the effects of the particle.

With the classical Worm-Like chain model[3], the amplitude of motion of the DNA-particle complex, take into account the persistence length of the DNA, is derived from the 〈RDNA2〉calculated for an isolated DNA molecule:

〈RDNA2〉= 2LL p - 2L 2p

In this second step we considering that the particle and the DNA molecule are statistically independent and in ignoring the effect of the substrate. We can simply infer R DNA with:

$R^2_{DNA} = \frac{3}{2} \langle R^2 _{||} \rangle - R^2{par} ^{1/2}$

[3] O. Kratky and G. Porod. Röntgenuntersuchung gelöster Fadenmoleküle. Recueil des Travaux Chimiques des Pays-Bas, 1949, 68(12) pp1106–1122, DOI: 10.1002/recl.19490681203

Results

Global effect:

Variation of Ionic strenght [4]: A first study was conducted on the effect of the ionic strength induced by surrounding ions in solution on the DNA persistence length (Lp ) which characterizes the DNA polymer rigidity. The extracted Lp values of HT-TPM measurement decrease from 55 to 30 nm when the ionic strength increases. A stronger decrease was observed in presence of divalent ions Mg2+ than with monovalent ions Na+.

This quantification of Lp dependence, on a large and strongly prospected range of ionic strengthes, tends to validate the theoretical approach proposed in 2006 by Manning[5] in presence of monovalent ions Na+.

[4] Brunet, A., Tardin, C., Salomé, L., Rousseau, P., Destainville, N., Manghi, M., Dependence of DNA persistence length on ionic strength of solutions with monovalent and divalent salts : a joint theory-experiment study, Macromolecules, 2015, 48 (11), pp 3641–3652, DOI : 10.1021/acs.macromol.5b00735.

[5] Manning G. S., The persistence length of DNA is reached from the persistence length of its null isomer through an internal electrostatic stretching force. Biophysical journal, 2006, 91(10) pp3607–16, DOI:  10.1529/biophysj.106.089029

Variation of temperature [6]: This study was conducted on the effect of the temperature on the DNA persistence length (Lp ) and the possibility to induced local formation od DNA denaturation bubble (local effect). For different DNA molecules, the persistence length is extracted by using the calibration curve obtained by an exact sampling simulation model [4] We show that in the physiologically relevant temperature range from 15 to 60°C, the temperature induces a variation of Lp similar to the expected one for a structurally conserved double stranded DNA that is Lp=κ/kBT with κ≅2.06×10−19 Jnm assuming Lp= 50 nm for dsDNA at 25°C.

Temperature dependence of Lp for DNA molecule of 2000 bp without A50 tract (blue dots) and 2000 bp with A50 (green dots), 2060 bp (orang dots), and 583 bp on a PEG surface (blue cross), as well as the results obtained by Geggier et al.[7] on a 2686 bp circular DNA, as well as those obtained by Driessen et al.[8] on a 685 bp linear DNA. Fits performed between 15 and 60°C by Lp=κ/kBT with kB= 1.38×10−23JK−1 and κ as a fitting parameter, are shown when possible. Data are represented with logarithmic scales for both axes. Error bars (95% confidence intervals) are displayed, if not, they are smaller than the symbol size

Above 60°C, the extracted Lp falls due to a complex combination of partial denaturation that is discussed largly in our paper as well as biochip disassembly

[6] Brunet, A., Salomé, L., Rousseau, P., Destainville, N., Manghi, M., Tardin, C., How does temperature impact the conformation of single DNA molecules below melting temperature?, Nucleic acids research, 2017, DOI : 10.1093/nar/gkx1285

[7] Geggier S., Kotlyar A., Vologodskii A., Temperature dependence of DNA persistence length, Nucleic acids research, 2011, 39, 1419–1426, 10.1093/nar/gkq932

[8] Driessen R.P.C., Sitters G., Laurens N., Moolenaar G.F., Wuite G.J.L., Goosen N., Dame R.T., Effect of temperature on the intrinsic flexibility of DNA and its interaction with architectural proteins, Biochemistry, 2014, 53, 6430–6438.

Local defect:

Intrisic bent [9]: A second project allows us to develop a method of evaluation and quantification of local DNA bending angles, induced either by specific intrinsic sequence, or by the binding of proteins on DNA. Constructs made of 575 base-pair DNAs with in-phase assemblies of one to seven sequences CAAAAAACGG was used. A theoretical description of the polymer chain, named "kinked Worm-Like Chain" was proposed which leads to a simple formulation of the end-to-end distance of DNA molecules allowing to extract local bend angles from HT-TPM measurement. As a result, we find that the sequence CAAAAAACGG induces a bend angle of 19 ± 4, in agreement with other value from the literature.

[9] Brunet, A., Chevalier, S., Destainville, N., Manghi, M., Rousseau, P., Salhi, M., Salomé, L., Tardin, C., Probing a label-free local bend in DNA by single molecule tethered particle motion, Nucleic acids research, 2015, 42 (11), pe 72(7) DOI : 10.1093/nar/gkv201

Local denaturation bubble formation ? In the denaturation study, we explore the influence of temperature on the mechanical characteristics of an internal sequence composed of 50 adenines (A50). Two DNA molecules of 2000 bp are chose, one containing a central core composed of a 50 A-tract (2000A) and another deprived of such a central core (2000wo). Out study reveals that the persistence length of 2000 bp long DNA lacking an A50 sequence remains at all temperatures slightly above for the almost identical 2000 bp long DNA containing a A50 sequence in the center of the molecule. The only differences between the two DNA molecules result from the presence of the A50 in the center of 2000A and the existence of two new 25 bp long sequences at each extremity of the 2000wo molecule without any sequence specificity able to induce structural peculiarities. This effect could result from the presence of either a more flexible DNA sequence or a local bend in the 2000A construct. At this stage and on this range of temperature variation we do not detect any temperature effect or validate the formation of local denaturation bubble.

• [1] Plénat, T., Tardin, C., Rousseau, P., Salomé, L., 2012. High-throughput single-molecule analysis of DNA-protein interactions by tethered particle motion. Nucleic Acids Research. 40 (12): e89.
[2] M. Manghi, C. Tardin, J. Baglio, P. Rousseau, L. Salomé, N. Destainville, 2010. Probing DNA conformational changes with high temporal resolution by tethered particle motion, Phys. Biol. 7, 046003.
[3] O. Kratky and G. Porod. Röntgenuntersuchung gelöster Fadenmoleküle. Recueil des Travaux Chimiques des Pays-Bas, 1949, 68(12) pp1106–1122, DOI: 10.1002/recl.19490681203
[4] Brunet, A., Tardin, C., Salomé, L., Rousseau, P., Destainville, N., Manghi, M., Dependence of DNA persistence length on ionic strength of solutions with monovalent and divalent salts: a joint theory-experiment study, Macromolecules, 2015, 48 (11), pp 3641–3652, DOI : 10.1021/acs.macromol.5b00735
[5] Manning G. S., The persistence length of DNA is reached from the persistence length of its null isomer through an internal electrostatic stretching force. Biophysical journal, 2006, 91(10) pp3607–16, DOI: 10.1529/biophysj.106.089029

[6] Brunet, A., Salomé, L., Rousseau, P., Destainville, N., Manghi, M., Tardin, C., How does temperature impact the conformation of single DNA molecules below melting temperature?, Nucleic acids research, 2017, DOI : 10.1093/nar/gkx1285
[7] Geggier S., Kotlyar A., Vologodskii A., Temperature dependence of DNA persistence length, Nucleic acids research, 2011, 39, 1419–1426, 10.1093/nar/gkq932
[8] Driessen R.P.C., Sitters G., Laurens N., Moolenaar G.F., Wuite G.J.L., Goosen N., Dame R.T., Effect of temperature on the intrinsic flexibility of DNA and its interaction with architectural proteins, Biochemistry, 2014, 53, 6430–6438.
[9] Brunet, A., Chevalier, S., Destainville, N., Manghi, M., Rousseau, P., Salhi, M., Salomé, L., Tardin, C., Probing a label-free local bend in DNA by single molecule tethered particle motion, Nucleic acids research, 2015, 42 (11), pe 72(7) DOI : 10.1093/nar/gkv201