Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Feb;17(2):999–1009. doi: 10.1128/mcb.17.2.999

The scs' boundary element: characterization of boundary element-associated factors.

C M Hart 1, K Zhao 1, U K Laemmli 1
PMCID: PMC231825  PMID: 9001253

Abstract

Boundary elements are thought to define the peripheries of chromatin domains and to restrict enhancer-promoter interactions to their target genes within their domains. We previously characterized a cDNA encoding the BEAF-32A protein (32A), which binds with high affinity to the scs' boundary element from the Drosophila melanogaster 87A7 hsp70 locus. Here, we report a second protein, BEAF-32B, that differs from 32A only in its amino terminus. Unlike 32A, it has the same DNA binding specificity as the complete BEAF activity affinity purified from Drosophila. We characterize three domains in these proteins. Heterocomplex formation is mediated by their identical carboxy-terminal domains, and DNA binding is mediated by their unique amino-terminal domains. The identical middle domains of 32A and 32B are dispensable for the functions described here, although they may be important for boundary element function. 32A and 32B apparently form trimers, and the ratio of 32A to 32B varies at different loci on polytene chromosomes as judged by immunofluorescence. The scs' element contains a high- and low-affinity binding site for BEAF. We observed that interaction with the low-affinity site is facilitated by binding to the high-affinity site some 200 bp distant.

Full Text

The Full Text of this article is available as a PDF (687.4 KB).

Selected References

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

  1. Adachi Y., Laemmli U. K. Identification of nuclear pre-replication centers poised for DNA synthesis in Xenopus egg extracts: immunolocalization study of replication protein A. J Cell Biol. 1992 Oct;119(1):1–15. doi: 10.1083/jcb.119.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cai H., Levine M. Modulation of enhancer-promoter interactions by insulators in the Drosophila embryo. Nature. 1995 Aug 10;376(6540):533–536. doi: 10.1038/376533a0. [DOI] [PubMed] [Google Scholar]
  3. Carey M., Lin Y. S., Green M. R., Ptashne M. A mechanism for synergistic activation of a mammalian gene by GAL4 derivatives. Nature. 1990 May 24;345(6273):361–364. doi: 10.1038/345361a0. [DOI] [PubMed] [Google Scholar]
  4. Chung J. H., Whiteley M., Felsenfeld G. A 5' element of the chicken beta-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila. Cell. 1993 Aug 13;74(3):505–514. doi: 10.1016/0092-8674(93)80052-g. [DOI] [PubMed] [Google Scholar]
  5. Csink A. K., Henikoff S. Genetic modification of heterochromatic association and nuclear organization in Drosophila. Nature. 1996 Jun 6;381(6582):529–531. doi: 10.1038/381529a0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Farkas G., Udvardy A. Sequence of scs and scs' Drosophila DNA fragments with boundary function in the control of gene expression. Nucleic Acids Res. 1992 May 25;20(10):2604–2604. doi: 10.1093/nar/20.10.2604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Galloni M., Gyurkovics H., Schedl P., Karch F. The bluetail transposon: evidence for independent cis-regulatory domains and domain boundaries in the bithorax complex. EMBO J. 1993 Mar;12(3):1087–1097. doi: 10.1002/j.1460-2075.1993.tb05750.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Georgiev P. G., Corces V. G. The su(Hw) protein bound to gypsy sequences in one chromosome can repress enhancer-promoter interactions in the paired gene located in the other homolog. Proc Natl Acad Sci U S A. 1995 May 23;92(11):5184–5188. doi: 10.1073/pnas.92.11.5184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gerasimova T. I., Gdula D. A., Gerasimov D. V., Simonova O., Corces V. G. A Drosophila protein that imparts directionality on a chromatin insulator is an enhancer of position-effect variegation. Cell. 1995 Aug 25;82(4):587–597. doi: 10.1016/0092-8674(95)90031-4. [DOI] [PubMed] [Google Scholar]
  11. Geyer P. K., Corces V. G. DNA position-specific repression of transcription by a Drosophila zinc finger protein. Genes Dev. 1992 Oct;6(10):1865–1873. doi: 10.1101/gad.6.10.1865. [DOI] [PubMed] [Google Scholar]
  12. Gietz D., St Jean A., Woods R. A., Schiestl R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992 Mar 25;20(6):1425–1425. doi: 10.1093/nar/20.6.1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Glover D. M., Leibowitz M. H., McLean D. A., Parry H. Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles. Cell. 1995 Apr 7;81(1):95–105. doi: 10.1016/0092-8674(95)90374-7. [DOI] [PubMed] [Google Scholar]
  14. Holdridge C., Dorsett D. Repression of hsp70 heat shock gene transcription by the suppressor of hairy-wing protein of Drosophila melanogaster. Mol Cell Biol. 1991 Apr;11(4):1894–1900. doi: 10.1128/mcb.11.4.1894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jamrich M., Greenleaf A. L., Bautz E. K. Localization of RNA polymerase in polytene chromosomes of Drosophila melanogaster. Proc Natl Acad Sci U S A. 1977 May;74(5):2079–2083. doi: 10.1073/pnas.74.5.2079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kalos M., Fournier R. E. Position-independent transgene expression mediated by boundary elements from the apolipoprotein B chromatin domain. Mol Cell Biol. 1995 Jan;15(1):198–207. doi: 10.1128/mcb.15.1.198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Karch F., Galloni M., Sipos L., Gausz J., Gyurkovics H., Schedl P. Mcp and Fab-7: molecular analysis of putative boundaries of cis-regulatory domains in the bithorax complex of Drosophila melanogaster. Nucleic Acids Res. 1994 Aug 11;22(15):3138–3146. doi: 10.1093/nar/22.15.3138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Kermekchiev M., Pettersson M., Matthias P., Schaffner W. Every enhancer works with every promoter for all the combinations tested: could new regulatory pathways evolve by enhancer shuffling? Gene Expr. 1991 Apr;1(1):71–81. [PMC free article] [PubMed] [Google Scholar]
  21. Klehr D., Maass K., Bode J. Scaffold-attached regions from the human interferon beta domain can be used to enhance the stable expression of genes under the control of various promoters. Biochemistry. 1991 Feb 5;30(5):1264–1270. doi: 10.1021/bi00219a015. [DOI] [PubMed] [Google Scholar]
  22. Krämer A., Haars R., Kabisch R., Will H., Bautz F. A., Bautz E. K. Monoclonal antibody directed against RNA polymerase II of Drosophila melanogaster. Mol Gen Genet. 1980;180(1):193–199. doi: 10.1007/BF00267369. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Li Q., Stamatoyannopoulos G. Hypersensitive site 5 of the human beta locus control region functions as a chromatin insulator. Blood. 1994 Sep 1;84(5):1399–1401. [PubMed] [Google Scholar]
  25. Lupas A., Van Dyke M., Stock J. Predicting coiled coils from protein sequences. Science. 1991 May 24;252(5009):1162–1164. doi: 10.1126/science.252.5009.1162. [DOI] [PubMed] [Google Scholar]
  26. Pascal E., Tjian R. Different activation domains of Sp1 govern formation of multimers and mediate transcriptional synergism. Genes Dev. 1991 Sep;5(9):1646–1656. doi: 10.1101/gad.5.9.1646. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Poljak L., Seum C., Mattioni T., Laemmli U. K. SARs stimulate but do not confer position independent gene expression. Nucleic Acids Res. 1994 Oct 25;22(21):4386–4394. doi: 10.1093/nar/22.21.4386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Roseman R. R., Pirrotta V., Geyer P. K. The su(Hw) protein insulates expression of the Drosophila melanogaster white gene from chromosomal position-effects. EMBO J. 1993 Feb;12(2):435–442. doi: 10.1002/j.1460-2075.1993.tb05675.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rykowski M. C., Parmelee S. J., Agard D. A., Sedat J. W. Precise determination of the molecular limits of a polytene chromosome band: regulatory sequences for the Notch gene are in the interband. Cell. 1988 Aug 12;54(4):461–472. doi: 10.1016/0092-8674(88)90067-0. [DOI] [PubMed] [Google Scholar]
  31. Scott K. S., Geyer P. K. Effects of the su(Hw) insulator protein on the expression of the divergently transcribed Drosophila yolk protein genes. EMBO J. 1995 Dec 15;14(24):6258–6267. doi: 10.1002/j.1460-2075.1995.tb00316.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  33. Udvardy A., Maine E., Schedl P. The 87A7 chromomere. Identification of novel chromatin structures flanking the heat shock locus that may define the boundaries of higher order domains. J Mol Biol. 1985 Sep 20;185(2):341–358. doi: 10.1016/0022-2836(85)90408-5. [DOI] [PubMed] [Google Scholar]
  34. Vazquez J., Farkas G., Gaszner M., Udvardy A., Muller M., Hagstrom K., Gyurkovics H., Sipos L., Gausz J., Galloni M. Genetic and molecular analysis of chromatin domains. Cold Spring Harb Symp Quant Biol. 1993;58:45–54. doi: 10.1101/sqb.1993.058.01.008. [DOI] [PubMed] [Google Scholar]
  35. Vazquez J., Schedl P. Sequences required for enhancer blocking activity of scs are located within two nuclease-hypersensitive regions. EMBO J. 1994 Dec 15;13(24):5984–5993. doi: 10.1002/j.1460-2075.1994.tb06944.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wolffe A. P. Gene regulation. Insulating chromatin. Curr Biol. 1994 Jan 1;4(1):85–87. doi: 10.1016/s0960-9822(00)00022-1. [DOI] [PubMed] [Google Scholar]
  37. Zhao K., Hart C. M., Laemmli U. K. Visualization of chromosomal domains with boundary element-associated factor BEAF-32. Cell. 1995 Jun 16;81(6):879–889. doi: 10.1016/0092-8674(95)90008-x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES