Understanding the Connection between Epigenetic DNA Methylation and Nucleosome Positioning from Computer Simulations Guillem Portella1,2., Federica Battistini1,2., Modesto Orozco1,2,3* 1 Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain, 2 Joint IRB-BSC Program in Computational Biology, Barcelona, Spain, 3 Departament de Bioqu´ımica i Biologia Molecular, Universitat de Barcelona, Barcelona, Spain Abstract Cytosine methylation is one of the most important epigenetic marks that regulate the process of gene expression. Here, we have examined the effect of epigenetic DNA methylation on nucleosomal stability using molecular dynamics simulations and elastic deformation models. We found that methylation of CpG steps destabilizes nucleosomes, especially when these are placed in sites where the DNA minor groove faces the histone core. The larger stiffness of methylated CpG steps is a crucial factor behind the decrease in nucleosome stability. Methylation changes the positioning and phasing of the nucleosomal DNA, altering the accessibility of DNA to regulatory proteins, and accordingly gene functionality. Our theoretical calculations highlight a simple physical-based explanation on the foundations of epigenetic signaling. Citation: Portella G, Battistini F, Orozco M (2013) Understanding the Connection between Epigenetic DNA Methylation and Nucleosome Positioning from Computer Simulations. PLoS Comput Biol 9(11): e1003354. doi:10.1371/journal.pcbi.1003354 Editor: James M. Briggs, University of Houston, United States of America Received July 22, 2013; Accepted October 5, 2013; Published November 21, 2013 Copyright: ß 2013 Portella et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a Sara Borrell fellowship to GP, by MINECO-Spain BIO2012-32868, the European Research Council (ERC-Advanced Grant, MO), and the Fundacio´ n Bot´ın. MO is an ICREA Academia Researcher. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: email@example.com . These authors contributed equally to this work. Introduction In eukaryotic cells gene function is modulated by a myriad of epigenetic marks and interactions with signal molecules that control synergistically the production of RNA and proteins. Epigenetic marks are a set of heritable but reversible chemical changes of the DNA and histones that can trigger gene silencing and activation. One of the most important epigenetic marks is DNA cytosine methylation, which occurs in 60–90% of the CpG content in mammalian DNA. In fact, most (CpG)s, except those in regions with large tracts of CpG steps (‘‘CpG-islands’’), are methylated [1,2], and changes in the methylation pattern of DNA is a fingerprint of different pathologies, including cancer [3–10]. DNA methylation has a known role in gene expression regulation [11–13], but despite extensive work [14–24] its mechanism of action is not well understood , i.e. it is not clear whether and how the chemical properties of MeC impact gene expression regulation. A popular explanation suggests that it regulates the action of proteins containing methylated-DNA binding domains . However, the prevalence of DNA methylation and the magnitude of the changes in the methylation pattern occurring under pathological conditions points towards a more general mechanism [26,27]. A promising hypothesis to rationalize the biological impact of cytosine methylation is that it affects the accessibility of the DNA within chromatin by modulating intrinsic nucleosome positioning. Although several works suggest that methylated DNA increases nucleosome rigidity [28–30] and that it is less prone to wrap around nucleosomes than normal DNA [30–32], recent genomescale studies suggest that nucleosome-bound sequences are slightly enriched in methylated cytosines (MeC), which are placed in a subtle 10-base periodicity pattern . It is thus unclear whether methylation intrinsically favours or disfavours nucleosome formation, whether it leads or not to changes in nucleosome positioning or phasing, and what is the preferential location (if any) of MeC. To shed light on these questions, we have performed a theoretical analysis of the impact of CpG methylation on the structure and stability of the nucleosome. We find that methylation of CpG steps decreases the stability of the nucleosome. Such effect increases with the number of MeCs, depends on the position of the MeC with respect to the histone core, and can be explained from variations in the mechanical properties of methylated versus unmethylated DNAs. Our results reveal that methylation is sufficient to induce changes in phasing and/or positioning of the DNA around the nucleosome, which in turn might modify the accessibility of DNA sequences to proteins controlling gene expression. Our study helps understand the important role of methylation in gene expression regulation. Methods Molecular dynamics simulations and free energy calculations of fully solvated and neutralized mono-nucleosomes were carried on the X-ray structure with PDB code 1KX5 . To save computational cost we have removed the long histone tails protruding out from the core. We subjected the energy-minimized PLOS Computational Biology | www.ploscompbiol.org 1 November 2013 | Volume 9 | Issue 11 | e1003354 DNA Methylation and Nucleosome Positioning Author Summary In Eukaryotic cells, control of the patterns of DNA cytosine methylation – a mechanism that acts on top of the genetic code – plays a key role in the regulation of gene expression. The large prevalence of DNA methylation in vivo, suggests a connection between the physical properties of methylated and un-methylated DNA with the control of gene expression. In this work we investigate the physical implications of DNA methylation in nucleosomal DNA, in particular its preferred location with respect to the nucleosome core-particle and the consequences of DNA methylation for the accessibility of the genetic material. We find that methylated DNA is less prone to form nucleosomes due to a reduced elasticity, especially when all methyl groups are pointing outwards from the nucleosome core, and that multiple methylation could give rise to changes in nucleosome positioning. structure to 200 ns of MD simulation, and we used the last structure to introduce different number of CpG and methylated CpG steps in positions described in Table S1 in Text S1. After energy minimization and initial thermalization, we performed MD for 100 ns for the selected single mutations and 200 ns for the multiple mutations (see table 1 and the next section), gathering information concerning solvent interaction or solvent densities, energies of stacking and geometrical parameters. Differential binding free energies were computed using the thermodynamic integration method in its discrete formalism, exploiting a thermodynamic cycle sketched in figure 1B. In this method, the free energy between two states is computed by integration of the derivative of the energy of the system as function of the state parameter l, known as coupling parameter , which in our case describes either the methylated (e.g. l = 0) or the unmethylated state (l = 1). For each window we collected 9 estimates for SdG=dlTl by using 9 blocks of 100 ps, which were then integrated through the entire mutation pathway to obtain mutation free energies (with associated statistical errors). We measured the deformation energy for the methylated and un-methylated sequences using a mesoscopic energy model. This model describes the deformability along DNA helical parameters by an harmonic approximation, using the stiffness constants (ki) associated with the displacements with respect to the equilibrium values of the helical parameter [36,37]. The values for the parameters describing the equilibrium geometry and stiffness constants of naked DNA were derived from long atomistic MD simulations (.200 ns, as found in the ABC consortium database ) of a reduced number of short DNA duplexes in water. The parameters for methylated cytosine were extracted from Perez et al. . Full details on all computational methods and on the analysis performed are provided as SI text. Results/Discussion Free energy calculations indicate that nucleosomal DNA methylation disfavors nucleosome formation We first studied the change in the stability of a nucleosome particle (histone proteins and DNA) induced by replacing cytosines with 5-methylcytosines in CpG steps located at representative positions along the DNA (examples in Figure 1A, full list in Table S1 in Text S1) by means of the thermodynamic cycle shown in figure 1B. The starting conformations for our free energy calculations were obtained from a 200 ns molecular dynamics (MD) simulation of the nucleosome in physiological conditions, using as initial conformation the highest-resolution X-ray structure available of the nucleosome . We produced 18 different mutated nucleosome models, where each mutation consisted on placing a single CpG step at different locations where either the minor, or the major grooves face the histones (Figure 1A); these two types of positions explore widely different geometrical placements for MeC in the nucleosome . In addition, to study the effect of multiple methylations on nucleosomal stability we introduced several CpG steps simultaneously (see SI and Table S1 in Text S1). All the systems were extensively re-equilibrated prior to production runs. Nucleosomal and corresponding naked DNAs were used as starting points for TI calculations, where the reversible work associated with the methylation of the CpG step in nucleosomal and naked DNA was computed and processed to determine the change in nucleosome stability induced by cytosine methylation (see Figure 1B and Suppl. Information for details on all calculations performed in Text S1). MD/TI calculations yield a positive free energy variation in all cases, demonstrating that methylation of DNA decreases the stability of the nucleosome (Figure 2), in contradiction with recent genome-wide-association study (GWAS) , but in agreement Table 1. List of the molecular dynamics simulations performed in this work. Mutation type Single mutation 1–10 Initial equilibration + SGTI 10 ns equilibration + 10 ns SGTI Single mutation 11–18 10 ns equilibration + 10 ns SGTI Multiple mutations 20 ns equilibration + 10 ns SGTI Discrete Thermodynamic Integration 21 windows of 1 ns for each mutation, also for the reference state. 21 windows of 1 ns for each mutation, also for the reference state. 21 windows of 1 ns for each mutation, also for the reference state. MD simulations Nucleosome (un-methylated and methylated) 100 ns each 200 ns each (except Mixed2) MD simulations unbound state oligo fragments (un-methylated and methylated) 50 ns each 50 ns each (except Mixed2) For each mutation type, columns two and three describe the MD simulations that were performed to extract free energy differences. SGTI refers to slow growth thermodynamic integration method, see SI material methods. In columns four and five we list all the MD simulations that were carried out to characterize the effect of such mutation in terms of structural and energetic variations. doi:10.1371/journal.pcbi.1003354.t001 PLOS Computational Biology | www.ploscompbiol.org 2 November 2013 | Volume 9 | Issue 11 | e1003354 DNA Methylation and Nucleosome Positioning Figure 1. Mutation sites and thermodynamic cycle. (A) Example of two extreme cases of MeC positioning (spheres) along the nucleosomal DNA with respect to the histone core (in grey) used in this work: (top) the CpG step minor groove faces the histones; (bottom) the CpG major groove faces the histones. The methyl carbon is colored in green. The images on the right show a lateral view of the DNA and the protein as seen from the solvent. (B) Diagram of the thermodynamic cycle used to extract the free energy variation (DDGb (kJ/mol)) in nucleosome-DNA stability due to methylation of CpG steps. The calculations of the unbound reference state for the single mutations were performed on shorter DNA chains, using the nearest 3 neighbors of the CpG steps in the nucleosome sequence, and 4 bases to cap the duplex termini (59-CGAT and TACG-39). As the histone proteins are not affected by the cytosine methylation in the unbound state, they were not included in the calculations related to such state. In case of multiple methylations, large fragments of different length were used (further details in SI material). The free energy difference associated with the removal of the methyl group is calculated using discrete thermodynamic integration (DTI). The methyl group is shown as a green sphere. doi:10.1371/journal.pcbi.1003354.g001 with many previous biophysical studies [25,30,32,41,42]. The disagreement with the GWAS conclusions could be attributed to an uncertainty of up to four base pairs in MNase-degradation nucleosome footprinting, which is close to half a DNA helical turn, and to the cell-to-cell variability of nucleosome positioning and methylations maps . The MeC-mediated destabilization of nucleosome is cumulative for multiple methylations, and in some cases the expected destabilization is so large (more than 20 kJ/ mol) that it could challenge the entire stability of the nucleosome. Our MD/TI simulations also show that the effect of methylation Figure 2. Differential binding free energy (DDG bind. (kJ/mol)) of nucleosomal DNA. (A) Differential binding free energy values for single and multiple methylated CpG steps with respect to unmethylated sequences; methylations at major groove positions (blue) are better tolerated than at minor groove positions (light orange). Multiple methylations show a cumulative effect on the differential binding energy, following the trend of single methylations (methylation in major groove in dark blue, mixed major-minor groove in green, and minor groove in dark red). The exact location of each mutation is listed in Table S1 in Text S1. (B) Correlation between the variation in free energy (DDG (kJ/mol)) and the variation in elastic energy (DDE (kJ/mol)) for single (black dots) or multiple (red squares) methylated CpG steps in the nucleosomal DNA. doi:10.1371/journal.pcbi.1003354.g002 on nucleosome stability is phase/position-dependent (Figure 2). In general, major groove methylations (i.e. those of CpG steps that face the histones through the major groove) are much better tolerated than minor groove methylations (i.e. those of CpG steps that face the histones through the minor groove). These results indicate that nucleosomes are more stable when the methyl groups in MeCpG steps are placed pointing towards the histones and not to the solvent. Analysis of the large amount of MD/TI data presented here (Figure 2) shows that methylation is especially nucleosomedestabilizing at some specific positions, such as those located at 626 base steps from the nucleosome dyad position (mutations 10 and 16), where the nucleosome-bound DNA is characterized by a kinked geometry and a value of the roll angle (,27 deg. Fig. S1A in Text S1) that is widely different to the equilibrium value of MeCpG steps (,+14 deg.) [31,44]. In comparison, methylation has a significantly lower stability cost when happening at major groove positions, such as 211 and 21 base pair from dyad (mutations 9 and 12), where the roll of the nucleosome bound conformation (+10 deg.) is more compatible with the equilibrium geometry of MeCpG steps. The nucleosome destabilizing effect of cytosine methylation increases with the number of methylated cytosines, following the same position dependence as the single methylations. The multiple-methylation case reveals that each major groove meth- PLOS Computational Biology | www.ploscompbiol.org 3 November 2013 | Volume 9 | Issue 11 | e1003354 DNA Methylation and Nucleosome Positioning ylation destabilizes the nucleosome by around 1 kJ/mol (close to the average estimate of 2 kJ/mol obtained for from individual methylation studies), while each minor groove methylation destabilizes it by up to 5 kJ/mol (average free energy as single mutation is around 6 kJ/mol). This energetic position-dependence is the reverse of what was observed in a recent FRET/SAXS study . The differences can be attributed to the use of different ionic conditions and different sequences: a modified Widom-601 sequence of 157 bp, which already contains multiple CpG steps in mixed orientations, and which could assume different positioning due to the introduction of new CpG steps and by effect of the methylation. The analysis of our trajectories reveals a larger root mean square deviation (RMSD) and fluctuation (RMSF; see Figures S2– S3 in Text S1) for the methylated nucleosomes, but failed to detect any systematic change in DNA geometry or in intermolecular DNA-histone energy related to methylation (Fig. S1B, S1C, S4–S6 in Text S1). The hydrophobic effect should favor orientation of the methyl group out from the solvent but this effect alone is not likely to justify the positional dependent stability changes in Figure 2, as the differential solvation of the methyl groups in the bound and unbound states is only in the order of a fraction of a water molecule (Figure S5 in Text S1). We find however, a reasonable correlation between methylation-induced changes in hydrogen bond and stacking interactions of the bases and the change in nucleosome stability (see Figure S6 in Text S1). This markers, albeit at a low frequency, and have high levels of DNA methylation of genes that undergo age-associated DNA hypermethylation. These tumors have been described as CIMP-low or CIMP2 [35,36]. We did not find an association between IGFBP7 DNA hypermethylation and KRAS mutations when we excluded tumors with mutant BRAF (P value = 0.85). In agreement with Figure 5. IGFBP7 promoter DNA methylation in primary colorectal cancers. (A) Genomic map of IGFBP7 promoter-associated CpG island, transcription start site (TSS) and exon 1 based on the UCSC genome browser (March 2006 assembly). The location of CpG sites interrogated by the Illumina GoldenGate DNA methylation assay is indicated by vertical arrows. (B) DNA methylation levels of the two CpG dinucelotides in the IGFBP7 promoter CpG island. b-values of each CpG site in 10 tumors (five CIMP2 tumors with wild-type BRAF and five CIMP+ tumors with mutant BRAF, black bars) and adjacent non-tumor tissues (gray bars) are listed. (C) IGFBP7 promoter DNA methylation box plots of 235 human colorectal tumors stratified by BRAF mutation status (left) and CIMP+ status (right) at the IGFBP7 P371 locus. In the box plots, the ends of the box are the 25th and 75th quartiles. The line within the box identifies the median b-value. The whiskers above and below the box extend to at most 1.5 times the IQR. The CIMP status of each colorectal tumor sample is determined as described in the Materials and Methods section. doi:10.1371/journal.pone.0008357.g005 PLoS ONE | www.plosone.org 6 December 2009 | Volume 4 | Issue 12 | e8357 Analysis of CIMP and BRAFV600E these observations, we found that DNA hypermethylation of IGFBP7 is mostly present in colorectal cancer cell lines which harbor BRAFV600E and show frequent DNA methylation of the five-gene CIMP-specific marker panel previously described (Figure 6). Real-time RT-PCR analysis of colorectal cancer cell lines showed that IGFBP7 mRNA expression was inversely related to DNA hypermethylation, as cell lines with IGFBP7 hypermethylation showed little or no IGFBP7 gene expression (Figure 6). Among the CIMP2 cells we examined, only COLO 320DM showed DNA hypermethylation of the IGFBP7 CpG island promoter with minimal level of expression. In retrospect, this unique characteristic of COLO 320DM cells compared to the other CIMP2 cell lines might have enabled these cells to tolerate mutant BRAF overexpression, and may explain our difficulties in obtaining BRAFV600E expressing clones in other colorectal cancer cell line such as Caco-2. Discussion CIMP in colorectal cancer provides a unique opportunity to study molecular mechanisms that lead to epigenetic changes in cancer and the contributions of these changes in the development of the disease [3,37]. The distinct features found in CIMP are important clues in understanding this phenotype [3,5,7,8]. Particularly striking is the extremely tight association between CIMP and BRAFV600E . Mechanisms linking epigenetic (CIMP) and genetic (BRAF mutation) events and the temporal sequence in which these two events take place have attracted interest . In this study, by using the high-throughput Illumina GoldenGate DNA methylation platform, we investigated the link between CIMP and BRAFV600E in colorectal cancer. We first tested whether expression of BRAFV600E causes DNA hypermethylation by stably expressing BRAFV600E in the CIMP- negative, BRAF wild-type COLO 320DM colorectal cancer cell line. We have examined DNA methylation changes in 100 CIMPassociated CpG sites, and found that BRAFV600E is not sufficient to induce DNA hypermethylation at these sites. One caveat of our system is that BRAFV600E could play a role in inducing DNA methylation only early in colorectal tumorigenesis, as BRAF mutations have been described at the earliest stage of tumor development [10,38,39]. It is possible that a unique set of genetic and/or epigenetic changes that occurred in COLO 320DM cells might have created an unfavorable environment for BRAFV600E to induce DNA hypermethylation. Experiments similar to those described above using Caco-2 cells, which also show CIMP– and carry BRAF-wild type, were not successful. We were not able to obtain any stably transfected clones that exhibited detectable levels of BRAFV600E (data not shown). Sustained BRAFV600E expression might be incompatible with Caco-2 cell proliferation due to cellular senescence or apoptosis induced by BRAFV600E. It is noteworthy that our RT-PCR analysis showed the robust expression of IGFBP7, a mediator of BRAFV600E-induced senescence or apoptosis, in Caco-2 cells in contrast to COLO 320DM cells. Previously, we described CIMP-associated methylation of MLH1 as the underlying basis for mismatch repair deficiency (MSI+) in sporadic colorectal cancer . Minoo et al. reported MLH1 DNA methylation upon stable transfection of BRAFV600E into the NCM460 cell line . In our system, we did not detect such an increase in MLH1 DNA methylation (data not shown). Figure 6. Analysis of DNA methylation and mRNA expression of IGFBP7 in colorectal cancer cell lines. Quantitative real-time RT-PCR analysis of IGFBP7 expression. IGFBP7 expression levels are presented relative to PCNA expression. The error bars indicate the standard deviation of technical triplicate measurements. The number of methylated loci among the five CIMP markers and mutation status of BRAF and KRAS listed in Figure 1 are provided. doi:10.1371/journal.pone.0008357.g006 PLoS ONE | www.plosone.org 7 December 2009 | Volume 4 | Issue 12 | e8357 Analysis of CIMP and BRAFV600E Moreover, of the 33 BRAFV600E primary tumors we examined only 42% (14/33) showed MLH1 DNA hypermethylation. Therefore, BRAFV600E may affect DNA hypermethylation of MLH1 but only in certain circumstances. Interestingly, in the proposed serrated pathway to CIMP+ tumors, both BRAF mutations and CIMP+ have been observed in early precursor lesions, whereas MSI+ has not [6,10,41]. Thus, inactivation of MLH1 might occur at a later stage of tumor development. Minoo and colleagues observed the DNA hypermethylation of CDKN2A and 15 other CIMP-associated markers (IGFBP7 was not examined) in parent NCM460 cells, which limited their ability to study further the role of BRAFV600E inducing CIMP in their experimental system . Intriguingly, we observed that the overall DNA methylation level of the CIMP-specific loci in our stably transfected cells increases as a function of cell passage. It is interesting to note that a selection drug in cultured cells has been described to result in changes in global chromatin structure , and a similar process may be associated with our observations here. In addition, we found relatively large inter-clonal (among different clones) variation in DNA methylation levels in our transfection experiments (Figures 2B and 3), with an average R2 correlation calculated based on four EVCs of 0.8860.01 (6 s.d.). Our average intra-clonal (within clones at different passages) R2 correlation is 0.9760.01 and the R2 correlation between technical replicates in Illumina GoldenGate DNA methylation analysis is 0.9860.02 . Consequently, we found some large differences in DNA methylation at several loci even among control clones (Figures 2B and 3). This emphasizes the importance of using multiple clones for this type of studies. Alternatively, the strong association between CIMP and BRAFV600E might arise if CIMP specifically provides a favorable cellular context for BRAFV600E to promote tumorigenesis. In the second set of experiments, we determined genes whose DNA hypermethylation was tightly linked to BRAFV600E and CIMP+ in colorectal cancer. Intriguingly, we observed CIMP-dependent DNA hypermethylation and transcriptional inactivation of IGFBP7, which has been shown to mediate BRAFV600E-induced cellular senescence and apoptosis . BRAFV600E has been shown to induce cellular senescence in cultured and primary human cells [30,31], as well as mouse model . Oncogene- induced senescence (OIS) has been recognized as an important tumor suppressor mechanism . In order for BRAFV600E to promote its oncogenic effects, additional cooperative events are required to bypass senescence . Recently, the molecular basis of BRAFV600E-induced senescence and apoptosis has been studied in detail. Wajapeyee et al. identified IGFBP7 as a mediator of BRAFV600E-induced senescence in human primary fibroblasts using a genome-wide shRNA screen. Their subsequent findings suggest that IGFBP7 expression is both necessary and sufficient to induce senescence and apoptosis in human primary melanocytes and melanoma, respectively. Moreover, they observed loss of IGFBP7 in primary BRAFV600E-positive melanoma samples and concluded that silencing of IGFBP7 expression is a critical step in the development of a melanoma harboring BRAFV600E . extração do DNA) 3ª análise (9 meses após a 280,00 379,17 587,92 619,17 103,25 105,42 96,67 142,92 495,00 476,67 309,58 306,67 extração do DNA) F= Freezer; G= Geladeira. As comparações das médias foram realizadas utilizando-se três diferenças mínimas significativas (dms), sendo: dms 1= 155,726 para comparar médias entre tempos de análise (coluna); dms 2= 169,287 para comparar médias entre freezer e geladeira, na mesma técnica e tecido; dms 3= 1936,132 para comparar médias entre técnicas de extração no mesmo ambiente (freezer ou geladeira), no mesmo tecido ou em tecidos diferentes. Coeficiente de variação = 37%. Não houve diferença significativa entre as médias da quantidade de DNA, nos três períodos de análises, nos três tecidos usados e nos dois métodos de conservação. Também não houve diferença entre as médias dos dois métodos de conservação no mesmo tecido e na mesma técnica de extração. Para o mesmo tecido, houve diferença entre as técnicas de extração no sangue, conservado tanto em freezer quanto em geladeira. A técnica permanente forneceu maior quantidade de DNA. Isso pode ser explicado pelo fato de na técnica permanente a extração ser feita preferencialmente em linfócitos (colhidos com pipeta Pasteur após centrifugação do sangue), enquanto que na técnica rápida utiliza-se sangue total, portanto, maior quantidade de hemácias (células anucleadas). As médias obtidas para a quantidade de contaminação no DNA, fornecidas pela comparação com o nível de DNA puro que é dado pela relação 260/280nm na leitura feita pelo espectrofotômetro, são mostradas na Tab. 2. O 112 Arq. Bras. Med. Vet. Zootec. v.56, n.1, p.111-115, 2004 Comparação entre métodos de estocagem de DNA. valor 1,8 representa um DNA livre de contaminação, e abaixo dessa estimativa considera-se que há contaminação por proteínas (Sambrook et. al., 1989). Não houve diferença no nível de contaminação do DNA ao longo dos períodos de análises. A técnica que demonstrou menor índice de contaminação do DNA foi a permanente para sangue, confirmando a eficiência da proteinase K (Moore, 1993). Os maiores índices de contaminação do DNA foram observados nas extrações de DNA de sêmen, e provavelmente correspondem às proteínas da gema de ovo, utilizadas na preservação desse material. Tais achados mostraram a necessidade de adaptação das técnicas para a utilização de sêmen preservado em gema de ovo. Ajustes na diluição da amostra de sêmen, nas quantidades das soluções de lise e neutralizante, bem como no tempo de incubação da amostra, forneceram resultados satisfatórios. Quanto aos padrões de amplificação, todas as técnicas de extração de DNA mostraram bons resultados em todos períodos de análises. Houve amplificação dos extratos, não sendo observadas diferenças entre as formas de conservação, como mostrado na Fig. 1 (A,B,C e D) Tabela 2. Médias do nível de contaminação por proteína (mg/ml) do DNA segundo a técnica de extração, tecido usado, local de armazenamento e período para análise após a extração do DNA Tecido Sangue Sêmen Pêlos Técnica Rápida Permanente Rápida Permanente Rápida Permanente Conservação FGFGFGFG F G F G 1ª análise (3 meses após a 1,2393 1,2936 1,4462 1,4785 0,9033 0,8770 0,8290 0,7585 1,3059 1,3877 1,4203 1,3689 extração do DNA) 2ª análise (6 meses após a 1,0858 1,112 1,3690 1,3761 0,9195 0,8464 0,6565 0,6033 1,2741 1,2560 extração do DNA) 1,2037 1,2246 3ª análise (9 meses após a 1,1174 1,1550 1,5173 1,5153 0,7861 0,8488 0,6190 0,7385 1,1819 1,2027 1,1191 1,1489 extração do DNA) Conservação F= Freezer; G= Geladeira. As comparações das médias foram realizadas utilizando três diferenças mínimas significativas (dms), sendo: dms 1= 0,0832 para comparar médias entre tempos de análise (coluna); dms 2= 0,0995 para comparar médias entre freezer e geladeira, na mesma técnica e tecido; dms 3= 0,1104 para comparar médias entre técnicas de extração no mesmo ambiente (freezer ou geladeira), no mesmo tecido ou em tecidos diferentes. Coeficiente de Variação (CV) = 6,5%. PM 1 2 3 4 5 6 A PM 1 2 3 4 5 6 B PM 1 2 3 4 5 6 PM 1 2 3 4 5 6 CD Figura 1. Amplificações com o primer RM 29, primeira análise (3 meses após a extração), DNA conservado em freezer. A – Técnica permanente de extração de DNA para sangue. B – Técnica rápida de extração de DNA para sangue. C – Técnica permanente de extração de DNA para pêlos. D – Técnica rápida de extração de DNA para pêlos - padrão de peso molecular com amostras dos touros (PM). Arq. Bras. Med. Vet. Zootec. v.56, n.1, p.111-115, 2004 113 Coelho et al. Como já mencionado, devido ao nível de contaminantes protéicos provenientes da gema de ovo usada como preservativo, só se obteve um bom padrão de amplificação para o DNA extraído de sêmen após modificações nas técnicas (Fig. 2). Desse modo, conseguiu-se obter maior quantidade de DNA genômico bovino e boa amplificação. PM 1 2 3 4 56 Figura 2. Eletroforese da amplificação com primer RM 29, primeira análise. DNA conservado em geladeira - padrão de peso molecular, com amostras dos touros (PM). Este estudo permitiu a comparação de algumas técnicas de extração de DNA rotineiramente usadas em laboratórios, apontando as vantagens e desvantagens de cada uma delas quanto à quantidade e qualidade do extrato obtido. Ao final do estudo, pode-se concluir que, para um período de nove meses, não há diferença entre os métodos de conservação em freezer (-20ºC) ou em geladeira (4ºC). Também não foram observadas diferenças na quantidade de DNA e nível de contaminação protéica, sendo que o DNA extraído pelos métodos considerados rápidos mostrou-se eficiente para amplificações pela PCR durante os nove meses de estudos. A técnica permanente de extração de DNA para sangue foi a que forneceu maior quantidade de DNA com menor índice de contaminação. Para a extração de DNA de amostras de sêmen envasado conservado em gema de ovo, foram necessárias modificações nas técnicas originais, de modo a fornecer resultados satisfatórios. Palavras-chave: DNA, métodos de extração, amplificação ABSTRACT DNA samples of six bovines obtained from three tissues (blood, semen and hair) were extracted using two different techniques. After the extraction procedures the samples were divided in six fractions. Three were stored at -20°C and three at 4°C. Every three months one sample of each tissue/extraction procedure was analyzed in spectrophotometer, to determine the quantity of the DNA and the extract was amplified using the primer RM 29. No differences in the DNA quantity or in the level of protein contamination among the three periods of analyses were observed. All the DNA extracted by quick extraction technique showed good amplification patterns during the nine months, meaning that this technique can be used in laboratory routine instead of the permanent extraction technique. The extract obtained from blood, using the permanent extraction technique, showed the higher quantity of DNA with the smaller index of protein contamination. The high quantity of protein contamination found in the semen samples preserved in egg yolk demanded modifications in both extraction techniques. After that the results were positive, showing good amplification patterns. Keywords: DNA, extraction methods, amplification REFERÊNCIAS BIBLIOGRÁFICAS BARENDSE, W.; ARNUTAGE, S.M.; SHALOM, A. et al. A genetic linkage map of the bovine genome. Nature Gen.,v.6, p.227-234, 1994. CATTANEO, C.; CRAIG, O.E.; JAMES, N.T. et. al. Comparison of three DNA extraction methods on bone and blood stains up to 43 years old and amplification of three different gene sequences. J. Forensic Sci., v.42, p.1126-1135, 1997. 114 Arq. Bras. Med. Vet. Zootec. v.56, n.1, p.111-115, 2004 Comparação entre métodos de estocagem de DNA. CRAIN, P.F. Preparation and enzymatic hydrolysis of DNA and RNA for mass spectrometry. Methods Enzymol., v.193, p.782790, 1990. GALLAGHER, S.R.Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. Current protocols in molecular biology. Suppl. 28 CPMB, appendix 3D, A.3D.1A.3D.8, 1994. LEFF, L.G.; DANA, J.R.; McARTHUR, J.V. et al. Comparison of methods of DNA extraction from stream sediments. Appl. Envirom. Microbiol., v.61, p.1141-1143, 1995. LEWIS, H.A.; STEWART-HAYNES, J. A simple method for DNA extraction from leucocytes for use in PCR. Biotechniques, v.13, p.522-523,1992. MOORE, D. Preparation and analysis of DNA. Current protocols in molecular biology, Suppl. 24, 25 CPMB, c.2, 2.0.5 – 2.2.2, 1993. SAMBROOK, J.; FRITSCH, E.F.; MANIATIS, T. Molecular cloning: a laboratory manual. New York: Cold Spring Harbor Laboratory Press, 1989. p.E.5-E.7. SAMPAIO, I.B.M., Estatística aplicada à experimentação animal. Belo Horizonte: Fundação de Ensino e Pesquisa em Medicina Veterinária e Zootecnia, 1998. 221p. Arq. Bras. Med. Vet. Zootec. v.56, n.1, p.111-115, 2004 115 Understanding the connection between epigenetic DNA methylation and nucleosome positioning from computer simulations.