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Book of Abstracts: Albany 2011

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

Exploring the DNA Binding Specificities of Homeodomain Complexes Using SELEX-sequencing

Hox genes encode an evolutionarily conserved set of homeodomain-containing transcriptional regulators that play critical roles in the development of all metazoans. Although first discovered in Drosophila because of their role in anterior (A)-posterior (P) axial patterning, these genes are now known to assign morphological identities along the AP axes in both vertebrates and invertebrates (1). Hox proteins typically bind to degenerate AT-rich DNA sequences. This low degree of sequence specificity in vitro contrasts with the highly gene-specific regulation Hox proteins carry out in vivo. Complicating the Hox specificity problem is that this family of proteins, which are encoded by eight Hox paralogs in Drosophila and 39 Hox genes in vertebrates, all bind to very similar DNA sequences via identical DNA-contacting residues in their homeodomains (2). One way in which Hox proteins achieve a higher degree of DNA binding specificity is to bind cooperatively with cofactors. One such cofactor is a heterodimer composed of Extradenticle (Exd; Pbx in vertebrates) and its binding partner Homothorax (Hth; Meis in vertebrates), both homeodomain proteins (2). Together, Exd-Hth bind cooperatively with Hox proteins, allowing them to recognize features of the DNA that cannot be read in the absence of these cofactors.

To explore the DNA binding specificities of the Hox transcription factors and their cofactor Exd, we have combined the traditional method of SELEX (Systematic Evolution of Ligands by Exponential Enrichment), which can be used to select DNA binding sites, with next generation sequencing technology (e.g. Illumina sequencing). This combination, termed SELEX-seq, allows the identification of the complete repertoire of binding sites for any Hox-Exd combination after only a small number of SELEX rounds. Subtle differences in DNA binding preferences among the Hox-Exd complexes can be clearly observed using this technology. In general Hox-Exd complexes seem to use a modular binding site architecture, in which an octomer core sequence determines the DNA signature motif for various Hox-Exd dimers while flanking nucleotides adjust the absolute affinity of a signature motif. Importantly, these motifs can explain certain aspects of in vivo Hox function, and our findings suggest that signature motif selectivity is achieved in part by Hox-Exd recognition of structural features of the DNA.

References

  1. T. Iimura, O. Pourquie, Dev. Growth Differ. 49, 265-275 (2007).
  2. R.S. Mann, K.M. Lelli, R. Joshi, Curr. Top. Dev. Biol. 88, 63-101 (2009).

Matthew Slattery1,4
Todd R. Riley2,3
Namiko Abe 1
Harmen J. Bussemaker2,3,*
Richard S. Mann1,*

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

ph: (773)-834-0074
fx: (773)-834-2877
mgslatte@gmail.com