Protein-DNA interaction mapping using genomic tiling path microarrays in Drosophila

Abstract

We demonstrate the use of a chromosomal walk (or "tiling path") printed as DNA microarrays for mapping protein-DNA interactions across large regions of contiguous genomic DNA in Drosophila melanogaster. Microarrays were constructed with genomic DNA fragments 430-920 bp in length, covering 2.9 million base pairs of the Adh-cactus region of chromosome 2 and 85,000 base pairs of the 82F region of chromosome 3. We performed DNA localization mapping for the heterochromatin protein HP1 and for the sequence-specific GAGA transcription factor, producing a comprehensive, high-resolution map of in vivo protein-DNA interactions throughout these regions of the Drosophila genome.

Fig. 1.

Tiling path construction for Adh–cactus and 82F chromosomal regions, and theoretical distributions for tiling path microarray results for a protein–DNA interaction experiment. (a) Polytene chromosomes 2L and 3R, with regions used for microarray construction indicated by square black boxes. (b) Design of tiling path. The thick stippled line represents genomic template DNA, arrows indicate forward (f) and reverse (r) primers, and thin lines below represent amplified overlapping DNA fragments printed on tiling path arrays. (c) A monotonic binding profile expected if binding occurs in DNA sequences at a single chromosomal site. (d) A multimodal binding profile expected if binding occurs in DNA sequences at multiple chromosomal sites in close proximity to one another. (e) Binding profile expected in the case of cross-hybridization to DNA elsewhere in the genome that is either near a binding site(s) or is itself bound.

Fig. 2.

GAF binding profiles. (a) GAF binding profiles across the entire 2.9-Mb Adh–cactus region. (b) GAF binding profiles in the 82F region. (c) Example of monotonic GAF binding profile in the CG11860 gene. Peak signal is seen in the first exon and intron of CG11860. Three GAGAG/CTCTC consensus binding sites are in the 5′ UTR of CG11860. (d) Example of a multimodal GAF binding profile, showing multiple binding sites of GAF throughout the Imaginal disk growth factor (Idgf) gene region. (e) Example of a pattern likely due to cross-hybridization. Two half monotonic curves are arranged as a near mirror image of one another with sharply decreasing signal at the ends of a blood transposable element at 2L:15329890 to 15339590. Black diamonds, GAGAG/CTCTC consensus binding sites; solid bars, exons; lines connecting solid bars, introns; gray bars, transposons. Standard errors are shown.

Fig. 3.

HP1 binding profiles. Profiles displayed as in Fig. 2. (a) HP1 binding profiles across the entire 2.9 Mb Adh–cactus region. (b) HP1 binding profiles in the 82F region. (c) Binding of HP1 in the crinkled gene. (d) An example of clear cross-hybridization from remote sequences associated with HP1 binding.