Dataset Information

The Use of Mononucleosome Immunoprecipitation for Analysis of Combinatorial Histone Post-translational Modifications and Purification of Nucleosome-Interacting Proteins.

ABSTRACT: The nucleosome is the principal structural unit of chromatin. Although many studies focus on individual histone post-translational modifications (PTMs) in isolation, it is important to recognize that multiple histone PTMs can function together or cross-regulate one another within the nucleosome context. In addition, different modifications or histone-binding surfaces can synergize to stabilize the binding of nuclear factors to nucleosomes. To facilitate these types of studies, we present here a step-by-step protocol for isolating high yields of mononucleosomes for biochemical analyses. Furthermore, we discuss differences and variations of the basic protocol used in different publications and characterize the relative abundance of selected histone PTMs and chromatin-binding proteins in the different chromatin fractions obtained by this method.

SUBMITTER: Khan KA

PROVIDER: S-EPMC7225312 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

The Use of Mononucleosome Immunoprecipitation for Analysis of Combinatorial Histone Post-translational Modifications and Purification of Nucleosome-Interacting Proteins.

Khan Kashif Aziz KA Ng Marlee K MK Cheung Peter P

Frontiers in cell and developmental biology 20200508

The nucleosome is the principal structural unit of chromatin. Although many studies focus on individual histone post-translational modifications (PTMs) in isolation, it is important to recognize that multiple histone PTMs can function together or cross-regulate one another within the nucleosome context. In addition, different modifications or histone-binding surfaces can synergize to stabilize the binding of nuclear factors to nucleosomes. To facilitate these types of studies, we present here a ...[more]

PMID: 32457909

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Project description:Chromatin immunoprecipitation coupled to next-generation sequencing (ChIP-seq) has served as the central method for the study of histone modifications for the past decade. In ChIP-seq analyses, antibodies selectively capture nucleosomes bearing a modification of interest and the associated DNA is then mapped to the genome to determine the distribution of the mark. This approach has several important drawbacks: (i) ChIP interpretation necessitates the assumption of perfect antibody specificity, despite growing evidence that this is often not the case. (ii) Common methods for evaluating antibody specificity in other formats have little or no bearing on specificity within a ChIP experiment. (iii) Uncalibrated ChIP is reported as relative enrichment, which is biologically meaningless outside the experimental reference frame defined by a discrete immunoprecipitation (IP), thus preventing facile comparison across experimental conditions or modifications. (iv) Differential library amplification and loading onto next-generation sequencers, as well as computational normalization, can further compromise quantitative relationships that may exist between samples. Consequently, the researcher is presented with a series of potential pitfalls and is blind to nearly all of them. Here we provide a detailed protocol for internally calibrated ChIP (ICeChIP), a method we recently developed to resolve these problems by spike-in of defined nucleosomal standards within a ChIP procedure. This protocol is optimized for specificity and quantitative power, allowing for measurement of antibody specificity and absolute measurement of histone modification density (HMD) at genomic loci on a biologically meaningful scale enabling unambiguous comparisons. We provide guidance on optimal conditions for next-generation sequencing (NGS) and instructions for data analysis. This protocol takes between 17 and 18 h, excluding time for sequencing or bioinformatic analysis. The ICeChIP procedure enables accurate measurement of histone post-translational modifications (PTMs) genome-wide in mammalian cells as well as Drosophila melanogaster and Caenorhabditis elegans, indicating suitability for use in eukaryotic cells more broadly.

Dataset Information

The Use of Mononucleosome Immunoprecipitation for Analysis of Combinatorial Histone Post-translational Modifications and Purification of Nucleosome-Interacting Proteins.

Publications

The Use of Mononucleosome Immunoprecipitation for Analysis of Combinatorial Histone Post-translational Modifications and Purification of Nucleosome-Interacting Proteins.

Similar Datasets

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