ComparisonofDamtaggingandchromatinimmunoprecipitationastoolsfor the identi¢cation of the binding sites for S. pombe CENP-C Sara Holland, Dimitris Ioannou, Steven Haines & William R. A. Brown* Institute of Genetics, School of Biology, Queen’s Medical Centre, Nottingham, NG7 2UH, UK; Tel: þ44(0)1158-493244; Fax: þ44(0)1159-709906; E-mail: William.brown@nottingham.ac.uk *Correspondence Received 16 September 2004. Received in revised form and accepted for publication by Adrian Sumner 18 November 2004 Key words: centromere, ChiP, chromatin, DAM tagging, Schizosaccharomyces Abstract We have established the identity of the Schizosaccharomyces pombe homologue of vertebrate CENP-C and Saccharomyces cerevisiae MIF2p and have used it to compare Dam tagging and chromatin immunoprecipitation (ChiP) as tools for the mapping of protein binding sites on DNA. ChiP shows that S. pombe CENP-C binds to the central core and inner repeats of the S. pombe centromere. It binds weakly, however, to the outer repeats. The binding pattern is thus similar to that of S. pombe CENP-A. Dam-tagged S. pombe CENP-C, however, methylates the entire centromere and 5 kb of flanking DNA. This comparison suggests that Dam tagging is less precise as a tool for mapping DNA binding sites than ChiP. We have also used the Dam tagging technique to address the question of whether there is any CENP-C binding to the ribosomal DNA in S. pombe and find none. Introduction The analysis of many problems in molecular genetics requires the mapping of DNA protein interactions in vivo. The standard technique for this type of mapping is chromatin immunopreci- pitation (ChiP; Orlando et al. 1997, Strutt & Paro 1999). ChiP uses immunoprecipitation of chromatin and quantitation of the precipitated DNA to identify sequences that are specifically bound by the protein antigen. The mechanistic understanding of many areas of biology relies heavily upon the results from just one techni- que. Given the indirect nature of ChiP and the fundamental importance of this type of map- ping, it would be useful to have an alternative methodology. Van Steensel and colleagues have suggested the use of the N 6 adenine methylase domain of the pro- tein encoded by the E. coli dam gene as a tag with which to identify protein binding sequences within eukaryotic genomes (van Steensel & Heniko¡ 2000; van Steensel et al. 2001). Dam methyl transferase methylates adenine residues within the sequence GATC. Eucaryotic DNA is not usually methy- lated at this residue. The restriction enzyme DpnI only cuts at methylated G me ATC and correspond- ingly does not cleave most eucaryotic DNA. Bind- ing of a protein tagged with the dam methylase would be expected to methylate the adjacent sequences and thus confer upon them sensitivity to cleavage by DpnI. The use of Dam tags in this way has been used to map binding sites for various tran- scription factors in Drosophila (Orian et al. 2003). Chromosome Research 13: 73–83, 2005. 73 # 2005 Springer. Printed in the Netherlands