Once conceived as an inert scaffold to package feet of DNA into each cell, chromatin is now recognized to have a multiplicity of functions in genome regulation. The basic building block of chromatin, the nucleosome, comprises an octamer of histone proteins wrapped by ~146 basepairs of DNA. Histone proteins are subject to over one hundred known post-translational modifications, including acetylation, methylation, ADP-ribosylation, ubiquitination, and phosphorylation [1
]. These modifications occur on the side chains of specific residues in the histone tails and cores and functionally impact transcription, replication, recombination, and repair. Accordingly, chromatin modifying proteins can show cell type specific phenotypes in gain- or loss-of-function studies [1
]. Lysine acetylation and methylation are both reversible processes thanks to the activities of histone deacetylases and recently discovered demethylases [1
]. Both of these enzyme families are implicated in a range of developmental and physiologic pathways as well as in malignancy [1
Our ability to interrogate chromatin structure has been transformed in the past few years by a convergence of advances in chromatin antibody development and genomic technologies. Since its introduction 20 years ago, the chromatin immunoprecipitation (ChIP) assay has gradually become a mainstay for chromatin biology experimentation [7
]. This procedure uses specific antibodies to enrich genomic DNA associated with a particular histone modification or chromosomal protein in vivo
. The enriched DNA can then be interrogated by PCR (ChIP-PCR), microarray hybridization (ChIP-chip), or high-throughput sequencing (ChIP-Seq). PCR can be used to query a limited number of genomic positions – e.g., a small panel of known promoters. In contrast, high-density microarrays with probes placed at regular intervals (‘tiling arrays’) can be used to query large domains such as chromosomes or the whole genome, although the latter can be costly (reviewed in [10
]). More recently, next-generation sequencing platforms have been applied to chromatin state mapping [11
]. These new technologies can sequence ChIP DNA at sufficient depth to enable accurate and high-resolution whole genome analysis. Several technical advantages, including rapid throughput, high resolution, better genome coverage and the ability to interrogate comparatively small amounts of ChIP DNA, make the sequencing approach particularly powerful.
The power of genomic technologies to inform accurately on biology is contingent on high quality reagents. A vast number of chromatin modifications and structural proteins have been identified to date, and an immense number of antibody reagents have been raised against these epitopes. However, the specificity of these reagents and their efficacy in ChIP may vary widely depending on the different epitopes, antibody sources, and bleeds. As with any chromatin assay, precise standardization of these starting reagents is essential for effective genome-scale characterization.
Genome-scale chromatin assays also require large numbers of cells, and homogeneity of the input population is critical for clarity of data interpretation. Because of this requirement, most experiments to date have focused on cell lines that can be readily expanded in culture. However, recent studies have leveraged techniques in stem cell biology, in vitro
differentiation and cell sorting to obtain populations of high biological interest. Genome-wide maps for a number of chromatin marks have now been reported for human and mouse embryonic stem (ES) cells, neural progenitor cells, fibroblasts, hepatocytes and T-cells [11