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. 1993 Oct 25;21(21):4886–4892. doi: 10.1093/nar/21.21.4886

Dissection of the methyl-CpG binding domain from the chromosomal protein MeCP2.

X Nan 1, R R Meehan 1, A Bird 1
PMCID: PMC311401  PMID: 8177735

Abstract

MeCP2 is a chromosomal protein which binds to DNA that is methylated at CpG. In situ immunofluorescence in mouse cells has shown that the protein is most concentrated in pericentromeric heterochromatin, suggesting that MeCP2 may play a role in the formation of inert chromatin. Here we have isolated a minimal methyl-CpG binding domain (MBD) from MeCP2. MBD is 85 amino acids in length, and binds exclusively to DNA that contains one or more symmetrically methylated CpGs. MBD has negligable non-specific affinity for DNA, confirming that non-specific and methyl-CpG specific binding domains of MeCP2 are distinct. In vitro footprinting indicates that MBD binding can protect a 12 nucleotide region surrounding a methyl-CpG pair, with an approximate dissociation constant of 10(-9) M.

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Selected References

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  1. Antequera F., Macleod D., Bird A. P. Specific protection of methylated CpGs in mammalian nuclei. Cell. 1989 Aug 11;58(3):509–517. doi: 10.1016/0092-8674(89)90431-5. [DOI] [PubMed] [Google Scholar]
  2. Blatter E. E., Ebright Y. W., Ebright R. H. Identification of an amino acid-base contact in the GCN4-DNA complex by bromouracil-mediated photocrosslinking. Nature. 1992 Oct 15;359(6396):650–652. doi: 10.1038/359650a0. [DOI] [PubMed] [Google Scholar]
  3. Boyes J., Bird A. DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein. Cell. 1991 Mar 22;64(6):1123–1134. doi: 10.1016/0092-8674(91)90267-3. [DOI] [PubMed] [Google Scholar]
  4. Churchill M. E., Suzuki M. 'SPKK' motifs prefer to bind to DNA at A/T-rich sites. EMBO J. 1989 Dec 20;8(13):4189–4195. doi: 10.1002/j.1460-2075.1989.tb08604.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ellenberger T. E., Brandl C. J., Struhl K., Harrison S. C. The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: crystal structure of the protein-DNA complex. Cell. 1992 Dec 24;71(7):1223–1237. doi: 10.1016/s0092-8674(05)80070-4. [DOI] [PubMed] [Google Scholar]
  6. Hendrickson W., Schleif R. A dimer of AraC protein contacts three adjacent major groove regions of the araI DNA site. Proc Natl Acad Sci U S A. 1985 May;82(10):3129–3133. doi: 10.1073/pnas.82.10.3129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Johnson K. R., Lehn D. A., Reeves R. Alternative processing of mRNAs encoding mammalian chromosomal high-mobility-group proteins HMG-I and HMG-Y. Mol Cell Biol. 1989 May;9(5):2114–2123. doi: 10.1128/mcb.9.5.2114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Lamb P., McKnight S. L. Diversity and specificity in transcriptional regulation: the benefits of heterotypic dimerization. Trends Biochem Sci. 1991 Nov;16(11):417–422. doi: 10.1016/0968-0004(91)90167-t. [DOI] [PubMed] [Google Scholar]
  9. Lewis J. D., Meehan R. R., Henzel W. J., Maurer-Fogy I., Jeppesen P., Klein F., Bird A. Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA. Cell. 1992 Jun 12;69(6):905–914. doi: 10.1016/0092-8674(92)90610-o. [DOI] [PubMed] [Google Scholar]
  10. Lewis J., Bird A. DNA methylation and chromatin structure. FEBS Lett. 1991 Jul 22;285(2):155–159. doi: 10.1016/0014-5793(91)80795-5. [DOI] [PubMed] [Google Scholar]
  11. Meehan R. R., Lewis J. D., Bird A. P. Characterization of MeCP2, a vertebrate DNA binding protein with affinity for methylated DNA. Nucleic Acids Res. 1992 Oct 11;20(19):5085–5092. doi: 10.1093/nar/20.19.5085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Meehan R. R., Lewis J. D., McKay S., Kleiner E. L., Bird A. P. Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs. Cell. 1989 Aug 11;58(3):499–507. doi: 10.1016/0092-8674(89)90430-3. [DOI] [PubMed] [Google Scholar]
  13. Meehan R., Lewis J., Cross S., Nan X., Jeppesen P., Bird A. Transcriptional repression by methylation of CpG. J Cell Sci Suppl. 1992;16:9–14. doi: 10.1242/jcs.1992.supplement_16.2. [DOI] [PubMed] [Google Scholar]
  14. Miller O. J., Schnedl W., Allen J., Erlanger B. F. 5-Methylcytosine localised in mammalian constitutive heterochromatin. Nature. 1974 Oct 18;251(5476):636–637. doi: 10.1038/251636a0. [DOI] [PubMed] [Google Scholar]
  15. Pfeifer G. P., Riggs A. D. Chromatin differences between active and inactive X chromosomes revealed by genomic footprinting of permeabilized cells using DNase I and ligation-mediated PCR. Genes Dev. 1991 Jun;5(6):1102–1113. doi: 10.1101/gad.5.6.1102. [DOI] [PubMed] [Google Scholar]
  16. Pfeifer G. P., Tanguay R. L., Steigerwald S. D., Riggs A. D. In vivo footprint and methylation analysis by PCR-aided genomic sequencing: comparison of active and inactive X chromosomal DNA at the CpG island and promoter of human PGK-1. Genes Dev. 1990 Aug;4(8):1277–1287. doi: 10.1101/gad.4.8.1277. [DOI] [PubMed] [Google Scholar]
  17. Reeves R., Nissen M. S. The A.T-DNA-binding domain of mammalian high mobility group I chromosomal proteins. A novel peptide motif for recognizing DNA structure. J Biol Chem. 1990 May 25;265(15):8573–8582. [PubMed] [Google Scholar]
  18. Selker E. U. DNA methylation and chromatin structure: a view from below. Trends Biochem Sci. 1990 Mar;15(3):103–107. doi: 10.1016/0968-0004(90)90193-f. [DOI] [PubMed] [Google Scholar]
  19. Smith D. B., Davern K. M., Board P. G., Tiu W. U., Garcia E. G., Mitchell G. F. Mr 26,000 antigen of Schistosoma japonicum recognized by resistant WEHI 129/J mice is a parasite glutathione S-transferase. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8703–8707. doi: 10.1073/pnas.83.22.8703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  21. Zhang X. Y., Hörz W. Nucleosomes are positioned on mouse satellite DNA in multiple highly specific frames that are correlated with a diverged subrepeat of nine base-pairs. J Mol Biol. 1984 Jun 15;176(1):105–129. doi: 10.1016/0022-2836(84)90384-x. [DOI] [PubMed] [Google Scholar]

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