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. 1997 Aug;17(8):4397–4405. doi: 10.1128/mcb.17.8.4397

A nucleosome positioned in the distal promoter region activates transcription of the human U6 gene.

W Stünkel 1, I Kober 1, K H Seifart 1
PMCID: PMC232294  PMID: 9234698

Abstract

To investigate the consequences of chromatin reconstitution for transcription of the human U6 gene, we assembled nucleosomes on both plasmids and linear DNA fragments containing the U6 gene. Initial experiments with DNA fragments revealed that U6 sequences located between the distal sequence element (DSE) and the proximal sequence element (PSE) lead to the positioning of a nucleosome partially encompassing these promoter elements. Furthermore, indirect end-labelling analyses of the reconstituted U6 wild-type plasmids showed strong micrococcal nuclease cuts near the DSE and PSE, indicating that a nucleosome is located between these elements. To investigate the influence that nucleosomes exert on U6 transcription, we used two different experimental approaches for chromatin reconstitution, both of which resulted in the observation that transcription of the U6 wild-type gene was enhanced after chromatin assembly. To ensure that the facilitated transcription of the nucleosomal templates is in fact due to a positioned nucleosome, we constructed mutants of the U6 gene in which the sequences between the DSE and PSE were progressively deleted. In contrast to what was observed with the wild-type genes, transcription of these deletion mutants was significantly inhibited when they were packaged into nucleosomes.

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Selected References

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

  1. Almer A., Hörz W. Nuclease hypersensitive regions with adjacent positioned nucleosomes mark the gene boundaries of the PHO5/PHO3 locus in yeast. EMBO J. 1986 Oct;5(10):2681–2687. doi: 10.1002/j.1460-2075.1986.tb04551.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bouvet P., Dimitrov S., Wolffe A. P. Specific regulation of Xenopus chromosomal 5S rRNA gene transcription in vivo by histone H1. Genes Dev. 1994 May 15;8(10):1147–1159. doi: 10.1101/gad.8.10.1147. [DOI] [PubMed] [Google Scholar]
  3. Brown S. A., Imbalzano A. N., Kingston R. E. Activator-dependent regulation of transcriptional pausing on nucleosomal templates. Genes Dev. 1996 Jun 15;10(12):1479–1490. doi: 10.1101/gad.10.12.1479. [DOI] [PubMed] [Google Scholar]
  4. Burnol A. F., Margottin F., Huet J., Almouzni G., Prioleau M. N., Méchali M., Sentenac A. TFIIIC relieves repression of U6 snRNA transcription by chromatin. Nature. 1993 Apr 1;362(6419):475–477. doi: 10.1038/362475a0. [DOI] [PubMed] [Google Scholar]
  5. Cairns B. R., Kim Y. J., Sayre M. H., Laurent B. C., Kornberg R. D. A multisubunit complex containing the SWI1/ADR6, SWI2/SNF2, SWI3, SNF5, and SNF6 gene products isolated from yeast. Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1950–1954. doi: 10.1073/pnas.91.5.1950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Camerini-Otero R. D., Sollner-Webb B., Felsenfeld G. The organization of histones and DNA in chromatin: evidence for an arginine-rich histone kernel. Cell. 1976 Jul;8(3):333–347. doi: 10.1016/0092-8674(76)90145-8. [DOI] [PubMed] [Google Scholar]
  7. Côté J., Quinn J., Workman J. L., Peterson C. L. Stimulation of GAL4 derivative binding to nucleosomal DNA by the yeast SWI/SNF complex. Science. 1994 Jul 1;265(5168):53–60. doi: 10.1126/science.8016655. [DOI] [PubMed] [Google Scholar]
  8. Drew H. R., Travers A. A. DNA bending and its relation to nucleosome positioning. J Mol Biol. 1985 Dec 20;186(4):773–790. doi: 10.1016/0022-2836(85)90396-1. [DOI] [PubMed] [Google Scholar]
  9. Godde J. S., Wolffe A. P. Nucleosome assembly on CTG triplet repeats. J Biol Chem. 1996 Jun 21;271(25):15222–15229. doi: 10.1074/jbc.271.25.15222. [DOI] [PubMed] [Google Scholar]
  10. Gottesfeld J. M. DNA sequence-directed nucleosome reconstitution on 5S RNA genes of Xenopus laevis. Mol Cell Biol. 1987 May;7(5):1612–1622. doi: 10.1128/mcb.7.5.1612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grunstein M. Histone function in transcription. Annu Rev Cell Biol. 1990;6:643–678. doi: 10.1146/annurev.cb.06.110190.003235. [DOI] [PubMed] [Google Scholar]
  12. Hayes J. J., Wolffe A. P. Histones H2A/H2B inhibit the interaction of transcription factor IIIA with the Xenopus borealis somatic 5S RNA gene in a nucleosome. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1229–1233. doi: 10.1073/pnas.89.4.1229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hayes J. J., Wolffe A. P. Preferential and asymmetric interaction of linker histones with 5S DNA in the nucleosome. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6415–6419. doi: 10.1073/pnas.90.14.6415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Henry R. W., Ma B., Sadowski C. L., Kobayashi R., Hernandez N. Cloning and characterization of SNAP50, a subunit of the snRNA-activating protein complex SNAPc. EMBO J. 1996 Dec 16;15(24):7129–7136. [PMC free article] [PubMed] [Google Scholar]
  15. Imbalzano A. N., Kwon H., Green M. R., Kingston R. E. Facilitated binding of TATA-binding protein to nucleosomal DNA. Nature. 1994 Aug 11;370(6489):481–485. doi: 10.1038/370481a0. [DOI] [PubMed] [Google Scholar]
  16. Jackson J. R., Benyajati C. DNA-histone interactions are sufficient to position a single nucleosome juxtaposing Drosophila Adh adult enhancer and distal promoter. Nucleic Acids Res. 1993 Feb 25;21(4):957–967. doi: 10.1093/nar/21.4.957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jahn D., Wingender E., Seifart K. H. Transcription complexes for various class III genes differ in parameters of formation and stability towards salt. J Mol Biol. 1987 Jan 20;193(2):303–313. doi: 10.1016/0022-2836(87)90221-x. [DOI] [PubMed] [Google Scholar]
  18. Knezetic J. A., Luse D. S. The presence of nucleosomes on a DNA template prevents initiation by RNA polymerase II in vitro. Cell. 1986 Apr 11;45(1):95–104. doi: 10.1016/0092-8674(86)90541-6. [DOI] [PubMed] [Google Scholar]
  19. Kunkel G. R. RNA polymerase III transcription of genes that lack internal control regions. Biochim Biophys Acta. 1991 Jan 17;1088(1):1–9. doi: 10.1016/0167-4781(91)90146-d. [DOI] [PubMed] [Google Scholar]
  20. Kwon H., Imbalzano A. N., Khavari P. A., Kingston R. E., Green M. R. Nucleosome disruption and enhancement of activator binding by a human SW1/SNF complex. Nature. 1994 Aug 11;370(6489):477–481. doi: 10.1038/370477a0. [DOI] [PubMed] [Google Scholar]
  21. Laskey R. A., Mills A. D., Morris N. R. Assembly of SV40 chromatin in a cell-free system from Xenopus eggs. Cell. 1977 Feb;10(2):237–243. doi: 10.1016/0092-8674(77)90217-3. [DOI] [PubMed] [Google Scholar]
  22. Linxweiler W., Hörz W. Reconstitution of mononucleosomes: characterization of distinct particles that differ in the position of the histone core. Nucleic Acids Res. 1984 Dec 21;12(24):9395–9413. doi: 10.1093/nar/12.24.9395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lorch Y., LaPointe J. W., Kornberg R. D. Nucleosomes inhibit the initiation of transcription but allow chain elongation with the displacement of histones. Cell. 1987 Apr 24;49(2):203–210. doi: 10.1016/0092-8674(87)90561-7. [DOI] [PubMed] [Google Scholar]
  24. Lu Q., Wallrath L. L., Elgin S. C. The role of a positioned nucleosome at the Drosophila melanogaster hsp26 promoter. EMBO J. 1995 Oct 2;14(19):4738–4746. doi: 10.1002/j.1460-2075.1995.tb00155.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mattaj I. W., Lienhard S., Jiricny J., De Robertis E. M. An enhancer-like sequence within the Xenopus U2 gene promoter facilitates the formation of stable transcription complexes. Nature. 1985 Jul 11;316(6024):163–167. doi: 10.1038/316163a0. [DOI] [PubMed] [Google Scholar]
  26. Murphy S., Yoon J. B., Gerster T., Roeder R. G. Oct-1 and Oct-2 potentiate functional interactions of a transcription factor with the proximal sequence element of small nuclear RNA genes. Mol Cell Biol. 1992 Jul;12(7):3247–3261. doi: 10.1128/mcb.12.7.3247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ossipow V., Tassan J. P., Nigg E. A., Schibler U. A mammalian RNA polymerase II holoenzyme containing all components required for promoter-specific transcription initiation. Cell. 1995 Oct 6;83(1):137–146. doi: 10.1016/0092-8674(95)90242-2. [DOI] [PubMed] [Google Scholar]
  28. Owen-Hughes T., Utley R. T., Côté J., Peterson C. L., Workman J. L. Persistent site-specific remodeling of a nucleosome array by transient action of the SWI/SNF complex. Science. 1996 Jul 26;273(5274):513–516. doi: 10.1126/science.273.5274.513. [DOI] [PubMed] [Google Scholar]
  29. Piña B., Barettino D., Truss M., Beato M. Structural features of a regulatory nucleosome. J Mol Biol. 1990 Dec 20;216(4):975–990. doi: 10.1016/S0022-2836(99)80015-1. [DOI] [PubMed] [Google Scholar]
  30. Piña B., Brüggemeier U., Beato M. Nucleosome positioning modulates accessibility of regulatory proteins to the mouse mammary tumor virus promoter. Cell. 1990 Mar 9;60(5):719–731. doi: 10.1016/0092-8674(90)90087-u. [DOI] [PubMed] [Google Scholar]
  31. Ptashne M. Gene regulation by proteins acting nearby and at a distance. Nature. 1986 Aug 21;322(6081):697–701. doi: 10.1038/322697a0. [DOI] [PubMed] [Google Scholar]
  32. Quivy J. P., Becker P. B. The architecture of the heat-inducible Drosophila hsp27 promoter in nuclei. J Mol Biol. 1996 Feb 23;256(2):249–263. doi: 10.1006/jmbi.1996.0083. [DOI] [PubMed] [Google Scholar]
  33. Rhodes D. Structural analysis of a triple complex between the histone octamer, a Xenopus gene for 5S RNA and transcription factor IIIA. EMBO J. 1985 Dec 16;4(13A):3473–3482. doi: 10.1002/j.1460-2075.1985.tb04106.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Richard-Foy H., Hager G. L. Sequence-specific positioning of nucleosomes over the steroid-inducible MMTV promoter. EMBO J. 1987 Aug;6(8):2321–2328. doi: 10.1002/j.1460-2075.1987.tb02507.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Roberts S., Colbert T., Hahn S. TFIIIC determines RNA polymerase III specificity at the TATA-containing yeast U6 promoter. Genes Dev. 1995 Apr 1;9(7):832–842. doi: 10.1101/gad.9.7.832. [DOI] [PubMed] [Google Scholar]
  36. Schild C., Claret F. X., Wahli W., Wolffe A. P. A nucleosome-dependent static loop potentiates estrogen-regulated transcription from the Xenopus vitellogenin B1 promoter in vitro. EMBO J. 1993 Feb;12(2):423–433. doi: 10.1002/j.1460-2075.1993.tb05674.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shimamura A., Tremethick D., Worcel A. Characterization of the repressed 5S DNA minichromosomes assembled in vitro with a high-speed supernatant of Xenopus laevis oocytes. Mol Cell Biol. 1988 Oct;8(10):4257–4269. doi: 10.1128/mcb.8.10.4257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shpigelman E. S., Trifonov E. N., Bolshoy A. CURVATURE: software for the analysis of curved DNA. Comput Appl Biosci. 1993 Aug;9(4):435–440. doi: 10.1093/bioinformatics/9.4.435. [DOI] [PubMed] [Google Scholar]
  39. Shrader T. E., Crothers D. M. Effects of DNA sequence and histone-histone interactions on nucleosome placement. J Mol Biol. 1990 Nov 5;216(1):69–84. doi: 10.1016/S0022-2836(05)80061-0. [DOI] [PubMed] [Google Scholar]
  40. Stein A. Reconstitution of chromatin from purified components. Methods Enzymol. 1989;170:585–603. doi: 10.1016/0076-6879(89)70066-5. [DOI] [PubMed] [Google Scholar]
  41. Stünkel W., Kober I., Kauer M., Taimor G., Seifart K. H. Human TFIIIA alone is sufficient to prevent nucleosomal repression of a homologous 5S gene. Nucleic Acids Res. 1995 Jan 11;23(1):109–116. doi: 10.1093/nar/23.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Tsukiyama T., Becker P. B., Wu C. ATP-dependent nucleosome disruption at a heat-shock promoter mediated by binding of GAGA transcription factor. Nature. 1994 Feb 10;367(6463):525–532. doi: 10.1038/367525a0. [DOI] [PubMed] [Google Scholar]
  44. Tsukiyama T., Daniel C., Tamkun J., Wu C. ISWI, a member of the SWI2/SNF2 ATPase family, encodes the 140 kDa subunit of the nucleosome remodeling factor. Cell. 1995 Dec 15;83(6):1021–1026. doi: 10.1016/0092-8674(95)90217-1. [DOI] [PubMed] [Google Scholar]
  45. Tsukiyama T., Wu C. Purification and properties of an ATP-dependent nucleosome remodeling factor. Cell. 1995 Dec 15;83(6):1011–1020. doi: 10.1016/0092-8674(95)90216-3. [DOI] [PubMed] [Google Scholar]
  46. Wall G., Varga-Weisz P. D., Sandaltzopoulos R., Becker P. B. Chromatin remodeling by GAGA factor and heat shock factor at the hypersensitive Drosophila hsp26 promoter in vitro. EMBO J. 1995 Apr 18;14(8):1727–1736. doi: 10.1002/j.1460-2075.1995.tb07162.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wang W., Côté J., Xue Y., Zhou S., Khavari P. A., Biggar S. R., Muchardt C., Kalpana G. V., Goff S. P., Yaniv M. Purification and biochemical heterogeneity of the mammalian SWI-SNF complex. EMBO J. 1996 Oct 1;15(19):5370–5382. [PMC free article] [PubMed] [Google Scholar]
  48. Wang Y. H., Griffith J. Expanded CTG triplet blocks from the myotonic dystrophy gene create the strongest known natural nucleosome positioning elements. Genomics. 1995 Jan 20;25(2):570–573. doi: 10.1016/0888-7543(95)80061-p. [DOI] [PubMed] [Google Scholar]
  49. Wilson C. J., Chao D. M., Imbalzano A. N., Schnitzler G. R., Kingston R. E., Young R. A. RNA polymerase II holoenzyme contains SWI/SNF regulators involved in chromatin remodeling. Cell. 1996 Jan 26;84(2):235–244. doi: 10.1016/s0092-8674(00)80978-2. [DOI] [PubMed] [Google Scholar]
  50. 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]
  51. Yoon J. B., Roeder R. G. Cloning of two proximal sequence element-binding transcription factor subunits (gamma and delta) that are required for transcription of small nuclear RNA genes by RNA polymerases II and III and interact with the TATA-binding protein. Mol Cell Biol. 1996 Jan;16(1):1–9. doi: 10.1128/mcb.16.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]

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