Members of transcription factor families typically have similar DNA binding specificities yet execute unique functions in vivo. Transcription factors often bind DNA as multiprotein complexes, raising the possibility that complex formation might modify their DNA binding specificities. To test this hypothesis, we developed an experimental and computational platform, SELEX-seq, that can be used to determine the relative affinities to any DNA sequence for any transcription factor complex. Applying this method to all eight Drosophila Hox proteins, we show that they obtain novel recognition properties when they bind DNA with the dimeric cofactor Extradenticle-Homothorax (Exd). Exd-Hox specificities group into three main classes that obey Hox gene collinearity rules and DNA structure predictions suggest that anterior and posterior Hox proteins prefer DNA sequences with distinct minor groove topographies. Together, these data suggest that emergent DNA recognition properties revealed by interactions with cofactors contribute to transcription factor specificities in vivo.
Bibliographical noteFunding Information:
We thank the members of the Bussemaker, Honig, Mann, and Rohs labs for comments and feedback during the course of these studies; B. Snyder for programming support; W. Shin and C. Hawkins for early contributions to the analysis of SELEX-seq data; A. Boyanov for sequencing support; and K. Lelli for constructs. This work was supported by NIH grants U54CA121852, R01GM054510, R01HG003008, R01GM030518, and P50GM071508, a John Simon Guggenheim Foundation Fellowship, Columbia University's RISE program, USC start-up funds, the USC-Technion Visiting Fellows Program, and an Andrew Viterbi Fellowship. Author contributions: M.S. designed and executed the SELEX experiments and contributed to the analysis of the SELEX data. T.R. designed and executed the analysis of the SELEX-seq data. P.L. carried out and analyzed Monte Carlo simulations. N.A. carried out later rounds of SELEX and EMSA-based validation experiments. P.G.-A. analyzed Ubx ChIP data. I.D. executed high-throughput DNA shape analysis. T.Z. developed high-throughput DNA shape prediction method. R.R. and B.H. supervised and designed DNA shape analyses. H.J.B. supervised and designed the analysis of the SELEX-seq data. R.S.M. supervised and designed SELEX experiments; contributed to the analysis of the SELEX data and shape analyses.