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    • ISSN 0975-6299 Vol 3/Issue 1/Jan – Mar 2012 International Journal of Pharma and Bio Sciences RESEARCH ARTICLE BIOINFORMATICSCOMPUTATIONAL 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*21 Bioinformatics Laboratory, Department of Botany, University School of Sciences, Gujarat University, Ahmedabad- 380 009.2 Division of Medicinal Chemistry and Pharmacogenomics, Department of Cancer Biology, The Gujarat Cancer & Research Institute (GCRI), Ahmedabad- 380 016. RAKESH M. RAWAL Division of Medicinal Chemistry and Pharmacogenomics, Department of Cancer Biology, The Gujarat Cancer & Research Institute (GCRI), Ahmedabad- 380 016. 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. This article can be downloaded from www.ijpbs.net B - 581
    • ISSN 0975-6299 Vol 3/Issue 1/Jan – Mar 2012KEYWORDS Core histone tail domain, Linker DNA, CpG island, Molecular docking, Isocontour-basedelectrostatic potential map.INTRODUCTION Chromatin is a large macromolecular crosslinking6, quantitative hydrodynamic7 and gelcomplex composed of an array of nucleosome electrophoresis experiments8.assembled by histones and other nuclearproteins. About 146 bp of DNA wraps around the CpG islands, defined as genomic regionshistone octamer in a left-handed superhelical of more than 200 bases with a G+C content of atfashion with each octamer consisting of 2 copies least 50% and a ratio of observed to statisticallyeach of the core histones H2A, H2B, H3 and H4 expected CpG frequencies of atleast 0.6,1 . A stretch of about 10-80 bp free DNA called typically occurs at or near the transcription start‘linker DNA’ connects the adjacent nucleosomes. site (TSS) or even extend over exon1 of genes9.Histone H1, also known as ‘linker histone’ binds CpG islands methylation is positively associatedto the linker DNA but does not form part of the with the interference of gene transcription bynucleosome thereby facilitates sealing off the impeding transcipting factor binding or bynucleosome at the location where DNA enters bringing about chromatin alterations. It isand leaves2. The core histone tail domains generally recognized that DNA hypermethylationthrough its post-translational modification (PTM) is involved in transcriptional repression. Someaid in the intracellular signals mediated exceptions to this mechanism exist, for e.g.transduction to organize functional states of hypermethylation of CpG island of the KAI1chromatin to initiate cellular processes such as metastasis suppressor gene could not downDNA replication or transcription. It has been regulate its expression in invasive and metastaticdemonstrated that flexible histone tails are human cancers10. Thus, it is still disputed thatcrucial players in the mitotic chromosome CpG island methylation precedes the assemblycondensation3 and the positive correlation of of repressive chromatin or a coordinatedchromosome assembly with the tail PTMs (e.g. regulatory mechanism exist11. We know thatphosphorylation of H3 at Ser10)4. Hence, core histone tail PTMs also promote gene silencing byhistone tails through its PTM serves as a target recruiting epigenetic enzymes. However, thefor binding of ancillary proteins or other wild-type p16INK4a allele in HCT116 colorectalenzymatic functions and these processes alter tumor cells is independently silenced by histonethe structure of core histone tails. Experiments H3 lysine 9 (H3-K9) methylation without anyon circular dichroism showed that N tails (N- DNA methylation12.terminal region of the tail) of H3 and H4 arehighly organized as DNA bound conformations Recent studies exhibited that the majorityand half of their residues were observed as α- of core histone tail domains interacts withhelical segment in contrast to random-coil extranucleosomal linker DNA13. It has beenconformation confined by H2A and H2B tails, elucidated that low nucleosome occupancy isrespectively5. Moreover, the tails are interacting observed at TSS and it is intrinsically encoded inwith DNA and protein within the condensed fiber the DNA sequence. For example, the locationsat defined locations and in the presence of of TATA element were found to be occupiedphysiological salts as revealed in the oligosomes outside a stably positioned nucleosome but not This article can be downloaded from www.ijpbs.net B - 582
    • ISSN 0975-6299 Vol 3/Issue 1/Jan – Mar 2012upon nucleosome in yeast genome14. On the CpG island predictionother hand, CpG islands are known to be the The von Hippel-Lindau tumor suppressor genenucleosome-destabilizing elements and this sequence was scanned for the distribution ofdestabilization is to enable transcriptional CpG island(s) using CpG Island Searcher20 withactivator Sp1 to bind to promoter binding sites the parameters set as lower limits: percentage ofwithout any nucleosome remodeling. In the G and C bases (%GC) = 55%, ratio of observedpresent study, the interaction of core histone tail to statistically expected CpG frequenciesdomains (H3 and H4) with CpG island was (ObsCpG/ExpCpG) = 0.65, frequency of basescarried out computationally15. It should be noted in the island = 500 bp and gap between adjacentthat CpG island localizes in the linker DNA due islands = 100 bp.to the fact that CpG island is always observedupstream of TSS and/or overlap within the DNA structure modelingpromoter, if so, CpG island cannot be observed Canonical B-DNA structure was constructedover nucleosome and forms a part of linker DNA. using 3D-DART (3DNA-Driven DNA AnalysisMoreover, the sequence-dependent anisotropic and Rebuilding Tool) web server21. The CpGnucleosome positioning signatures (CG island (5’-3’ sequence) was given as input. 3D-dinucleotides) does not facilitate that CpG DART exclusively uses 3DNA software suite22clusters can be wrapped over nucleosome16. which comprised of different modules to performHere, we investigated that whether CpG island the computational modeling. The ‘fiber’ modulemethylation and core histone tail PTM (H3 and initially developed a canonical B-DNA structureH4’s arginines and lysines are methylated) are and a corresponding base pair (step) parametercoordinated process and if so, preferential file was generated using ‘find_pair’ and ‘analyze’physical interaction of both of these methylated modules. The parameter file was thenbiomolecules exhibited? We report that these subsequently utilized to introduce ‘local’ andinteractions were found to be electrostatically ‘global’ bends in the DNA structure with defaultfavored in the docked conformations of histone settings of roll, tilt and twist. Finally, the DNAtails (both H3 and H4) with normal B-DNA structure file in PDB format was returned with thecontaining CpG dinucleotides (unmethylated help of ‘rebuild’ module of 3DNA.CpG island) and unfavoured interaction wasobserved if one of the associative partners is Structure manipulation and energymethylated as dictated by the isocontour-based minimizationelectrostatic potential maps. Recovered PDB file of nucleosome core particle (entry code: 1KX5) was manually spliced intoMATERIALS AND METHODS chains of histones viz. H3, H4, H2A and H2B. The H3 and H4 chains (chains ‘A’ and ‘B’) wereNucleotide sequence and protein structure separately saved in PDB file format with the helpretrieval of text editor. Methylation involves the addition ofThe sequence of von Hippel-Lindau tumor CH3 group in arginine and lysine residues insuppressor gene of Homo sapiens (accession protein as well as cytosines in the DNA molecule.number: NC_000003.11) was retrieved from YASARA View23 was extensively used toNational Center for Biotechnology Information perform this operation. ‘Build’ utility was(NCBI) Gene database17. The crystal structure of employed to introduce methyl group andthe nucleosome core particle (entry code: subsequently the bond orders were corrected1KX5)18 was obtained from RCSB Protein Data using ‘Adjust bond order’ utility. The resultantBank (PDB)19. structure files were then energy minimized using YASARA Energy minimization server24. This This article can be downloaded from www.ijpbs.net B - 583
    • ISSN 0975-6299 Vol 3/Issue 1/Jan – Mar 2012structure manipulation step was carried out for of van der Waals, electrostatic, desolvation andeach arginine and lysine residues in the H3 and restraint violation energies together with buriedH4 structures. Similarly, cytosines in the surface area.canonical B-DNA was ‘computationallymethylated’ with the same approach. PQR files and electrostatic potential map generationComputational docking To study the influence of electrostatics on DNA-The docking simulations were executed using protein complex (docked structures), continuumHADDOCK (High Ambiguity Driven biomolecular electrostatic approach was chosen. This can beDOCKing) engine25 with the in-house generated achieved by APBS (Automated Poisson-structure files. The structure files can be Boltzmann Solver)26 for which PQR files werecategorized into two types: unmodified and required as inputs. Hence, the dockedstructurally modified files. Structurally unmodified complexes in PDB format were converted intofiles represent the chains of H3 and H4 (protein PQR format using PDB2PQR server27. PQRfiles) and the 3D-DART generated canonical B- format embodies the replacement of occupancyDNA structure (DNA file) whereas modified files column in a PDB file (‘P’) with the atomic chargeinclude the structurally manipulated files of (‘Q’) and the temperature factor column with thechains H3 and H4 (From here, it will be called as atomic radius (‘R’). The inputted PDB files were‘methylated H3’ and ‘methylated H4’ as initially screened for heavy atoms (if missing, itapplicable; protein files) and structurally will be rebuild) and hydrogens were added andmanipulated CpG island (From here, it will be optimized followed by assignment of atomicknown as ‘methylated CpG island’; DNA file). parameters using force-fields such asMolecular docking was performed with each CHARMM22, AMBER99 (selected in the APBSDNA and protein files iteratively. The active sites input generation) or PARSE. At last, PDB2PQR(arginines, methylated arginines, lysines, server results were further requested to generatemethylated lysines in the protein files and APBS input file (electrostatic potential grid map)cytosines and methylated cytosines in the DNA in grid data explorer (.dx) format. Molecularfiles, respectively) were specified and passive Graphics Laboratory (MGL) Tools of Scrippsresidues were automatically defined around the Research Institute28 was utilized to developactive sites. The specification of active and isocontours of the electrostatic potential mappassive residues takes the form of experimental built from the docked structure files. MGL Toolsdata to drive docking and these data were comes up with shipped APBS widget toconverted into ambiguous interaction restraints accomplish this task and it is primarily used for(AIRS) by HADDOCK and subsequently visualization and analysis of molecular structures.generates the topology of the molecules inputted.Docking protocol consists of three stages viz. a RESULTS AND DISCUSSIONrigid-body energy minimization, a semi-flexible The docking simulation was carried outrefinement in torsional angle space and a using HADDOCK program with the followingfinishing refinement in explicit solvent. After molecular inputs: DNA containing CpG island,execution of each of these stages, the docked methylated CpG island (addition of CH3 group atconformations are scored and ranked by the cytosines), native (unmethylated or unmodified)scoring function to facilitate the selection of the chains of H3 and H4 (generated frombest conformations and subsequently employed nucleosome core particle (1KX5) using textin the next stage. The best docked conformers editor) and methylated H3 and H4 (addition ofcan be recovered by inspection of HADDOCK CH3 group at arginines and lysines), respectively.score which takes into account the weighted sum This article can be downloaded from www.ijpbs.net B - 584
    • ISSN 0975-6299 Vol 3/Issue 1/Jan – Mar 2012Prior to docking, all the structure files were Upon examining the scoring functions ofenergy minimized using YASARA energy HADDOCK score, binding and electrostaticminimization server. All the structure files were energies, minute atomic manipulations indocked in an iterative fashion (i.e each DNA file methylated structural files (both methylated DNAwith each protein file) and the binding efficiency and protein) did not altered the dockingwas evaluated using HADDOCK score and conformations rather atomic restraints werebinding energy term. 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. This article can be downloaded from www.ijpbs.net B - 585
    • ISSN 0975-6299 Vol 3/Issue 1/Jan – Mar 2012 Table 1. Docking result of biomolecules H3 tail – H3 tail – Methylated H3 Methylated H3 tail CpG island methylated tail – – methylated CpG CpG island CpG island island HADDOCK -18.5 +/- 20.7 -127.9 +/- 7.5 -16.5 +/- 11.7 -183.1 +/- 11.7 score Binding energy -170841.00 -1349.54 -178775.00 -1293.91 (KJ/mol) Electrostatic energy -653.6 +/- 45.5 -645.6 +/- 41.3 -797.4 +/- 38.0 -759.8 +/- 79.7 (KJ/mol) H4 tail – H4 tail – Methylated H4 Methylated H4 tail CpG island methylated tail – – methylated CpG CpG island CpG island island HADDOCK 18.2 +/- 8.9 -133.9 +/- 8.9 16.4 +/- 6.6 -101.8 +/- 4.9 score Binding energy -192429.00 -1219.68 -182923.00 -900.103 (KJ/mol) Electrostatic energy -619.1 +/- 13.0 -559.7 +/- 35.5 -572.5 +/- 33.9 -441.8 +/- 52.1 (KJ/mol)-16.5, -18.5, -127.9 and -183.1 KJ/mol. charged in physiological conditions.However, the binding energies were better for Electrostatic potential map for all of the aboveCpG island (unmethylated) with H3 chain. The docked conformations were developed usingelectrostatic interactions did not disclosed APBS approach. All the docked complexesinformation for preferential interaction as the were initially converted into ‘chargedenergy values were localized around -653. 6, - coordinates’ using PDB2PQR and subsequently,645.6, -759.8 and -797.4 KJ/mol. Similar grid files symbolizing the electrostatic potentialsetback aroused for chain H4 with its entire grid map were generated in order to developDNA interacting partners. We found an isocontour map. As methyl group confers aimportant insight that there is no fair interaction charge of -1, methylated arginines and lysinesif both the DNA and the protein partners were in H3 and H4 chains and methylated cytosinesmethylated as revealed in the binding energy in CpG containing DNA will tend to have aterm itself. The binding energy was found to be cluster of negative charge on its surface and agreater (-1293.91 KJ/mol for methylated H3- clear picture of charged clusters at themethylated CpG island conformation and - molecular interface will show the type of900.103 KJ/mol for methylated H4-methylated interaction.CpG island conformation) when compared to itscorresponding methylated and unmethylated Isocontour of electrostatic map for all thedocked conformations (Figure 1). computationally developed complexes were We know that biomolecular complexes generated at an isocontour value of -1.000such as DNA and H3 and H4 chains are KbTec-1 (APBS writes dimensionless units) using This article can be downloaded from www.ijpbs.net B - 586
    • ISSN 0975-6299 Vol 3/Issue 1/Jan – Mar 2012APBS widget of MGL Tools where the grid data with an isocontour surface of -1 showed thatexplorer (.dx) format was provided as input. The there is even distribution of negatively charged‘-1’ negatively charged clusters were observed clusters at the molecular interface in theas molecular surface with the remaining regions complexes of CpG island with H3 and H4in spine view. The negatively charged cluster chains. Figure 2Isocontour-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 DNA13. But it is still unknown that whether anelectrostatically favored making room for interaction will be facilitated if the linker DNAgeometrically preferred biomolecular association. contains CpG (both methylated andHowever, docked complexes in which one of the unmethylated) dinucleotides runs. Irvine et al.,interacting partners is methylated was failed to 2002 confirmed that the methylation of DNA hadshow such electrostatic interaction. The a local effect on transcription29. They alsoisocontour surface of these complexes disclosed showed that acetylated histones were found tothat the charged surfaces were unevenly be associated with unmethylated DNA and weredistributed at the interface site (Figure 2). This nearly absent from methylated DNA regions.finding envisioned us to rethink the strategy of Hence, we came to a conclusion that thesecharge-charge interactions as usually observed methylation effects were local and there is noin electrostatically associative biomolecules. preferential interaction if both the partners Angelov et al., 2001 showed that core (histone tails and DNA) were methylated. Wehistone tail domains bind preferentially to ‘linker’ also found that DNA normally being a negatively This article can be downloaded from www.ijpbs.net B - 587
    • ISSN 0975-6299 Vol 3/Issue 1/Jan – Mar 2012charged biomolecule, if methylated, it additives simulations and isocontour-based electrostaticthe negativity of the DNA thereby eliminating the potential map. We emphasize that theserole of methylated histones tails to interact association was found to be unfavored in termsphysically. The CpG island being one amongst of electrostatics when one of the interactingthe nucleosome-destabilizing elements, cannot partner is methylated. We conclude that theseobserved to be wrapped around the histone interactions will have a local effect impartingoctamer due to the sequence anisotropic negative charged regions which wasreasons. It still has to be uncovered that this accumulated at the molecular interface of DNA-sequence anisotropic nature of CpG histone tail and the physical interaction wasdinculeotides remains the same for amino acids much favored only in their native formsin the histone tails as similar to its core octamer. (unmethylated DNA and/or normal runs of basesWe hereby confirmed that the methylated zones in linker DNA with unmethylated histone tails).in the biomolecules will have a local effect and We hope that these results will help to elucidateare likely to be mutually exclusive events. the exact molecular interaction in physiological conditions.CONCLUSION ACKNOWLEDGEMENT In vitro experiments revealed that corehistone tail domains preferentially interact with S. Prasanth Kumar is thankful to Dr. Sawsanlinker DNA. In the present study, the Khuri, Bioinformatics Senior Scientist, Universitybiomolecular associations between H3 and H4 of Miami for her critical comments and helpfultails with CpG containing linker DNA were discussions.computationally studied with the help of dockingREFERENCES1. Luger K, Mader AW, Richmond RK, 5. Baneres JL, Martin A and Parello J, The N Sargent DF and Richmond TJ, Crystal tails of histones H3 and H4 adopt a highly structure of the nucleosome core particle structured conformation in the nucleosome. at 2.8 Å resolution. Nature, 389(6648): J. Mol. Biol., 273(3): 503-508, (1997). 251-260, (1997). 6. Stefanovsky VYu, Dimitrov SI, Russanova2. Berezney R and Jeon KW, Structural and VR, Angelov D and Pashev IG, Laser- functional organization of the nuclear induced crosslinking of histones to DNA in matrix, 1st Edn, Vol 2, Academic chromatin and core particles: implications Press :214-217, (1995). in studying histone–DNA interactions. Nucl.3. de la Barre AE, Gerson V, Gout S, Acids Res. 17(23), 10069-10081, (1989). Creaven M, Allis CD and Dimitrov S, Core 7. Fletcher TM and Hansen JC, Core histone histone N-termini play an essential role in tail domains mediate oligonucleosome mitotic chromosome condensation. EMBO folding and nucleosomal DNA organization J., 19: 379-391, (2000). through distinct molecular mechanics. J.4. Nowak SJ and Corces VG, Biol. Chem. 270(43): 25359-25362, (1995). Phosphorylation of histone H3: a balancing 8. Hansen JC, Tse C and Wolffe AP, act between chromosome condensation Structure and function of the core histone and transcriptional activation. Trends N-termini: more than meets the eye. Genet., 20(4): 214-220, (2004) Biochem. 37(51): 17637-17641, (1998). This article can be downloaded from www.ijpbs.net B - 588
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