The specificity of protein-DNA crosslinking by formaldehyde: in vitro and in Drosophila embryos

Abstract

Formaldehyde crosslinking has been widely used to study binding of specific proteins to DNA elements in intact cells. However, previous studies have not determined if this crosslinker preserves the bona fide pattern of DNA binding. Here we show that formaldehyde crosslinking of Drosophila embryos maps an interaction of the transcription factor Zeste to a known target element in the Ultrabithorax promoter. This data agrees broadly with previous mapping of the same Zeste binding sites by in vivo UV crosslinking, though the formaldehyde method does give a low, possibly artifactual signal on other DNA fragments that is not detected by the UV method. We also demonstrate, using an in vitro assay, that formaldehyde crosslinking accurately reflects the DNA binding specificities of both Zeste and a second transcription factor, Eve. The crosslinking reagent methylene blue is shown to preserve DNA binding specificity in vitro as well. Our results suggest that crosslinking by formaldehyde, and possibly also by methylene blue, provide an accurate guide to the interaction of proteins with their high affinity target sites in cells.

Targeting of Arabidopsis KNL2 to Centromeres Depends on the Conserved CENPC-k Motif in Its C Terminus

“…However, according to our ChIP data (Figure 4), in vivo KNL2 is preferentially associated to the centromeric repeat pAL1. Since formaldehyde crosslinking, used for ChIP, can occur at the protein-DNA interface and reflect the in vivo binding of a specific factor to its cognate DNA sites (Toth and Biggin, 2000), we suggest that KNL2 interacts with the centromeric DNA directly.…”

Forced binding of the origin of replication complex to chromosomal sites inDrosophilaS2 cells creates an origin of replication

“…Although this is likely to be a technical artefact caused by the spatial arrangements of components in the complex and the way that they are crosslinked (Toth and Biggin, 2000), it was decided that the primary construct used for further testing should the Orc5 fusion because this could be demonstrated to load all necessary components.…”

Genome-wide identification of in vivoDrosophilaEngrailed-binding DNA fragments and related target genes

“…We present a similar approach, performed on a larger scale, leading to a genome-wide view of direct Engrailed-binding loci in embryos. UV light or formaldehyde crosslinking are currently used to purify protein-DNA complexes, and to isolate specific binding fragments (Graba et al, 1992;Serrano et al, 1995;Serrano et al, 1997;Liang and Biggin, 1998;Cavalli et al, 1999;Toth and Biggin, 2000;Weinmann et al, 2001;Weinmann et al, 2002). However, UV light is believed to be more efficient in fixing proteins that are directly bound to DNA (Toth and Biggin, 2000).…”