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This article can be downloaded from www.ijpbs.net

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ISSN 0975-6299 Vol 3/Issue 1/Jan – Mar 2012

RESEARCH ARTICLE

International Journal of Pharma and Bio Sciences

COMPUTATIONAL STUDIES ON THE INTERACTION OF CORE HISTONE TAIL

DOMAINS WITH CpG ISLAND

S. PRASANTH KUMAR1, RAVI G. KAPOPARA1,YOGESH T. JASRAI1 AND RAKESH M. RAWAL*2

1Bioinformatics Laboratory, Department of Botany, University School of Sciences, Gujarat University,

Ahmedabad- 380 009. 2Division of Medicinal Chemistry and Pharmacogenomics, Department of Cancer Biology, The Gujarat

Cancer & Research Institute (GCRI), Ahmedabad- 380 016.

BIOINFORMATICS

ABSTRACT

It has been elucidated through in vitro studies that core histone tail domains preferentially interact with linker DNA. In the present study, we studied these interactions computationally using molecular docking and isocontour-based electrostatic map approach in order to identify the domains and regions of H3 and H4 tails and DNA contributing for the physical associativeness. We also explored the interaction made by the linker DNA containing methylated CpG dinucleotides (CpG island) with the normal and post-translational modified histone tails. We report that these interactions are electrostatically unfavored if one of the biomolecular partners is methylated thereby, negatively charged zones of DNA and histone tails are required to be absent nearby.

RAKESH M. RAWAL Division of Medicinal Chemistry and Pharmacogenomics, Department of Cancer

Biology, The Gujarat Cancer & Research Institute (GCRI), Ahmedabad- 380 016.


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KEYWORDS

Core histone tail domain, Linker DNA, CpG island, Molecular docking, Isocontour-based electrostatic potential map.

INTRODUCTION

Chromatin is a large macromolecular complex composed of an array of nucleosome assembled by histones and other nuclear proteins. About 146 bp of DNA wraps around the histone octamer in a left-handed superhelical fashion with each octamer consisting of 2 copies each of the core histones H2A, H2B, H3 and H4 1. A stretch of about 10-80 bp free DNA called ‘linker DNA’ connects the adjacent nucleosomes. Histone H1, also known as ‘linker histone’ binds to the linker DNA but does not form part of the nucleosome thereby facilitates sealing off the nucleosome at the location where DNA enters and leaves2. The core histone tail domains through its post-translational modification (PTM) aid in the intracellular signals mediated transduction to organize functional states of chromatin to initiate cellular processes such as DNA replication or transcription. It has been demonstrated that flexible histone tails are crucial players in the mitotic chromosome condensation3 and the positive correlation of chromosome assembly with the tail PTMs (e.g. phosphorylation of H3 at Ser10)4. Hence, core histone tails through its PTM serves as a target for binding of ancillary proteins or other enzymatic functions and these processes alter the structure of core histone tails. Experiments on circular dichroism showed that N tails (N-terminal region of the tail) of H3 and H4 are highly organized as DNA bound conformations and half of their residues were observed as α-helical segment in contrast to random-coil conformation confined by H2A and H2B tails, respectively5. Moreover, the tails are interacting with DNA and protein within the condensed fiber at defined locations and in the presence of physiological salts as revealed in the oligosomes

crosslinking6, quantitative hydrodynamic7 and gel electrophoresis experiments8.

CpG islands, defined as genomic regions of more than 200 bases with a G+C content of at least 50% and a ratio of observed to statistically expected CpG frequencies of atleast 0.6, typically occurs at or near the transcription start site (TSS) or even extend over exon1 of genes9. CpG islands methylation is positively associated with the interference of gene transcription by impeding transcipting factor binding or by bringing about chromatin alterations. It is generally recognized that DNA hypermethylation is involved in transcriptional repression. Some exceptions to this mechanism exist, for e.g. hypermethylation of CpG island of the KAI1 metastasis suppressor gene could not down regulate its expression in invasive and metastatic human cancers10. Thus, it is still disputed that CpG island methylation precedes the assembly of repressive chromatin or a coordinated regulatory mechanism exist11. We know that histone tail PTMs also promote gene silencing by recruiting epigenetic enzymes. However, the wild-type p16INK4a allele in HCT116 colorectal tumor cells is independently silenced by histone H3 lysine 9 (H3-K9) methylation without any DNA methylation12.

Recent studies exhibited that the majority of core histone tail domains interacts with extranucleosomal linker DNA13. It has been elucidated that low nucleosome occupancy is observed at TSS and it is intrinsically encoded in the DNA sequence. For example, the locations of TATA element were found to be occupied outside a stably positioned nucleosome but not


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upon nucleosome in yeast genome14. On the other hand, CpG islands are known to be the nucleosome-destabilizing elements and this destabilization is to enable transcriptional activator Sp1 to bind to promoter binding sites without any nucleosome remodeling. In the present study, the interaction of core histone tail domains (H3 and H4) with CpG island was carried out computationally15. It should be noted that CpG island localizes in the linker DNA due to the fact that CpG island is always observed upstream of TSS and/or overlap within the promoter, if so, CpG island cannot be observed over nucleosome and forms a part of linker DNA. Moreover, the sequence-dependent anisotropic nucleosome positioning signatures (CG dinucleotides) does not facilitate that CpG clusters can be wrapped over nucleosome16. Here, we investigated that whether CpG island methylation and core histone tail PTM (H3 and H4’s arginines and lysines are methylated) are coordinated process and if so, preferential physical interaction of both of these methylated biomolecules exhibited? We report that these interactions were found to be electrostatically favored in the docked conformations of histone tails (both H3 and H4) with normal B-DNA containing CpG dinucleotides (unmethylated CpG island) and unfavoured interaction was observed if one of the associative partners is methylated as dictated by the isocontour-based electrostatic potential maps.

MATERIALS AND METHODS Nucleotide sequence and protein structure retrieval The sequence of von Hippel-Lindau tumor suppressor gene of Homo sapiens (accession number: NC_000003.11) was retrieved from National Center for Biotechnology Information (NCBI) Gene database17. The crystal structure of the nucleosome core particle (entry code: 1KX5)18 was obtained from RCSB Protein Data Bank (PDB)19.

CpG island prediction The von Hippel-Lindau tumor suppressor gene sequence was scanned for the distribution of CpG island(s) using CpG Island Searcher20 with the parameters set as lower limits: percentage of G and C bases (%GC) = 55%, ratio of observed to statistically expected CpG frequencies (ObsCpG/ExpCpG) = 0.65, frequency of bases in the island = 500 bp and gap between adjacent islands = 100 bp. DNA structure modeling Canonical B-DNA structure was constructed using 3D-DART (3DNA-Driven DNA Analysis and Rebuilding Tool) web server21. The CpG island (5’-3’ sequence) was given as input. 3D-DART exclusively uses 3DNA software suite22 which comprised of different modules to perform the computational modeling. The ‘fiber’ module initially developed a canonical B-DNA structure and a corresponding base pair (step) parameter file was generated using ‘find_pair’ and ‘analyze’ modules. The parameter file was then subsequently utilized to introduce ‘local’ and ‘global’ bends in the DNA structure with default settings of roll, tilt and twist. Finally, the DNA structure file in PDB format was returned with the help of ‘rebuild’ module of 3DNA. Structure manipulation and energy minimization Recovered PDB file of nucleosome core particle (entry code: 1KX5) was manually spliced into chains of histones viz. H3, H4, H2A and H2B. The H3 and H4 chains (chains ‘A’ and ‘B’) were separately saved in PDB file format with the help of text editor. Methylation involves the addition of CH3 group in arginine and lysine residues in protein as well as cytosines in the DNA molecule. YASARA View23 was extensively used to perform this operation. ‘Build’ utility was employed to introduce methyl group and subsequently the bond orders were corrected using ‘Adjust bond order’ utility. The resultant structure files were then energy minimized using YASARA Energy minimization server24. This


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structure manipulation step was carried out for each arginine and lysine residues in the H3 and H4 structures. Similarly, cytosines in the canonical B-DNA was ‘computationally methylated’ with the same approach. Computational docking The docking simulations were executed using HADDOCK (High Ambiguity Driven biomolecular DOCKing) engine25 with the in-house generated structure files. The structure files can be categorized into two types: unmodified and structurally modified files. Structurally unmodified files represent the chains of H3 and H4 (protein files) and the 3D-DART generated canonical B-DNA structure (DNA file) whereas modified files include the structurally manipulated files of chains H3 and H4 (From here, it will be called as ‘methylated H3’ and ‘methylated H4’ as applicable; protein files) and structurally manipulated CpG island (From here, it will be known as ‘methylated CpG island’; DNA file). Molecular docking was performed with each DNA and protein files iteratively. The active sites (arginines, methylated arginines, lysines, methylated lysines in the protein files and cytosines and methylated cytosines in the DNA files, respectively) were specified and passive residues were automatically defined around the active sites. The specification of active and passive residues takes the form of experimental data to drive docking and these data were converted into ambiguous interaction restraints (AIRS) by HADDOCK and subsequently generates the topology of the molecules inputted. Docking protocol consists of three stages viz. a rigid-body energy minimization, a semi-flexible refinement in torsional angle space and a finishing refinement in explicit solvent. After execution of each of these stages, the docked conformations are scored and ranked by the scoring function to facilitate the selection of the best conformations and subsequently employed in the next stage. The best docked conformers can be recovered by inspection of HADDOCK score which takes into account the weighted sum

of van der Waals, electrostatic, desolvation and restraint violation energies together with buried surface area. PQR files and electrostatic potential map generation To study the influence of electrostatics on DNA-protein complex (docked structures), continuum electrostatic approach was chosen. This can be achieved by APBS (Automated Poisson-Boltzmann Solver)26 for which PQR files were required as inputs. Hence, the docked complexes in PDB format were converted into PQR format using PDB2PQR server27. PQR format embodies the replacement of occupancy column in a PDB file (‘P’) with the atomic charge (‘Q’) and the temperature factor column with the atomic radius (‘R’). The inputted PDB files were initially screened for heavy atoms (if missing, it will be rebuild) and hydrogens were added and optimized followed by assignment of atomic parameters using force-fields such as CHARMM22, AMBER99 (selected in the APBS input generation) or PARSE. At last, PDB2PQR server results were further requested to generate APBS input file (electrostatic potential grid map) in grid data explorer (.dx) format. Molecular Graphics Laboratory (MGL) Tools of Scripps Research Institute28 was utilized to develop isocontours of the electrostatic potential map built from the docked structure files. MGL Tools comes up with shipped APBS widget to accomplish this task and it is primarily used for visualization and analysis of molecular structures.

RESULTS AND DISCUSSION The docking simulation was carried out

using HADDOCK program with the following molecular inputs: DNA containing CpG island, methylated CpG island (addition of CH3 group at cytosines), native (unmethylated or unmodified) chains of H3 and H4 (generated from nucleosome core particle (1KX5) using text editor) and methylated H3 and H4 (addition of CH3 group at arginines and lysines), respectively.


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Prior to docking, all the structure files were energy minimized using YASARA energy minimization server. All the structure files were docked in an iterative fashion (i.e each DNA file with each protein file) and the binding efficiency was evaluated using HADDOCK score and binding energy term.

Upon examining the scoring functions of HADDOCK score, binding and electrostatic energies, minute atomic manipulations in methylated structural files (both methylated DNA and protein) did not altered the docking conformations rather atomic restraints were observed (Table 1). The HADDOCK score was centered on a large pool of values for H3, i.e.

Figure 1

Docked conformation of H3 and H4 with CpG island containing DNA. A. H3-CpG island B. H4-CpG island C. H3-methylated CpG island D. H4-methylated CpG island E. Methylated H3-CpG island F. Methylated H4-CpG island

G. Methylated H3-methylated CpG island and H. Methylated H4-methylated CpG island.


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Table 1. Docking result of biomolecules

H3 tail – CpG island

H3 tail – methylated CpG island

Methylated H3 tail – CpG island

Methylated H3 tail – methylated CpG island

HADDOCK score

-18.5 +/- 20.7 -127.9 +/- 7.5 -16.5 +/- 11.7 -183.1 +/- 11.7

Binding energy (KJ/mol)

-170841.00 -1349.54 -178775.00 -1293.91

Electrostatic energy (KJ/mol)

-653.6 +/- 45.5 -645.6 +/- 41.3 -797.4 +/- 38.0 -759.8 +/- 79.7

H4 tail – CpG island

H4 tail – methylated CpG island

Methylated H4 tail – CpG island

Methylated H4 tail – methylated CpG island

HADDOCK score

18.2 +/- 8.9 -133.9 +/- 8.9 16.4 +/- 6.6 -101.8 +/- 4.9

Binding energy (KJ/mol)

-192429.00 -1219.68 -182923.00 -900.103

Electrostatic energy (KJ/mol)

-619.1 +/- 13.0 -559.7 +/- 35.5 -572.5 +/- 33.9 -441.8 +/- 52.1

-16.5, -18.5, -127.9 and -183.1 KJ/mol. However, the binding energies were better for CpG island (unmethylated) with H3 chain. The electrostatic interactions did not disclosed information for preferential interaction as the energy values were localized around -653. 6, -645.6, -759.8 and -797.4 KJ/mol. Similar setback aroused for chain H4 with its entire DNA interacting partners. We found an important insight that there is no fair interaction if both the DNA and the protein partners were methylated as revealed in the binding energy term itself. The binding energy was found to be greater (-1293.91 KJ/mol for methylated H3-methylated CpG island conformation and -900.103 KJ/mol for methylated H4-methylated CpG island conformation) when compared to its corresponding methylated and unmethylated docked conformations (Figure 1).

We know that biomolecular complexes such as DNA and H3 and H4 chains are

charged in physiological conditions. Electrostatic potential map for all of the above docked conformations were developed using APBS approach. All the docked complexes were initially converted into ‘charged coordinates’ using PDB2PQR and subsequently, grid files symbolizing the electrostatic potential grid map were generated in order to develop isocontour map. As methyl group confers a charge of -1, methylated arginines and lysines in H3 and H4 chains and methylated cytosines in CpG containing DNA will tend to have a cluster of negative charge on its surface and a clear picture of charged clusters at the molecular interface will show the type of interaction.

Isocontour of electrostatic map for all the computationally developed complexes were generated at an isocontour value of -1.000 KbTec

-1 (APBS writes dimensionless units) using


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APBS widget of MGL Tools where the grid data explorer (.dx) format was provided as input. The ‘-1’ negatively charged clusters were observed as molecular surface with the remaining regions in spine view. The negatively charged cluster

with an isocontour surface of -1 showed that there is even distribution of negatively charged clusters at the molecular interface in the complexes of CpG island with H3 and H4 chains.

Figure 2 Isocontour-based electrostatic potential map of biomolecular complexes. A. H3- CpG island B. H4- CpG island C. H3-methylated CpG island and D. H4-methylated CpG island. Electrostatic interactions were much favored when both of the molecules were unmethylated (A, B) while it is unfavored if one of the interacting molecules was methylated (C, D). Similar scheme of interactions were observed in complexes of H3 and H4 with CpG island

provided a single associative partner is methylated.

Hence, the interaction in these complexes was electrostatically favored making room for geometrically preferred biomolecular association. However, docked complexes in which one of the interacting partners is methylated was failed to show such electrostatic interaction. The isocontour surface of these complexes disclosed that the charged surfaces were unevenly distributed at the interface site (Figure 2). This finding envisioned us to rethink the strategy of charge-charge interactions as usually observed in electrostatically associative biomolecules.

Angelov et al., 2001 showed that core histone tail domains bind preferentially to ‘linker’

DNA13. But it is still unknown that whether an interaction will be facilitated if the linker DNA contains CpG (both methylated and unmethylated) dinucleotides runs. Irvine et al., 2002 confirmed that the methylation of DNA had a local effect on transcription29. They also showed that acetylated histones were found to be associated with unmethylated DNA and were nearly absent from methylated DNA regions. Hence, we came to a conclusion that these methylation effects were local and there is no preferential interaction if both the partners (histone tails and DNA) were methylated. We also found that DNA normally being a negatively


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charged biomolecule, if methylated, it additives the negativity of the DNA thereby eliminating the role of methylated histones tails to interact physically. The CpG island being one amongst the nucleosome-destabilizing elements, cannot observed to be wrapped around the histone octamer due to the sequence anisotropic reasons. It still has to be uncovered that this sequence anisotropic nature of CpG dinculeotides remains the same for amino acids in the histone tails as similar to its core octamer. We hereby confirmed that the methylated zones in the biomolecules will have a local effect and are likely to be mutually exclusive events.

CONCLUSION

In vitro experiments revealed that core

histone tail domains preferentially interact with linker DNA. In the present study, the biomolecular associations between H3 and H4 tails with CpG containing linker DNA were computationally studied with the help of docking

simulations and isocontour-based electrostatic potential map. We emphasize that these association was found to be unfavored in terms of electrostatics when one of the interacting partner is methylated. We conclude that these interactions will have a local effect imparting negative charged regions which was accumulated at the molecular interface of DNA-histone tail and the physical interaction was much favored only in their native forms (unmethylated DNA and/or normal runs of bases in linker DNA with unmethylated histone tails). We hope that these results will help to elucidate the exact molecular interaction in physiological conditions.

ACKNOWLEDGEMENT S. Prasanth Kumar is thankful to Dr. Sawsan Khuri, Bioinformatics Senior Scientist, University of Miami for her critical comments and helpful discussions.

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