The Current State of Chromatin Immunoprecipitation

Collas, Philippe

doi:10.1007/s12033-009-9239-8

Cited by 216 publications

(120 citation statements)

References 119 publications

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“…Retrieval of the crosslinked complexes and analysis of the associated DNA then gave birth to the ChIP assay (14,39,40), which has become ubiquitous in the chromatin field in a multitude of variations (41)(42)(43)(44)(45)(46)(47)(48)(49). Although ChIP assays performed without crosslinking have proven valuable for analyses of stable chromatin complexes (50), crosslinking has made it possible to identify interactions that would not otherwise withstand the isolation procedure.…”

Section: Capture Of Protein-dna Complexesmentioning

confidence: 99%

Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes

Hoffman

Frey

Smith

et al. 2015

Journal of Biological Chemistry

336

261

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Formaldehyde has been used for decades to probe macromolecular structure and function and to trap complexes, cells, and tissues for further analysis. Formaldehyde crosslinking is routinely employed for detection and quantification of protein-DNA interactions, interactions between chromatin proteins, and interactions between distal segments of the chromatin fiber. Despite widespread use and a rich biochemical literature, important aspects of formaldehyde behavior in cells have not been well described. Here, we highlight features of formaldehyde chemistry relevant to its use in analyses of chromatin complexes, focusing on how its properties may influence studies of chromatin structure and function.Prior to its use in the chromatin field, formaldehyde use had a long history in a number of fields, including vaccine production (1, 2) and histology (3). In this review, we focus on its use in chromatin immunoprecipitation approaches and protein-protein interaction studies applied to understand the location and abundance of transcription factor binding along DNA. A recent complementary perspective highlights gaps in knowledge with a particular focus on how formaldehyde crosslinking data have been used to interpret aspects of chromatin three-dimensional organization (4). Here, we briefly review prior work describing formaldehyde reactivity toward proteins, DNA, and their constituent monomers. This information provides a basis for understanding how formaldehyde functions in widely used assays in the chromatin field, and conversely, highlights less well understood aspects of formaldehyde behavior in cells. These issues are of significance for designing crosslinkingbased studies as well as for properly interpreting the resulting data. The analysis of formaldehyde-fixed chromatin has provided fundamental insights into where and when regulatory factors associate with the DNA template in vivo, but it in general does not provide unambiguous information about chromatin binding kinetics. A major goal of ongoing work is to understand kinetic and thermodynamic aspects of chromatin complex assembly at single copy loci in vivo. Development of experimental strategies to achieve these goals will require a deeper and more comprehensive understanding of the effects mediated by formaldehyde in cells.The following discussion provides a framework for understanding aspects of formaldehyde function when used to trap macromolecular complexes in cells, with the main features shown in Fig. 1. Beginning with basic chemical reactivity, this review will explore the ability of formaldehyde to crosslink with proteins and DNA to form protein-protein or protein-DNA complexes, common molecular quenchers, and the potential for crosslink reversal. Progress in capturing crosslinked complexes will also be discussed with an emphasis on the impact of a better understanding of formaldehyde chemistry in vivo. Basic ChemistryFormaldehyde is the smallest aldehyde, an electrophilic molecule susceptible to chemical attack by a wide range of nucleophilic species of ...

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Section: Capture Of Protein-dna Complexesmentioning

confidence: 99%

Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes

Hoffman

Frey

Smith

et al. 2015

Journal of Biological Chemistry

336

261

View full text Add to dashboard Cite

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“…Yet how these processes occur to regulate gene expression in the cell is not fully understood. Chromatin immunoprecipitation (ChIP) assays are conventional and widely used techniques to investigate protein-DNA interactions at specific locations in the genome 7 . Briefly, the standard ChIP protocol consists of fixation and lysis of the cells of interest before chromatin fragmentation.…”

Section: Introductionmentioning

confidence: 99%

Chromatin immunoprecipitation in microfluidic droplets: towards fast and cheap analyses

et al. 2017

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Genetic organization is governed by the interaction of DNA with histone proteins, and differential modifications of these proteins is a fundamental mechanism of gene regulation. Histone modifications are primarily studied through chromatin immunoprecipitation (ChIP) assays, however conventional ChIP procedures are time consuming, laborious and require a large number of cells. Here we report for the first time the development of ChIP in droplets based on a microfluidic platform combining nanoliter droplets, magnetic beads (MB) and magnetic tweezers (MT). The droplet approach enabled compartmentalization and improved mixing, while reducing the consumption of samples and reagents in an integrated workflow. Anti-histone antibodies grafted to MB were used as a solid support to capture and transfer the target chromatin from droplets to droplets in order to perform chromatin immunoprecipitation, washing, elution and purification of DNA. We designed a new ChIP protocol to investigate four different types of modified histones with known roles in gene activation or repression. We evaluated the performances of this new ChIP in droplet assay in comparison with conventional methods. The proposed technology dramatically reduces analytical time from a few days to 7 hours, simplifies the ChIP protocol and decreases the number of cells required by 100 fold while maintaining a high degree of sensitivity and specificity. Therefore this droplet-based ChIP assay represents a new, highly advantageous and convenient approach to epigenetic analyses.

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“…Simplistically, when the amount of fluorescence from immunoprecipitated DNA is greater than the input DNA, the presence of the protein or modification of interest can be inferred. For histone modifications, an antibody specific to the modification of interest can be used or alternatively histone-modifying enzymes that are associated with chromatin can be targeted (Collas 2010). Typically, in these types of studies, antibodies directed to histone tail modification, which include methylation and/or acetylation, are used.…”

Section: Chips With Everythingmentioning

confidence: 99%

Make way for the ‘next generation’: application and prospects for genome-wide, epigenome-specific technologies in endocrine research

Emes

Farrell²

2012

Journal of Molecular Endocrinology

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Epigenetic changes, which target DNA and associated histones, can be described as a pivotal mechanism of interaction between genes and the environment. The field of epigenomics aims to detect and interpret epigenetic modifications at the whole genome level. These approaches have the potential to increase resolution of epigenetic changes to the single base level in multiple disease states or across a population of individuals. Identification and comparison of the epigenomic landscape has challenged our understanding of the regulation of phenotype. Additionally, inclusion of these marks as biomarkers in the early detection or progression monitoring of disease is providing novel avenues for future biomedical research. Cells of the endocrine organs, which include pituitary, thyroid, thymus, pancreas ovary and testes, have been shown to be susceptible to epigenetic alteration, leading to both local and systemic changes often resulting in lifethreatening metabolic disease. As with other cell types and populations, endocrine cells are susceptible to tumour development, which in turn may have resulted from aberration of epigenetic control. Techniques including highthroughput sequencing and array-based analysis to investigate these changes have rapidly emerged and are continually evolving. Here, we present a review of these methods and their promise to influence our studies on the epigenome for endocrine research and perhaps to uncover novel therapeutic options in disease states.

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The Current State of Chromatin Immunoprecipitation

Cited by 216 publications

References 119 publications

Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes

Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes

Chromatin immunoprecipitation in microfluidic droplets: towards fast and cheap analyses

Make way for the ‘next generation’: application and prospects for genome-wide, epigenome-specific technologies in endocrine research

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