title: DNA-binding sequence specificity of DUX4.
abstract: BACKGROUND
  Misexpression of the double homeodomain transcription factor DUX4 results in facioscapulohumeral muscular dystrophy (FSHD). A DNA-binding consensus with two tandem TAAT motifs based on chromatin IP peaks has been discovered; however, the consensus has multiple variations (flavors) of unknown relative activity. In addition, not all peaks have this consensus, and the Pitx1 promoter, the first DUX4 target sequence mooted, has a different TAAT-rich sequence. Furthermore, it is not known whether and to what extent deviations from the consensus affect DNA-binding affinity and transcriptional activation potential.
  
  
  RESULTS
  Here, we take both unbiased and consensus sequence-driven approaches to determine the DNA-binding specificity of DUX4 and its tolerance to mismatches at each site within its consensus sequence. We discover that the best binding and the greatest transcriptional activation are observed when the two TAAT motifs are separated by a C residue. The second TAAT motif in the consensus sequence is actually (T/C)AAT. We find that a T is preferred here. DUX4 has no transcriptional activity on "half-sites", i.e., those bearing only a single TAAT motif. We further find that DUX4 does not bind to the TAATTA motif in the Pitx1 promoter, that Pitx1 sequences have no competitive band shift activity, and that the Pitx1 sequence is transcriptionally inactive, calling into question PITX1 as a DUX4 target gene. Finally, by multimerizing binding sites, we find that DUX4 transcriptional activation demonstrates tremendous synergy and that at low DNA concentrations, at least two motifs are necessary to detect a transcriptional response.
  
  
  CONCLUSIONS
  These studies illuminate the DNA-binding sequence preferences of DUX4.
authors.affiliation: Lillehei Heart Institute, University of Minnesota, Minneapolis, MN 55455 USA ; Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455 USA.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455 USA.
authors.forename: John K
authors.initials: JK
authors.lastname: Lee
authors.affiliation: Lillehei Heart Institute, University of Minnesota, Minneapolis, MN 55455 USA ; Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455 USA.
authors.forename: Erik A
authors.initials: EA
authors.lastname: Toso
authors.affiliation: Department of Biomedical Sciences, University of Minnesota Medical School, Duluth, MN 55812 USA.
authors.forename: Joslynn S
authors.initials: JS
authors.lastname: Lee
authors.affiliation: Lillehei Heart Institute, University of Minnesota, Minneapolis, MN 55455 USA ; Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455 USA ; Research Center, Dongnam Institute of Radiological & Medical Sciences (DIRAMS), Busan, South Korea.
authors.forename: Si Ho
authors.initials: SH
authors.lastname: Choi
authors.affiliation: Department of Biomedical Sciences, University of Minnesota Medical School, Duluth, MN 55812 USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Slattery
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455 USA.
authors.forename: Hideki
authors.initials: H
authors.lastname: Aihara
authors.affiliation: Lillehei Heart Institute, University of Minnesota, Minneapolis, MN 55455 USA ; Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455 USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Kyba
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D016384:Consensus Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D057809:HEK293 Cells; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D059372:Nucleotide Motifs; D051761:Paired Box Transcription Factors / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D052156:SELEX Aptamer Technique; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
chemical_list: C121257:DUX4L1 protein, human; D018398:Homeodomain Proteins; D051761:Paired Box Transcription Factors; C101485:homeobox protein PITX1; D004247:DNA
keywords: DUX4; FSHD; Facioscapulohumeral muscular dystrophy; SELEX
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