195 Oral - Techniques and Technology Session
Saturday April 09, 4:45 PM - 5:00 PM

Authors:
Siqian Feng; Richard Mann

Affiliation: Jerome L. Greene Science Center, Columbia University, New York, NY

Keywords:
k. next-generation sequencing; b. transcription initiation/elongation/termination

A major drawback of chromatin immunoprecipitation (ChIP) based techniques is the lack of cell type-specificity. To overcome this limitation, we developed SpyChIP to identify sites of cell type-specific transcription factor (TF) occupancy in native physiological contexts without tissue dissociation or nuclei sorting. SpyChIP takes advantage of a specific covalent isopeptide bond that rapidly forms between the 15 amino acid SpyTag and its binding partner, the 17 kD protein SpyCatcher. In SpyChIP, the target TF is fused with SpyTag by genome engineering, and an epitope tagged SpyCatcher is expressed by the Gal4/UAS system in cell populations of interest, where it covalently links to SpyTag-TF. Cell type-specific ChIP results are obtained by performing ChIP against the epitope on SpyCatcher using chromatin prepared from whole tissue.

Using SpyChIP, we characterized the genome-wide binding profiles of the Hox protein Ultrabithorax (Ubx) in two non-overlapping domains of the Drosophila haltere imaginal disc. Our results revealed extensive region-specific Ubx-DNA binding events, thus highlighting the significance of cell type-specific ChIP results and the limitations of whole tissue ChIP approaches. Analysis of SpyChIP results for Ubx provided novel insights into the relationship between chromatin accessibility and Ubx-DNA binding, as well as different mechanisms Ubx employs to regulate different downstream cis-regulatory modules (CRMs). We envision that the covalent bond between SpyTag and SpyCatcher will have wide-spread in vivo applications, and our demonstration of SpyChIP sets the stage for carrying out many other cell type-specific characterizations and manipulations in vivo that were previously unachievable.