The Current State of Chromatin Immunoprecipitation (ChIP) from FFPE Tissues
Cancer cells accumulate epigenomic aberrations that contribute to cancer initiation and progression by altering both the genomic stability and the expression of genes. The awareness of such alterations could improve our understanding of cancer dynamics and the identification of new therapeutic strategies and biomarkers to refine tumor classification and treatment. Formalin fixation and paraffin embedding (FFPE) is the gold standard to preserve both tissue integrity and organization, and, in the last decades, a huge number of biological samples have been archived all over the world following this procedure. Recently, new chromatin immunoprecipitation (ChIP) techniques have been developed to allow the analysis of histone post-translational modifications (PTMs) and transcription factor (TF) distribution in FFPE tissues.
The application of ChIP to genome-wide chromatin studies using real archival samples represents an unprecedented opportunity to conduct retrospective clinical studies thanks to the possibility of accessing large cohorts of samples and their associated diagnostic records. However, although recent attempts to standardize have been made, fixation and storage conditions of clinical specimens are still extremely variable and can affect the success of chromatin studies. The procedures introduced in the last few years dealt with this problem proponing successful strategies to obtain high-resolution ChIP profiles from FFPE archival samples. In this review, we compare the different FFPE-ChIP techniques, highlighting their strengths, limitations, common features, and peculiarities, as well as pitfalls and caveats related to ChIP studies in FFPE samples, in order to facilitate their application.
Epi-Decoder: Decoding the Local Proteome of a Genomic Locus by Massive Parallel Chromatin Immunoprecipitation Combined with DNA-Barcode Sequencing
The genome in a eukaryotic cell is packaged into chromatin and regulated by chromatin-binding and chromatin-modifying factors. Many of these factors and their complexes have been identified before, but how each genomic locus interacts with its surrounding proteins in the nucleus over time and in changing conditions remains poorly described. Measuring protein-DNA interactions at a specific locus in the genome is challenging and current techniques such as capture of a locus followed by mass spectrometry require high levels of enrichment. Epi-Decoder, a method developed in budding yeast, enables systematic decoding of the proteome of a single genomic locus of interest without the need for locus enrichment.
Instead, Epi-Decoder uses massive parallel chromatin immunoprecipitation of tagged proteins combined with barcoding a genomic locus and counting of coimmunoprecipitated barcodes by DNA sequencing (TAG-ChIP-Barcode-Seq). In this scenario, DNA barcode counts serve as a quantitative readout for protein binding of each tagged protein to the barcoded locus. Epi-Decoder can be applied to determine the protein-DNA interactions at a wide range of genomic loci, such as coding genes, noncoding genes, and intergenic regions. Furthermore, Epi-Decoder provides the option to study protein-DNA interactions upon changing cellular and/or genetic conditions. In this protocol, we describe in detail how to construct Epi-Decoder libraries and how to perform an Epi-Decoder analysis.
Transcription Factor Chromatin Immunoprecipitation in Endothelial Cells
Interactions between DNA and proteins are crucial for the regulation of gene expression. Chromatin immunoprecipitation (ChIP) is a powerful technique that allows the study of specific protein-DNA interactions in cultured cells and fresh or fixed tissue. Chromatin is isolated and sheared, and antibodies against the protein(s) of interest are used to isolate specific protein-DNA complexes. Subsequent analysis by real-time polymerase chain reaction (qPCR) or next-generation sequencing (NGS) allows identification and quantification of the co-purified DNA fragments, and NGS also gives insight into the genomic binding sites of a protein. Here we describe a cross-linking ChIP (X-ChIP) protocol, based around the example of a myc-tagged Proline-Rich Homeodomain (PRH) protein expressed in human umbilical vein endothelial cells. We also describe how to analyse specific known or suspected binding sites using quantitative PCR as well as how to analyse genome-wide binding from ChIP sequencing data.
Chromatin Immunoprecipitation in Chloroplasts
Chromatin is the genetic material assembled by nucleic acids (including DNA and RNA) and proteins. The biological functions of chromatin are highly dependent on the interaction between DNA (and/or RNA) and proteins that bind to it. Chromatin immunoprecipitation (ChIP) is a powerful technique for evaluating these interactions and has been widely used to characterize the functions of nuclear proteins. However, its application in identifying plant organellar chromatin-binding proteins is lagging. This article describes the method for analyzing the association of chloroplast-localized proteins with the chloroplast genome. 2022 Wiley Periodicals LLC. Basic Protocol 1: Chloroplast isolation Basic Protocol 2: Crosslinking of DNA-Protein complexes Basic Protocol 3: Chromatin isolation and preparation Support Protocol: Bead-antibody complex preparation Basic Protocol 4: Immunoprecipitation and washes Basic Protocol 5: DNA preparation Basic Protocol 6: Analysis of results.
Chromatin Immunoprecipitation dataset of H3ac and H3K27me3 histone marks followed by DNA sequencing of Medicago truncatula embryos during control and heat stress conditions to decipher epigenetic regulation of desiccation tolerance acquisition
Desiccation tolerance (DT) is one of the most important processes that seeds need to acquire during seed maturation because it will ensure survival until seeds have favourable conditions for germinating. Moreover, in the current climate warming context, heat stress and its impact on seed maturation and quality has been increasingly studied by the scientific community. Even if the transcriptomic changes enrolled in DT acquisition and seed heat stress response are fairly known, its epigenetic control has not yet been investigated. Medicago truncatula is a model legume for studying seed molecular mechanisms, which is known to display a delay in the acquisition of seed maturation mechanisms under heat conditions, except for desiccation acquisition. Our aim was to evaluate the role of two histone marks during embryo development under control and heat stress conditions on seed maturation processes, including the DT acquisition.
EpiQuik Chromatin Immunoprecipitation (ChIP) Kit
EpiQuik Tissue Chromatin Immunoprecipitation (ChIP) Kit
Immunoprecipitation (IP) Kit
Immunoprecipitation Kit (OKRA00051)
|OKRA00051||Aviva Systems Biology||1 kit||1248 EUR|
Topoisomerase II Immunoprecipitation Kit
|TG1035||TopoGen||>50 analyses||657.6 EUR|
Immunoprecipitation Starter Pack 2 x 2mL
|17600235||Scientific Laboratory Supplies||EACH||757.2 EUR|
Immunoprecipitation Kit: Protein A-Agarose
|BS688||Bio Basic||20Rxn, 20prep||242.7 EUR|
Immunoprecipitation Kit: Protein A-Sepharose
|BS690||Bio Basic||20Rxn, 20prep||237.48 EUR|
EpiQuik Methylated DNA Immunoprecipitation Kit
HDAC2 Immunoprecipitation (IP) & Activity Assay Kit
HDAC1 Immunoprecipitation (IP) & Activity Assay Kit
HDAC3 Immunoprecipitation (IP) & Activity Assay Kit
Immunoprecipitation Kit: Protein A/G PLUS-Agarose
|BS689||Bio Basic||20Rxn, 20prep||237.48 EUR|
EpiQuik Tissue Methylated DNA Immunoprecipitation Kit
EpiQuik Hydroxymethylated DNA Immunoprecipitation (hMeDIP) Kit
Monoclonal Anti-Rabbit IgG (Light-chain) IgG (adsorbed, for immunoprecipitation/WB)
|20160-UL||Alpha Diagnostics||0.5 ml||270 EUR|
Monoclonal Anti-Rabbit IgG (Light-chain) IgG-HRP Conj (adsorbed, for immunoprecipitation/WB)
|20160-HRP||Alpha Diagnostics||0.5 ml||315.6 EUR|
These histone marks have either repressive (H3K27me3) or inducible (H3ac) effects on gene transcription, respectively corresponding to markers of packed and accessible chromatins. We identified all genomic regions bound to the H3K27me3 histones at four developmental stages and to the H3ac histones at the two earlier developmental stages during seed maturation, from seed filling to mature dry seeds, collected under optimal and heat stress conditions in the model legume, Medicago truncatula (reference genotype A17). A list of genes and promoters potentially linked to these two histone marks is reported and could provide clues about the epigenetic regulation of seed maturation between control and heat stress conditions, including the desiccation tolerance acquisition.
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