Book of Abstracts: Albany 2011

category image Albany 2011
Conversation 17
June 14-18 2011
©Adenine Press (2010)

Dimerization with Extradenticle Unlocks Latent DNA Binding Specificities that Discriminate Hox Proteins in Drosophila

Hox proteins constitute a subclass of the large homeodomain family of transcription factors. They play a crucial role in body plan and tissue specification throughout the animal kingdom (1). In the transcriptional network of the cell, each Hox protein targets a distinct set of genes. Little is currently understood about the differences in DNA binding specificity that underlie these functional differences. In particular, Hox monomers have been shown to bind DNA with similar sequence preferences (2,3). We developed a method (“SELEX-seq”) that couples in vitro selection of pools of random DNA with massively parallel sequencing. In combination with a novel computational method for analyzing the data, based on a biophysical model, it allows us to quantify relative binding affinities at unprecedented resolution for all 12-mer sequences. Application of SELEX-seq to dimers of each of the eight Drosophila Hox proteins with the co-factor Extradenticle (Exd) revealed the DNA binding specificities for this family of homeodomain proteins. Only in the context of the Exd-Hox complex is the identity of the Hox protein manifested, through major differences in how base identities in the core of the binding site are recognized. Our analysis reveals that most Hox-Exd dimers have a unique DNA recognition signature. These differences in binding specificity in vitro may go a long way towards explaining the functional differences between the Hox proteins observed in vivo.

We present a novel method, based on a biophysical model of the SELEX procedure, which is capable of estimating binding affinities between a protein or protein complex of interest and all oligonucleotide sequences of a given length with great accuracy. Relative affinities are obtained by comparing the sequence composition of later rounds to that of the initial round. In this comparison our method takes into account the significant sequence biases in the initial pool of dsDNA molecules. While the higher sequence counts associated with the later rounds of SELEX may be preferable in terms of statistical accuracy, the accumulation of PCR-amplification noise and binding-site saturation leads to systematic error in the estimated affinities. We developed a procedure that uses the earlier and later rounds together, and thereby obtains affinities that are both accurate and precise.

The unprecedented depth of the sequencing data allowed us to systematically determine the effective length of the Exd-Hox dimer. While a 12nt degenerate consensus binding motif emerged for all Hox identities, we found that there exist striking differences in how different Exd-Hox dimers interact with the central hexanucleotide of the binding site. By quantifying the differences in affinity for each class of binding motif, we were able to identify a unique DNA recognition signature for each of the Exd-Hox dimers.


  1. S. Banerjee-Basu, A.D. Baxevanis, Nucleic Acids Res. 29, 3258–3269 (2001).
  2. M.F. Berger, G. Badis, A.R. Gehrke, S. Talukder, A.A. Philippakis, L. Pena-Castillo, T.M. Alleyne, S. Mnaimneh, O.B. Botvinnik, et al., Cell 133, 1266-1276 (2008).
  3. M.B. Noyes, R.G. Christensen, A. Wakabayashi, G.D. Stormo, M.H. Brodsky, S.A. Wolfe, Cell 133, 1277–1289 (2008).

Todd R. Riley 1,2,*
Matthew Slattery3, 4
Namiko Abe3
Harmen J. Bussemaker1,2,*
Richard S. Mann3,*

1 Columbia University
Department of Biological Sciences
1212 Amsterdam Avenue
New York, NY, 10027
2 Columbia University
Center for Computational Biology and Bioinformatics
1130 St Nicholas Avenue
New York, NY, 10032
3 Department of Biochemistry and Molecular Biophysics
Columbia University
701 West 168th Street HHSC 1104
New York, NY 10032
4 Current address:
Institute for Genomics and Systems Biology
University of Chicago
900 East 57th Street KCBD 10115
Chicago, IL 60637

Ph: 7329794414
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