Global Identification of Enhancers Using CHIP. P.T. Spellman 1, M.C. Holmes 1, M.D. Biggin 3, G.M. Rubin 2. 1) Dept MCB, Univ. California Berkeley, Berkeley, CA; 2) Howard Hughes Medical Institute, Berkeley, CA; 3) Life Sciences Dvision, Lawrence Berkeley National Laboratory, Berkeley, CA.
We are developing a high-throughput assay system for global characterization of Drosophila promoters. This system utilizes in vivo crosslinking and chromatin immunoprecipitation methods to isolate DNA sequences bound by transcription factors. DNA regions that co-immunoprecipitate with these sequence-specific DNA-binding factors are then assayed using DNA microarrays.
Microarrays can be used to measure the relative abundance of different sequences within a mixture of nucleic acid sequences by competitively hybridizing differentially labeled populations of nucleic acids. We constructed a microarray covering 3.6 Mb of Drosophila sequence (the Adh, Ubx, and Ant regions). Pairs of PCR primers spaced 1 Kb apart were designed, synthesized, and used to amplify the 3,600 fragments covering the three genomic regions from BAC templates. We are testing the sensitivity of this system. Early results indicate that fluorescently labeled DNA from 2-20ng of genomic DNA can be specifically and accurately detected on these microarrays.
We have performed chromatin immunoprecipitation studies using the transcription factor TRF1 for which some in vivo binding targets are known. Briefly, specific protein-DNA complexes are created in vivo by subjecting cells to a crosslinking agent, such as formaldehyde. The chromatin is purified, fragmented, and applied to beads containing antibodies to a DNA-binding protein of interest. DNA fragments isolated in the immunoprecipitations are eluted, purified, and assayed, in this protocol after PCR amplification, by microarray hybridization. Early data =confirmed known results that TRF1 binds to genomic regions that contain tRNA genes. We will present further studies showing results of TRF1, Zeste, and Adf-1.