title: Crystal structure and DNA binding of the homeodomain of the stem cell transcription factor Nanog.
abstract: The transcription factor Nanog is an upstream regulator in early mammalian development and a key determinant of pluripotency in embryonic stem cells. Nanog binds to promoter elements of hundreds of target genes and regulates their expression by an as yet unknown mechanism. Here, we report the crystal structure of the murine Nanog homeodomain (HD) and analysis of its interaction with a DNA element derived from the Tcf3 promoter. Two Nanog amino acid pairs, unique among HD sequences, appear to affect the mechanism of nonspecific DNA recognition as well as maintain the integrity of the structural scaffold. To assess selective DNA recognition by Nanog, we performed electrophoretic mobility shift assays using a panel of modified DNA binding sites and found that Nanog HD preferentially binds the TAAT(G/T)(G/T) motif. A series of rational mutagenesis experiments probing the role of six variant residues of Nanog on its DNA binding function establish their role in affecting binding affinity but not binding specificity. Together, the structural and functional evidence establish Nanog as a distant member of a Q50-type HD despite having considerable variation at the sequence level.
authors.affiliation: Laboratory of Structural Biochemistry, Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore. jauchr@gis.a-star.edu.sg
authors.forename: Ralf
authors.initials: R
authors.lastname: Jauch
authors.forename: Calista Keow Leng
authors.initials: CK
authors.lastname: Ng
authors.forename: Kumar Singh
authors.initials: KS
authors.lastname: Saikatendu
authors.forename: Raymond C
authors.initials: RC
authors.lastname: Stevens
authors.forename: Prasanna R
authors.initials: PR
authors.lastname: Kolatkar
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000737:chemistry*; D018398:Homeodomain Proteins / Q000737:chemistry*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000071317:Nanog Homeobox Protein; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
chemical_list: D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse