Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Jul;15(7):3848–3856. doi: 10.1128/mcb.15.7.3848

Molecular analysis of the distal enhancer of the mouse alpha-fetoprotein gene.

J H Millonig 1, J A Emerson 1, J M Levorse 1, S M Tilghman 1
PMCID: PMC230624  PMID: 7540720

Abstract

The mouse alpha-fetoprotein (AFP) gene is transcribed at high levels in the visceral endoderm of the yolk sac and fetal liver and at much lower rates in the endoderm of the fetal gut. Expression of the gene in vivo requires the presence of at least one of three enhancers which lie in its 5' flanking region. In this report, we establish that the most distal AFP enhancer directed consistent expression of a linked AFP minigene in all three endodermal tissues in transgenic mice. The enhancer is composed of three domains, each of which is essential for full enhancer function by transient transfection assays. DNase I footprinting identified three regions of the enhancer which are protected by human hepatoma nuclear extracts, one of which corresponded to a consensus site for HNF-3 binding. Site-directed mutations in this site caused a 10-fold reduction in enhancer function by transient transfection. In transgenic mice, however, the mutation resulted in sporadic expression of the transgene, dependent on the site of integration. A similar acquisition of position-dependent sporadic expression of the transgene was observed with a mutation in a second protein binding site, despite the fact that this mutation had very little effect on enhancer function as assessed by transient transfection. These studies underscore the value of examining the functions of specific protein binding sites in vivo.

Full Text

The Full Text of this article is available as a PDF (542.7 KB).

Selected References

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

  1. Andrews G. K., Janzen R. G., Tamaoki T. Stability of alpha-fetoprotein messenger RNA in mouse yolk sac. Dev Biol. 1982 Jan;89(1):111–116. doi: 10.1016/0012-1606(82)90299-8. [DOI] [PubMed] [Google Scholar]
  2. Ang S. L., Wierda A., Wong D., Stevens K. A., Cascio S., Rossant J., Zaret K. S. The formation and maintenance of the definitive endoderm lineage in the mouse: involvement of HNF3/forkhead proteins. Development. 1993 Dec;119(4):1301–1315. doi: 10.1242/dev.119.4.1301. [DOI] [PubMed] [Google Scholar]
  3. Auffray C., Rougeon F. Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur J Biochem. 1980 Jun;107(2):303–314. doi: 10.1111/j.1432-1033.1980.tb06030.x. [DOI] [PubMed] [Google Scholar]
  4. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bernier D., Thomassin H., Allard D., Guertin M., Hamel D., Blaquière M., Beauchemin M., LaRue H., Estable-Puig M., Bélanger L. Functional analysis of developmentally regulated chromatin-hypersensitive domains carrying the alpha 1-fetoprotein gene promoter and the albumin/alpha 1-fetoprotein intergenic enhancer. Mol Cell Biol. 1993 Mar;13(3):1619–1633. doi: 10.1128/mcb.13.3.1619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Buzard G., Locker J. The transcription control region of the rat alpha-fetoprotein gene. DNA sequence and homology studies. DNA Seq. 1990;1(1):33–48. doi: 10.3109/10425179009041345. [DOI] [PubMed] [Google Scholar]
  7. Clark K. L., Halay E. D., Lai E., Burley S. K. Co-crystal structure of the HNF-3/fork head DNA-recognition motif resembles histone H5. Nature. 1993 Jul 29;364(6436):412–420. doi: 10.1038/364412a0. [DOI] [PubMed] [Google Scholar]
  8. Costa R. H., Grayson D. R., Darnell J. E., Jr Multiple hepatocyte-enriched nuclear factors function in the regulation of transthyretin and alpha 1-antitrypsin genes. Mol Cell Biol. 1989 Apr;9(4):1415–1425. doi: 10.1128/mcb.9.4.1415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Costa R. H., Grayson D. R., Xanthopoulos K. G., Darnell J. E., Jr A liver-specific DNA-binding protein recognizes multiple nucleotide sites in regulatory regions of transthyretin, alpha 1-antitrypsin, albumin, and simian virus 40 genes. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3840–3844. doi: 10.1073/pnas.85.11.3840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Courtois G., Baumhueter S., Crabtree G. R. Purified hepatocyte nuclear factor 1 interacts with a family of hepatocyte-specific promoters. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7937–7941. doi: 10.1073/pnas.85.21.7937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dziadek M. A., Andrews G. K. Tissue specificity of alpha-fetoprotein messenger RNA expression during mouse embryogenesis. EMBO J. 1983;2(4):549–554. doi: 10.1002/j.1460-2075.1983.tb01461.x. [DOI] [PMC free article] [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. Feuerman M. H., Godbout R., Ingram R. S., Tilghman S. M. Tissue-specific transcription of the mouse alpha-fetoprotein gene promoter is dependent on HNF-1. Mol Cell Biol. 1989 Oct;9(10):4204–4212. doi: 10.1128/mcb.9.10.4204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Friedman A. D., Landschulz W. H., McKnight S. L. CCAAT/enhancer binding protein activates the promoter of the serum albumin gene in cultured hepatoma cells. Genes Dev. 1989 Sep;3(9):1314–1322. doi: 10.1101/gad.3.9.1314. [DOI] [PubMed] [Google Scholar]
  15. Giese K., Grosschedl R. LEF-1 contains an activation domain that stimulates transcription only in a specific context of factor-binding sites. EMBO J. 1993 Dec;12(12):4667–4676. doi: 10.1002/j.1460-2075.1993.tb06155.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Godbout R., Ingram R. S., Tilghman S. M. Fine-structure mapping of the three mouse alpha-fetoprotein gene enhancers. Mol Cell Biol. 1988 Mar;8(3):1169–1178. doi: 10.1128/mcb.8.3.1169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Godbout R., Ingram R., Tilghman S. M. Multiple regulatory elements in the intergenic region between the alpha-fetoprotein and albumin genes. Mol Cell Biol. 1986 Feb;6(2):477–487. doi: 10.1128/mcb.6.2.477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Godbout R., Tilghman S. M. Configuration of the alpha-fetoprotein regulatory domain during development. Genes Dev. 1988 Aug;2(8):949–956. doi: 10.1101/gad.2.8.949. [DOI] [PubMed] [Google Scholar]
  19. Grosschedl R., Marx M. Stable propagation of the active transcriptional state of an immunoglobulin mu gene requires continuous enhancer function. Cell. 1988 Nov 18;55(4):645–654. doi: 10.1016/0092-8674(88)90223-1. [DOI] [PubMed] [Google Scholar]
  20. Hammer R. E., Krumlauf R., Camper S. A., Brinster R. L., Tilghman S. M. Diversity of alpha-fetoprotein gene expression in mice is generated by a combination of separate enhancer elements. Science. 1987 Jan 2;235(4784):53–58. doi: 10.1126/science.2432657. [DOI] [PubMed] [Google Scholar]
  21. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  22. Jackson D. A., Hassan A. B., Errington R. J., Cook P. R. Visualization of focal sites of transcription within human nuclei. EMBO J. 1993 Mar;12(3):1059–1065. doi: 10.1002/j.1460-2075.1993.tb05747.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Jenuwein T., Grosschedl R. Complex pattern of immunoglobulin mu gene expression in normal and transgenic mice: nonoverlapping regulatory sequences govern distinct tissue specificities. Genes Dev. 1991 Jun;5(6):932–943. doi: 10.1101/gad.5.6.932. [DOI] [PubMed] [Google Scholar]
  25. Johnson P. F., Landschulz W. H., Graves B. J., McKnight S. L. Identification of a rat liver nuclear protein that binds to the enhancer core element of three animal viruses. Genes Dev. 1987 Apr;1(2):133–146. doi: 10.1101/gad.1.2.133. [DOI] [PubMed] [Google Scholar]
  26. Jones D. H., Howard B. H. A rapid method for site-specific mutagenesis and directional subcloning by using the polymerase chain reaction to generate recombinant circles. Biotechniques. 1990 Feb;8(2):178–183. [PubMed] [Google Scholar]
  27. Kellum R., Schedl P. A position-effect assay for boundaries of higher order chromosomal domains. Cell. 1991 Mar 8;64(5):941–950. doi: 10.1016/0092-8674(91)90318-s. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Kuo C. J., Conley P. B., Chen L., Sladek F. M., Darnell J. E., Jr, Crabtree G. R. A transcriptional hierarchy involved in mammalian cell-type specification. Nature. 1992 Jan 30;355(6359):457–461. doi: 10.1038/355457a0. [DOI] [PubMed] [Google Scholar]
  30. Lai E., Prezioso V. R., Smith E., Litvin O., Costa R. H., Darnell J. E., Jr HNF-3A, a hepatocyte-enriched transcription factor of novel structure is regulated transcriptionally. Genes Dev. 1990 Aug;4(8):1427–1436. doi: 10.1101/gad.4.8.1427. [DOI] [PubMed] [Google Scholar]
  31. Lai E., Prezioso V. R., Tao W. F., Chen W. S., Darnell J. E., Jr Hepatocyte nuclear factor 3 alpha belongs to a gene family in mammals that is homologous to the Drosophila homeotic gene fork head. Genes Dev. 1991 Mar;5(3):416–427. doi: 10.1101/gad.5.3.416. [DOI] [PubMed] [Google Scholar]
  32. Landschulz W. H., Johnson P. F., Adashi E. Y., Graves B. J., McKnight S. L. Isolation of a recombinant copy of the gene encoding C/EBP. Genes Dev. 1988 Jul;2(7):786–800. doi: 10.1101/gad.2.7.786. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Mendel D. B., Hansen L. P., Graves M. K., Conley P. B., Crabtree G. R. HNF-1 alpha and HNF-1 beta (vHNF-1) share dimerization and homeo domains, but not activation domains, and form heterodimers in vitro. Genes Dev. 1991 Jun;5(6):1042–1056. doi: 10.1101/gad.5.6.1042. [DOI] [PubMed] [Google Scholar]
  36. Nicosia A., Monaci P., Tomei L., De Francesco R., Nuzzo M., Stunnenberg H., Cortese R. A myosin-like dimerization helix and an extra-large homeodomain are essential elements of the tripartite DNA binding structure of LFB1. Cell. 1990 Jun 29;61(7):1225–1236. doi: 10.1016/0092-8674(90)90687-a. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Piña B., Brüggemeier U., Beato M. Nucleosome positioning modulates accessibility of regulatory proteins to the mouse mammary tumor virus promoter. Cell. 1990 Mar 9;60(5):719–731. doi: 10.1016/0092-8674(90)90087-u. [DOI] [PubMed] [Google Scholar]
  39. Sasaki H., Hogan B. L. Differential expression of multiple fork head related genes during gastrulation and axial pattern formation in the mouse embryo. Development. 1993 May;118(1):47–59. doi: 10.1242/dev.118.1.47. [DOI] [PubMed] [Google Scholar]
  40. Sladek F. M., Zhong W. M., Lai E., Darnell J. E., Jr Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily. Genes Dev. 1990 Dec;4(12B):2353–2365. doi: 10.1101/gad.4.12b.2353. [DOI] [PubMed] [Google Scholar]
  41. Stief A., Winter D. M., Strätling W. H., Sippel A. E. A nuclear DNA attachment element mediates elevated and position-independent gene activity. Nature. 1989 Sep 28;341(6240):343–345. doi: 10.1038/341343a0. [DOI] [PubMed] [Google Scholar]
  42. Taylor I. C., Workman J. L., Schuetz T. J., Kingston R. E. Facilitated binding of GAL4 and heat shock factor to nucleosomal templates: differential function of DNA-binding domains. Genes Dev. 1991 Jul;5(7):1285–1298. doi: 10.1101/gad.5.7.1285. [DOI] [PubMed] [Google Scholar]
  43. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Thomassin H., Hamel D., Bernier D., Guertin M., Belanger L. Molecular cloning of two C/EBP-related proteins that bind to the promoter and the enhancer of the alpha 1-fetoprotein gene. Further analysis of C/EBP beta and C/EBP gamma. Nucleic Acids Res. 1992 Jun 25;20(12):3091–3098. doi: 10.1093/nar/20.12.3091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tilghman S. M., Belayew A. Transcriptional control of the murine albumin/alpha-fetoprotein locus during development. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5254–5257. doi: 10.1073/pnas.79.17.5254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tyner A. L., Godbout R., Compton R. S., Tilghman S. M. The ontogeny of alpha-fetoprotein gene expression in the mouse gastrointestinal tract. J Cell Biol. 1990 Apr;110(4):915–927. doi: 10.1083/jcb.110.4.915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Vogt T. F., Compton R. S., Scott R. W., Tilghman S. M. Differential requirements for cellular enhancers in stem and differentiated cells. Nucleic Acids Res. 1988 Jan 25;16(2):487–500. doi: 10.1093/nar/16.2.487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Weigel D., Jürgens G., Küttner F., Seifert E., Jäckle H. The homeotic gene fork head encodes a nuclear protein and is expressed in the terminal regions of the Drosophila embryo. Cell. 1989 May 19;57(4):645–658. doi: 10.1016/0092-8674(89)90133-5. [DOI] [PubMed] [Google Scholar]
  49. Zhang D. E., Ge X., Rabek J. P., Papaconstantinou J. Functional analysis of the trans-acting factor binding sites of the mouse alpha-fetoprotein proximal promoter by site-directed mutagenesis. J Biol Chem. 1991 Nov 5;266(31):21179–21185. [PubMed] [Google Scholar]

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

RESOURCES