Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
DNA Shape Patterns and Binding Specificities for Drosophila Hox Proteins
The Hox family of transcription factors is essential for the patterning of the anterior-posterior axis, stem cell maintenance, and motor neuron specification in Drosophila (1-3). This family of transcription factors carries out highly specific gene regulation; however, the source of specificity for binding of Hox proteins to DNA is poorly understood. Hox proteins, such as Ultrabithorax (Ubx) and Sex combs reduced (Scr), bind as monomers to degenerate DNA sequences with little specificity. In the presence of the homeodomain-containing cofactors Extradenticle (Exd; Pbx in vertebrates) and Homothorax (Hth; Meis in vertebrates), Hox proteins bind DNA sequences with a high degree of specificity, in part by recognizing sequence-dependent DNA structural features, such as minor groove shape and electrostatic potential (4).
In order to determine DNA structural features that are associated with specific binding to Hox proteins, we are employing hydroxyl radical cleavage chemistry to probe the structures of DNA sequences that have been identified as high-affinity binding sites by the Mann and Bussemaker labs using the SELEX-seq technique. The hydroxyl radical cleavage pattern depends on the local solvent accessibility of each nucleotide in a duplex DNA molecule, thereby yielding a representation of the sequence-dependent shape of a DNA molecule. We compare our results with Monte Carlo-based computational predictions of DNA structure made by the Rohs and Honig laboratories (5). We also use the ORChID (OH Radical Cleavage Intensity Database) and ORChID2 algorithms developed by our laboratory (6) to compute the sequence-dependent structure of SELEX-seq-identified binding sites.
In previous work our laboratory used ORChID to show that similar DNA structures can arise from different DNA sequences (6). This result suggests that nucleotide sequence similarity alone may not be enough to recognize all classes of Hox binding sites. By experimentally determining the shapes of DNA sequences that bind to Hox proteins, and then comparing our experimental structural results to Monte Carlo- and ORChID-based computational predictions of structure, we can identify DNA shape-based binding preferences for Hox transcription factors.
Dana R. Holcomb
Department of Chemistry