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Nature Reviews. Genetics Oct 2009Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique for genome-wide profiling of DNA-binding proteins, histone modifications or nucleosomes.... (Review)
Review
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique for genome-wide profiling of DNA-binding proteins, histone modifications or nucleosomes. Owing to the tremendous progress in next-generation sequencing technology, ChIP-seq offers higher resolution, less noise and greater coverage than its array-based predecessor ChIP-chip. With the decreasing cost of sequencing, ChIP-seq has become an indispensable tool for studying gene regulation and epigenetic mechanisms. In this Review, I describe the benefits and challenges in harnessing this technique with an emphasis on issues related to experimental design and data analysis. ChIP-seq experiments generate large quantities of data, and effective computational analysis will be crucial for uncovering biological mechanisms.
Topics: Animals; Chromatin Immunoprecipitation; Computational Biology; DNA-Binding Proteins; Epigenesis, Genetic; Humans; Nucleosomes; Sequence Analysis, DNA
PubMed: 19736561
DOI: 10.1038/nrg2641 -
Cell Cycle (Georgetown, Tex.) 2014Many biologically significant processes, such as cell differentiation and cell cycle progression, gene transcription and DNA replication, chromosome stability and... (Review)
Review
Many biologically significant processes, such as cell differentiation and cell cycle progression, gene transcription and DNA replication, chromosome stability and epigenetic silencing etc. depend on the crucial interactions between cellular proteins and DNA. Chromatin immunoprecipitation (ChIP) is an important experimental technique for studying interactions between specific proteins and DNA in the cell and determining their localization on a specific genomic locus. In recent years, the combination of ChIP with second generation DNA-sequencing technology (ChIP-seq) allows precise genomic functional assay. This review addresses the important applications of ChIP-seq with an emphasis on its role in genome-wide mapping of transcription factor binding sites, the revelation of underlying molecular mechanisms of differential gene regulation that are governed by specific transcription factors, and the identification of epigenetic marks. Furthermore, we also describe the ChIP-seq data analysis workflow and a perspective for the exciting potential advancement of ChIP-seq technology in the future.
Topics: Binding Sites; Chromatin Immunoprecipitation; Epigenesis, Genetic; Gene Expression Profiling; Histones; Humans; Sequence Analysis, DNA; Transcription Factors
PubMed: 25486472
DOI: 10.4161/15384101.2014.949201 -
Genome Research Sep 2012Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) has become a valuable and widely used approach for mapping the genomic...
Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) has become a valuable and widely used approach for mapping the genomic location of transcription-factor binding and histone modifications in living cells. Despite its widespread use, there are considerable differences in how these experiments are conducted, how the results are scored and evaluated for quality, and how the data and metadata are archived for public use. These practices affect the quality and utility of any global ChIP experiment. Through our experience in performing ChIP-seq experiments, the ENCODE and modENCODE consortia have developed a set of working standards and guidelines for ChIP experiments that are updated routinely. The current guidelines address antibody validation, experimental replication, sequencing depth, data and metadata reporting, and data quality assessment. We discuss how ChIP quality, assessed in these ways, affects different uses of ChIP-seq data. All data sets used in the analysis have been deposited for public viewing and downloading at the ENCODE (http://encodeproject.org/ENCODE/) and modENCODE (http://www.modencode.org/) portals.
Topics: Animals; Chromatin Immunoprecipitation; Databases, Genetic; Genome; Genomics; Guidelines as Topic; High-Throughput Nucleotide Sequencing; Histones; Humans; Internet; Transcription Factors
PubMed: 22955991
DOI: 10.1101/gr.136184.111 -
Genome Biology 2008We present Model-based Analysis of ChIP-Seq data, MACS, which analyzes data generated by short read sequencers such as Solexa's Genome Analyzer. MACS empirically models... (Comparative Study)
Comparative Study
We present Model-based Analysis of ChIP-Seq data, MACS, which analyzes data generated by short read sequencers such as Solexa's Genome Analyzer. MACS empirically models the shift size of ChIP-Seq tags, and uses it to improve the spatial resolution of predicted binding sites. MACS also uses a dynamic Poisson distribution to effectively capture local biases in the genome, allowing for more robust predictions. MACS compares favorably to existing ChIP-Seq peak-finding algorithms, and is freely available.
Topics: Algorithms; Cell Line, Tumor; Chromatin Immunoprecipitation; Hepatocyte Nuclear Factor 3-alpha; Humans; Models, Genetic; Oligonucleotide Array Sequence Analysis
PubMed: 18798982
DOI: 10.1186/gb-2008-9-9-r137 -
Methods in Molecular Biology (Clifton,... 2022Non-alcoholic steatohepatitis (NASH) is an advanced form of non-alcoholic fatty liver disease characterized by hepatosteatosis, liver cell injury, and inflammation. The...
Non-alcoholic steatohepatitis (NASH) is an advanced form of non-alcoholic fatty liver disease characterized by hepatosteatosis, liver cell injury, and inflammation. The pathogenesis of NASH involves dysregulated transcription of genes involved in critical processes in the liver, including metabolic homeostasis and inflammation. Chromatin immunoprecipitation (ChIP) utilizes antibody-mediated immunoprecipitation followed by the detection of associated DNA fragments via real-time PCR or high-throughput sequencing to quantitatively profile the interactions of proteins of interest with functional chromatin elements. Here, we present a detailed protocol to study the interactions of DNA and chromatin-associated proteins (e.g., transcription factors, co-activators, co-repressors, and chromatin modifiers) and modified histones (e.g., acetylated and methylated) in isolated primary mouse hepatocytes and mouse liver. The application of these methods can enable the identification of molecular mechanisms that underpin dysregulated hepatic processes in NASH.
Topics: Animals; Chromatin; Chromatin Immunoprecipitation; Hepatocytes; Liver; Mice; Non-alcoholic Fatty Liver Disease
PubMed: 35212993
DOI: 10.1007/978-1-0716-2128-8_13 -
BioTechniques Dec 2004Association between proteins and DNA is crucial for many vital cellular functions such as gene transcription, DNA replication and recombination, repair, segregation,... (Review)
Review
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.
Topics: Chromatin Immunoprecipitation; DNA; DNA-Binding Proteins
PubMed: 15597545
DOI: 10.2144/04376RV01 -
Journal of Biomedicine & Biotechnology 2010
Topics: Chromatin Immunoprecipitation; Epigenomics; Genomic Structural Variation; Human Genome Project; Humans; Information Storage and Retrieval; Polymorphism, Single Nucleotide; Sequence Analysis, DNA; Sequence Analysis, RNA; Statistics as Topic
PubMed: 21512588
DOI: 10.1155/2010/370710 -
International Journal of Molecular... Jan 2022Cancer cells accumulate epigenomic aberrations that contribute to cancer initiation and progression by altering both the genomic stability and the expression of genes.... (Review)
Review
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.
Topics: Animals; Chromatin Immunoprecipitation; Epigenomics; High-Throughput Nucleotide Sequencing; Humans; Paraffin Embedding; Tissue Fixation
PubMed: 35163027
DOI: 10.3390/ijms23031103 -
Molecules (Basel, Switzerland) Nov 2021Chromatin is a dynamic structure comprising of DNA and proteins. Its unique nature not only help to pack the DNA tightly within the cell but also is pivotal in... (Review)
Review
Chromatin is a dynamic structure comprising of DNA and proteins. Its unique nature not only help to pack the DNA tightly within the cell but also is pivotal in regulating gene expression DNA replication. Furthermore it also protects the DNA from being damaged. Various proteins are involved in making a specific complex within a chromatin and the knowledge about these interacting partners is helpful to enhance our understanding about the pathophysiology of various chromatin associated diseases. Moreover, it could also help us to identify new drug targets and design more effective remedies. Due to the existence of chromatin in different forms under various physiological conditions it is hard to develop a single strategy to study chromatin associated proteins under all conditions. In our current review, we tried to provide an overview and comparative analysis of the strategies currently adopted to capture the DNA bounded protein complexes and their mass spectrometric identification and quantification. Precise information about the protein partners and their function in the DNA-protein complexes is crucial to design new and more effective therapeutic molecules against chromatin associated diseases.
Topics: Binding Sites; Chromatin; Chromatin Immunoprecipitation; Chromatin Immunoprecipitation Sequencing; DNA-Binding Proteins; Euchromatin; Heterochromatin; Humans; Proteome; Proteomics; Telomere-Binding Proteins
PubMed: 34771102
DOI: 10.3390/molecules26216694 -
Life Science Alliance Dec 2023Existing monitoring approaches in heart transplantation lack the sensitivity to provide deep molecular assessments to guide management, or require endomyocardial biopsy,...
Existing monitoring approaches in heart transplantation lack the sensitivity to provide deep molecular assessments to guide management, or require endomyocardial biopsy, an invasive and blind procedure that lacks the precision to reliably obtain biopsy samples from diseased sites. This study examined plasma cell-free DNA chromatin immunoprecipitation sequencing (cfChIP-seq) as a noninvasive proxy to define molecular gene sets and sources of tissue injury in heart transplant patients. In healthy controls and in heart transplant patients, cfChIP-seq reliably detected housekeeping genes. cfChIP-seq identified differential gene signals of relevant immune and nonimmune molecular pathways that were predominantly down-regulated in immunosuppressed heart transplant patients compared with healthy controls. cfChIP-seq also identified cell-free DNA tissue sources. Compared with healthy controls, heart transplant patients demonstrated greater cell-free DNA from tissue types associated with heart transplant complications, including the heart, hematopoietic cells, lungs, liver, and vascular endothelium. cfChIP-seq may therefore be a reliable approach to profile dynamic assessments of molecular pathways and sources of tissue injury in heart transplant patients.
Topics: Humans; Heart Transplantation; Chromatin Immunoprecipitation; Heart; Chromatin Immunoprecipitation Sequencing; Cell-Free Nucleic Acids
PubMed: 37730434
DOI: 10.26508/lsa.202302003