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. 1994 Aug;14(8):5076–5087. doi: 10.1128/mcb.14.8.5076

One exon of the human LSF gene includes conserved regions involved in novel DNA-binding and dimerization motifs.

M K Shirra 1, Q Zhu 1, H C Huang 1, D Pallas 1, U Hansen 1
PMCID: PMC359026  PMID: 8035790

Abstract

The transcription factor LSF, identified as a HeLa protein that binds the simian virus 40 late promoter, recognizes direct repeats with a center-to-center spacing of 10 bp. The characterization of two human cDNAs, representing alternatively spliced mRNAs, provides insight into the unusual DNA-binding and oligomerization properties of LSF. The sequence of the full-length LSF is identical to that of the transcription factors alpha CP2 and LBP-1c and has similarity to the Drosophila transcription factor Elf-1/NTF-1. Using an epitope-counting method, we show that LSF binds DNA as a homodimer. LSF-ID, which is identical to LBP-1d, contains an in-frame internal deletion of 51 amino acids resulting from alternative mRNA splicing. Unlike LSF, LSF-ID did not bind LSF DNA-binding sites. Furthermore, LSF-ID did not affect the binding of LSF to DNA, suggesting that the two proteins do not interact. Of three short regions with a high degree of homology between LSF and Elf-1/NTF-1, LSF-ID lacks two, which are predicted to form beta-strands. Double amino acid substitutions in each of these regions eliminated specific DNA-binding activity, similarly to the LSF-ID deletion. The dimerization potential of these mutants was measured both by the ability to inhibit the binding of LSF to DNA and by direct protein-protein interaction studies. Mutations in one homology region, but not the other, functionally eliminated dimerization.

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Selected References

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  1. Aebersold R. H., Leavitt J., Saavedra R. A., Hood L. E., Kent S. B. Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc Natl Acad Sci U S A. 1987 Oct;84(20):6970–6974. doi: 10.1073/pnas.84.20.6970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  3. Aruffo A., Seed B. Molecular cloning of a CD28 cDNA by a high-efficiency COS cell expression system. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8573–8577. doi: 10.1073/pnas.84.23.8573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Attardi L. D., Tjian R. Drosophila tissue-specific transcription factor NTF-1 contains a novel isoleucine-rich activation motif. Genes Dev. 1993 Jul;7(7B):1341–1353. doi: 10.1101/gad.7.7b.1341. [DOI] [PubMed] [Google Scholar]
  5. Bickmore W. A., Oghene K., Little M. H., Seawright A., van Heyningen V., Hastie N. D. Modulation of DNA binding specificity by alternative splicing of the Wilms tumor wt1 gene transcript. Science. 1992 Jul 10;257(5067):235–237. doi: 10.1126/science.1321494. [DOI] [PubMed] [Google Scholar]
  6. Bray S. J., Burke B., Brown N. H., Hirsh J. Embryonic expression pattern of a family of Drosophila proteins that interact with a central nervous system regulatory element. Genes Dev. 1989 Aug;3(8):1130–1145. doi: 10.1101/gad.3.8.1130. [DOI] [PubMed] [Google Scholar]
  7. Cooney A. J., Tsai S. Y., O'Malley B. W., Tsai M. J. Chicken ovalbumin upstream promoter transcription factor (COUP-TF) dimers bind to different GGTCA response elements, allowing COUP-TF to repress hormonal induction of the vitamin D3, thyroid hormone, and retinoic acid receptors. Mol Cell Biol. 1992 Sep;12(9):4153–4163. doi: 10.1128/mcb.12.9.4153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Copertino D. W., Hallick R. B. Group II twintron: an intron within an intron in a chloroplast cytochrome b-559 gene. EMBO J. 1991 Feb;10(2):433–442. doi: 10.1002/j.1460-2075.1991.tb07965.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dombroski B. A., Mathias S. L., Nanthakumar E., Scott A. F., Kazazian H. H., Jr Isolation of an active human transposable element. Science. 1991 Dec 20;254(5039):1805–1808. doi: 10.1126/science.1662412. [DOI] [PubMed] [Google Scholar]
  10. Dynlacht B. D., Attardi L. D., Admon A., Freeman M., Tjian R. Functional analysis of NTF-1, a developmentally regulated Drosophila transcription factor that binds neuronal cis elements. Genes Dev. 1989 Nov;3(11):1677–1688. doi: 10.1101/gad.3.11.1677. [DOI] [PubMed] [Google Scholar]
  11. Freedman L. P., Towers T. L. DNA binding properties of the vitamin D3 receptor zinc finger region. Mol Endocrinol. 1991 Dec;5(12):1815–1826. doi: 10.1210/mend-5-12-1815. [DOI] [PubMed] [Google Scholar]
  12. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  13. Gogos J. A., Hsu T., Bolton J., Kafatos F. C. Sequence discrimination by alternatively spliced isoforms of a DNA binding zinc finger domain. Science. 1992 Sep 25;257(5078):1951–1955. doi: 10.1126/science.1290524. [DOI] [PubMed] [Google Scholar]
  14. Green M. R. Pre-mRNA splicing. Annu Rev Genet. 1986;20:671–708. doi: 10.1146/annurev.ge.20.120186.003323. [DOI] [PubMed] [Google Scholar]
  15. Ho Y. S., Mahoney M. E., Wulff D. L., Rosenberg M. Identification of the DNA binding domain of the phage lambda cII transcriptional activator and the direct correlation of cII protein stability with its oligomeric forms. Genes Dev. 1988 Feb;2(2):184–195. doi: 10.1101/gad.2.2.184. [DOI] [PubMed] [Google Scholar]
  16. Hope I. A., Struhl K. GCN4, a eukaryotic transcriptional activator protein, binds as a dimer to target DNA. EMBO J. 1987 Sep;6(9):2781–2784. doi: 10.1002/j.1460-2075.1987.tb02573.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Huang H. C., Sundseth R., Hansen U. Transcription factor LSF binds two variant bipartite sites within the SV40 late promoter. Genes Dev. 1990 Feb;4(2):287–298. doi: 10.1101/gad.4.2.287. [DOI] [PubMed] [Google Scholar]
  18. Kaelin W. G., Jr, Pallas D. C., DeCaprio J. A., Kaye F. J., Livingston D. M. Identification of cellular proteins that can interact specifically with the T/E1A-binding region of the retinoblastoma gene product. Cell. 1991 Feb 8;64(3):521–532. doi: 10.1016/0092-8674(91)90236-r. [DOI] [PubMed] [Google Scholar]
  19. Kim C. H., Heath C., Bertuch A., Hansen U. Specific stimulation of simian virus 40 late transcription in vitro by a cellular factor binding the simian virus 40 21-base-pair repeat promoter element. Proc Natl Acad Sci U S A. 1987 Sep;84(17):6025–6029. doi: 10.1073/pnas.84.17.6025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lim L. C., Fang L., Swendeman S. L., Sheffery M. Characterization of the molecularly cloned murine alpha-globin transcription factor CP2. J Biol Chem. 1993 Aug 25;268(24):18008–18017. [PubMed] [Google Scholar]
  21. Lim L. C., Swendeman S. L., Sheffery M. Molecular cloning of the alpha-globin transcription factor CP2. Mol Cell Biol. 1992 Feb;12(2):828–835. doi: 10.1128/mcb.12.2.828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Link D. C., Gutkind S. J., Robbins K. C., Ley T. J. Characterization of the 5' untranslated region of the human c-fgr gene and identification of the major myelomonocytic c-fgr promoter. Oncogene. 1992 May;7(5):877–884. [PubMed] [Google Scholar]
  23. Manley J. L., Fire A., Cano A., Sharp P. A., Gefter M. L. DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3855–3859. doi: 10.1073/pnas.77.7.3855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nasim F. H., Spears P. A., Hoffmann H. M., Kuo H. C., Grabowski P. J. A Sequential splicing mechanism promotes selection of an optimal exon by repositioning a downstream 5' splice site in preprotachykinin pre-mRNA. Genes Dev. 1990 Jul;4(7):1172–1184. doi: 10.1101/gad.4.7.1172. [DOI] [PubMed] [Google Scholar]
  25. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  26. Pabo C. O., Sauer R. T. Transcription factors: structural families and principles of DNA recognition. Annu Rev Biochem. 1992;61:1053–1095. doi: 10.1146/annurev.bi.61.070192.005201. [DOI] [PubMed] [Google Scholar]
  27. Perisic O., Xiao H., Lis J. T. Stable binding of Drosophila heat shock factor to head-to-head and tail-to-tail repeats of a conserved 5 bp recognition unit. Cell. 1989 Dec 1;59(5):797–806. doi: 10.1016/0092-8674(89)90603-x. [DOI] [PubMed] [Google Scholar]
  28. Principaud E., Spohr G. Xenopus laevis c-myc I and II genes: molecular structure and developmental expression. Nucleic Acids Res. 1991 Jun 11;19(11):3081–3088. doi: 10.1093/nar/19.11.3081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sauer R. T., Smith D. L., Johnson A. D. Flexibility of the yeast alpha 2 repressor enables it to occupy the ends of its operator, leaving the center free. Genes Dev. 1988 Jul;2(7):807–816. doi: 10.1101/gad.2.7.807. [DOI] [PubMed] [Google Scholar]
  30. Seed B., Aruffo A. Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid immunoselection procedure. Proc Natl Acad Sci U S A. 1987 May;84(10):3365–3369. doi: 10.1073/pnas.84.10.3365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Smith C. W., Patton J. G., Nadal-Ginard B. Alternative splicing in the control of gene expression. Annu Rev Genet. 1989;23:527–577. doi: 10.1146/annurev.ge.23.120189.002523. [DOI] [PubMed] [Google Scholar]
  32. Smith D. L., Johnson A. D. A molecular mechanism for combinatorial control in yeast: MCM1 protein sets the spacing and orientation of the homeodomains of an alpha 2 dimer. Cell. 1992 Jan 10;68(1):133–142. doi: 10.1016/0092-8674(92)90212-u. [DOI] [PubMed] [Google Scholar]
  33. Sorger P. K., Nelson H. C. Trimerization of a yeast transcriptional activator via a coiled-coil motif. Cell. 1989 Dec 1;59(5):807–813. doi: 10.1016/0092-8674(89)90604-1. [DOI] [PubMed] [Google Scholar]
  34. Sundseth R., Hansen U. Activation of RNA polymerase II transcription by the specific DNA-binding protein LSF. Increased rate of binding of the basal promoter factor TFIIB. J Biol Chem. 1992 Apr 15;267(11):7845–7855. [PubMed] [Google Scholar]
  35. Suzuki M. SPXX, a frequent sequence motif in gene regulatory proteins. J Mol Biol. 1989 May 5;207(1):61–84. doi: 10.1016/0022-2836(89)90441-5. [DOI] [PubMed] [Google Scholar]
  36. Yoon J. B., Li G., Roeder R. G. Characterization of a family of related cellular transcription factors which can modulate human immunodeficiency virus type 1 transcription in vitro. Mol Cell Biol. 1994 Mar;14(3):1776–1785. doi: 10.1128/mcb.14.3.1776. [DOI] [PMC free article] [PubMed] [Google Scholar]

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