NORMAL GENOMIC

• Apuzzo S, Abdelhakim A, Fortin AS, Gros P
• Cross-talk between the paired domain and the homeodomain of Pax3: DNAbinding by each domain causes a structural change in the other domain,supporting interdependence for DNA Binding.
• J Biol Chem. 2004; 279: 33601-12
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• The Pax3 protein has two DNA binding domains, a Paired domain (PD) and apaired-type Homeo domain (HD). Although the PD and HD can bind to cognateDNA sequences when expressed individually, genetic and biochemical dataindicate that the two domains are functionally interdependent in intactPax3. The mechanistic basis of this functional interdependence is unknownand was studied by protease sensitivity. Pax3 was modified by the creationof Factor Xa cleavage sites at discrete locations in the PD, the HD, andin the linker segment joining the PD and the HD (Xa172, Xa189, and Xa216)in individual Pax3 mutants. The effect of Factor Xa insertions on proteinstability and on DNA binding by the PD and the HD was measured usingspecific target site sequences. Independent insertions at position 100 inthe linker separating the first from the second helix-turn-helix motif ofthe PD and at position 216 immediately upstream of the HD were found to bereadily accessible to Factor Xa cleavage. The effect of DNA binding by thePD or the HD on accessibility of Factor Xa sites inserted in the same orin the other domain was monitored and quantitated for multiple mutantsbearing different numbers of Xa sites at each position. In general, DNAbinding reduced accessibility of all sites, suggesting a more compact andless solvent-exposed structure of DNA-bound versus DNA-free Pax3. Resultsof dose response and time course experiments were consistent and showedthat DNA binding by the PD not only caused a local structural change inthe PD but also caused a conformational change in the HD (P3OPT binding toXa216 mutants); similarly, DNA binding by the HD also caused aconformational change in the PD (P2 binding to Xa100 mutants). Theseresults provide a structural basis for the functional interdependence ofthe two DNA binding domains of Pax3.

• Aishima J, Wolberger C
• Insights into nonspecific binding of homeodomains from a structure ofMATalpha2 bound to DNA.
• Proteins. 2003; 51: 544-51
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• The 2.1-A resolution crystal structure of the MATalpha2 homeodomain boundto DNA reveals the unexpected presence of two nonspecifically bound alpha2homeodomains, in addition to the two alpha2 homeodomains bound tocanonical alpha2 binding sites. One of the extra homeodomains makes fewbase-specific contacts, while the other extra homeodomain binds to DNA ina previously unobserved manner. This unusually bound homeodomain isrotated on the DNA, making possible major groove contacts by side-chainsthat normally do not contact the DNA. This alternate docking may representone way in which homeodomains sample nonspecific DNA sequences.

• van Dongen MJ, Cederberg A, Carlsson P, Enerback S, Wikstrom M
• Solution structure and dynamics of the DNA-binding domain of theadipocyte-transcription factor FREAC-11.
• J Mol Biol. 2000; 296: 351-9
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• Transcription factors of the forkhead type share a highly conservedDNA-binding domain of about 100 amino acid residues. FREAC-11, expressedin adipocytes, belongs to this class. Here, we report on NMR studies thatestablished the three-dimensional structure of the FREAC-11, DNA-bindingdomain. Although apparent similarities to the structures of other memberswithin the forkhead family are observed, the structure also reveals someremarkable differences. Along with the complementary dynamics, the dataprovide insight into the fundamentals of sequence specificity within ahighly conserved motif.

• Carr A, Biggin MD
• A comparison of in vivo and in vitro DNA-binding specificities suggests anew model for homeoprotein DNA binding in Drosophila embryos.
• EMBO J. 1999; 18: 1598-608
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• Little is known about the range of DNA sequences bound by transcriptionfactors in vivo. Using a sensitive UV cross-linking technique, we showthat three classes of homeoprotein bind at significant levels to themajority of genes in Drosophila embryos. The three classes bind withspecificities different from each other; however, their levels of bindingon any single DNA fragment differ by no more than 5- to 10-fold. Onactively transcribed genes, there is a good correlation between the invivo DNA-binding specificity of each class and its in vitro DNA-bindingspecificity. In contrast, no such correlation is seen on inactive orweakly transcribed genes. These genes are bound poorly in vivo, eventhough they contain many high affinity homeoprotein-binding sites. Basedon these results, we suggest how the in vivo pattern of homeoprotein DNAbinding is determined.

• Rupert PB, Daughdrill GW, Bowerman B, Matthews BW
• A new DNA-binding motif in the Skn-1 binding domain-DNA complex.
• Nat Struct Biol. 1998; 5: 484-91
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• The DNA-binding domain of Skn-1, a developmental transcription factor thatspecifies mesoderm in C. elegans, is shown by X-ray crystallography tohave a novel fold in which a compact, monomeric, four-helix unit organizestwo DNA-contact elements. At the C-terminus, a helix extends from thedomain to occupy the major groove of DNA in a manner similar to bZipproteins. Skn-1, however, lacks the leucine zipper found in all bZips.Additional contacts with the DNA are made by a short basic segment at theN-terminus of the domain, reminiscent of the 'homeodomain arm'.

• Justice MC, Hogan BP, Vershon AK
• Homeodomain-DNA interactions of the Pho2 protein are promoter-dependent.
• Nucleic Acids Res. 1997; 25: 4730-9
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• The homeodomain (HD) is a conserved sequence-specific DNA-binding motiffound in many eukaryotic transcriptional regulatory proteins. Despite thewealth of in vitro data on the mechanism HD proteins use to bind DNA,comparatively little is known about the roles of individual residues inthese domains in vivo . The Saccharomyces cerevisiae Pho2 protein containsa HD that shares significant sequence identity with the DrosophilaEngrailed protein. We have used the co-crystal structure of Engrailed as amodel to predict how Pho2 might contact DNA and have examined howindividual residues of the Pho2 HD contribute to transcriptionalactivation in vivo and to DNA binding in vitro. Our results demonstratethat Pho2 and Engrailed share many similar DNA-binding characteristics.However, our results also show that some highly conserved residues, whichcontact the DNA in many HD structures, make relatively small contributionsto the DNA-binding affinity and in vivo activity of the Pho2 protein. Wealso show that the N-terminal arm of the Pho2 HD is a critical componentin determining the DNA-binding specificity of the protein and that therequirements for residues in the N-terminal arm are promoter-dependent forPho2 transcriptional activation and DNA binding.

• Stepchenko AG, Polianovskii OL
• [Interaction of Oct-proteins with DNA].
• Mol Biol (Mosk). 1996; 30: 503-13

• Chen CY, Schwartz RJ
• Recruitment of the tinman homolog Nkx-2.5 by serum response factoractivates cardiac alpha-actin gene transcription.
• Mol Cell Biol. 1996; 16: 6372-84
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• We recently showed that the cardiogenic homeodomain factor Nkx-2.5 servedas a positive acting accessory factor for serum response factor (SRF) andthat together they provided strong transcriptional activation of thecardiac alpha-actin promoter, depending upon intact serum responseelements (SREs) (C. Y. Chen, J. Croissant, M. Majesky, S. Topouz, T.McQuinn, M. J. Frankovsky, and R. J. Schwartz, Dev. Genet. 19:119-130,1996). As shown here, Nkx-2.5 and SRF collaborated to activate theendogenous murine cardiac alpha-actin gene in 10T1/2 fibroblasts by amechanism in which SRF recruited Nkx-2.5 to the alpha-actin promoter.Activation of a truncated promoter consisting of the proximal alpha-actinSRE1 occurred even when Nkx-2.5 DNA-binding activity was blocked by apoint mutation in the third helix of its homeodomain. Investigation ofprotein-protein interactions showed that Nkx-2.5 was bound to SRF in theabsence of DNA in soluble protein complexes retrieved from cardiac myocytenuclei but could also be detected in coassociated binding complexes on theproximal SRE1. Recruitment of Nkx-2.5 to an SRE depended upon SRFDNA-binding activity and was blocked by the dominant negative SRFpm1mutant, which allowed for dimerization of SRF monomers but prevented DNAbinding. Interactive regions shared by Nkx-2.5 and SRF were mapped toN-terminal/helix I and helix II/helix III regions of the Nkx-2.5homeodomain and to the N-terminal extension of the MADS box. Our studysuggests that physical association between Nkx-2.5 and SRF is one way thatcardiac specified genes are activated in cardiac cell lineages.

• Zilliacus J, Wright AP, Carlstedt-Duke J, Gustafsson JA
• Structural determinants of DNA-binding specificity by steroid receptors.
• Mol Endocrinol. 1995; 9: 389-400

• McClellan JA
• Osmium tetroxide modification and the study of DNA-protein interactions.
• Methods Mol Biol. 1994; 30: 97-112

• Billeter M, Neri D, Otting G, Qian YQ, Wuthrich K
• Precise vicinal coupling constants 3JHN alpha in proteins from nonlinearfits of J-modulated [15N,1H]-COSY experiments.
• J Biomol NMR. 1992; 2: 257-74
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• Improved experimental schemes for the recently introduced J-modulated[15N,1H]-correlation experiment for measurements of the homonuclear amideproton-C alpha proton vicinal coupling constants, 3JHN alpha, in uniformly15N-labeled proteins are described, and a nonlinear fit procedure ispresented for quantitative evaluation of 3JHN alpha. The method was firsttested with the N-terminal DNA-binding domain of the 434 repressor (M =7.3 kDa), where at 13 degrees C precise values of 3JHN alpha in the range2.0-9.5 Hz were obtained for all residues with resolved 15N-1H crosspeaks. It was then applied to the Antennapedia homeodomain complexed to asynthetic 14-base pair DNA fragment (molecular weight of the complexapproximately 18 kDa). The 3JHN alpha values measured were found to be inexcellent agreement with those predicted from the secondary structure ofthis protein in the complex.

• Joliot AH, Triller A, Volovitch M, Pernelle C, Prochiantz A
• alpha-2,8-Polysialic acid is the neuronal surface receptor of antennapediahomeobox peptide.
• New Biol. 1991; 3: 1121-34
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• A synthetic peptide that is 60 amino acids in length and corresponds tothe homeobox sequence of antennapedia protein (pAntp) is specifically andefficiently captured by neurons in culture and conveyed to their nuclei.The internalization process is followed by a strong induction of neuronalmorphological differentiation. In the study described here, all treatmentsmasking or removing the alpha-2,8-polysialic acid (PSA) chains specific tothe neuronal cell adhesion molecule (NCAM) were found to block thepenetration of pAntp and abolish its morphogenetic effects. Structuralcomparison between PSA and double-stranded DNA suggests that a sequence ofeight sialic acid residues can mimic one large groove of the DNA. Wepropose that this structural similarity is the basis for the property ofNCAM polysialic acid to participate in the internalization of the homeboxpolypeptide.

• Lim VI, Mazanov AL
• Tertiary structure for palindromic regions of DNA.
• FEBS Lett. 1978; 88: 118-23