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. 1996 Feb 1;15(3):562–568.

A dominant chromatin-opening activity in 5' hypersensitive site 3 of the human beta-globin locus control region.

J Ellis 1, K C Tan-Un 1, A Harper 1, D Michalovich 1, N Yannoutsos 1, S Philipsen 1, F Grosveld 1
PMCID: PMC449974  PMID: 8599939

Abstract

Single-copy human beta-globin transgenes are very susceptible to suppression by position effects of surrounding closed chromatin. However, these position effects are overcome by a 20 kbp DNA fragment containing the locus control region (LCR). Here we show that the 6.5 kbp microlocus LCR cassette reproducibly directs full expression from independent single-copy beta-globin transgenes. By testing individual DNase I-hypersensitive sites (HS) present in the microlocus cassette, we demonstrate that the 1.5 kbp 5'HS2 enhancer fragment does not direct beta-globin expression from single-copy transgenes. In contrast, the 1.9 kbp 5'HS3 fragment directs beta-globin expression in five independent single-copy transgenic mouse lines. Moreover, the 5'HS3 core element and beta-globin proximal promoter sequences are DNase I hypersensitive in fetal liver nuclei of these expressing transgenic lines. Taken together, these results demonstrate that LCR activity is the culmination of at least two separable functions including: (i) a novel activity located in 5'HS3 that dominantly opens and remodels chromatin structure; and (ii) a recessive enhancer activity residing in 5'HS2. We postulate that the different elements of the LCR form a 'holocomplex' that interacts with the individual globin genes.

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Selected References

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  1. Antoniou M., deBoer E., Habets G., Grosveld F. The human beta-globin gene contains multiple regulatory regions: identification of one promoter and two downstream enhancers. EMBO J. 1988 Feb;7(2):377–384. doi: 10.1002/j.1460-2075.1988.tb02824.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berry M., Grosveld F., Dillon N. A single point mutation is the cause of the Greek form of hereditary persistence of fetal haemoglobin. Nature. 1992 Aug 6;358(6386):499–502. doi: 10.1038/358499a0. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Curtin P. T., Liu D. P., Liu W., Chang J. C., Kan Y. W. Human beta-globin gene expression in transgenic mice is enhanced by a distant DNase I hypersensitive site. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7082–7086. doi: 10.1073/pnas.86.18.7082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dillon N., Grosveld F. Human gamma-globin genes silenced independently of other genes in the beta-globin locus. Nature. 1991 Mar 21;350(6315):252–254. doi: 10.1038/350252a0. [DOI] [PubMed] [Google Scholar]
  6. Dillon N., Grosveld F. Transcriptional regulation of multigene loci: multilevel control. Trends Genet. 1993 Apr;9(4):134–137. doi: 10.1016/0168-9525(93)90208-y. [DOI] [PubMed] [Google Scholar]
  7. Driscoll M. C., Dobkin C. S., Alter B. P. Gamma delta beta-thalassemia due to a de novo mutation deleting the 5' beta-globin gene activation-region hypersensitive sites. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7470–7474. doi: 10.1073/pnas.86.19.7470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Engel J. D. Developmental regulation of human beta-globin gene transcription: a switch of loyalties? Trends Genet. 1993 Sep;9(9):304–309. doi: 10.1016/0168-9525(93)90248-g. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Forrester W. C., Thompson C., Elder J. T., Groudine M. A developmentally stable chromatin structure in the human beta-globin gene cluster. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1359–1363. doi: 10.1073/pnas.83.5.1359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Gaensler K. M., Kitamura M., Kan Y. W. Germ-line transmission and developmental regulation of a 150-kb yeast artificial chromosome containing the human beta-globin locus in transgenic mice. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11381–11385. doi: 10.1073/pnas.90.23.11381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Groudine M., Kohwi-Shigematsu T., Gelinas R., Stamatoyannopoulos G., Papayannopoulou T. Human fetal to adult hemoglobin switching: changes in chromatin structure of the beta-globin gene locus. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7551–7555. doi: 10.1073/pnas.80.24.7551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gumucio D. L., Heilstedt-Williamson H., Gray T. A., Tarlé S. A., Shelton D. A., Tagle D. A., Slightom J. L., Goodman M., Collins F. S. Phylogenetic footprinting reveals a nuclear protein which binds to silencer sequences in the human gamma and epsilon globin genes. Mol Cell Biol. 1992 Nov;12(11):4919–4929. doi: 10.1128/mcb.12.11.4919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Hebbes T. R., Clayton A. L., Thorne A. W., Crane-Robinson C. Core histone hyperacetylation co-maps with generalized DNase I sensitivity in the chicken beta-globin chromosomal domain. EMBO J. 1994 Apr 15;13(8):1823–1830. doi: 10.1002/j.1460-2075.1994.tb06451.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. 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]
  24. Kollias G., Wrighton N., Hurst J., Grosveld F. Regulated expression of human A gamma-, beta-, and hybrid gamma beta-globin genes in transgenic mice: manipulation of the developmental expression patterns. Cell. 1986 Jul 4;46(1):89–94. doi: 10.1016/0092-8674(86)90862-7. [DOI] [PubMed] [Google Scholar]
  25. Lee D. Y., Hayes J. J., Pruss D., Wolffe A. P. A positive role for histone acetylation in transcription factor access to nucleosomal DNA. Cell. 1993 Jan 15;72(1):73–84. doi: 10.1016/0092-8674(93)90051-q. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Mastrangelo I. A., Courey A. J., Wall J. S., Jackson S. P., Hough P. V. DNA looping and Sp1 multimer links: a mechanism for transcriptional synergism and enhancement. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5670–5674. doi: 10.1073/pnas.88.13.5670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Moi P., Kan Y. W. Synergistic enhancement of globin gene expression by activator protein-1-like proteins. Proc Natl Acad Sci U S A. 1990 Nov;87(22):9000–9004. doi: 10.1073/pnas.87.22.9000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ney P. A., Sorrentino B. P., Lowrey C. H., Nienhuis A. W. Inducibility of the HS II enhancer depends on binding of an erythroid specific nuclear protein. Nucleic Acids Res. 1990 Oct 25;18(20):6011–6017. doi: 10.1093/nar/18.20.6011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Peterson K. R., Clegg C. H., Huxley C., Josephson B. M., Haugen H. S., Furukawa T., Stamatoyannopoulos G. Transgenic mice containing a 248-kb yeast artificial chromosome carrying the human beta-globin locus display proper developmental control of human globin genes. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7593–7597. doi: 10.1073/pnas.90.16.7593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Philipsen S., Pruzina S., Grosveld F. The minimal requirements for activity in transgenic mice of hypersensitive site 3 of the beta globin locus control region. EMBO J. 1993 Mar;12(3):1077–1085. doi: 10.1002/j.1460-2075.1993.tb05749.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Philipsen S., Talbot D., Fraser P., Grosveld F. The beta-globin dominant control region: hypersensitive site 2. EMBO J. 1990 Jul;9(7):2159–2167. doi: 10.1002/j.1460-2075.1990.tb07385.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pruzina S., Hanscombe O., Whyatt D., Grosveld F., Philipsen S. Hypersensitive site 4 of the human beta globin locus control region. Nucleic Acids Res. 1991 Apr 11;19(7):1413–1419. doi: 10.1093/nar/19.7.1413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Reitman M., Lee E., Westphal H., Felsenfeld G. An enhancer/locus control region is not sufficient to open chromatin. Mol Cell Biol. 1993 Jul;13(7):3990–3998. doi: 10.1128/mcb.13.7.3990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rivier D. H., Pillus L. Silencing speaks up. Cell. 1994 Mar 25;76(6):963–966. doi: 10.1016/0092-8674(94)90373-5. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Spanopoulou E., Giguere V., Grosveld F. Transcriptional unit of the murine Thy-1 gene: different distribution of transcription initiation sites in brain. Mol Cell Biol. 1988 Sep;8(9):3847–3856. doi: 10.1128/mcb.8.9.3847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Strouboulis J., Dillon N., Grosveld F. Developmental regulation of a complete 70-kb human beta-globin locus in transgenic mice. Genes Dev. 1992 Oct;6(10):1857–1864. doi: 10.1101/gad.6.10.1857. [DOI] [PubMed] [Google Scholar]
  41. Su W., Jackson S., Tjian R., Echols H. DNA looping between sites for transcriptional activation: self-association of DNA-bound Sp1. Genes Dev. 1991 May;5(5):820–826. doi: 10.1101/gad.5.5.820. [DOI] [PubMed] [Google Scholar]
  42. Talbot D., Collis P., Antoniou M., Vidal M., Grosveld F., Greaves D. R. A dominant control region from the human beta-globin locus conferring integration site-independent gene expression. Nature. 1989 Mar 23;338(6213):352–355. doi: 10.1038/338352a0. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. 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]
  46. 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]
  47. Wallrath L. L., Lu Q., Granok H., Elgin S. C. Architectural variations of inducible eukaryotic promoters: preset and remodeling chromatin structures. Bioessays. 1994 Mar;16(3):165–170. doi: 10.1002/bies.950160306. [DOI] [PubMed] [Google Scholar]

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