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. 1996 Jun;16(6):2906–2912. doi: 10.1128/mcb.16.6.2906

Analysis of mice containing a targeted deletion of beta-globin locus control region 5' hypersensitive site 3.

B A Hug 1, R L Wesselschmidt 1, S Fiering 1, M A Bender 1, E Epner 1, M Groudine 1, T J Ley 1
PMCID: PMC231284  PMID: 8649401

Abstract

To examine the function of murine beta-globin locus region (LCR) 5' hypersensitive site 3 (HS3) in its native chromosomal context, we deleted this site from the mouse germ line by using homologous recombination techniques. Previous experiments with human 5' HS3 in transgenic models suggested that this site independently contains at least 50% of total LCR activity and that it interacts preferentially with the human gamma-globin genes in embryonic erythroid cells. However, in this study, we demonstrate that deletion of murine 5' HS3 reduces expression of the linked embryonic epsilon y- and beta H 1-globin genes only minimally in yolk sac-derived erythroid cells and reduces output of the linked adult beta (beta major plus beta minor) globin genes by approximately 30% in adult erythrocytes. When the selectable marker PGK-neo cassette was left within the HS3 region of the LCR, a much more severe phenotype was observed at all developmental stages, suggesting that PGK-neo interferes with LCR activity when it is retained within the LCR. Collectively, these results suggest that murine 5' HS3 is not required for globin gene switching; importantly, however, it is required for approximately 30% of the total LCR activity associated with adult beta-globin gene expression in adult erythrocytes.

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Selected References

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  1. 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]
  2. Bungert J., Davé U., Lim K. C., Lieuw K. H., Shavit J. A., Liu Q., Engel J. D. Synergistic regulation of human beta-globin gene switching by locus control region elements HS3 and HS4. Genes Dev. 1995 Dec 15;9(24):3083–3096. doi: 10.1101/gad.9.24.3083. [DOI] [PubMed] [Google Scholar]
  3. Chada K., Magram J., Costantini F. An embryonic pattern of expression of a human fetal globin gene in transgenic mice. Nature. 1986 Feb 20;319(6055):685–689. doi: 10.1038/319685a0. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Dhar V., Nandi A., Schildkraut C. L., Skoultchi A. I. Erythroid-specific nuclease-hypersensitive sites flanking the human beta-globin domain. Mol Cell Biol. 1990 Aug;10(8):4324–4333. doi: 10.1128/mcb.10.8.4324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  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., Tan-Un K. C., Harper A., Michalovich D., Yannoutsos N., Philipsen S., Grosveld F. A dominant chromatin-opening activity in 5' hypersensitive site 3 of the human beta-globin locus control region. EMBO J. 1996 Feb 1;15(3):562–568. [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Fiering S., Epner E., Robinson K., Zhuang Y., Telling A., Hu M., Martin D. I., Enver T., Ley T. J., Groudine M. Targeted deletion of 5'HS2 of the murine beta-globin LCR reveals that it is not essential for proper regulation of the beta-globin locus. Genes Dev. 1995 Sep 15;9(18):2203–2213. doi: 10.1101/gad.9.18.2203. [DOI] [PubMed] [Google Scholar]
  11. Fiering S., Kim C. G., Epner E. M., Groudine M. An "in-out" strategy using gene targeting and FLP recombinase for the functional dissection of complex DNA regulatory elements: analysis of the beta-globin locus control region. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8469–8473. doi: 10.1073/pnas.90.18.8469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  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. 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]
  16. 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]
  17. 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]
  18. Hardison R., Xu J., Jackson J., Mansberger J., Selifonova O., Grotch B., Biesecker J., Petrykowska H., Miller W. Comparative analysis of the locus control region of the rabbit beta-like gene cluster: HS3 increases transient expression of an embryonic epsilon-globin gene. Nucleic Acids Res. 1993 Mar 11;21(5):1265–1272. doi: 10.1093/nar/21.5.1265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hug B. A., Moon A. M., Ley T. J. Structure and function of the murine beta-globin locus control region 5' HS-3. Nucleic Acids Res. 1992 Nov 11;20(21):5771–5778. doi: 10.1093/nar/20.21.5771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jiménez G., Gale K. B., Enver T. The mouse beta-globin locus control region: hypersensitive sites 3 and 4. Nucleic Acids Res. 1992 Nov 11;20(21):5797–5803. doi: 10.1093/nar/20.21.5797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Kulozik A. E., Bail S., Bellan-Koch A., Bartram C. R., Kohne E., Kleihauer E. The proximal element of the beta globin locus control region is not functionally required in vivo. J Clin Invest. 1991 Jun;87(6):2142–2146. doi: 10.1172/JCI115246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Li Q., Zhou B., Powers P., Enver T., Stamatoyannopoulos G. Primary structure of the goat beta-globin locus control region. Genomics. 1991 Mar;9(3):488–499. doi: 10.1016/0888-7543(91)90415-b. [DOI] [PubMed] [Google Scholar]
  25. 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]
  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. Moon A. M., Ley T. J. Functional properties of the beta-globin locus control region in K562 erythroleukemia cells. Blood. 1991 May 15;77(10):2272–2284. [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. 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]
  31. Shehee W. R., Loeb D. D., Adey N. B., Burton F. H., Casavant N. C., Cole P., Davies C. J., McGraw R. A., Schichman S. A., Severynse D. M. Nucleotide sequence of the BALB/c mouse beta-globin complex. J Mol Biol. 1989 Jan 5;205(1):41–62. doi: 10.1016/0022-2836(89)90363-x. [DOI] [PubMed] [Google Scholar]
  32. Soriano P., Montgomery C., Geske R., Bradley A. Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell. 1991 Feb 22;64(4):693–702. doi: 10.1016/0092-8674(91)90499-o. [DOI] [PubMed] [Google Scholar]
  33. Starck J., Sarkar R., Romana M., Bhargava A., Scarpa A. L., Tanaka M., Chamberlain J. W., Weissman S. M., Forget B. G. Developmental regulation of human gamma- and beta-globin genes in the absence of the locus control region. Blood. 1994 Sep 1;84(5):1656–1665. [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. 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]
  37. Trudel M., Magram J., Bruckner L., Costantini F. Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice. Mol Cell Biol. 1987 Nov;7(11):4024–4029. doi: 10.1128/mcb.7.11.4024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Ulrich M. J., Ley T. J. Function of normal and mutated gamma-globin gene promoters in electroporated K562 erythroleukemia cells. Blood. 1990 Feb 15;75(4):990–999. [PubMed] [Google Scholar]
  40. Whitelaw E., Tsai S. F., Hogben P., Orkin S. H. Regulated expression of globin chains and the erythroid transcription factor GATA-1 during erythropoiesis in the developing mouse. Mol Cell Biol. 1990 Dec;10(12):6596–6606. doi: 10.1128/mcb.10.12.6596. [DOI] [PMC free article] [PubMed] [Google Scholar]

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