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. 1999 Aug 1;27(15):3009–3017. doi: 10.1093/nar/27.15.3009

An origin of bidirectional DNA replication is located within a CpG island at the 3" end of the chicken lysozyme gene.

L Phi-van 1, W H Strätling 1
PMCID: PMC148524  PMID: 10454594

Abstract

We previously identified a broad initiation zone of DNA replication at the chicken lysozyme gene locus. However, the existence of a highly preferred origin of bidirectional replication (OBR), often found in initiation zones, remained elusive. In order to re-examine this issue we used a competitive PCR assay to determine the abundance of closely spaced genomic segments in a 1 kb size fraction of nascent DNA. A sharp peak of nascent strand abundance occurred at the 3" end of the gene, where initiation events were 17 times more frequent than upstream of the gene. This primary initiation site, active in lysozyme expressing myelomonocytic HD11 cells and non-expressing hepatic DU249 cells, was found to reside within an unusually located CpG island. While most CpG islands are found at the 5" end of genes, the lysozyme gene island extends from the 3" end of the second intron and includes approximately 1.2 kb of 3" flanking DNA. As diagnosed by methylation-sensitive restriction enzymes, the island is largely non-methylated in HD11 cells, DU249 cells and inactive chicken erythrocytes. Furthermore, a DNase I hypersensitive site (HS) that is composed of two subsites separated by approximately 100 bp, was localised very close to the segment with the highest initiation activity. Our results suggest that the non-methylated CpG island and the HS provide an accessible chromatin structure for the lysozyme gene origin of replication.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abdurashidova G., Riva S., Biamonti G., Giacca M., Falaschi A. Cell cycle modulation of protein-DNA interactions at a human replication origin. EMBO J. 1998 May 15;17(10):2961–2969. doi: 10.1093/emboj/17.10.2961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Antequera F., Bird A. Number of CpG islands and genes in human and mouse. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11995–11999. doi: 10.1073/pnas.90.24.11995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Antequera F., Boyes J., Bird A. High levels of de novo methylation and altered chromatin structure at CpG islands in cell lines. Cell. 1990 Aug 10;62(3):503–514. doi: 10.1016/0092-8674(90)90015-7. [DOI] [PubMed] [Google Scholar]
  4. Barlow D. P. Gametic imprinting in mammals. Science. 1995 Dec 8;270(5242):1610–1613. doi: 10.1126/science.270.5242.1610. [DOI] [PubMed] [Google Scholar]
  5. Beug H., von Kirchbach A., Döderlein G., Conscience J. F., Graf T. Chicken hematopoietic cells transformed by seven strains of defective avian leukemia viruses display three distinct phenotypes of differentiation. Cell. 1979 Oct;18(2):375–390. doi: 10.1016/0092-8674(79)90057-6. [DOI] [PubMed] [Google Scholar]
  6. Bird A. P. CpG-rich islands and the function of DNA methylation. Nature. 1986 May 15;321(6067):209–213. doi: 10.1038/321209a0. [DOI] [PubMed] [Google Scholar]
  7. Brown J. A., Holmes S. G., Smith M. M. The chromatin structure of Saccharomyces cerevisiae autonomously replicating sequences changes during the cell division cycle. Mol Cell Biol. 1991 Oct;11(10):5301–5311. doi: 10.1128/mcb.11.10.5301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Burhans W. C., Vassilev L. T., Caddle M. S., Heintz N. H., DePamphilis M. L. Identification of an origin of bidirectional DNA replication in mammalian chromosomes. Cell. 1990 Sep 7;62(5):955–965. doi: 10.1016/0092-8674(90)90270-o. [DOI] [PubMed] [Google Scholar]
  9. Cheng L., Kelly T. J. Transcriptional activator nuclear factor I stimulates the replication of SV40 minichromosomes in vivo and in vitro. Cell. 1989 Nov 3;59(3):541–551. doi: 10.1016/0092-8674(89)90037-8. [DOI] [PubMed] [Google Scholar]
  10. Chuang L. S., Ian H. I., Koh T. W., Ng H. H., Xu G., Li B. F. Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1. Science. 1997 Sep 26;277(5334):1996–2000. doi: 10.1126/science.277.5334.1996. [DOI] [PubMed] [Google Scholar]
  11. Clark S. J., Harrison J., Paul C. L., Frommer M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 1994 Aug 11;22(15):2990–2997. doi: 10.1093/nar/22.15.2990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Delgado S., Gómez M., Bird A., Antequera F. Initiation of DNA replication at CpG islands in mammalian chromosomes. EMBO J. 1998 Apr 15;17(8):2426–2435. doi: 10.1093/emboj/17.8.2426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Diffley J. F., Cocker J. H., Dowell S. J., Rowley A. Two steps in the assembly of complexes at yeast replication origins in vivo. Cell. 1994 Jul 29;78(2):303–316. doi: 10.1016/0092-8674(94)90299-2. [DOI] [PubMed] [Google Scholar]
  14. Diviacco S., Norio P., Zentilin L., Menzo S., Clementi M., Biamonti G., Riva S., Falaschi A., Giacca M. A novel procedure for quantitative polymerase chain reaction by coamplification of competitive templates. Gene. 1992 Dec 15;122(2):313–320. doi: 10.1016/0378-1119(92)90220-j. [DOI] [PubMed] [Google Scholar]
  15. Fritton H. P., Igo-Kemenes T., Nowock J., Strech-Jurk U., Theisen M., Sippel A. E. DNase I-hypersensitive sites in the chromatin structure of the lysozyme gene in steroid hormone target and non-target cells. Biol Chem Hoppe Seyler. 1987 Feb;368(2):111–119. doi: 10.1515/bchm3.1987.368.1.111. [DOI] [PubMed] [Google Scholar]
  16. Gardiner-Garden M., Frommer M. CpG islands in vertebrate genomes. J Mol Biol. 1987 Jul 20;196(2):261–282. doi: 10.1016/0022-2836(87)90689-9. [DOI] [PubMed] [Google Scholar]
  17. Giacca M., Zentilin L., Norio P., Diviacco S., Dimitrova D., Contreas G., Biamonti G., Perini G., Weighardt F., Riva S. Fine mapping of a replication origin of human DNA. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7119–7123. doi: 10.1073/pnas.91.15.7119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Goethe R., Phi-van L. Evidence for an enhanced transcription-dependent de novo synthesis of C/EBPbeta in the LPS activation of the chicken lysozyme gene. J Leukoc Biol. 1997 Mar;61(3):367–374. doi: 10.1002/jlb.61.3.367. [DOI] [PubMed] [Google Scholar]
  19. Goldman M. A., Holmquist G. P., Gray M. C., Caston L. A., Nag A. Replication timing of genes and middle repetitive sequences. Science. 1984 May 18;224(4650):686–692. doi: 10.1126/science.6719109. [DOI] [PubMed] [Google Scholar]
  20. Gögel E., Längst G., Grummt I., Kunkel E., Grummt F. Mapping of replication initiation sites in the mouse ribosomal gene cluster. Chromosoma. 1996 Apr;104(7):511–518. doi: 10.1007/BF00352115. [DOI] [PubMed] [Google Scholar]
  21. Heintz N. H., Hamlin J. L. An amplified chromosomal sequence that includes the gene for dihydrofolate reductase initiates replication within specific restriction fragments. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4083–4087. doi: 10.1073/pnas.79.13.4083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hendrich B., Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins. Mol Cell Biol. 1998 Nov;18(11):6538–6547. doi: 10.1128/mcb.18.11.6538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hewish D. R., Burgoyne L. A. Chromatin sub-structure. The digestion of chromatin DNA at regularly spaced sites by a nuclear deoxyribonuclease. Biochem Biophys Res Commun. 1973 May 15;52(2):504–510. doi: 10.1016/0006-291x(73)90740-7. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Jakobovits E. B., Bratosin S., Aloni Y. A nucleosome-free region in SV40 minichromosomes. Nature. 1980 May 22;285(5762):263–265. doi: 10.1038/285263a0. [DOI] [PubMed] [Google Scholar]
  26. Jones P. L., Veenstra G. J., Wade P. A., Vermaak D., Kass S. U., Landsberger N., Strouboulis J., Wolffe A. P. Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat Genet. 1998 Jun;19(2):187–191. doi: 10.1038/561. [DOI] [PubMed] [Google Scholar]
  27. Kelly R. E., DeRose M. L., Draper B. W., Wahl G. M. Identification of an origin of bidirectional DNA replication in the ubiquitously expressed mammalian CAD gene. Mol Cell Biol. 1995 Aug;15(8):4136–4148. doi: 10.1128/mcb.15.8.4136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kobayashi T., Rein T., DePamphilis M. L. Identification of primary initiation sites for DNA replication in the hamster dihydrofolate reductase gene initiation zone. Mol Cell Biol. 1998 Jun;18(6):3266–3277. doi: 10.1128/mcb.18.6.3266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Langlois A. J., Lapis K., Ishizaki R., Beard J. W., Bolognesi D. P. Isolation of a transplantable cell line induced by the MC29 avian leukosis virus. Cancer Res. 1974 Jun;34(6):1457–1464. [PubMed] [Google Scholar]
  30. Leonhardt H., Page A. W., Weier H. U., Bestor T. H. A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. Cell. 1992 Nov 27;71(5):865–873. doi: 10.1016/0092-8674(92)90561-p. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Macleod D., Ali R. R., Bird A. An alternative promoter in the mouse major histocompatibility complex class II I-Abeta gene: implications for the origin of CpG islands. Mol Cell Biol. 1998 Aug;18(8):4433–4443. doi: 10.1128/mcb.18.8.4433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Nan X., Ng H. H., Johnson C. A., Laherty C. D., Turner B. M., Eisenman R. N., Bird A. Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature. 1998 May 28;393(6683):386–389. doi: 10.1038/30764. [DOI] [PubMed] [Google Scholar]
  35. Pemov A., Bavykin S., Hamlin J. L. Attachment to the nuclear matrix mediates specific alterations in chromatin structure. Proc Natl Acad Sci U S A. 1998 Dec 8;95(25):14757–14762. doi: 10.1073/pnas.95.25.14757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Phi-van L., Sellke C., von Bodenhausen A., Strätling W. H. An initiation zone of chromosomal DNA replication at the chicken lysozyme gene locus. J Biol Chem. 1998 Jul 17;273(29):18300–18307. doi: 10.1074/jbc.273.29.18300. [DOI] [PubMed] [Google Scholar]
  37. Razin A., Cedar H. DNA methylation and genomic imprinting. Cell. 1994 May 20;77(4):473–476. doi: 10.1016/0092-8674(94)90208-9. [DOI] [PubMed] [Google Scholar]
  38. Riggs A. D., Pfeifer G. P. X-chromosome inactivation and cell memory. Trends Genet. 1992 May;8(5):169–174. doi: 10.1016/0168-9525(92)90219-t. [DOI] [PubMed] [Google Scholar]
  39. Saragosti S., Moyne G., Yaniv M. Absence of nucleosomes in a fraction of SV40 chromatin between the origin of replication and the region coding for the late leader RNA. Cell. 1980 May;20(1):65–73. doi: 10.1016/0092-8674(80)90235-4. [DOI] [PubMed] [Google Scholar]
  40. Simpson R. T. Nucleosome positioning can affect the function of a cis-acting DNA element in vivo. Nature. 1990 Jan 25;343(6256):387–389. doi: 10.1038/343387a0. [DOI] [PubMed] [Google Scholar]
  41. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  42. Strätling W. H., Dölle A., Sippel A. E. Chromatin structure of the chicken lysozyme gene domain as determined by chromatin fractionation and micrococcal nuclease digestion. Biochemistry. 1986 Jan 28;25(2):495–502. doi: 10.1021/bi00350a033. [DOI] [PubMed] [Google Scholar]
  43. Tazi J., Bird A. Alternative chromatin structure at CpG islands. Cell. 1990 Mar 23;60(6):909–920. doi: 10.1016/0092-8674(90)90339-g. [DOI] [PubMed] [Google Scholar]
  44. Toniolo D., D'Urso M., Martini G., Persico M., Tufano V., Battistuzzi G., Luzzatto L. Specific methylation pattern at the 3' end of the human housekeeping gene for glucose 6-phosphate dehydrogenase. EMBO J. 1984 Sep;3(9):1987–1995. doi: 10.1002/j.1460-2075.1984.tb02080.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Vassilev L., Johnson E. M. An initiation zone of chromosomal DNA replication located upstream of the c-myc gene in proliferating HeLa cells. Mol Cell Biol. 1990 Sep;10(9):4899–4904. doi: 10.1128/mcb.10.9.4899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Weitzel J. M., Buhrmester H., Strätling W. H. Chicken MAR-binding protein ARBP is homologous to rat methyl-CpG-binding protein MeCP2. Mol Cell Biol. 1997 Sep;17(9):5656–5666. doi: 10.1128/mcb.17.9.5656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wu C. The 5' ends of Drosophila heat shock genes in chromatin are hypersensitive to DNase I. Nature. 1980 Aug 28;286(5776):854–860. doi: 10.1038/286854a0. [DOI] [PubMed] [Google Scholar]
  48. Wutz A., Smrzka O. W., Schweifer N., Schellander K., Wagner E. F., Barlow D. P. Imprinted expression of the Igf2r gene depends on an intronic CpG island. Nature. 1997 Oct 16;389(6652):745–749. doi: 10.1038/39631. [DOI] [PubMed] [Google Scholar]
  49. Yoon Y., Sanchez J. A., Brun C., Huberman J. A. Mapping of replication initiation sites in human ribosomal DNA by nascent-strand abundance analysis. Mol Cell Biol. 1995 May;15(5):2482–2489. doi: 10.1128/mcb.15.5.2482. [DOI] [PMC free article] [PubMed] [Google Scholar]

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