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. 1998 Jun 15;26(12):2849–2858. doi: 10.1093/nar/26.12.2849

Stochastic, stage-specific mechanisms account for the variegation of a human globin transgene.

T A Graubert 1, B A Hug 1, R Wesselschmidt 1, C L Hsieh 1, T M Ryan 1, T M Townes 1, T J Ley 1
PMCID: PMC147660  PMID: 9611227

Abstract

The random insertion of transgenes into the genomic DNA of mice usually leads to widely variable levels of expression in individual founder lines. To study the mechanisms that cause variegation, we designed a transgene that we expected to variegate, which consisted of a beta-globin locus control region 5' HS-2 linked in tandem to a tagged human beta-globin gene (into which a Lac-Z cassette had been inserted). All tested founder lines exhibited red blood cell-specific expression, but levels of expression varied >1000-fold from the lowest to the highest expressing line. Most of the variation in levels of expression appeared to reflect differences in the percentage of cells in the peripheral blood that expressed the transgene, which ranged from 0.3% in the lowest expressing line to 88% in the highest; the level of transgene expression per cell varied no more than 10-fold from the lowest to the highest expressing line. These differences in expression levels could not be explained by the location of transgene integration, by an effect of beta-galactosidase on red blood cell survival, by the half life of the beta-galactosidase enzyme or by the age of the animals. The progeny of all early erythroid progenitors (BFU-E colony-forming cells) exhibited the same propensity to variegate in methylcellulose-based cultures, suggesting that the decision to variegate occurs after the BFU-E stage of erythroid differentiation. Collectively, these data suggest that variegation in levels of transgene expression are due to local, integration site-dependent phenomena that alter the probability that a transgene will be expressed in an appropriate cell; however, these local effects have a minimal impact on the transgene's activity in the cells that initiate transcription.

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Selected References

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

  1. Behringer R. R., Hammer R. E., Brinster R. L., Palmiter R. D., Townes T. M. Two 3' sequences direct adult erythroid-specific expression of human beta-globin genes in transgenic mice. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7056–7060. doi: 10.1073/pnas.84.20.7056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boyes J., Felsenfeld G. Tissue-specific factors additively increase the probability of the all-or-none formation of a hypersensitive site. EMBO J. 1996 May 15;15(10):2496–2507. [PMC free article] [PubMed] [Google Scholar]
  3. Caterina J. J., Ciavatta D. J., Donze D., Behringer R. R., Townes T. M. Multiple elements in human beta-globin locus control region 5' HS 2 are involved in enhancer activity and position-independent, transgene expression. Nucleic Acids Res. 1994 Mar 25;22(6):1006–1011. doi: 10.1093/nar/22.6.1006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Chada K., Magram J., Raphael K., Radice G., Lacy E., Costantini F. Specific expression of a foreign beta-globin gene in erythroid cells of transgenic mice. 1985 Mar 28-Apr 3Nature. 314(6009):377–380. doi: 10.1038/314377a0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Cynshi O., Shimonaka Y., Higuchi M., Imai N., Suzuki H., Togashi M., Okamoto M. T., Hirashima K. Effects of recombinant human erythropoietin on haemolytic anaemia in mice. Br J Haematol. 1990 Nov;76(3):414–419. doi: 10.1111/j.1365-2141.1990.tb06377.x. [DOI] [PubMed] [Google Scholar]
  8. Dobie K. W., Lee M., Fantes J. A., Graham E., Clark A. J., Springbett A., Lathe R., McClenaghan M. Variegated transgene expression in mouse mammary gland is determined by the transgene integration locus. Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6659–6664. doi: 10.1073/pnas.93.13.6659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Festenstein R., Tolaini M., Corbella P., Mamalaki C., Parrington J., Fox M., Miliou A., Jones M., Kioussis D. Locus control region function and heterochromatin-induced position effect variegation. Science. 1996 Feb 23;271(5252):1123–1125. doi: 10.1126/science.271.5252.1123. [DOI] [PubMed] [Google Scholar]
  12. Fiering S. N., Roederer M., Nolan G. P., Micklem D. R., Parks D. R., Herzenberg L. A. Improved FACS-Gal: flow cytometric analysis and sorting of viable eukaryotic cells expressing reporter gene constructs. Cytometry. 1991;12(4):291–301. doi: 10.1002/cyto.990120402. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Garrick D., Sutherland H., Robertson G., Whitelaw E. Variegated expression of a globin transgene correlates with chromatin accessibility but not methylation status. Nucleic Acids Res. 1996 Dec 15;24(24):4902–4909. doi: 10.1093/nar/24.24.4902. [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. Guy L. G., Kothary R., Wall L. Position effects in mice carrying a lacZ transgene in cis with the beta-globin LCR can be explained by a graded model. Nucleic Acids Res. 1997 Nov 1;25(21):4400–4407. doi: 10.1093/nar/25.21.4400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kollias G., Hurst J., deBoer E., Grosveld F. The human beta-globin gene contains a downstream developmental specific enhancer. Nucleic Acids Res. 1987 Jul 24;15(14):5739–5747. doi: 10.1093/nar/15.14.5739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lichter P., Cremer T., Borden J., Manuelidis L., Ward D. C. Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum Genet. 1988 Nov;80(3):224–234. doi: 10.1007/BF01790090. [DOI] [PubMed] [Google Scholar]
  20. Liu D., Chang J. C., Moi P., Liu W., Kan Y. W., Curtin P. T. Dissection of the enhancer activity of beta-globin 5' DNase I-hypersensitive site 2 in transgenic mice. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3899–3903. doi: 10.1073/pnas.89.9.3899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Magis W., Fiering S., Groudine M., Martin D. I. An upstream activator of transcription coordinately increases the level and epigenetic stability of gene expression. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13914–13918. doi: 10.1073/pnas.93.24.13914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Magram J., Chada K., Costantini F. Developmental regulation of a cloned adult beta-globin gene in transgenic mice. Nature. 1985 May 23;315(6017):338–340. doi: 10.1038/315338a0. [DOI] [PubMed] [Google Scholar]
  23. Magram J., Niederreither K., Costantini F. Beta-globin enhancers target expression of a heterologous gene to erythroid tissues of transgenic mice. Mol Cell Biol. 1989 Oct;9(10):4581–4584. doi: 10.1128/mcb.9.10.4581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McDevitt M. A., Fujiwara Y., Shivdasani R. A., Orkin S. H. An upstream, DNase I hypersensitive region of the hematopoietic-expressed transcription factor GATA-1 gene confers developmental specificity in transgenic mice. Proc Natl Acad Sci U S A. 1997 Jul 22;94(15):7976–7981. doi: 10.1073/pnas.94.15.7976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Milot E., Strouboulis J., Trimborn T., Wijgerde M., de Boer E., Langeveld A., Tan-Un K., Vergeer W., Yannoutsos N., Grosveld F. Heterochromatin effects on the frequency and duration of LCR-mediated gene transcription. Cell. 1996 Oct 4;87(1):105–114. doi: 10.1016/s0092-8674(00)81327-6. [DOI] [PubMed] [Google Scholar]
  26. Moon A. M., Ley T. J. Conservation of the primary structure, organization, and function of the human and mouse beta-globin locus-activating regions. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7693–7697. doi: 10.1073/pnas.87.19.7693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Moreau P., Hen R., Wasylyk B., Everett R., Gaub M. P., Chambon P. The SV40 72 base repair repeat has a striking effect on gene expression both in SV40 and other chimeric recombinants. Nucleic Acids Res. 1981 Nov 25;9(22):6047–6068. doi: 10.1093/nar/9.22.6047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nolan G. P., Fiering S., Nicolas J. F., Herzenberg L. A. Fluorescence-activated cell analysis and sorting of viable mammalian cells based on beta-D-galactosidase activity after transduction of Escherichia coli lacZ. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2603–2607. doi: 10.1073/pnas.85.8.2603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pinkel D., Straume T., Gray J. W. Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci U S A. 1986 May;83(9):2934–2938. doi: 10.1073/pnas.83.9.2934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Robertson G., Garrick D., Wilson M., Martin D. I., Whitelaw E. Age-dependent silencing of globin transgenes in the mouse. Nucleic Acids Res. 1996 Apr 15;24(8):1465–1471. doi: 10.1093/nar/24.8.1465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Robertson G., Garrick D., Wu W., Kearns M., Martin D., Whitelaw E. Position-dependent variegation of globin transgene expression in mice. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5371–5375. doi: 10.1073/pnas.92.12.5371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Schmitz F. J., Werner E. Optimization of flow-cytometric discrimination between reticulocytes and erythrocytes. Cytometry. 1986 Sep;7(5):439–444. doi: 10.1002/cyto.990070508. [DOI] [PubMed] [Google Scholar]
  34. Stamatoyannopoulos G., Kurnit D. M., Papayannopoulou T. Stochastic expression of fetal hemoglobin in adult erythroid cells. Proc Natl Acad Sci U S A. 1981 Nov;78(11):7005–7009. doi: 10.1073/pnas.78.11.7005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sutherland H. G., Martin D. I., Whitelaw E. A globin enhancer acts by increasing the proportion of erythrocytes expressing a linked transgene. Mol Cell Biol. 1997 Mar;17(3):1607–1614. doi: 10.1128/mcb.17.3.1607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Townes T. M., Lingrel J. B., Chen H. Y., Brinster R. L., Palmiter R. D. Erythroid-specific expression of human beta-globin genes in transgenic mice. EMBO J. 1985 Jul;4(7):1715–1723. doi: 10.1002/j.1460-2075.1985.tb03841.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tuan D., London I. M. Mapping of DNase I-hypersensitive sites in the upstream DNA of human embryonic epsilon-globin gene in K562 leukemia cells. Proc Natl Acad Sci U S A. 1984 May;81(9):2718–2722. doi: 10.1073/pnas.81.9.2718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Walters M. C., Fiering S., Eidemiller J., Magis W., Groudine M., Martin D. I. Enhancers increase the probability but not the level of gene expression. Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):7125–7129. doi: 10.1073/pnas.92.15.7125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Walters M. C., Magis W., Fiering S., Eidemiller J., Scalzo D., Groudine M., Martin D. I. Transcriptional enhancers act in cis to suppress position-effect variegation. Genes Dev. 1996 Jan 15;10(2):185–195. doi: 10.1101/gad.10.2.185. [DOI] [PubMed] [Google Scholar]
  42. Weintraub H. Formation of stable transcription complexes as assayed by analysis of individual templates. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5819–5823. doi: 10.1073/pnas.85.16.5819. [DOI] [PMC free article] [PubMed] [Google Scholar]

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