MOLECULAR BIOTECHNOLOGY Volume 20, 2002 Analysis of Protein–Chromatin Interactions 1 1 Molecular Biotechnology 2002 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2002/20:1/1-15/$14.50 *Author to whom all correspondence and reprint requests should be addressed: Dr. Simon P. Chandler, Sangamo Biosciences Inc., Point Richmond Tech Center II, 501 Canal Blvd., Suite A100, Richmond, CA 04804. Email: schandler @sangamo.com. REVIEW Abstract The analysis of protein interactions with chromatin is vital for the understanding of DNA sequence recog- nition in vivo. Chromatin binding requires the interaction of proteins with DNA lying on the macromolecu- lar protein surface of nucleosomes, a situation that can alter factor binding characteristics substantially when compared with naked DNA. It is therefore important to study these protein–DNA interactions in the context of a chromatin substrate, the more physiologically relevant binding situation. In this article we review tech- niques used in the investigation of protein interactions with defined nucleosomal templates. Index Entries: Nucleosome, binding; EMSA; footprinting; transcription factors; chromatin immunopre- cipitation. 1. Introduction Eukaryotic DNA is packaged in the nucleus into chromatin. Chromatin compaction is achieved by the wrapping of the DNA onto a protein complex resulting in the fundamental repeating unit of chro- matin, the nucleosome (1). This compaction results in compartmentalization of the nucleus that facili- tates the activities of DNA metabolism and tran- scription by spatially separating active vs silenced regions of the genome (2,3). The chromatin must allow compaction of a large mass of DNA although retaining trans-acting factor sites in an exposed and accessible state (1,4). The nucleosome consists of a histone octamer made up of two histone H2A/H2B heterodimers bound to a tetramer core of histones H3 and H4 (Fig. 1A). DNA is wrapped onto this core octamer in two superhelical turns with a periodicity of ~10 bp, giving a total of 146 bp of DNA in intimate association with the core histones (1,5,6). Addi- tional DNA is present beyond the entry and exit positions of the core particle: This is spatially less well organized and is referred to as linker region sequences. This is the position of linker histone binding and represents the DNA that links adja- cent nucleosomes in a tandem array (Fig. 1B) (1,6–8). Specific sequences within a core particle are therefore oriented toward or away from the octamer surface, allowing increased binding specificity or occlusion of binding for transcrip- tion factors in consequence of the underlying chromatin structure (9,10). This means that pro- teins that have high affinity for purified DNA tar- get sites in vitro may have much altered binding characteristics when presented with DNA targets in a chromatin environment (10). Nucleosome mobility can also have an effect on protein bind- ing by masking DNA sites owing to translational movement along the DNA (1,11,12). It is there- fore important to study DNA–protein interactions in the context of chromatin. It is clear from the Methods for the Analysis of Protein–Chromatin Interactions Sarah J. Brickwood, Fiona A. Myers and Simon P. Chandler*