Advanced methods for the analysis of chromatin-associated proteins

Abstract

DNA-protein interactions are central to gene expression and chromatin regulation and have become one of the main focus areas of the ENCODE consortium. Advances in mass spectrometry and associated technologies have facilitated studies of these interactions, revealing many novel DNA-interacting proteins and histone posttranslational modifications. Proteins interacting at a single locus or at multiple loci have been targeted in these recent studies, each requiring a separate analytical strategy for isolation and analysis of DNA-protein interactions. The enrichment of target chromatin fractions occurs via a number of methods including immunoprecipitation, affinity purification, and hybridization, with the shared goal of using proteomics approaches as the final readout. The result of this is a number of exciting new tools, with distinct strengths and limitations that can enable highly robust and novel chromatin studies when applied appropriately. The present review compares and contrasts these methods to help the reader distinguish the advantages of each approach.

Keywords: DNA-protein interactions; affinity purification; chromatin; chromatin enrichment; mass spectrometry.

Fig. 1.

Illustration of chromatin enrichment using chromatin immunoprecipitation (ChIP)-like methods. A: modified chromatin immunopurification (mChIP) uses histones with tandem affinity purification (TAP) tags to enrich local chromatin fragments. DNA shearing can be accomplished mechanically through sonication or enzymatically through micrococcal nuclease or DNase I digestion. B: chromatin-interacting protein in mass spectrometry (ChIP-MS). HTB-tags capable of undergoing endogenous biotinylation are attached to a target DNA-binding protein and used to capture a chromatin fragment by using streptavidin-coated magnetic beads. C: chromatin proteomics (ChroP). Nontreated chromatin can be sheared through micrococcal nuclease digestion, or cross-linked chromatin can be mechanically sheared through sonication. Chromatin fragments are enriched with antibodies against trimethylated histone variants.

Fig. 2.

Illustration of chromatin enrichment using locus-based methods. A: Proteomics of isolated chromatin segments (PICh). Cross-linked chromatin is sheared mechanically through sonication and captured using probes containing locked nucleic acids (LNA). Probes hybridize to telomere regions. B: chromatin affinity purification with mass spectrometry (ChAP-MS). A LexA binding site is genetically engineered immediately upstream of the GAL1 gene. A plasmid constitutively expressing LexA-PrA is introduced and used for affinity-based purification. Cross-linked chromatin is mechanically sheared through sonication. C: transcription activator-like-ChAP-MS (TAL-ChAP-MS). A plasmid constitutively expressing TAL-PrA, capable of binding a unique 18 nt sequence upstream of the GAL1 gene, is introduced and used for affinity-based purification.