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. 1993 Jul;13(7):3990–3998. doi: 10.1128/mcb.13.7.3990

An enhancer/locus control region is not sufficient to open chromatin.

M Reitman 1, E Lee 1, H Westphal 1, G Felsenfeld 1
PMCID: PMC359948  PMID: 8321206

Abstract

To study the way in which an enhancer/locus control region (LCR) activates chromatin, we examined transgenic mice carrying various combinations of the chicken beta A-globin gene coding region, promoter, and 3' enhancer/LCR. We compared lines carrying only the coding region and enhancer R (E) and only the coding region and promoter (P) with those containing all three elements (PE). We have shown previously that all PE mice transcribe the transgene in a copy number-dependent manner while the P mice do not express their transgene. In the current study, we examined chromatin activation by monitoring formation of erythroid-specific hypersensitive sites at the promoter and enhancer. We found that all of the PE lines but none of the P lines show hypersensitivity. In contrast, only three of six E lines are hypersensitive (two strongly and one weakly), demonstrating position dependence of this transgene. The two E lines with strong hypersensitive sites were found also to have RNA complementary to the transgene, presumably starting from an adjacent adventitious mouse promoter. In all of these lines, we found a correlation between erythroid-specific hypersensitivity and erythroid-specific general DNase I sensitivity, an indicator of regional chromatin activation. The results support a mutual interaction model for the mechanism of chromatin opening by LCRs in which the enhancer/LCR and promoter must cooperate in order to generate open chromatin. The data are not consistent with a dominant enhancer model in which the enhancer/LCR can open chromatin autonomously.

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Selected References

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

  1. Allen N. D., Cran D. G., Barton S. C., Hettle S., Reik W., Surani M. A. Transgenes as probes for active chromosomal domains in mouse development. Nature. 1988 Jun 30;333(6176):852–855. doi: 10.1038/333852a0. [DOI] [PubMed] [Google Scholar]
  2. Aparicio O. M., Billington B. L., Gottschling D. E. Modifiers of position effect are shared between telomeric and silent mating-type loci in S. cerevisiae. Cell. 1991 Sep 20;66(6):1279–1287. doi: 10.1016/0092-8674(91)90049-5. [DOI] [PubMed] [Google Scholar]
  3. Archer T. K., Lefebvre P., Wolford R. G., Hager G. L. Transcription factor loading on the MMTV promoter: a bimodal mechanism for promoter activation. Science. 1992 Mar 20;255(5051):1573–1576. doi: 10.1126/science.1347958. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Behringer R. R., Ryan T. M., Palmiter R. D., Brinster R. L., Townes T. M. Human gamma- to beta-globin gene switching in transgenic mice. Genes Dev. 1990 Mar;4(3):380–389. doi: 10.1101/gad.4.3.380. [DOI] [PubMed] [Google Scholar]
  6. Bonnerot C., Grimber G., Briand P., Nicolas J. F. Patterns of expression of position-dependent integrated transgenes in mouse embryo. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6331–6335. doi: 10.1073/pnas.87.16.6331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Caterina J. J., Ryan T. M., Pawlik K. M., Palmiter R. D., Brinster R. L., Behringer R. R., Townes T. M. Human beta-globin locus control region: analysis of the 5' DNase I hypersensitive site HS 2 in transgenic mice. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1626–1630. doi: 10.1073/pnas.88.5.1626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cheng T., Polmar S. K., Kazazian H. H., Jr Isolation and characterization of modified globin messenger ribonucleic acid from erythropoietic mouse spleen. J Biol Chem. 1974 Mar 25;249(6):1781–1786. [PubMed] [Google Scholar]
  9. Choi O. R., Engel J. D. Developmental regulation of beta-globin gene switching. Cell. 1988 Oct 7;55(1):17–26. doi: 10.1016/0092-8674(88)90005-0. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Ellis J., Talbot D., Dillon N., Grosveld F. Synthetic human beta-globin 5'HS2 constructs function as locus control regions only in multicopy transgene concatamers. EMBO J. 1993 Jan;12(1):127–134. doi: 10.1002/j.1460-2075.1993.tb05638.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Enver T., Raich N., Ebens A. J., Papayannopoulou T., Costantini F., Stamatoyannopoulos G. Developmental regulation of human fetal-to-adult globin gene switching in transgenic mice. Nature. 1990 Mar 22;344(6264):309–313. doi: 10.1038/344309a0. [DOI] [PubMed] [Google Scholar]
  14. Evans T., Felsenfeld G., Reitman M. Control of globin gene transcription. Annu Rev Cell Biol. 1990;6:95–124. doi: 10.1146/annurev.cb.06.110190.000523. [DOI] [PubMed] [Google Scholar]
  15. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  16. Felsenfeld G. Chromatin as an essential part of the transcriptional mechanism. Nature. 1992 Jan 16;355(6357):219–224. doi: 10.1038/355219a0. [DOI] [PubMed] [Google Scholar]
  17. Foley K. P., Engel J. D. Individual stage selector element mutations lead to reciprocal changes in beta- vs. epsilon-globin gene transcription: genetic confirmation of promoter competition during globin gene switching. Genes Dev. 1992 May;6(5):730–744. doi: 10.1101/gad.6.5.730. [DOI] [PubMed] [Google Scholar]
  18. Forrester W. C., Epner E., Driscoll M. C., Enver T., Brice M., Papayannopoulou T., Groudine M. A deletion of the human beta-globin locus activation region causes a major alteration in chromatin structure and replication across the entire beta-globin locus. Genes Dev. 1990 Oct;4(10):1637–1649. doi: 10.1101/gad.4.10.1637. [DOI] [PubMed] [Google Scholar]
  19. Forrester W. C., Takegawa S., Papayannopoulou T., Stamatoyannopoulos G., Groudine M. Evidence for a locus activation region: the formation of developmentally stable hypersensitive sites in globin-expressing hybrids. Nucleic Acids Res. 1987 Dec 23;15(24):10159–10177. doi: 10.1093/nar/15.24.10159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fort P., Marty L., Piechaczyk M., el Sabrouty S., Dani C., Jeanteur P., Blanchard J. M. Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family. Nucleic Acids Res. 1985 Mar 11;13(5):1431–1442. doi: 10.1093/nar/13.5.1431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fraser P., Hurst J., Collis P., Grosveld F. DNaseI hypersensitive sites 1, 2 and 3 of the human beta-globin dominant control region direct position-independent expression. Nucleic Acids Res. 1990 Jun 25;18(12):3503–3508. doi: 10.1093/nar/18.12.3503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gottschling D. E., Aparicio O. M., Billington B. L., Zakian V. A. Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription. Cell. 1990 Nov 16;63(4):751–762. doi: 10.1016/0092-8674(90)90141-z. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Grosveld F., van Assendelft G. B., Greaves D. R., Kollias G. Position-independent, high-level expression of the human beta-globin gene in transgenic mice. Cell. 1987 Dec 24;51(6):975–985. doi: 10.1016/0092-8674(87)90584-8. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Hanscombe O., Whyatt D., Fraser P., Yannoutsos N., Greaves D., Dillon N., Grosveld F. Importance of globin gene order for correct developmental expression. Genes Dev. 1991 Aug;5(8):1387–1394. doi: 10.1101/gad.5.8.1387. [DOI] [PubMed] [Google Scholar]
  27. Hayes J. J., Wolffe A. P. The interaction of transcription factors with nucleosomal DNA. Bioessays. 1992 Sep;14(9):597–603. doi: 10.1002/bies.950140905. [DOI] [PubMed] [Google Scholar]
  28. Jackson P. D., Evans T., Nickol J. M., Felsenfeld G. Developmental modulation of protein binding to beta-globin gene regulatory sites within chicken erythrocyte nuclei. Genes Dev. 1989 Dec;3(12A):1860–1873. doi: 10.1101/gad.3.12a.1860. [DOI] [PubMed] [Google Scholar]
  29. Jaenisch R. Transgenic animals. Science. 1988 Jun 10;240(4858):1468–1474. doi: 10.1126/science.3287623. [DOI] [PubMed] [Google Scholar]
  30. Jiménez G., Griffiths S. D., Ford A. M., Greaves M. F., Enver T. Activation of the beta-globin locus control region precedes commitment to the erythroid lineage. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10618–10622. doi: 10.1073/pnas.89.22.10618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kim C. G., Epner E. M., Forrester W. C., Groudine M. Inactivation of the human beta-globin gene by targeted insertion into the beta-globin locus control region. Genes Dev. 1992 Jun;6(6):928–938. doi: 10.1101/gad.6.6.928. [DOI] [PubMed] [Google Scholar]
  32. King C. R., Piatigorsky J. Alternative RNA splicing of the murine alpha A-crystallin gene: protein-coding information within an intron. Cell. 1983 Mar;32(3):707–712. doi: 10.1016/0092-8674(83)90056-9. [DOI] [PubMed] [Google Scholar]
  33. Kioussis D., Vanin E., deLange T., Flavell R. A., Grosveld F. G. Beta-globin gene inactivation by DNA translocation in gamma beta-thalassaemia. Nature. 1983 Dec 15;306(5944):662–666. doi: 10.1038/306662a0. [DOI] [PubMed] [Google Scholar]
  34. Lawson G. M., Knoll B. J., March C. J., Woo S. L., Tsai M. J., O'Malley B. W. Definition of 5' and 3' structural boundaries of the chromatin domain containing the ovalbumin multigene family. J Biol Chem. 1982 Feb 10;257(3):1501–1507. [PubMed] [Google Scholar]
  35. Lee M. S., Garrard W. T. Uncoupling gene activity from chromatin structure: promoter mutations can inactivate transcription of the yeast HSP82 gene without eliminating nucleosome-free regions. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9166–9170. doi: 10.1073/pnas.89.19.9166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lowrey C. H., Bodine D. M., Nienhuis A. W. Mechanism of DNase I hypersensitive site formation within the human globin locus control region. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):1143–1147. doi: 10.1073/pnas.89.3.1143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Orkin S. H. Globin gene regulation and switching: circa 1990. Cell. 1990 Nov 16;63(4):665–672. doi: 10.1016/0092-8674(90)90133-y. [DOI] [PubMed] [Google Scholar]
  38. Radice G., Costantini F. Tissue-specific DNase I hypersensitive sites in a foreign globin gene in transgenic mice. Nucleic Acids Res. 1986 Dec 22;14(24):9765–9780. doi: 10.1093/nar/14.24.9765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Reitman M., Felsenfeld G. Developmental regulation of topoisomerase II sites and DNase I-hypersensitive sites in the chicken beta-globin locus. Mol Cell Biol. 1990 Jun;10(6):2774–2786. doi: 10.1128/mcb.10.6.2774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Reitman M., Lee E., Westphal H., Felsenfeld G. Site-independent expression of the chicken beta A-globin gene in transgenic mice. Nature. 1990 Dec 20;348(6303):749–752. doi: 10.1038/348749a0. [DOI] [PubMed] [Google Scholar]
  41. Reuter G., Spierer P. Position effect variegation and chromatin proteins. Bioessays. 1992 Sep;14(9):605–612. doi: 10.1002/bies.950140907. [DOI] [PubMed] [Google Scholar]
  42. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  43. Schmid A., Fascher K. D., Hörz W. Nucleosome disruption at the yeast PHO5 promoter upon PHO5 induction occurs in the absence of DNA replication. Cell. 1992 Nov 27;71(5):853–864. doi: 10.1016/0092-8674(92)90560-y. [DOI] [PubMed] [Google Scholar]
  44. Smith R. D., Yu J., Annunziato A., Seale R. L. beta-Globin gene family in murine erythroleukemia cells resides within two chromatin domains differing in higher order structure. Biochemistry. 1984 Jun 19;23(13):2970–2976. doi: 10.1021/bi00308a019. [DOI] [PubMed] [Google Scholar]
  45. Talbot D., Grosveld F. The 5'HS2 of the globin locus control region enhances transcription through the interaction of a multimeric complex binding at two functionally distinct NF-E2 binding sites. EMBO J. 1991 Jun;10(6):1391–1398. doi: 10.1002/j.1460-2075.1991.tb07659.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Talbot D., Philipsen S., Fraser P., Grosveld F. Detailed analysis of the site 3 region of the human beta-globin dominant control region. EMBO J. 1990 Jul;9(7):2169–2177. doi: 10.1002/j.1460-2075.1990.tb07386.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Townes T. M., Behringer R. R. Human globin locus activation region (LAR): role in temporal control. Trends Genet. 1990 Jul;6(7):219–223. doi: 10.1016/0168-9525(90)90182-6. [DOI] [PubMed] [Google Scholar]
  48. Tuan D. Y., Solomon W. B., London I. M., Lee D. P. An erythroid-specific, developmental-stage-independent enhancer far upstream of the human "beta-like globin" genes. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2554–2558. doi: 10.1073/pnas.86.8.2554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tuan D., Solomon W., Li Q., London I. M. The "beta-like-globin" gene domain in human erythroid cells. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6384–6388. doi: 10.1073/pnas.82.19.6384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Weintraub H., Beug H., Groudine M., Graf T. Temperature-sensitive changes in the structure of globin chromatin in lines of red cell precursors transformed by ts-AEV. Cell. 1982 Apr;28(4):931–940. doi: 10.1016/0092-8674(82)90072-1. [DOI] [PubMed] [Google Scholar]
  51. 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]

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