Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Feb 1;25(3):511–517. doi: 10.1093/nar/25.3.511

Structural analysis of mouse rDNA: coincidence between nuclease hypersensitive sites, DNA curvature and regulatory elements in the intergenic spacer.

G Längst 1, T Schätz 1, J Langowski 1, I Grummt 1
PMCID: PMC146485  PMID: 9016589

Abstract

We have analyzed the chromatin structure of mouse ribosomal RNA genes (rDNA) by partial digestion of genomic DNA with micrococcal nuclease (MNase), DNase I and identified hypersensitive sites by indirect end-labeling. This analysis has revealed defined regions of nuclease hypersensitivity in the intergenic spacer which in turn coincide with regulatory elements. Hypersensitive sites map to the transcription initiation site, the enhancer repeats, the spacer promoter and two sequence elements which coincide with amplification-promoting sequences. Analysis of the DNA curvature by computer modeling uncovered a striking correlation between sequence-directed structural features of regulatory regions and the position of nuclease hypersensitive sites. Moreover, we demonstrate that nucleosomes are specifically positioned upstream and downstream of the transcription start site. In vitro studies using chromatin assembled in the presence of Drosophila embryo extracts show that binding of the transcription termination factor TTF-I to the upstream terminator mediates this specific nucleosome positioning at the rDNA promoter in an ATP- dependent fashion.

Full Text

The Full Text of this article is available as a PDF (152.3 KB).

Selected References

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

  1. Becker P. B., Wu C. Cell-free system for assembly of transcriptionally repressed chromatin from Drosophila embryos. Mol Cell Biol. 1992 May;12(5):2241–2249. doi: 10.1128/mcb.12.5.2241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bolshoy A., McNamara P., Harrington R. E., Trifonov E. N. Curved DNA without A-A: experimental estimation of all 16 DNA wedge angles. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2312–2316. doi: 10.1073/pnas.88.6.2312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cacchione S., De Santis P., Foti D., Palleschi A., Savino M. Periodical polydeoxynucleotides and DNA curvature. Biochemistry. 1989 Oct 31;28(22):8706–8713. doi: 10.1021/bi00448a006. [DOI] [PubMed] [Google Scholar]
  4. Calladine C. R., Drew H. R., McCall M. J. The intrinsic curvature of DNA in solution. J Mol Biol. 1988 May 5;201(1):127–137. doi: 10.1016/0022-2836(88)90444-5. [DOI] [PubMed] [Google Scholar]
  5. Conconi A., Widmer R. M., Koller T., Sogo J. M. Two different chromatin structures coexist in ribosomal RNA genes throughout the cell cycle. Cell. 1989 Jun 2;57(5):753–761. doi: 10.1016/0092-8674(89)90790-3. [DOI] [PubMed] [Google Scholar]
  6. Culotta V., Sollner-Webb B. Sites of topoisomerase I action on X. laevis ribosomal chromatin: transcriptionally active rDNA has an approximately 200 bp repeating structure. Cell. 1988 Feb 26;52(4):585–597. doi: 10.1016/0092-8674(88)90471-0. [DOI] [PubMed] [Google Scholar]
  7. Dammann R., Lucchini R., Koller T., Sogo J. M. Transcription in the yeast rRNA gene locus: distribution of the active gene copies and chromatin structure of their flanking regulatory sequences. Mol Cell Biol. 1995 Oct;15(10):5294–5303. doi: 10.1128/mcb.15.10.5294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Elgin S. C. The formation and function of DNase I hypersensitive sites in the process of gene activation. J Biol Chem. 1988 Dec 25;263(36):19259–19262. [PubMed] [Google Scholar]
  9. Foe V. E., Wilkinson L. E., Laird C. D. Comparative organization of active transcription units in Oncopeltus fasciatus. Cell. 1976 Sep;9(1):131–146. doi: 10.1016/0092-8674(76)90059-3. [DOI] [PubMed] [Google Scholar]
  10. Garel A., Axel R. Selective digestion of transcriptionally active ovalbumin genes from oviduct nuclei. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3966–3970. doi: 10.1073/pnas.73.11.3966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gross D. S., Garrard W. T. Nuclease hypersensitive sites in chromatin. Annu Rev Biochem. 1988;57:159–197. doi: 10.1146/annurev.bi.57.070188.001111. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Hagerman P. J. Sequence-directed curvature of DNA. Annu Rev Biochem. 1990;59:755–781. doi: 10.1146/annurev.bi.59.070190.003543. [DOI] [PubMed] [Google Scholar]
  14. Labhart P., Koller T. Structure of the active nucleolar chromatin of Xenopus laevis Oocytes. Cell. 1982 Feb;28(2):279–292. doi: 10.1016/0092-8674(82)90346-4. [DOI] [PubMed] [Google Scholar]
  15. Lewin B. Chromatin and gene expression: constant questions, but changing answers. Cell. 1994 Nov 4;79(3):397–406. doi: 10.1016/0092-8674(94)90249-6. [DOI] [PubMed] [Google Scholar]
  16. Lucchini R., Sogo J. M. Replication of transcriptionally active chromatin. Nature. 1995 Mar 16;374(6519):276–280. doi: 10.1038/374276a0. [DOI] [PubMed] [Google Scholar]
  17. Macgregor H. C., del Pino E. M. Ribosomal gene amplification in multinucleate oocytes of the egg brooding hylid frog Flectonotus pygmaeus. Chromosoma. 1982;85(4):475–488. doi: 10.1007/BF00327344. [DOI] [PubMed] [Google Scholar]
  18. Marilley M., Pasero P., Got C. Molecular dissection of a specific nuclear domain: the chromatin region of the ribosomal gene cluster in Xenopus laevis. Exp Cell Res. 1992 Sep;202(1):87–97. doi: 10.1016/0014-4827(92)90407-y. [DOI] [PubMed] [Google Scholar]
  19. Mathis D. J., Gorovsky M. A. Subunit structure of rDNA-containing chromatin. Biochemistry. 1976 Feb 24;15(4):750–755. doi: 10.1021/bi00649a005. [DOI] [PubMed] [Google Scholar]
  20. McKee B. D., Handel M. A. Sex chromosomes, recombination, and chromatin conformation. Chromosoma. 1993 Jan;102(2):71–80. doi: 10.1007/BF00356023. [DOI] [PubMed] [Google Scholar]
  21. Moss T., Stefanovsky V. Y. Promotion and regulation of ribosomal transcription in eukaryotes by RNA polymerase I. Prog Nucleic Acid Res Mol Biol. 1995;50:25–66. doi: 10.1016/s0079-6603(08)60810-7. [DOI] [PubMed] [Google Scholar]
  22. Paalman M. H., Henderson S. L., Sollner-Webb B. Stimulation of the mouse rRNA gene promoter by a distal spacer promoter. Mol Cell Biol. 1995 Aug;15(8):4648–4656. doi: 10.1128/mcb.15.8.4648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Palen T. E., Cech T. R. Chromatin structure at the replication origins and transcription-initiation regions of the ribosomal RNA genes of Tetrahymena. Cell. 1984 Apr;36(4):933–942. doi: 10.1016/0092-8674(84)90043-6. [DOI] [PubMed] [Google Scholar]
  24. Petes T. D. Unequal meiotic recombination within tandem arrays of yeast ribosomal DNA genes. Cell. 1980 Mar;19(3):765–774. doi: 10.1016/s0092-8674(80)80052-3. [DOI] [PubMed] [Google Scholar]
  25. Pruitt S. C., Grainger R. M. A mosaicism in the higher order structure of Xenopus oocyte nucleolar chromatin prior to and during ribosomal gene transcription. Cell. 1981 Mar;23(3):711–720. doi: 10.1016/0092-8674(81)90434-7. [DOI] [PubMed] [Google Scholar]
  26. Pérez-Martín J., Timmis K. N., de Lorenzo V. Co-regulation by bent DNA. Functional substitutions of the integration host factor site at sigma 54-dependent promoter Pu of the upper-TOL operon by intrinsically curved sequences. J Biol Chem. 1994 Sep 9;269(36):22657–22662. [PubMed] [Google Scholar]
  27. Reeves R. Ribosomal genes of Xenopus laevis: evidence of nucleosomes in transcriptionally active chromatin. Science. 1976 Oct 29;194(4264):529–532. doi: 10.1126/science.973136. [DOI] [PubMed] [Google Scholar]
  28. Sandaltzopoulos R., Blank T., Becker P. B. Transcriptional repression by nucleosomes but not H1 in reconstituted preblastoderm Drosophila chromatin. EMBO J. 1994 Jan 15;13(2):373–379. doi: 10.1002/j.1460-2075.1994.tb06271.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Scheer U., Sommerville J., Bustin M. Injected histone antibodies interfere with transcription of lampbrush chromosome loops in oocytes of Pleurodeles. J Cell Sci. 1979 Dec;40:1–20. doi: 10.1242/jcs.40.1.1. [DOI] [PubMed] [Google Scholar]
  30. Schroth G. P., Siino J. S., Cooney C. A., Th'ng J. P., Ho P. S., Bradbury E. M. Intrinsically bent DNA flanks both sides of an RNA polymerase I transcription start site. Both regions display novel electrophoretic mobility. J Biol Chem. 1992 May 15;267(14):9958–9964. [PubMed] [Google Scholar]
  31. Smid A., Finsterer M., Grummt I. Limited proteolysis unmasks specific DNA-binding of the murine RNA polymerase I-specific transcription termination factor TTFI. J Mol Biol. 1992 Oct 5;227(3):635–647. doi: 10.1016/0022-2836(92)90213-4. [DOI] [PubMed] [Google Scholar]
  32. Spear B. B., Gall J. G. Independent control of ribosomal gene replication in polytene chromosomes of Drosophila melanogaster. Proc Natl Acad Sci U S A. 1973 May;70(5):1359–1363. doi: 10.1073/pnas.70.5.1359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Thomas G. H., Elgin S. C. Protein/DNA architecture of the DNase I hypersensitive region of the Drosophila hsp26 promoter. EMBO J. 1988 Jul;7(7):2191–2201. doi: 10.1002/j.1460-2075.1988.tb03058.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Udvardy A., Louis C., Han S., Schedl P. Ribosomal RNA genes of Drosophila melanogaster have a novel chromatin structure. J Mol Biol. 1984 May 15;175(2):113–130. doi: 10.1016/0022-2836(84)90470-4. [DOI] [PubMed] [Google Scholar]
  35. VanWye J. D., Bronson E. C., Anderson J. N. Species-specific patterns of DNA bending and sequence. Nucleic Acids Res. 1991 Oct 11;19(19):5253–5261. doi: 10.1093/nar/19.19.5253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Voelkel-Meiman K., Keil R. L., Roeder G. S. Recombination-stimulating sequences in yeast ribosomal DNA correspond to sequences regulating transcription by RNA polymerase I. Cell. 1987 Mar 27;48(6):1071–1079. doi: 10.1016/0092-8674(87)90714-8. [DOI] [PubMed] [Google Scholar]
  37. Wegner M., Helftenbein E., Müller F., Meinecke M., Müller S., Grummt F. Identification of an amplification promoting DNA sequence from the hypotrichous ciliate Stylonychia lemnae. Nucleic Acids Res. 1989 Nov 11;17(21):8783–8802. doi: 10.1093/nar/17.21.8783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Weintraub H., Groudine M. Chromosomal subunits in active genes have an altered conformation. Science. 1976 Sep 3;193(4256):848–856. doi: 10.1126/science.948749. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES