ChIP-seq and beyond: new and improved methodologies to detect and characterize protein-DNA interactions.

Chromatin immunoprecipitation experiments followed by sequencing (ChIP-seq) detect protein-DNA binding events and chemical modifications of histone proteins. Challenges in the standard ChIP-seq protocol have motivated recent enhancements in this approach, such as reducing the number of cells that are required and increasing the resolution. Complementary experimental approaches - for example, DNaseI hypersensitive site mapping and analysis of chromatin interactions that are mediated by particular proteins - provide additional information about DNA-binding proteins and their function. These data are now being used to identify variability in the functions of DNA-binding proteins across genomes and individuals. In this Review, I describe the latest advances in methods to detect and functionally characterize DNA-bound proteins.

Protein-DNA binding in high-resolution.

Recent advances in experimental and computational methodologies are enabling ultra-high resolution genome-wide profiles of protein-DNA binding events. For example, the ChIP-exo protocol precisely characterizes protein-DNA cross-linking patterns by combining chromatin immunoprecipitation (ChIP) with 5' → 3' exonuclease digestion. Similarly, deeply sequenced chromatin accessibility assays (e.g. DNase-seq and ATAC-seq) enable the detection of protected footprints at protein-DNA binding sites. With these techniques and others, we have the potential to characterize the individual nucleotides that interact with transcription factors, nucleosomes, RNA polymerases and other regulatory proteins in a particular cellular context. In this review, we explain the experimental assays and computational analysis methods that enable high-resolution profiling of protein-DNA binding events. We discuss the challenges and opportunities associated with such approaches.

Sequence census methods for functional genomics.

Next-generation sequencing technologies are beginning to facilitate genome sequencing. But in addition, new applications and new assay concepts have emerged that are vastly increasing our ability to understand genome function.

The first decade of estrogen receptor cistromics in breast cancer.

The advent of genome-wide transcription factor profiling has revolutionized the field of breast cancer research. Estrogen receptor α (ERα), the major drug target in hormone receptor-positive breast cancer, has been known as a key transcriptional regulator in tumor progression for over 30 years. Even though this function of ERα is heavily exploited and widely accepted as an Achilles heel for hormonal breast cancer, only since the last decade we have been able to understand how this transcription factor is functioning on a genome-wide scale. Initial ChIP-on-chip (chromatin immunoprecipitation coupled with tiling array) analyses have taught us that ERα is an enhancer-associated factor binding to many thousands of sites throughout the human genome and revealed the identity of a number of directly interacting transcription factors that are essential for ERα action. More recently, with the development of massive parallel sequencing technologies and refinements thereof in sample processing, a genome-wide interrogation of ERα has become feasible and affordable with unprecedented data quality and richness. These studies have revealed numerous additional biological insights into ERα behavior in cell lines and especially in clinical specimens. Therefore, what have we actually learned during this first decade of cistromics in breast cancer and where may future developments in the field take us?

Chromatin immunoprecipitation assay.

Association between proteins and DNA is crucial for many vital cellular functions such as gene transcription, DNA replication and recombination, repair, segregation, chromosomal stability, cell cycle progression, and epigenetic silencing. It is important to know the genomic targets of DNA-binding proteins and the mechanisms by which they control and guide gene regulation pathways and cellular proliferation. Chromatin immunoprecipitation (ChIP) is an important technique in the study of protein-gene interactions. Using ChIP, DNA-protein interactions are studied within the context of the cell. The basic steps in this technique are fixation, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. Although ChIP is a very versatile tool, the procedure requires the optimization of reaction conditions. Several modifications to the original ChIP technique have been published to improve the success and to enhance the utility of this procedure. This review addresses the critical parameters and the variants of ChiP as well as the different analytical tools that can be combined with ChIP to enable better understanding of DNA-protein interactions in vivo.

The current state of chromatin immunoprecipitation.

The biological significance of interactions of nuclear proteins with DNA in the context of gene expression, cell differentiation, or disease has immensely been enhanced by the advent of chromatin immunoprecipitation (ChIP). ChIP is a technique whereby a protein of interest is selectively immunoprecipitated from a chromatin preparation to determine the DNA sequences associated with it. ChIP has been widely used to map the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin modifying enzymes on the genome or on a given locus. In spite of its power, ChIP has for a long time remained a cumbersome procedure requiring large numbers of cells. These limitations have sparked the development of modifications to shorten the procedure, simplify sample handling and make ChIP amenable to small numbers of cells. In addition, the combination of ChIP with DNA microarray and high-throughput sequencing technologies has in recent years enabled the profiling of histone modification, histone variants, and transcription factor occupancy on a genome-wide scale. This review highlights the variations on the theme of the ChIP assay, the various detection methods applied downstream of ChIP, and examples of their application.

The state-of-the-art of chromatin immunoprecipitation.

The biological significance of interactions of nuclear proteins with DNA in the context of gene expression, cell differentiation, or disease has immensely been enhanced by the advent of chromatin immunoprecipitation (ChIP). ChIP is a technique whereby a protein of interest is selectively immunoprecipitated from a chromatin preparation to determine the DNA sequences associated with it. ChIP has been widely used to map the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes on the genome or on a given locus. In spite of its power, ChIP has for a long time remained a cumbersome procedure requiring large number of cells. These limitations have sparked the development of modifications to shorten the procedure, simplify the sample handling, and make the ChIP amenable to small number of cells. In addition, the combination of ChIP with DNA microarray, paired-end ditag, and high-throughput sequencing technologies has in recent years enabled the profiling of histone modifications and transcription factor occupancy on a genome-wide scale. This review highlights the variations on the theme of the ChIP assay, the various detection methods applied downstream of ChIP, and examples of their application.

Applications of DNA tiling arrays for whole-genome analysis.

DNA microarrays are a well-established technology for measuring gene expression levels. Microarrays designed for this purpose use relatively few probes for each gene and are biased toward known and predicted gene structures. Recently, high-density oligonucleotide-based whole-genome microarrays have emerged as a preferred platform for genomic analysis beyond simple gene expression profiling. Potential uses for such whole-genome arrays include empirical annotation of the transcriptome, chromatin-immunoprecipitation-chip studies, analysis of alternative splicing, characterization of the methylome (the methylation state of the genome), polymorphism discovery and genotyping, comparative genome hybridization, and genome resequencing. Here we review different whole-genome microarray designs and applications of this technology to obtain a wide variety of genomic scale information.