Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Feb;15(2):1123–1135. doi: 10.1128/mcb.15.2.1123

Dissecting a locus control region: facilitation of enhancer function by extended enhancer-flanking sequences.

B J Aronow 1, C A Ebert 1, M T Valerius 1, S S Potter 1, D A Wiginton 1, D P Witte 1, J J Hutton 1
PMCID: PMC232021  PMID: 7823928

Abstract

Using transgenic mice, we have defined novel gene regulatory elements, termed "facilitators." These elements bilaterally flank, by up to 1 kb, a 200-bp T-cell-specific enhancer domain in the human adenosine deaminase (ADA) gene. Facilitators were essential for gene copy-proportional and integration site-independent reporter expression in transgenic thymocytes, but they had no effect on the enhancer in transfected T cells. Both segments were required. Individual segments had no activity. A lack of facilitator function caused positional susceptibility and prevented DNase I-hypersensitive site formation at the enhancer. The segments were required to be at opposed ends of the enhancer, and they could not be grouped together. Reversing the orientation of a facilitator segment caused a partial loss of function, suggesting involvement of a stereospecific chromatin structure. trans-acting factor access to enhancer elements was modeled by exposing nuclei to a restriction endonuclease. The enhancer domain was accessible to the 4-cutter DpnII in a tissue- and cell-type-specific fashion. However, unlike DNase I hypersensitivity and gene expression, accessibility to the endonuclease could occur without the facilitator segments, suggesting that an accessible chromatin domain is an intermediate state in the activational pathway. These results suggest that facilitators (i) are distinct from yet positionally constrained to the enhancer, (ii) participate in a chromatin structure transition that is necessary for the DNase I hypersensitivity and the transcriptional activating function of the enhancer, and (iii) act after cell-type-specific accessibility to the enhancer sequences is established by factors that do not require the facilitators to be present.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

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

  1. Affara N., Fleming J., Goldfarb P. S., Black E., Thiele B., Harrison P. R. Analysis of chromatin changes associated with the expression of globin and non-globin genes in cell hybrids between erythroid and other cells. Nucleic Acids Res. 1985 Aug 12;13(15):5629–5644. doi: 10.1093/nar/13.15.5629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aronow B. J., Silbiger R. N., Dusing M. R., Stock J. L., Yager K. L., Potter S. S., Hutton J. J., Wiginton D. A. Functional analysis of the human adenosine deaminase gene thymic regulatory region and its ability to generate position-independent transgene expression. Mol Cell Biol. 1992 Sep;12(9):4170–4185. doi: 10.1128/mcb.12.9.4170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aronow B., Lattier D., Silbiger R., Dusing M., Hutton J., Jones G., Stock J., McNeish J., Potter S., Witte D. Evidence for a complex regulatory array in the first intron of the human adenosine deaminase gene. Genes Dev. 1989 Sep;3(9):1384–1400. doi: 10.1101/gad.3.9.1384. [DOI] [PubMed] [Google Scholar]
  4. Barton M. C., Madani N., Emerson B. M. The erythroid protein cGATA-1 functions with a stage-specific factor to activate transcription of chromatin-assembled beta-globin genes. Genes Dev. 1993 Sep;7(9):1796–1809. doi: 10.1101/gad.7.9.1796. [DOI] [PubMed] [Google Scholar]
  5. Blaese R. M., Culver K. W. Gene therapy for primary immunodeficiency disease. Immunodefic Rev. 1992;3(4):329–349. [PubMed] [Google Scholar]
  6. Blom van Assendelft G., Hanscombe O., Grosveld F., Greaves D. R. The beta-globin dominant control region activates homologous and heterologous promoters in a tissue-specific manner. Cell. 1989 Mar 24;56(6):969–977. doi: 10.1016/0092-8674(89)90630-2. [DOI] [PubMed] [Google Scholar]
  7. Bode J., Kohwi Y., Dickinson L., Joh T., Klehr D., Mielke C., Kohwi-Shigematsu T. Biological significance of unwinding capability of nuclear matrix-associating DNAs. Science. 1992 Jan 10;255(5041):195–197. doi: 10.1126/science.1553545. [DOI] [PubMed] [Google Scholar]
  8. Bonifer C., Hecht A., Saueressig H., Winter D. M., Sippel A. E. Dynamic chromatin: the regulatory domain organization of eukaryotic gene loci. J Cell Biochem. 1991 Oct;47(2):99–108. doi: 10.1002/jcb.240470203. [DOI] [PubMed] [Google Scholar]
  9. Carter K. C., Bowman D., Carrington W., Fogarty K., McNeil J. A., Fay F. S., Lawrence J. B. A three-dimensional view of precursor messenger RNA metabolism within the mammalian nucleus. Science. 1993 Feb 26;259(5099):1330–1335. doi: 10.1126/science.8446902. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Clark-Adams C. D., Norris D., Osley M. A., Fassler J. S., Winston F. Changes in histone gene dosage alter transcription in yeast. Genes Dev. 1988 Feb;2(2):150–159. doi: 10.1101/gad.2.2.150. [DOI] [PubMed] [Google Scholar]
  12. Cockerill P. N., Garrard W. T. Chromosomal loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. Cell. 1986 Jan 31;44(2):273–282. doi: 10.1016/0092-8674(86)90761-0. [DOI] [PubMed] [Google Scholar]
  13. Cockerill P. N., Garrard W. T. Chromosomal loop anchorage sites appear to be evolutionarily conserved. FEBS Lett. 1986 Aug 11;204(1):5–7. doi: 10.1016/0014-5793(86)81377-1. [DOI] [PubMed] [Google Scholar]
  14. Cockerill P. N., Yuen M. H., Garrard W. T. The enhancer of the immunoglobulin heavy chain locus is flanked by presumptive chromosomal loop anchorage elements. J Biol Chem. 1987 Apr 15;262(11):5394–5397. [PubMed] [Google Scholar]
  15. Crowder C. M., Merlie J. P. Stepwise activation of the mouse acetylcholine receptor delta- and gamma-subunit genes in clonal cell lines. Mol Cell Biol. 1988 Dec;8(12):5257–5267. doi: 10.1128/mcb.8.12.5257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Durrin L. K., Mann R. K., Kayne P. S., Grunstein M. Yeast histone H4 N-terminal sequence is required for promoter activation in vivo. Cell. 1991 Jun 14;65(6):1023–1031. doi: 10.1016/0092-8674(91)90554-c. [DOI] [PubMed] [Google Scholar]
  17. Eissenberg J. C., Elgin S. C. Boundary functions in the control of gene expression. Trends Genet. 1991 Oct;7(10):335–340. doi: 10.1016/0168-9525(91)90424-o. [DOI] [PubMed] [Google Scholar]
  18. Ford A. M., Bennett C. A., Healy L. E., Navarro E., Spooncer E., Greaves M. F. Immunoglobulin heavy-chain and CD3 delta-chain gene enhancers are DNase I-hypersensitive in hemopoietic progenitor cells. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3424–3428. doi: 10.1073/pnas.89.8.3424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Forrester W. C., Novak U., Gelinas R., Groudine M. Molecular analysis of the human beta-globin locus activation region. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5439–5443. doi: 10.1073/pnas.86.14.5439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Fraser P., Pruzina S., Antoniou M., Grosveld F. Each hypersensitive site of the human beta-globin locus control region confers a different developmental pattern of expression on the globin genes. Genes Dev. 1993 Jan;7(1):106–113. doi: 10.1101/gad.7.1.106. [DOI] [PubMed] [Google Scholar]
  22. Gasser S. M., Laemmli U. K. Cohabitation of scaffold binding regions with upstream/enhancer elements of three developmentally regulated genes of D. melanogaster. Cell. 1986 Aug 15;46(4):521–530. doi: 10.1016/0092-8674(86)90877-9. [DOI] [PubMed] [Google Scholar]
  23. Georgopoulos K., van den Elsen P., Bier E., Maxam A., Terhorst C. A T cell-specific enhancer is located in a DNase I-hypersensitive area at the 3' end of the CD3-delta gene. EMBO J. 1988 Aug;7(8):2401–2407. doi: 10.1002/j.1460-2075.1988.tb03085.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Giese K., Cox J., Grosschedl R. The HMG domain of lymphoid enhancer factor 1 bends DNA and facilitates assembly of functional nucleoprotein structures. Cell. 1992 Apr 3;69(1):185–195. doi: 10.1016/0092-8674(92)90129-z. [DOI] [PubMed] [Google Scholar]
  25. Greaves D. R., Wilson F. D., Lang G., Kioussis D. Human CD2 3'-flanking sequences confer high-level, T cell-specific, position-independent gene expression in transgenic mice. Cell. 1989 Mar 24;56(6):979–986. doi: 10.1016/0092-8674(89)90631-4. [DOI] [PubMed] [Google Scholar]
  26. Grosveld F., Antoniou M., van Assendelft G. B., de Boer E., Hurst J., Kollias G., MacFarlane F., Wrighton N. The regulation of expression of human beta-globin genes. Prog Clin Biol Res. 1987;251:133–144. [PubMed] [Google Scholar]
  27. 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]
  28. Groudine M., Linial M. Chromatin structure and gene expression in germ line and somatic cells. Adv Exp Med Biol. 1986;205:205–243. doi: 10.1007/978-1-4684-5209-9_10. [DOI] [PubMed] [Google Scholar]
  29. Han M., Kim U. J., Kayne P., Grunstein M. Depletion of histone H4 and nucleosomes activates the PHO5 gene in Saccharomyces cerevisiae. EMBO J. 1988 Jul;7(7):2221–2228. doi: 10.1002/j.1460-2075.1988.tb03061.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Hirschhorn J. N., Brown S. A., Clark C. D., Winston F. Evidence that SNF2/SWI2 and SNF5 activate transcription in yeast by altering chromatin structure. Genes Dev. 1992 Dec;6(12A):2288–2298. doi: 10.1101/gad.6.12a.2288. [DOI] [PubMed] [Google Scholar]
  31. Jenuwein T., Forrester W. C., Qiu R. G., Grosschedl R. The immunoglobulin mu enhancer core establishes local factor access in nuclear chromatin independent of transcriptional stimulation. Genes Dev. 1993 Oct;7(10):2016–2032. doi: 10.1101/gad.7.10.2016. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Kellum R., Schedl P. A group of scs elements function as domain boundaries in an enhancer-blocking assay. Mol Cell Biol. 1992 May;12(5):2424–2431. doi: 10.1128/mcb.12.5.2424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Knezetic J. A., Jacob G. A., Luse D. S. Assembly of RNA polymerase II preinitiation complexes before assembly of nucleosomes allows efficient initiation of transcription on nucleosomal templates. Mol Cell Biol. 1988 Aug;8(8):3114–3121. doi: 10.1128/mcb.8.8.3114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Laemmli U. K., Käs E., Poljak L., Adachi Y. Scaffold-associated regions: cis-acting determinants of chromatin structural loops and functional domains. Curr Opin Genet Dev. 1992 Apr;2(2):275–285. doi: 10.1016/s0959-437x(05)80285-0. [DOI] [PubMed] [Google Scholar]
  37. Lake R. A., Wotton D., Owen M. J. A 3' transcriptional enhancer regulates tissue-specific expression of the human CD2 gene. EMBO J. 1990 Oct;9(10):3129–3136. doi: 10.1002/j.1460-2075.1990.tb07510.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Lang G., Mamalaki C., Greenberg D., Yannoutsos N., Kioussis D. Deletion analysis of the human CD2 gene locus control region in transgenic mice. Nucleic Acids Res. 1991 Nov 11;19(21):5851–5856. doi: 10.1093/nar/19.21.5851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Li Q. L., Zhou B., Powers P., Enver T., Stamatoyannopoulos G. Beta-globin locus activation regions: conservation of organization, structure, and function. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8207–8211. doi: 10.1073/pnas.87.21.8207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Lohr D. Chromatin structure and regulation of the eukaryotic regulatory gene GAL80. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10628–10632. doi: 10.1073/pnas.90.22.10628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Lu Q., Wallrath L. L., Granok H., Elgin S. C. (CT)n (GA)n repeats and heat shock elements have distinct roles in chromatin structure and transcriptional activation of the Drosophila hsp26 gene. Mol Cell Biol. 1993 May;13(5):2802–2814. doi: 10.1128/mcb.13.5.2802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Ma X. J., Goodridge A. G. Nutritional regulation of nucleosomal structure at the chicken malic enzyme promoter in liver. Nucleic Acids Res. 1992 Oct 11;20(19):4997–5002. doi: 10.1093/nar/20.19.4997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. McKnight R. A., Shamay A., Sankaran L., Wall R. J., Hennighausen L. Matrix-attachment regions can impart position-independent regulation of a tissue-specific gene in transgenic mice. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6943–6947. doi: 10.1073/pnas.89.15.6943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. McPherson C. E., Shim E. Y., Friedman D. S., Zaret K. S. An active tissue-specific enhancer and bound transcription factors existing in a precisely positioned nucleosomal array. Cell. 1993 Oct 22;75(2):387–398. doi: 10.1016/0092-8674(93)80079-t. [DOI] [PubMed] [Google Scholar]
  46. Mirkovitch J., Gasser S. M., Laemmli U. K. Relation of chromosome structure and gene expression. Philos Trans R Soc Lond B Biol Sci. 1987 Dec 15;317(1187):563–574. doi: 10.1098/rstb.1987.0081. [DOI] [PubMed] [Google Scholar]
  47. Mirkovitch J., Gasser S. M., Laemmli U. K. Scaffold attachment of DNA loops in metaphase chromosomes. J Mol Biol. 1988 Mar 5;200(1):101–109. doi: 10.1016/0022-2836(88)90336-1. [DOI] [PubMed] [Google Scholar]
  48. Palmiter R. D., Brinster R. L. Germ-line transformation of mice. Annu Rev Genet. 1986;20:465–499. doi: 10.1146/annurev.ge.20.120186.002341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Peterson D. O. Alterations in chromatin structure associated with glucocorticoid-induced expression of endogenous mouse mammary tumor virus genes. Mol Cell Biol. 1985 May;5(5):1104–1110. doi: 10.1128/mcb.5.5.1104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Phi-Van L., von Kries J. P., Ostertag W., Strätling W. H. The chicken lysozyme 5' matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes. Mol Cell Biol. 1990 May;10(5):2302–2307. doi: 10.1128/mcb.10.5.2302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Pinkert C. A., Ornitz D. M., Brinster R. L., Palmiter R. D. An albumin enhancer located 10 kb upstream functions along with its promoter to direct efficient, liver-specific expression in transgenic mice. Genes Dev. 1987 May;1(3):268–276. doi: 10.1101/gad.1.3.268. [DOI] [PubMed] [Google Scholar]
  52. Roth S. Y., Shimizu M., Johnson L., Grunstein M., Simpson R. T. Stable nucleosome positioning and complete repression by the yeast alpha 2 repressor are disrupted by amino-terminal mutations in histone H4. Genes Dev. 1992 Mar;6(3):411–425. doi: 10.1101/gad.6.3.411. [DOI] [PubMed] [Google Scholar]
  53. Ryan T. M., Behringer R. R., Martin N. C., Townes T. M., Palmiter R. D., Brinster R. L. A single erythroid-specific DNase I super-hypersensitive site activates high levels of human beta-globin gene expression in transgenic mice. Genes Dev. 1989 Mar;3(3):314–323. doi: 10.1101/gad.3.3.314. [DOI] [PubMed] [Google Scholar]
  54. 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]
  55. Simpson R. T. Nucleosome positioning: occurrence, mechanisms, and functional consequences. Prog Nucleic Acid Res Mol Biol. 1991;40:143–184. doi: 10.1016/s0079-6603(08)60841-7. [DOI] [PubMed] [Google Scholar]
  56. 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]
  57. 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]
  58. Vettese-Dadey M., Walter P., Chen H., Juan L. J., Workman J. L. Role of the histone amino termini in facilitated binding of a transcription factor, GAL4-AH, to nucleosome cores. Mol Cell Biol. 1994 Feb;14(2):970–981. doi: 10.1128/mcb.14.2.970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Witte D. P., Wiginton D. A., Hutton J. J., Aronow B. J. Coordinate developmental regulation of purine catabolic enzyme expression in gastrointestinal and postimplantation reproductive tracts. J Cell Biol. 1991 Oct;115(1):179–190. doi: 10.1083/jcb.115.1.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Workman J. L., Buchman A. R. Multiple functions of nucleosomes and regulatory factors in transcription. Trends Biochem Sci. 1993 Mar;18(3):90–95. doi: 10.1016/0968-0004(93)90160-o. [DOI] [PubMed] [Google Scholar]
  61. Workman J. L., Kingston R. E. Nucleosome core displacement in vitro via a metastable transcription factor-nucleosome complex. Science. 1992 Dec 11;258(5089):1780–1784. doi: 10.1126/science.1465613. [DOI] [PubMed] [Google Scholar]
  62. Wustmann G., Szidonya J., Taubert H., Reuter G. The genetics of position-effect variegation modifying loci in Drosophila melanogaster. Mol Gen Genet. 1989 Jun;217(2-3):520–527. doi: 10.1007/BF02464926. [DOI] [PubMed] [Google Scholar]
  63. Xing Y., Johnson C. V., Dobner P. R., Lawrence J. B. Higher level organization of individual gene transcription and RNA splicing. Science. 1993 Feb 26;259(5099):1326–1330. doi: 10.1126/science.8446901. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES