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“All in All It's Just Another Brick in the Wall” Cooperative Effects of Cytosine Methylation on DNA Structure and Dynamics Cécilia Hognon, Vanessa Besancenot, Arnaud Gruez, Stephanie Grandemange, and Antonio Monari J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b05835 • Publication Date (Web): 31 Jul 2019 Downloaded from pubs.acs.org on August 2, 2019

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The Journal of Physical Chemistry

“All in All It’s Just Another Brick in the Wall” Cooperative Effects of Cytosine Methylation on DNA Structure and Dynamics Cécilia Hognon,a Vanessa Besancenot,b Arnaud Gruez,c Stephanie Grandemange,b* Antonio Monaria* Université de Lorraine and CNRS, UMR 7019 LPCT, F-54000 Nancy, France. b Université de Lorraine and CNRS, UMR 7039 CRAN, F-54000 Nancy, France. C Université de Lorraine and CNRS, UMR 7356 IMOPA, F-54000 Nancy, France a

AUTHOR INFORMATION Corresponding Author [email protected] [email protected]

ABSTRACT The behavior of the structural parameters of DNA considering different level of methylation in CpG islands is studied by means of full-atom molecular dynamics simulations and electronic circular dichroism, both in an artificial model system and in a gene promoter sequence. It is demonstrated that methylation although intrinsically brings quite local perturbations may, if its level is high enough, induce cooperative effects that strongly modify the DNA backbone torsional parameters altering the helicity as compared to the non-methylated case. Since methylation of CpG island is correlated with the regulation of gene expression, understanding the structural modifications induced in DNA is crucial to characterize all the fine equilibria into play in epigenetics phenomena.

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INTRODUCTION The role of nucleic acids in storing the genetic information while allowing its faithful replication during cells mitosis, as well as its decoding and transcription culminating in protein synthesis, is widely recognized. The molecular bases underlying this phenomenon have been firmly established since the seminal discover of the DNA double helix structure in the fifties, setting the stage for the molecular genetic sciences.1 However, and in addition to the former, understanding the fine regulations taking place at cellular level also requires the comprehension of the molecular factors leading to the regulation of gene expressions2–7 and its variability as a result of the cellular cycle or as a response to stress or external stimuli.8,9 The latter represents the domain of epigenetic, i.e. the study of the different reversible modification of DNA composition or structure leading to the regulation of the gene expression.10–12 As such, epigenetics is related to one of the most fundamental processes in biology, while the deregulation in gene expression has been linked to the development of different debilitating diseases including cancers13–16 and neurodegeneration.17–20 Indeed, in a vast amount of cancer cell lines the global transcriptome21,22 and proteome23–25 are highly deregulated as compared to healthy ones, with the overexpression of oncogenes and the inhibition of tumor suppressor genes. Thus, the precise understanding of epigenetics mechanisms could allow the possibility to design specifically tailored epigenetics therapy, targeting in a personalized way the factors related to tumor resistance or progression. However, even if in principle promising, epigenetics therapies are plagued with a plethora of harmful side-effects, mainly due to a global deregulation of gene expressions, that seriously limit their applicability.26–28 From a molecular point of view epigenetics modulations are performed via a series of reversible chemical and structural modification of the DNA structure, particularly in correspondence of the gene regulating sequences.29 By far the most common and celebrated of such modification, as 2 ACS Paragon Plus Environment

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presented in Figure 1, is the methylation of cytosine (dC) nucleobases to produce 5-methyl-cytosine (5mC).30–34 The former chemical modification takes place mostly in guanine and cytosine rich regions of the genome known as CpG islands. Cytosine methylation is normally associated to gene silencing, i.e. to the repression of its expression, that can be related either to a modified interaction with the corresponding transcription factors or to a subsequent reorganization of the DNA spatial arrangement limiting the accessibility of the gene. Cytosine methylation level is maintained in cell via a rather complex interplay between specific enzymes such as the DNA methyltransferase (DNMT) class, and the DNA lesion repair machinery. Even if fundamental, DNA methylation is not the only epigenetics regulatory factor, other factors involving both nucleic acids and proteins such as chromatine remodeling, histone acetylation, or post-trasductional modifications have to be considered. The complexity of epigenetics phenomena and the subtle network of cross-talks and regulations between different factors necessary to assure its maintenance is one of the reasons behind the modest success of epigenetics therapies so far, since the latter have mostly targeted the DNMT enzymes without proper selectivity towards specific genes.35

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B

C

C*

C

5’ - CGCGCGCGCGCGCGCGCGCG - 3’ 3’ - GCGCGCGCGCGCGCGCGCGC - 5’

D

5’ - CGCGCGCGCGC*GCGCGCGCG - 3’ 3’ - GCGCGCGCGCGCGCGCGCGC - 5’

E 5’ - CGC*GC*GC*GC*GC*GC*GC*GC*GC*G - 3’

3’ - GCGCGCGCGCGCGCGCGCGC - 5’

F 5’ - CGC*GC*GC*GC*GC*GC*GC*GC*GC*G - 3’ 3’ - GC*GC*GC*GC*GC*GC*GC*GC*GC*GC - 5’

Figure 1. Molecular sketch of cytosine (A) and 5mC (B). The model strands used for the subsequent molecular simulations are also reported: un-methylated (C), singly-methylated (D), hemi-methylated (E), and fully methylated (F). Note that the position of 5mC is indicated in red as C*.

As already underlined the biological role of the DNA methylation is exerted also via the modification of the intrinsic structural properties of DNA double strands that may lead to chromatin rearrangements. As such it is fundamental to characterize precisely at a molecular and atomistic level the subtle modifications induced by methylation in DNA intrinsic dynamics and stability. Indeed, the passage from dC to 5mC may seam a rather innocent modification that should have only marginal effects on the DNA properties since it should not impact neither the p-stacking properties of the nucleobases nor the Watson and Crick hydrogen bond pairing.36,37 However, DNA inherent flexibility and its complex macromolecular structure may lead to the appearance of complicated conformational landscapes as the result of the presence of damages in its sequence.38 Furthermore, even slightly different lesions may give raise to totally different structural properties and conformational flexibility.39 Finally one has also to consider that in biological relevant situations DNA methylation takes place in or intergenic or intrageneic with CpG islands, leading to the accumulation of the chemical modifications in spatially close regions of the DNA sequence, that in turn may induce the 4 ACS Paragon Plus Environment

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coupling of the individual deformations leading to a cooperative and cumulative effect as it was evidenced in case of oxidative DNA cluster lesions.40,41 The role of DNA methylation, and the related change in the DNA mechanical properties, is indeed fundamental in assuring epigenetics regulation.42–47 Some important computational studies have been performed concerning from the one side the interaction between DNA and histones, and hence nucleosomal compaction and the role of methylation,48 and on the other side the interaction with specific enzymes and the sensitivity to methylation experienced by transcription factors.49 In addition, combined molecular force assay, single-molecule spectroscopy, and molecular dynamic simulations have pinpointed the role of methylation in affecting DNA strand separation.50 As a matter of fact methylation has also been shown to significantly alter the interaction of DNA with different materials such as graphene quantum dots.51 In this contribution we rely on extend full atom molecular dynamics (MD) to unravel at an atomistic level the effects of DNA methylation on both model poly(dCdG) strands (Figure 1) and a methylated consensus region extracted from the adenomatous polyposis coli (APC) gene promoter, whose methylation status has been largely described while its aberration is correlated to cancer development affecting different organs and including breast, lungs, and digestive tract.52–55 In addition electronic circular dichroism (ECD) is used to confirm the presence of non-negligible structural modification of DNA.56–62 This will allow to point out the fundamental effects of methylation on the native B-DNA structure and the alteration of its native structure, hence filling a gap in the understanding of the molecular, and in a certain instance mechanical, epigenetics factors. Our results clearly underline the fact that DNA methylation induces important conformational effects that are particularly captured by significant differences observed in the backbone structure and flexibility. Indeed, even though the effect of one single methylation is rather marginal the cooperativity in CpG islands induces modifications that are easily confirmed by the observed change of 5 ACS Paragon Plus Environment


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the ECD profile. The differences in the intrinsic properties of methylated and non-methylated DNA sequences may help setting the base to understand on the one side the differential interactions with transcription factors and on the other one the perturbation of the coiling leading to chromatin remodeling.44,47 EXPERIMENTAL AND COMPUTATIONAL DETAILS To characterize the impact of 5-mC on the global structural and dynamical properties of DNA we performed classical molecular dynamics on a 20 base pairs model B-DNA double strand of general sequence poly-(dC)-(dG) and on the 17 base pairs consensus sequence of the APC promoting gene (Figure 1), following different methylation patterns, namely fully methylated, hemimethylated or singly-methylated. All the systems have been built and set-up using the NAB utility distributed in the AmberTools package. DNA was described using the amberf99 force field 63 including the bsc1 corrections.64,65 The force field for 5mC was parameterized coherently with the amber force field, following the usual Amber antechamber procedure. The Restricted Electrostatic Potential (RESP)66 procedure was used consistently, the ground state geometry of the model 5mC nucleotide was optimized at density functional theory (DFT) level using the standard 6-31G basis set and the B3LYP functional.67 (See SI) The different DNA strands have been solvated in a cubic water box described by TIP3P force field68,69 and K+ cations were added to ensure electroneutrality of the simulation box. The H Mass Repartition (HMR) algorithm has been used consistently to speed up the simulation and allow a more important sampling.70 HMR allows to increase the time step used to integrate Newton’s equations of motion to 4 fs by artificially scaling the mass of all non water hydrogen atoms from 1.008 Da to 3.024 Da. 6 ACS Paragon Plus Environment

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After an equilibration and thermalization of 36 ns, 500 ns for the model systems bearing different methylation levels and 200 ns for the APC promoting gene have been run in the NPT thermodynamic ensemble at the temperature of 300 K and the pressure of 1 atm. All MD simulations have been performed using the NAMD 2.12 code,71 and results have been analyzed and visualized with VMD72 and Curves+ suites.73 Circular dichroism (CD) was performed out using a Chirascan CD from Applied Photophysics. All the data have been collected at 20 °C with 0.5 nm intervals in the wavelength range of 180−320 nm, using a temperature-controlled chamber. A 0.01 cm cuvette containing 30 μL of DNA sample at 100 M was used for all the measurements. All the measurement were replicated at least three times, and sample spectra were corrected for buffer background by subtracting the average spectrum of buffer alone.

Figure 2. Time series of the RMSD for the different model DNA strands.



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RESULTS AND DISCUSSION In order to asses the effects of methylation on the DNA structural parameters in a systematic and rather unbiased way we first compare the behavior of a native alternating poly-(dC)-(dG) strand with different degrees of methylation (Figure 1). In the case of the non-methylated DNA we can observe a dynamic totally coherent with the one exhibited by B-DNA double strands with a rather small root mean square deviation (RMSD) as reported in Figure 2. The inclusion of only one 5mC positioned at the center of the strand, as expected, does not alter the RMSD, whose time series is almost superposed with the previous one, since the modification induced are of low magnitude and more importantly extremely local. On the contrary some differences appear when one consider the hemi-methylated model, i.e. the system in which one strand is totally methylated while the other one is left unchanged, is considered. As expected the discrepancy is also increased when a fully methylated DNA is considered. From the analysis of a global index such as the RMSD one may already infer the cumulative effects induced by methylation leading to an accumulation of structural deformation. However, more specific and local parameters are necessary to proper characterize the intrinsic behavior of poly-methylated DNA and more importantly its difference compared to the native B-DNA. As confirmed by our simulations the methyl group in 5mC is directed directly into the major groove of DNA. Hence, we expect to observe important modifications of the major groove parameters depth upon methylation.


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Figure 3. Time series of the major groove width in correspondence of the different bases for the 4 DNA model strands as defined in Figure 1. A) native DNA, b) singly-methylated, C) hemi-methylated, D) fully-methylated.

A

C O5’

P

O3’ C3’

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G G C 5mC

O5’ P O3’ C3’

O5’ P

Unmethylated Methylated

Figure 4. A) Definition of the torsional angle z. B) Superposition of the backbone of a DNA strand in correspondence of methylated and non-methylated sites showing the different orientation of z. C) Superposition of methylated and nonmethylated DNA strand highlighting the global effects on the DNA backbone.



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The time series along the dynamics of this parameter for the different model strands are reported in Figure 3. Indeed, an increase of the major groove depth can be observed, coherently with the presence of a more sterical hindered group and of backbone reorganization. Interestingly enough, once again this modification is quite local as observed for the single methylated strand in which the corresponding increase in the depth value is observed only in correspondence of 5mC. While the other structural parameters describing the base coupling are almost unaltered upon methylation, as can be seen from the data reported in SI, the backbone torsional parameters are much more affected and globally induce an important deviation of the backbone helicity from the one of the native B-DNA as illustrated pictorially in Figure 4. In particular it appears that the most affected parameters is the one phosphate torsional angle z defined in Figure 4A, that leads to a different orientation of the sugar and phosphate moieties (Figure 4B) and reflects on a global distortion of the helicity (Figure 4C). A

B

C

D

Figure 5. Distribution of the average z angle over all the bases of a single strand for the un-methylated DNA (A), singlemethylated DNA (B), hemi-methylated DNA (C), and fully methylated DNA (D). In case of hemi-methylated and singlymethylated DNA the strand bearing 5mC is represented in red, while the ones having dC in black.


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In Figure 5 we report the distribution for the average value of the torsional angle z over all the sequence. We may observe that while in the case of the native B-DNA the value is centered around -100° while a large distribution peaking around 70° is observed for the methylated case. Interestingly in the case of the hemy-methylated system a very different behavior for the two strands is observed with the non-methylated one experiences a very sharp peak around -100° while the distribution for the methylated strand is wide and again centered around 70°. Interestingly in the case of a singly-methylated strand a double population emerges with two peaks at -100° and 100°. On the other hand no significant modification for the other parameters such as the torsional angle e is observed.

C

A

5’ - CGGCGCACGTGACCGCG - 3’ 3’ - GCCGCGTGCACTGGCGC- 5’

B

5’ - CGGC*GCAC*GTGACC*GCG - 3’ 3’ - GCC*GC*GTGCACTGGCGC- 5’

D

Figure 6. Sequence of the un-methylated (A) and methylated (B) APC promoting region. Distribution of the average torsional z angle for the un-methylated (C) and methylated (D) sequence, respectively.

The MD simulations on the model DNA strand have clearly confirmed that methylation induces cooperative structural modifications that strongly alter the helical parameters. However, those conclusions have been reported based on a model in which the methylation level is artificially high and hence could suffer from an over interpretation. In order to analyze a more biological relevant model we considered a fragment of the promoting sequence of the APC gene (Figure 6) that is composed of seventeen base pairs and harbors two CpG islands per strand. The same protocol as for the model system has been used, i.e.



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considering non-methylated, hemi-methylated, and fully-methylated strands. Importantly, the same strands have also been experimentally produced and considered for spectroscopical characterization From the analysis of the MD trajectories (Figure 6) we can infer that the behavior underlined for the model system is indeed confirmed and important modifications of the backbone torsional angle are present upon methylation or hemi-methylation involving the relevant CpG islands. In particular, and coherently with what was observed for the poly-(dC)-poly(dG) strands methylation is accompanied by the inversion of the torsions z and the increase of its distribution width and hence by the corresponding cumulative effects on the backbone properties. One technique of choice to highlight possible structural deformations in biological macromolecules is indeed ECD, due to its sensitivity able to monitor even slight changes in global arrangement of organized chiral structures. Hence, we performed ECD for the promoting gene strands having different methylation level and results are collected in Figure 7. The helical structure of DNA is evidenced in the ECD spectra by the negative peaks at around 210 nm and 250 nm, and the positive peak around 280 nm that constitute a very well-known signature of the excitonic coupling between the B-DNA bases. Methylation, on the other hand, has only a very limited effect on the values of the absorption maxima as also confirmed by the absorption spectrum reported in SI. This result is expected since the modifications on the DNA nucleobases induced by methylation are too small to allow for a significant changes of the excitonic coupling strength. On the other hand, the intensity of the ECD positive peak at around 280 nm clearly increases with the methylation level, while the absolute value of the intensity of the negative peak at 250 nm follows a less clear trend. Indeed, the continuum of wavelength shifts between maxima (210 nm, 250 nm and 280 nm) of non-methylated, hemi-methylated, and fully-methylated strands also reflects a DNA structural change. Such spectral signatures are coherent with a partial modification of the helical parameters of the B-DNA structure upon methylation and with a slightly more important rigidity of methylated DNA, as evidenced by the RMSD. Indeed, ECD spectra is particularly sensitive to modifications happening in macromolecules secondary structure and hence to all the structural parameters of DNA affecting its 12 ACS Paragon Plus Environment

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helicity. In the present case the inversion of the backbone angles provoked by methylation and evidenced in the MD simulations may correlate well with the observed spectral signature.

50

Unmethylated Hemimethylated (5’−3’) Hemimethylated (3’−5’) Methylated

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Δε/n x 104 (M−1.cm−1.n−1)

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30 20 10 0

−10

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Figure 7. Experimental ECD for the un-methylated, hemi-methylated, and fully-methylated APC promoting region.

CONCLUSIONS By resorting to extended full atom MD simulations we have clearly demonstrated that even if the inclusion of one single 5mC has little to no effect on the global structure, extended methylation of CpG islands clearly correlates with cooperative effects and hence significantly alters the DNA backbone torsional parameters, especially the z angle, in addition to modifying the major groove accessibility. The results have been confirmed both in model systems consisting of poly-(dC)-poly(dG) strands that have also allowed to underlined the differential effects related to either full- or hemy-methylation. Importantly, the same results have been confirmed in the case of a gene consensus sequence containing CpG islands whose methylation level has been varied accordingly. ECD on the gene sequence has also experimentally confirmed the alteration of the helicity compared to the ideal B-DNA upon methylation. Our work highlights, for the first time, the crucial and cooperative effects induced by methylation on the intrinsic structural parameters and DNA and hence can be fundamental in understanding the role played 13 ACS Paragon Plus Environment


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by methylation in regulating gene expression by modifying the intrinsic DNA flexibility and hence, at a larger scale, allowing not only preferential interactions with gene promoting factors, but also in possibly driving chromatin reorganization that is known to be a crucial epigenetic marker. In addition, by highlighting the differential dynamics of methylated DNA sequences of biological relevance, our work may also be fundamental to allow for a better rationalization or development of novel drugs for epigenetic treatment having DNA as a target. In the future we plan to extend the present study considering DNA methylation in coiled DNA, either by histones in nucleosomes or by histones-like (HU). Furthermore, and to allow a better one-to-one mapping between simulation and experience, from the one hand ECD will be simulated resorting to hybrid quantum mechanical molecular mechanics (QM/MM) approaches and on the other hand the structural of methylated DNA will be explored using small-angle neutron scattering (SANS) techniques. ASSOCIATED CONTENT Time series of the torsional parameters for the different model systems and the APC promoting gene, absorption spectrum of the methylated, hemi-methylated and non-methylated APC gene, force field parameters for the 5mC nucleotide. The Supporting Information is available free of charge on the ACS Publications website. ACKNOWLEDGMENT The authors acknowledge the LUE IMPACT “Biomolécules” for financial supports. C.H. Ph.D. scholarship is funded by the Université de Lorraine. Molecular Modeling and simulation was performed on the LPCT local computing resources.

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TOC GRAPHICS

fects ive Ef ion Cooperat e Methylat osin By e r u of Cyt t c D A stru ON DN and EC lations u im S MD 50 40

Unmethylated Hemimethylated (5’−3’) Hemimethylated (3’−5’) Methylated

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De/n x 10 (M .cm .n )

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