Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. 1995 Jan; 15(1): 129–140.
PMCID: PMC231920

DNA-binding specificity of the cut repeats from the human cut-like protein.


The Drosophila Cut and mammalian Cut-like proteins contain, in addition to the homeodomain, three other DNA-binding regions called Cut repeats. Cut-like proteins, therefore, belong to a distinct class of homeodomain proteins with multiple DNA-binding domains. In this study, we assessed the DNA-binding specificity of the human Cut repeats by performing PCR-mediated random oligonucleotide selection with glutathione S-transferase fusion proteins. Cut repeat 1, Cut repeat 3, and Cut repeat 3 plus the homeodomain selected related yet distinct sequences. Therefore, sequences selected by one of the fusion proteins were often, but not always, recognized by the other proteins. Consensus binding sites were derived for each fusion protein. In each case, however, some selected sequences diverged from the consensus but were confirmed to be high-affinity recognition sites by electrophoretic mobility shift assay. We conclude that Cut DNA-binding domains have broad, overlapping DNA-binding specificities. Determination of dissociation constants indicated that in addition to the core consensus, flanking sequences have a moderate but significant effect on sequence recognition. Evidence from electrophoretic mobility shift assay, DNase footprinting, and dissociation constant analyses strongly suggested that glutathione S-transferase/Cut fusion proteins bind to DNA as dimers. The implications of these findings are discussed in relation to the DNA-binding capabilities of Cut repeats. In contrast to other studies, we found that the human Cut-like protein does not preferably bind to a site that includes an ATTA homeodomain-binding motif. Here we demonstrate that the native human Cut-like protein recognizes more efficiently a site containing an ATCGAT core consensus flanked with G/C-rich sequences.

Full Text

The Full Text of this article is available as a PDF (784K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Aceto A, Caccuri AM, Sacchetta P, Bucciarelli T, Dragani B, Rosato N, Federici G, Di Ilio C. Dissociation and unfolding of Pi-class glutathione transferase. Evidence for a monomeric inactive intermediate. Biochem J. 1992 Jul 1;285(Pt 1):241–245. [PMC free article] [PubMed]
  • Andrés V, Chiara MD, Mahdavi V. A new bipartite DNA-binding domain: cooperative interaction between the cut repeat and homeo domain of the cut homeo proteins. Genes Dev. 1994 Jan;8(2):245–257. [PubMed]
  • Andres V, Nadal-Ginard B, Mahdavi V. Clox, a mammalian homeobox gene related to Drosophila cut, encodes DNA-binding regulatory proteins differentially expressed during development. Development. 1992 Oct;116(2):321–334. [PubMed]
  • Aufiero B, Neufeld EJ, Orkin SH. Sequence-specific DNA binding of individual cut repeats of the human CCAAT displacement/cut homeodomain protein. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7757–7761. [PMC free article] [PubMed]
  • Aurora R, Herr W. Segments of the POU domain influence one another's DNA-binding specificity. Mol Cell Biol. 1992 Feb;12(2):455–467. [PMC free article] [PubMed]
  • Barberis A, Superti-Furga G, Busslinger M. Mutually exclusive interaction of the CCAAT-binding factor and of a displacement protein with overlapping sequences of a histone gene promoter. Cell. 1987 Jul 31;50(3):347–359. [PubMed]
  • Baumhueter S, Mendel DB, Conley PB, Kuo CJ, Turk C, Graves MK, Edwards CA, Courtois G, Crabtree GR. HNF-1 shares three sequence motifs with the POU domain proteins and is identical to LF-B1 and APF. Genes Dev. 1990 Mar;4(3):372–379. [PubMed]
  • Blackwell TK, Kretzner L, Blackwood EM, Eisenman RN, Weintraub H. Sequence-specific DNA binding by the c-Myc protein. Science. 1990 Nov 23;250(4984):1149–1151. [PubMed]
  • Blackwell TK, Weintraub H. Differences and similarities in DNA-binding preferences of MyoD and E2A protein complexes revealed by binding site selection. Science. 1990 Nov 23;250(4984):1104–1110. [PubMed]
  • Blochlinger K, Bodmer R, Jan LY, Jan YN. Patterns of expression of cut, a protein required for external sensory organ development in wild-type and cut mutant Drosophila embryos. Genes Dev. 1990 Aug;4(8):1322–1331. [PubMed]
  • Blochlinger K, Bodmer R, Jack J, Jan LY, Jan YN. Primary structure and expression of a product from cut, a locus involved in specifying sensory organ identity in Drosophila. Nature. 1988 Jun 16;333(6174):629–635. [PubMed]
  • Blochlinger K, Jan LY, Jan YN. Transformation of sensory organ identity by ectopic expression of Cut in Drosophila. Genes Dev. 1991 Jul;5(7):1124–1135. [PubMed]
  • Blochlinger K, Jan LY, Jan YN. Postembryonic patterns of expression of cut, a locus regulating sensory organ identity in Drosophila. Development. 1993 Feb;117(2):441–450. [PubMed]
  • Bodmer R, Barbel S, Sheperd S, Jack JW, Jan LY, Jan YN. Transformation of sensory organs by mutations of the cut locus of D. melanogaster. Cell. 1987 Oct 23;51(2):293–307. [PubMed]
  • Bopp D, Burri M, Baumgartner S, Frigerio G, Noll M. Conservation of a large protein domain in the segmentation gene paired and in functionally related genes of Drosophila. Cell. 1986 Dec 26;47(6):1033–1040. [PubMed]
  • Dufort D, Nepveu A. The human cut homeodomain protein represses transcription from the c-myc promoter. Mol Cell Biol. 1994 Jun;14(6):4251–4257. [PMC free article] [PubMed]
  • Dynan WS, Tjian R. The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter. Cell. 1983 Nov;35(1):79–87. [PubMed]
  • Finney M, Ruvkun G, Horvitz HR. The C. elegans cell lineage and differentiation gene unc-86 encodes a protein with a homeodomain and extended similarity to transcription factors. Cell. 1988 Dec 2;55(5):757–769. [PubMed]
  • Frain M, Swart G, Monaci P, Nicosia A, Stämpfli S, Frank R, Cortese R. The liver-specific transcription factor LF-B1 contains a highly diverged homeobox DNA binding domain. Cell. 1989 Oct 6;59(1):145–157. [PubMed]
  • Freyd G, Kim SK, Horvitz HR. Novel cysteine-rich motif and homeodomain in the product of the Caenorhabditis elegans cell lineage gene lin-11. Nature. 1990 Apr 26;344(6269):876–879. [PubMed]
  • Galas DJ, Schmitz A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 1978 Sep;5(9):3157–3170. [PMC free article] [PubMed]
  • Gruss P, Walther C. Pax in development. Cell. 1992 May 29;69(5):719–722. [PubMed]
  • di Guan C, Li P, Riggs PD, Inouye H. Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein. Gene. 1988 Jul 15;67(1):21–30. [PubMed]
  • Harada R, Dufort D, Denis-Larose C, Nepveu A. Conserved cut repeats in the human cut homeodomain protein function as DNA binding domains. J Biol Chem. 1994 Jan 21;269(3):2062–2067. [PubMed]
  • Ingraham HA, Flynn SE, Voss JW, Albert VR, Kapiloff MS, Wilson L, Rosenfeld MG. The POU-specific domain of Pit-1 is essential for sequence-specific, high affinity DNA binding and DNA-dependent Pit-1-Pit-1 interactions. Cell. 1990 Jun 15;61(6):1021–1033. [PubMed]
  • Jack J, Dorsett D, Delotto Y, Liu S. Expression of the cut locus in the Drosophila wing margin is required for cell type specification and is regulated by a distant enhancer. Development. 1991 Nov;113(3):735–747. [PubMed]
  • Karlsson O, Thor S, Norberg T, Ohlsson H, Edlund T. Insulin gene enhancer binding protein Isl-1 is a member of a novel class of proteins containing both a homeo- and a Cys-His domain. Nature. 1990 Apr 26;344(6269):879–882. [PubMed]
  • Kissinger CR, Liu BS, Martin-Blanco E, Kornberg TB, Pabo CO. Crystal structure of an engrailed homeodomain-DNA complex at 2.8 A resolution: a framework for understanding homeodomain-DNA interactions. Cell. 1990 Nov 2;63(3):579–590. [PubMed]
  • Ko LJ, Engel JD. DNA-binding specificities of the GATA transcription factor family. Mol Cell Biol. 1993 Jul;13(7):4011–4022. [PMC free article] [PubMed]
  • Krämer H, Niemöller M, Amouyal M, Revet B, von Wilcken-Bergmann B, Müller-Hill B. lac repressor forms loops with linear DNA carrying two suitably spaced lac operators. EMBO J. 1987 May;6(5):1481–1491. [PMC free article] [PubMed]
  • Lee KA, Bindereif A, Green MR. A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing. Gene Anal Tech. 1988 Mar-Apr;5(2):22–31. [PubMed]
  • Liu S, Jack J. Regulatory interactions and role in cell type specification of the Malpighian tubules by the cut, Krüppel, and caudal genes of Drosophila. Dev Biol. 1992 Mar;150(1):133–143. [PubMed]
  • Liu S, McLeod E, Jack J. Four distinct regulatory regions of the cut locus and their effect on cell type specification in Drosophila. Genetics. 1991 Jan;127(1):151–159. [PMC free article] [PubMed]
  • Maina CV, Riggs PD, Grandea AG, 3rd, Slatko BE, Moran LS, Tagliamonte JA, McReynolds LA, Guan CD. An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein. Gene. 1988 Dec 30;74(2):365–373. [PubMed]
  • Mandal N, Su W, Haber R, Adhya S, Echols H. DNA looping in cellular repression of transcription of the galactose operon. Genes Dev. 1990 Mar;4(3):410–418. [PubMed]
  • McCutchan JH, Pagano JS. Enchancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J Natl Cancer Inst. 1968 Aug;41(2):351–357. [PubMed]
  • McGinnis W, Krumlauf R. Homeobox genes and axial patterning. Cell. 1992 Jan 24;68(2):283–302. [PubMed]
  • Mendel DB, Khavari PA, Conley PB, Graves MK, Hansen LP, Admon A, Crabtree GR. Characterization of a cofactor that regulates dimerization of a mammalian homeodomain protein. Science. 1991 Dec 20;254(5039):1762–1767. [PubMed]
  • Merika M, Orkin SH. DNA-binding specificity of GATA family transcription factors. Mol Cell Biol. 1993 Jul;13(7):3999–4010. [PMC free article] [PubMed]
  • Neufeld EJ, Skalnik DG, Lievens PM, Orkin SH. Human CCAAT displacement protein is homologous to the Drosophila homeoprotein, cut. Nat Genet. 1992 Apr;1(1):50–55. [PubMed]
  • 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. [PubMed]
  • Otting G, Qian YQ, Billeter M, Müller M, Affolter M, Gehring WJ, Wüthrich K. Protein--DNA contacts in the structure of a homeodomain--DNA complex determined by nuclear magnetic resonance spectroscopy in solution. EMBO J. 1990 Oct;9(10):3085–3092. [PMC free article] [PubMed]
  • Ruvkun G, Finney M. Regulation of transcription and cell identity by POU domain proteins. Cell. 1991 Feb 8;64(3):475–478. [PubMed]
  • Sacchetta P, Aceto A, Bucciarelli T, Dragani B, Santarone S, Allocati N, Di Ilio C. Multiphasic denaturation of glutathione transferase B1-1 by guanidinium chloride. Role of the dimeric structure on the flexibility of the active site. Eur J Biochem. 1993 Aug 1;215(3):741–745. [PubMed]
  • Scott MP, Tamkun JW, Hartzell GW., 3rd The structure and function of the homeodomain. Biochim Biophys Acta. 1989 Jul 28;989(1):25–48. [PubMed]
  • Skalnik DG, Strauss EC, Orkin SH. CCAAT displacement protein as a repressor of the myelomonocytic-specific gp91-phox gene promoter. J Biol Chem. 1991 Sep 5;266(25):16736–16744. [PubMed]
  • Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. [PubMed]
  • Sturm RA, Herr W. The POU domain is a bipartite DNA-binding structure. Nature. 1988 Dec 8;336(6199):601–604. [PubMed]
  • Superti-Furga G, Barberis A, Schaffner G, Busslinger M. The -117 mutation in Greek HPFH affects the binding of three nuclear factors to the CCAAT region of the gamma-globin gene. EMBO J. 1988 Oct;7(10):3099–3107. [PMC free article] [PubMed]
  • Superti-Furga G, Barberis A, Schreiber E, Busslinger M. The protein CDP, but not CP1, footprints on the CCAAT region of the gamma-globin gene in unfractionated B-cell extracts. Biochim Biophys Acta. 1989 Mar 1;1007(2):237–242. [PubMed]
  • TenHarmsel A, Austin RJ, Savenelli N, Biggin MD. Cooperative binding at a distance by even-skipped protein correlates with repression and suggests a mechanism of silencing. Mol Cell Biol. 1993 May;13(5):2742–2752. [PMC free article] [PubMed]
  • Treisman J, Harris E, Desplan C. The paired box encodes a second DNA-binding domain in the paired homeo domain protein. Genes Dev. 1991 Apr;5(4):594–604. [PubMed]
  • Treisman J, Harris E, Wilson D, Desplan C. The homeodomain: a new face for the helix-turn-helix? Bioessays. 1992 Mar;14(3):145–150. [PubMed]
  • Tymon AM, Kües U, Richardson WV, Casselton LA. A fungal mating type protein that regulates sexual and asexual development contains a POU-related domain. EMBO J. 1992 May;11(5):1805–1813. [PMC free article] [PubMed]
  • Valarché I, Tissier-Seta JP, Hirsch MR, Martinez S, Goridis C, Brunet JF. The mouse homeodomain protein Phox2 regulates Ncam promoter activity in concert with Cux/CDP and is a putative determinant of neurotransmitter phenotype. Development. 1993 Nov;119(3):881–896. [PubMed]
  • Verrijzer CP, Alkema MJ, van Weperen WW, Van Leeuwen HC, Strating MJ, van der Vliet PC. The DNA binding specificity of the bipartite POU domain and its subdomains. EMBO J. 1992 Dec;11(13):4993–5003. [PMC free article] [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)


Save items

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • PubMed
    PubMed citations for these articles

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...