Kromatinaren inmunoprezipitazioa (ChIP)

Chromatin structure plays a fundamental role in regulating processes that take place on the DNA, such as transcription, replication, and recombination, all of which occur on nucleosomal templates. Therefore, mapping the position of histones selectively modified and histone variants or non-histone components of chromatin in specific DNA sequences, can provide valuable insights into how these proteins (and their modifications) function in a chromatin context.
Chromatin Immunoprecipitation (Chip) is a biochemical method specially used to map the location of modified histones and other proteins in the genome in vivo. These experiments consist in the use of an antibody that recognizes and binds the protein of interest, not only in free solution, but also when assembled into chromatin.
Chip assay combines two basic steps. First, the cross-linking of DNA-binding proteins to DNA in vivo with formaldehyde of whole cells that fixes protein-protein and protein-DNA interactions, followed by immunoprecipitation of protein-DNA complexes with specific antibodies from sonicated extracts. Immunoprecipitated specific DNA sequences are then amplified by PCR to see whether they have been or not enriched in the samples (Kuo and Allis, 1999).

References:
Kuo MH, Allis CD.
In vivo cross-linking and immunoprecipitation for studying dynamic Protein:DNA associations in a chromatin environment.
Methods. 1999 Nov;19(3):425-33.

CpG irlen metilazioaren azterketa metilazioaren PCR espezifikoa (MSP) eta bisulfito-sekuantziazioa erabiliz

There is currently a wide range of methods designed to yield quantitative and qualitative information on genomic DNA methylation. The earliest approaches were concentrated on the study of overall levels of methyl-cytosine, but more recent efforts have focused on the study of the methylation status of specific DNA sequences. Particularly, optimization of the methods based on bisulfite modification of DNA (Clark et al. 1994) permits the analysis of limited CpGs based on differential methylation states (e.g. methylation-specific PCR) and allows very specific patterns of methylation to be revealed (bisulfite DNA sequencing).
Bisulfite converts unmethylated cytosine to uracil, while methylated cytosine does not react (Furuichi et al. 1970) (figure). This reaction constitutes the basis for discriminating between methylated and unmethylated DNA. Bisulfite transformation of DNA can be followed by several methods, including sequencing, methylation-specific PCR, combined bisulfite restriction assays, and others.

Methylation-specific PCR (MSP) is the most widely used technique for studying the methylation of CpG islands, which are common in the promoter regions of many genes. Cytosines in CpG islands are usually unmethylated in normal tissues, whereas they become methylated in the promoter sequences of genes associated with certain abnormal cellular processes such as cancer (Esteller et al. 2001). Methylation-specific PCR (Herman et al. 1996) is one of the most effective choices for investigating the methylation profile of these regions. The differences between methylated and unmethylated alleles that arise from sodium bisulfite treatment are the basis of methylation-specific PCR and are especially valuable in CpG islands because of the abundance of CpG sites.
Primer design is a critical and complex component of the procedure. Bisulfite-converted DNA strands are no longer complementary, so primer design must be customized for each DNA chain. The great sensitivity of the method allows determining the methylation status of small samples of DNA, including those from paraffin-embedded or microdissected tissues (Herman et al. 1996). However, resolution at the nucleotide level requires the sequencing of the PCR products, and quantitative data and identification of cellular heterogeneity in populations are still not possible with this technique.
Because of the versatility of the method, methylation-specific PCR has been widely proposed as a rapid and cost-effective clinical tool to use in the initial evaluation of a wide range of patients with specific allele-dependent diseases. For instance, methylation-specific PCR is currently applied to evaluate the methylation status of the CpG island of the SNRPN gene and, hence, for the rapid diagnosis of the
Prader-Willi and Angelman syndromes (Kosaki et al. 1997).

References:
Clark SJ, Harrison J, Paul CL, Frommer M.
High sensitivity mapping of methylated cytosines.
Nucleic Acids Res. 1994 Aug 11;22(15):2990-7.

Esteller M, Corn PG, Baylin SB, Herman JG.
A gene hypermethylation profile of human cancer.
Cancer Res. 2001 Apr 15;61(8):3225-9.Furuichi Y, Wataya Y, Hayatsu H, Ukita T.
Chemical modification of tRNA-Tyr-yeast with bisulfite. A new method to modify isopentenyladenosine residue.
Biochem Biophys Res Commun. 1970 Dec 9;41(5):1185-91.

Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB.
Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.
Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9821-6.

Kosaki K, McGinniss MJ, Veraksa AN, McGinnis WJ, Jones KL.
Prader-Willi and Angelman syndromes: diagnosis with a bisulfite-treated methylation-specific PCR method.
Am J Med Genet. 1997 Dec 19;73(3):308-13.

Erresoluzio Altuko Kapilare-Elektroforesia (HPCE)

DNAren metilazio globala

Global DNA methylation has been proposed as a molecular marker for a variety of biological processes such as cancer (Esteller et al. 2001, Piyathilake et al. 2001) and ontogenetic development in both plants (Fraga et al. 2000a) and animals (Jones and Wolffe, 1999). Methyl-cytosine concentration in genomic DNA is currently quantified by high-performance separation means (HPCE and HPLC) or by enzymatic/chemical approaches.
The development of Capillary Electrophoresis (CE) techniques present advantages over previous methodologies used to quantify the extent of DNA methylation. From its beginnings, CE has proved to be extremely helpful in separating various DNA components, including a number of base adducts (Norwood et al. 1993), and Fraga et al. (Fraga et al. 2000b) have reported the quantification of the relative methyl-cytosine content of the genomic DNA of plants using a High Performance Capillary Electrophoresis (HPCE) system to analyze acid-hydrolyzed genomic DNA. In this context, the separation and quantification of cytosine and methyl-cytosine are only possible by the use of an SDS micelle system.

In our laboratory we use a new approach to evaluate the relative degree of genomic DNA methylation through the quantification of 2'-deoxynucleosides. Detection and quantification of 5-methyl 2'-deoxycytidine in genomic DNA is performed using micellar high-performance capillary electrophoresis (HPCE) with UV-Vis detection. This approach has been demonstrated to be more sensitive and specific than other HPCE methods for the quantification of DNA methylation and also to be faster and cheaper than other HPLC-based methods. The detection and quantification of nucleosides through enzymatic hydrolysis notably increases the specificity of the technique and allows its exploitation in the analysis of poorly purified and/or concentrated DNA samples such as those obtained from meristematic plant regions and paraffin-embedded tissues (Fraga et al. 2002). The release of bases from DNA by chemical means involves the production of a complicated mixture of molecules that makes the detection and quantification of methyl-cytosine difficult when DNA is not sufficiently purified and/or concentrated. This problem can be avoided by enzymatic hydrolysis with Nuclease P1 and alkaline phosphatase to produce 2'-deoxymononucleosides, which are then fractionated using a modification of the previously described HPCE method. Nevertheless, almost no preparative analysis are possible with HPCE systems because of the low injection volumes involved.

DNA-proteina elkarrekintzak

DNA–protein interaction is a critical issue in a wide range of biological studies, specially in those related with chromatin structure and regulation of gene function. There are several established techniques for protein–DNA binding studies, among which electrophoretic mobility shift assays (EMSA) (Ceglarek and Revzin, 1989) are of particular importance. Although useful in many different protein–DNA binding studies, EMSA is not optimal for quantitative analysis, and the obtaining of accurate affinity constants by this method is a tedious task. In contrast, the calculation of protein–DNA affinities can be precisely obtained by calorimetric experiments (Nakatani et al. 2001) but the required equipment is not usually available in biochemistry and molecular biology laboratories. Alternatively, high-performance capillary electrophoresis (HPCE) provides the possibility of developing accurate and reproducible methods to quantitate protein–DNA affinities based on its ability to separate at high-resolution macromolecular complexes from their isolated interacting subunits. In fact, the quantification of ligand–DNA interactions by HPCE on untreated fused-silica capillaries has been widely reported (Tanaka and Terabe, 2002). But generally, DNA-interacting proteins are rich in basic amino acids, which cause adsorption to surfaces. This is in fact the major drawback of the previously described methods where adsorption by the charged sites of proteins on fixed, negatively charged sites on the capillary wall occurs.
A novel, rapid and simple capillary electrophoretic mobility shift assay (CEMSA) with laser-induced fluorescence (LIF) has been developed for the quantitative study of protein-DNA interactions (Fraga et al. 2003). This method is particularly useful for the study of basic proteins, the most common of the DNA-interacting proteins. To avoid protein stickiness to the capillary walls in our laboratory we use neutral polyacrylamide that requires the use of reverse polarity. Under these conditions, excellent separation of DNA and protein-DNA complexes is obtained without the requirement of a gel matrix, thereby allowing the easy and reliable quantification of protein-DNA affinities.

Histonen translazio-ondorengo aldaketen kuantifikazioa

Histone acetylation is the result of two enzymatic activities that opposes each other, the Histone Acetyltransferase (HAT) and Histone Deacetylase (HDAC) activities. Histone acetylation, together with other modifications of the chromatin, is the one responsible for the structural dynamic transitions that take place on the chromatin and the change of transcriptional activity state (Jenuwein and Allis, 2001). On the other hand, the N-end methylation of histones H3 and H4 seems to be more a static process, while the acetylation is considered more a dynamic process. Thus, the methylation is considered as an epigenetic mark more than a regulating signal (Jenuwein, 2001). The methylation of histones has been related mainly with the long term transcriptional repression of heterochromatin, but unlike the effect that global acetylation has on transcriptional activity, histone methylation affects transcription depending on which residue is the one modified and the degree of methylation (Lachner and Jenuwein, 2002).
Using a modification of a method previously described by Lindner et al. (Lindner et al. 1992) we are able to quantify the degrees of acetylation of histones H3 and H4, since the non-acetylated, mono-, di-, tri- and tetra-acetylated forms of histone H3 and histone H4 are separated using High Performance Capillary Electrophoresis (HPCE). In particular, the HPCE is performed using an uncoated fused-silica capillary and as resolving buffer a phosphate buffer with hydroxy-propyl-methylcellulose (HPM-cellulose) to avoid interactions of the extremely basic proteins with the wall of the capillary. With this technique we are also able to resolve methylation in histone H4, and this is why we obtain for each degree of acetylation a deployment of the corresponding tips related with a different degrees of methylation, as it is observed in the figure.

References:
Ceglarek JA, Revzin A.
Studies of DNA-protein interactions by gel electrophoresis.
Electrophoresis. 1989 May-Jun;10(5-6):360-5.

Esteller M, Fraga MF, Guo M, Garcia-Foncillas J, Hedenfalk I, Godwin AK, Trojan J, Vaurs-Barriere C, Bignon YJ, Ramus S, Benitez J, Caldes T, Akiyama Y, Yuasa Y, Launonen V, Canal MJ, Rodriguez R, Capella G, Peinado MA, Borg A, Aaltonen LA, Ponder BA, Baylin SB, Herman JG.
DNA methylation patterns in hereditary human cancers mimic sporadic tumorigenesis.
Hum Mol Genet. 2001 Dec 15;10(26):3001-7.

Fraga MF, Centeno ML, Valdes AE, Moncalean P, Canal MJ, Rodriguez R.
Genomic DNA methylation and polyamines levels as key processes in plant aging: applications for clonal multiplication of mature Pinus radiata trees, p. 495-506. In E. Ritter and S. Espinel (Eds.), Applications of Biotechnology to Forest Genetics. A.F. Albundia, Vitoria. 2000. (a)

Fraga MF, Rodriguez R, Canal MJ.
Rapid quantification of DNA methylation by high performance capillary electrophoresis.
Electrophoresis. 2000 Aug;21(14):2990-4. (b)

Fraga MF, Uriol E, Borja Diego L, Berdasco M, Esteller M, Canal MJ, Rodriguez R.
High-performance capillary electrophoretic method for the quantification of 5-methyl 2'-deoxycytidine in genomic DNA: application to plant, animal and human cancer tissues.
Electrophoresis. 2002 Jun;23(11):1677-81.

Fraga MF, Ballestar E, Esteller M.
Capillary electrophoresis-based method to quantitate DNA-protein interactions.
J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Jun 15;789(2):431-5.

Jenuwein T.
Re-SET-ting heterochromatin by histone methyltransferases.
Trends Cell Biol. 2001 Jun;11(6):266-73.

Jenuwein T, Allis CD.
Translating the histone code.
Science. 2001 Aug 10;293(5532):1074-80.

Jones PL, Wolffe AP.
Relationships between chromatin organization and DNA methylation in determining gene expression.
Semin Cancer Biol. 1999 Oct;9(5):339-47.

Lachner M, Jenuwein T.
The many faces of histone lysine methylation.
Curr Opin Cell Biol. 2002 Jun;14(3):286-98.

Lindner H, Helliger W, Dirschlmayer A, Jaquemar M, Puschendorf B.
High-performance capillary electrophoresis of core histones and their acetylated modified derivatives. Biochem J. 1992 283 (Pt 2) 467-71.

Nakatani K, Sando S, Kumasawa H, Kikuchi J, Saito I.
Recognition of guanine-guanine mismatches by the dimeric form of 2-amino-1,8-naphthyridine.
J Am Chem Soc. 2001 Dec 19;123(50):12650-7.

Norwood CB, Jackim E, Cheer S.
DNA adduct research with capillary electrophoresis.
Anal Biochem. 1993 Sep;213(2):194-9.

Piyathilake CJ, Frost AR, Bell WC, Oelschlager D, Weiss H, Johanning GL, Niveleau A, Heimburger DC, Grizzle WE.
Altered global methylation of DNA: an epigenetic difference in susceptibility for lung cancer is associated with its progression.
Hum Pathol. 2001 Aug;32(8):856-62.

Tanaka Y, Terabe S.
Estimation of binding constants by capillary electrophoresis.
J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Feb 25;768(1):81-92.

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