SELEX and missing phosphate contact analyses reveal flexibility within the AP-2 α protein:DNA binding complex

Author: Mohibullah Neeman   Donner Amy   Ippolito Joseph A.   Williams Trevor   Bosher J.M.   Chazaud C.   Mitchell P.J.   Moser M.   Moser M.   Nottoli T.   Schorle H.   West-Mays J.A.   Zhang J.   Means A.L.   Lüscher B.   Oulad-Abdelghani M.   Shen H.   Williams T.   Zeng Y.-X.   Jean D.   Turner B.C.   Bosher J.M.   Hennig G.   Mitchell P.J.   Fini M.E.   Somasundaram K.   Batsché E.   Gaubatz S.   Williams T.   Williams T.   Gee M.S.   Rosenberg A.H.   Ekker S.C.   Creaser P.   Schultz S.C.   Dixon W.J.   Wissmann A.   Sun L.   Koudelka G.B.   Le Cam E.   Aymami J.   Giraud-Panis M.-J.   Mills J.B.   Roll C.   Sigler P.B.   Imagawa M.   Mellentin-Michelotti J.   Bisgrove D.A.   Israël A.   Duan C.   Leask A.   Hyman S.E.   McPherson L.A.   Chen T.-T.   Snape A.M.  

Publisher: Oxford University Press

ISSN: 1362-4962

Source: Nucleic Acids Research, Vol.27, Iss.13, 1999-07, pp. : 2760-2769

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Abstract

The AP-2 family of transcription factors are defined by the presence of a novel DNA binding domain, termed a ‘basic helix–span–helix’ motif. The AP-2 genes regulate important aspects of vertebrate embryogenesis and have also been linked to the control of cell proliferation and tumorigenesis, but the cellular targets that the AP-2 proteins control are largely undefined. In particular, since only a limited number of sequences have previously been utilized to define the nature of the AP-2 binding site, the range of DNA sequences recognized by the AP-2 proteins remains unknown. We have therefore utilized a SELEX analysis to identify multiple new AP-2α binding sites. Moreover, we have devised a novel missing phosphate and nucleotide competition analysis to characterize the residues in the binding site required for AP-2α protein:DNA contact. These studies suggest that the AP-2α protein:DNA complex is flexible and indicate that AP-2α can bind three related sequence motifs: GCC N3 GGC, GCC N4 GGC and GCC N3/4 GGG. The availability of these more refined consensus sequences should assist in the identification of target genes for this critical transcription factor.

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