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. 1998 Jun 15;26(12):2935–2940. doi: 10.1093/nar/26.12.2935

Induction of XIST expression from the human active X chromosome in mouse/human somatic cell hybrids by DNA demethylation.

A V Tinker 1, C J Brown 1
PMCID: PMC147638  PMID: 9611238

Abstract

X chromosome inactivation occurs early in mammalian development to transcriptionally silence one of the pair of X chromosomes in females. The XIST RNA, a large untranslated RNA that is expressed solely from the inactive X chromosome, is implicated in the process of inactivation. As previous studies have shown that the XIST gene is methylated on the active X chromosome, we have treated a mouse/human somatic cell hybrid retaining an active human X chromosome with demethylating agents to determine whether expression of the human XIST gene could be induced. Stable expression of XIST was observed after several rounds of demethylation and stability of XIST expression correlated with the loss of methylation at the three sites analysed. We conclude that methylation is sufficient to inhibit expression of the XIST gene in somatic cell hybrids. No loss of expression was detected for eight other X-linked genes from the active X chromosome that was expressing XIST , suggesting that additional developmental or species-specific factors are required for the inactivation process.

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Selected References

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  1. Ariel M., Robinson E., McCarrey J. R., Cedar H. Gamete-specific methylation correlates with imprinting of the murine Xist gene. Nat Genet. 1995 Mar;9(3):312–315. doi: 10.1038/ng0395-312. [DOI] [PubMed] [Google Scholar]
  2. Ballabio A., Willard H. F. Mammalian X-chromosome inactivation and the XIST gene. Curr Opin Genet Dev. 1992 Jun;2(3):439–447. doi: 10.1016/s0959-437x(05)80155-8. [DOI] [PubMed] [Google Scholar]
  3. Beard C., Li E., Jaenisch R. Loss of methylation activates Xist in somatic but not in embryonic cells. Genes Dev. 1995 Oct 1;9(19):2325–2334. doi: 10.1101/gad.9.19.2325. [DOI] [PubMed] [Google Scholar]
  4. Brockdorff N., Ashworth A., Kay G. F., McCabe V. M., Norris D. P., Cooper P. J., Swift S., Rastan S. The product of the mouse Xist gene is a 15 kb inactive X-specific transcript containing no conserved ORF and located in the nucleus. Cell. 1992 Oct 30;71(3):515–526. doi: 10.1016/0092-8674(92)90519-i. [DOI] [PubMed] [Google Scholar]
  5. Brown C. J., Ballabio A., Rupert J. L., Lafreniere R. G., Grompe M., Tonlorenzi R., Willard H. F. A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome. Nature. 1991 Jan 3;349(6304):38–44. doi: 10.1038/349038a0. [DOI] [PubMed] [Google Scholar]
  6. Brown C. J., Carrel L., Willard H. F. Expression of genes from the human active and inactive X chromosomes. Am J Hum Genet. 1997 Jun;60(6):1333–1343. doi: 10.1086/515488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brown C. J., Flenniken A. M., Williams B. R., Willard H. F. X chromosome inactivation of the human TIMP gene. Nucleic Acids Res. 1990 Jul 25;18(14):4191–4195. doi: 10.1093/nar/18.14.4191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brown C. J., Hendrich B. D., Rupert J. L., Lafrenière R. G., Xing Y., Lawrence J., Willard H. F. The human XIST gene: analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus. Cell. 1992 Oct 30;71(3):527–542. doi: 10.1016/0092-8674(92)90520-m. [DOI] [PubMed] [Google Scholar]
  9. Brown C. J., Lafreniere R. G., Powers V. E., Sebastio G., Ballabio A., Pettigrew A. L., Ledbetter D. H., Levy E., Craig I. W., Willard H. F. Localization of the X inactivation centre on the human X chromosome in Xq13. Nature. 1991 Jan 3;349(6304):82–84. doi: 10.1038/349082a0. [DOI] [PubMed] [Google Scholar]
  10. Brown C. J., Robinson W. P. XIST expression and X-chromosome inactivation in human preimplantation embryos. Am J Hum Genet. 1997 Jul;61(1):5–8. doi: 10.1086/513914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Brown C. J., Willard H. F. The human X-inactivation centre is not required for maintenance of X-chromosome inactivation. Nature. 1994 Mar 10;368(6467):154–156. doi: 10.1038/368154a0. [DOI] [PubMed] [Google Scholar]
  12. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  13. Clemson C. M., McNeil J. A., Willard H. F., Lawrence J. B. XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure. J Cell Biol. 1996 Feb;132(3):259–275. doi: 10.1083/jcb.132.3.259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Daniels R., Zuccotti M., Kinis T., Serhal P., Monk M. XIST expression in human oocytes and preimplantation embryos. Am J Hum Genet. 1997 Jul;61(1):33–39. doi: 10.1086/513892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Duncan A. M., Macdonald A., Brown C. J., Wolff D., Willard H. F., Sutton B. Characterization of a small supernumerary ring X chromosome by fluorescence in situ hybridization. Am J Med Genet. 1993 Dec 1;47(8):1153–1156. doi: 10.1002/ajmg.1320470804. [DOI] [PubMed] [Google Scholar]
  16. Fisher E. M., Beer-Romero P., Brown L. G., Ridley A., McNeil J. A., Lawrence J. B., Willard H. F., Bieber F. R., Page D. C. Homologous ribosomal protein genes on the human X and Y chromosomes: escape from X inactivation and possible implications for Turner syndrome. Cell. 1990 Dec 21;63(6):1205–1218. doi: 10.1016/0092-8674(90)90416-c. [DOI] [PubMed] [Google Scholar]
  17. Gartler S. M., Goldman M. A. Reactivation of inactive X-linked genes. Dev Genet. 1994;15(6):504–514. doi: 10.1002/dvg.1020150609. [DOI] [PubMed] [Google Scholar]
  18. Goodfellow P., Pym B., Mohandas T., Shapiro L. J. The cell surface antigen locus, MIC2X, escapes X-inactivation. Am J Hum Genet. 1984 Jul;36(4):777–782. [PMC free article] [PubMed] [Google Scholar]
  19. Grant S. G., Chapman V. M. Mechanisms of X-chromosome regulation. Annu Rev Genet. 1988;22:199–233. doi: 10.1146/annurev.ge.22.120188.001215. [DOI] [PubMed] [Google Scholar]
  20. Haaf T., Werner P., Schmid M. 5-Azadeoxycytidine distinguishes between active and inactive X chromosome condensation. Cytogenet Cell Genet. 1993;63(3):160–168. doi: 10.1159/000133526. [DOI] [PubMed] [Google Scholar]
  21. Hansen R. S., Canfield T. K., Gartler S. M. Reverse replication timing for the XIST gene in human fibroblasts. Hum Mol Genet. 1995 May;4(5):813–820. doi: 10.1093/hmg/4.5.813. [DOI] [PubMed] [Google Scholar]
  22. Hendrich B. D., Brown C. J., Willard H. F. Evolutionary conservation of possible functional domains of the human and murine XIST genes. Hum Mol Genet. 1993 Jun;2(6):663–672. doi: 10.1093/hmg/2.6.663. [DOI] [PubMed] [Google Scholar]
  23. Hendrich B. D., Plenge R. M., Willard H. F. Identification and characterization of the human XIST gene promoter: implications for models of X chromosome inactivation. Nucleic Acids Res. 1997 Jul 1;25(13):2661–2671. doi: 10.1093/nar/25.13.2661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Herzing L. B., Romer J. T., Horn J. M., Ashworth A. Xist has properties of the X-chromosome inactivation centre. Nature. 1997 Mar 20;386(6622):272–275. doi: 10.1038/386272a0. [DOI] [PubMed] [Google Scholar]
  25. Holmquist G. P. Role of replication time in the control of tissue-specific gene expression. Am J Hum Genet. 1987 Feb;40(2):151–173. [PMC free article] [PubMed] [Google Scholar]
  26. Jones P. A., Taylor S. M., Mohandas T., Shapiro L. J. Cell cycle-specific reactivation of an inactive X-chromosome locus by 5-azadeoxycytidine. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1215–1219. doi: 10.1073/pnas.79.4.1215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kay G. F., Penny G. D., Patel D., Ashworth A., Brockdorff N., Rastan S. Expression of Xist during mouse development suggests a role in the initiation of X chromosome inactivation. Cell. 1993 Jan 29;72(2):171–182. doi: 10.1016/0092-8674(93)90658-d. [DOI] [PubMed] [Google Scholar]
  28. LYON M. F. Gene action in the X-chromosome of the mouse (Mus musculus L.). Nature. 1961 Apr 22;190:372–373. doi: 10.1038/190372a0. [DOI] [PubMed] [Google Scholar]
  29. Lee J. T., Strauss W. M., Dausman J. A., Jaenisch R. A 450 kb transgene displays properties of the mammalian X-inactivation center. Cell. 1996 Jul 12;86(1):83–94. doi: 10.1016/s0092-8674(00)80079-3. [DOI] [PubMed] [Google Scholar]
  30. Leppig K. A., Brown C. J., Bressler S. L., Gustashaw K., Pagon R. A., Willard H. F., Disteche C. M. Mapping of the distal boundary of the X-inactivation center in a rearranged X chromosome from a female expressing XIST. Hum Mol Genet. 1993 Jul;2(7):883–887. doi: 10.1093/hmg/2.7.883. [DOI] [PubMed] [Google Scholar]
  31. Lin C. S., Park T., Chen Z. P., Leavitt J. Human plastin genes. Comparative gene structure, chromosome location, and differential expression in normal and neoplastic cells. J Biol Chem. 1993 Feb 5;268(4):2781–2792. [PubMed] [Google Scholar]
  32. Luoh S. W., Jegalian K., Lee A., Chen E. Y., Ridley A., Page D. C. CpG islands in human ZFX and ZFY and mouse Zfx genes: sequence similarities and methylation differences. Genomics. 1995 Sep 20;29(2):353–363. doi: 10.1006/geno.1995.9994. [DOI] [PubMed] [Google Scholar]
  33. Marahrens Y., Panning B., Dausman J., Strauss W., Jaenisch R. Xist-deficient mice are defective in dosage compensation but not spermatogenesis. Genes Dev. 1997 Jan 15;11(2):156–166. doi: 10.1101/gad.11.2.156. [DOI] [PubMed] [Google Scholar]
  34. Migeon B. R. X-chromosome inactivation: molecular mechanisms and genetic consequences. Trends Genet. 1994 Jul;10(7):230–235. doi: 10.1016/0168-9525(94)90169-4. [DOI] [PubMed] [Google Scholar]
  35. Miller S. A., Dykes D. D., Polesky H. F. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988 Feb 11;16(3):1215–1215. doi: 10.1093/nar/16.3.1215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mondello C., Goodfellow P. J., Goodfellow P. N. Analysis of methylation of a human X located gene which escapes X inactivation. Nucleic Acids Res. 1988 Jul 25;16(14B):6813–6824. doi: 10.1093/nar/16.14.6813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Norris D. P., Patel D., Kay G. F., Penny G. D., Brockdorff N., Sheardown S. A., Rastan S. Evidence that random and imprinted Xist expression is controlled by preemptive methylation. Cell. 1994 Apr 8;77(1):41–51. doi: 10.1016/0092-8674(94)90233-x. [DOI] [PubMed] [Google Scholar]
  38. Panning B., Jaenisch R. DNA hypomethylation can activate Xist expression and silence X-linked genes. Genes Dev. 1996 Aug 15;10(16):1991–2002. doi: 10.1101/gad.10.16.1991. [DOI] [PubMed] [Google Scholar]
  39. Penny G. D., Kay G. F., Sheardown S. A., Rastan S., Brockdorff N. Requirement for Xist in X chromosome inactivation. Nature. 1996 Jan 11;379(6561):131–137. doi: 10.1038/379131a0. [DOI] [PubMed] [Google Scholar]
  40. Rack K. A., Chelly J., Gibbons R. J., Rider S., Benjamin D., Lafreniére R. G., Oscier D., Hendriks R. W., Craig I. W., Willard H. F. Absence of the XIST gene from late-replicating isodicentric X chromosomes in leukaemia. Hum Mol Genet. 1994 Jul;3(7):1053–1059. doi: 10.1093/hmg/3.7.1053. [DOI] [PubMed] [Google Scholar]
  41. Singer-Sam J., Keith D. H., Tani K., Simmer R. L., Shively L., Lindsay S., Yoshida A., Riggs A. D. Sequence of the promoter region of the gene for human X-linked 3-phosphoglycerate kinase. Gene. 1984 Dec;32(3):409–417. doi: 10.1016/0378-1119(84)90016-7. [DOI] [PubMed] [Google Scholar]
  42. Singer-Sam J., Riggs A. D. X chromosome inactivation and DNA methylation. EXS. 1993;64:358–384. doi: 10.1007/978-3-0348-9118-9_16. [DOI] [PubMed] [Google Scholar]
  43. Slim R., Levilliers J., Lüdecke H. J., Claussen U., Nguyen V. C., Gough N. M., Horsthemke B., Petit C. A human pseudoautosomal gene encodes the ANT3 ADP/ATP translocase and escapes X-inactivation. Genomics. 1993 Apr;16(1):26–33. doi: 10.1006/geno.1993.1135. [DOI] [PubMed] [Google Scholar]
  44. Tai H. H., Gordon J., McBurney M. W. Xist is expressed in female embryonal carcinoma cells with two active X chromosomes. Somat Cell Mol Genet. 1994 May;20(3):171–182. doi: 10.1007/BF02254758. [DOI] [PubMed] [Google Scholar]
  45. Willard H. F., Salz H. K. Remodelling chromatin with RNA. Nature. 1997 Mar 20;386(6622):228–229. doi: 10.1038/386228a0. [DOI] [PubMed] [Google Scholar]
  46. Xiong Z., Tsark W., Singer-Sam J., Riggs A. D. Differential replication timing of X-linked genes measured by a novel method using single-nucleotide primer extension. Nucleic Acids Res. 1998 Jan 15;26(2):684–686. doi: 10.1093/nar/26.2.684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yoshida I., Nishita Y., Mohandas T. K., Takagi N. Reactivation of an inactive human X chromosome introduced into mouse embryonal carcinoma cells by microcell fusion with persistent expression of XIST. Exp Cell Res. 1997 Feb 1;230(2):208–219. doi: 10.1006/excr.1996.3393. [DOI] [PubMed] [Google Scholar]
  48. Zuccotti M., Monk M. Methylation of the mouse Xist gene in sperm and eggs correlates with imprinted Xist expression and paternal X-inactivation. Nat Genet. 1995 Mar;9(3):316–320. doi: 10.1038/ng0395-316. [DOI] [PubMed] [Google Scholar]

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