ChIP‐based methods for the identification of long‐range chromatin interactions

Chromatin immunoprecipitation (ChIP) is an important technique for studying protein–DNA interactions. Whole genome ChIP methods have enjoyed much success, but are limited in that they cannot uncover important long‐range chromatin interactions. Chromosome conformation capture (3C) and related methods are capable of detecting remote chromatin interactions, but are tedious, have low signal‐to‐noise ratios, and are not genome‐wide. Although the addition of ChIP to 3C (ChIP–3C) would conceivably reduce noise and increase specificity for chromatin interaction detection, there are concerns that simple mixing of the ChIP and 3C protocols would lead to high levels of false positives. In this essay, we dissect current ChIP‐ and 3C‐based methodologies, discuss the models of specific as opposed to non‐specific chromatin interactions, and suggest approaches to separate specific chromatin complexes from non‐specific chromatin fragments. We conclude that the combination of sonication‐based chromatin fragmentation, ChIP‐based enrichment, chromatin proximity ligation and Paired‐End Tag ultra‐high‐throughput sequencing will be a winning implementation for genome‐wide, unbiased and de novo discovery of long‐range chromatin interactions, which will help to establish an emerging field for studying human chromatin interactomes and genome regulation networks in three‐dimensional spaces. J. Cell. Biochem. 107: 30–39, 2009. © 2009 Wiley‐Liss, Inc.     

Immediate chromatin immunoprecipitation and on-bead quantitative PCR analysis: a versatile and rapid ChIP procedure

Genome-wide chromatin immunoprecipitation (ChIP) studies have brought significant insight into the genomic localization of chromatin-associated proteins and histone modifications. The large amount of data generated by these analyses, however, require approaches that enable rapid validation and analysis of biological relevance. Furthermore, there are still protein and modification targets that are difficult to detect using standard ChIP methods. To address these issues, we developed an immediate chromatin immunoprecipitation procedure which we call ZipChip. ZipChip significantly reduces the time and increases sensitivity allowing for rapid screening of multiple loci. Here we describe how ZipChIP enables detection of histone modifications (H3K4 mono- and trimethylation) and two yeast histone demethylases, Jhd2 and Rph1, which were previously difficult to detect using standard methods. Furthermore, we demonstrate the versatility of ZipChIP by analyzing the enrichment of the histone deacetylase Sir2 at heterochromatin in yeast and enrichment of the chromatin remodeler, PICKLE, at euchromatin in Arabidopsis thaliana.     

Chromosome conformation capture technologies and their impact in understanding genome function

It has been more than a decade since the first chromosome conformation capture (3C) assay was described. The assay was originally devised to measure the frequency with which two genomic loci interact within the three-dimensional (3D) nuclear space. Over time, this method has evolved both qualitatively and quantitatively, from detection of pairwise interaction of two unique loci to generating maps for the global chromatin interactome. Combined with the analysis of the epigenetic chromatin context, these advances led to the unmasking of general genome folding principles. The evolution of 3C-based methods has been supported first by the revolution in ChIP and then by sequencing-based approaches, methods that were primarily tools to study the unidimensional genome. The gradual improvement of 3C-based methods illustrates how the field adapted to the need to gradually address more subtle questions, beginning with enquiries of reductionist nature to reach more holistic perspectives, as the technology advanced, in a process that is greatly improving our knowledge on genome behavior and regulation. Here, we describe the evolution of 3C and other 3C-based methods for the analysis of chromatin interactions, along with a brief summary of their contribution in uncovering the significance of the three-dimensional world within the nucleus. We also discuss their inherent limitations and caveats in order to provide a critical view of the power and the limits of this technology.     

Immunoprecipitation of native chromatin: NChIP

Chromatin immunoprecipitation (ChIP) is widely used in many fields to analyze the distribution of specific proteins, or their modified isoforms, across defined DNA domains. ChIP procedures fall into two main categories, namely, those that use native chromatin prepared by nuclease digestion (designated NChIP), and those that use chromatin in which DNA and proteins are crosslinked, either chemically or with UV light (designated XChIP). Each procedure has its own advantages and drawbacks. Here, we outline the methods currently in use in our laboratory to isolate and immunoprecipitate native chromatin from cultured cells, and to isolate and analyze immunoprecipitated protein and DNA.     

Two distinct methods analyzing chromatin structure using centrifugation and antibodies to modified histone H3: both provide similar chromatin states of the Rit1/Bcl11b gene

Chromatin state of a 2-Mb region harboring Rit1/Bcl11b on mouse chromosome 12 was examined using two distinct methods. One is ChIP assay examining the degree of enrichment with histone H3 methylated at lysine 9 (H3-mLys9) in chromatin and the other is H/E (heterochromatin/euchromatin) assay that measures a chromatin condensation state by using centrifugation. The ChIP assay showed that a 50-kb interval covering the gene and an upstream region constituted chromatin enriched with unmethylated H3-mLys9 in cells expressing Rit1 compared to cells not expressing Rit1. In contrast, regions other than the 50-kb interval did not show much difference in the enrichment between the two different types of cells. On the other hand, H/E assay of two expressing and two non-expressing tissues provided compatible fractionation patterns, suggesting that the chromatin condensation state detected by H/E assay is correlated with the chromatin state controlled by histone H3 tail modification linked to gene expression. These results indicate that the centrifugation-based H/E assay should provide a new approach to the regulation of chromatin structure with respect to its condensation state, complementing ChIP assays.     

A pipeline for the identification and characterization of chromatin modifications derived from ChIP-Seq datasets

The advent of massive parallel sequencing of immunopurified chromatin and its determinants has provided new avenues for researchers to map epigenome-wide changes and there is tremendous interest to uncover regulatory signatures to understand fundamental questions associated with chromatin structure and function. Indeed, the rapid development of large genome annotation projects has seen a resurgence in chromatin immunoprecipitation (ChIP) based protocols which are used to distinguish protein interactions coupled with large scale sequencing (Seq) to precisely map epigenome-wide interactions. Despite some of the great advances in our understanding of chromatin modifying complexes and their determinants, the development of ChIP-Seq technologies also pose specific demands on the integration of data for visualization, manipulation and analysis. In this article we discuss some of the considerations for experimental design planning, quality control, and bioinformatic analysis. The key aspects of post sequencing analysis are the identification of regions of interest, differentiation between biological conditions and the characterization of sequence differences for chromatin modifications. We provide an overview of best-practise approaches with background information and considerations of integrative analysis from ChIP-Seq experiments.