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The function of chromatin is to efficiently package DNA into a small volume to fit into the nucleus of a cell and protect the DNA structure and sequence. Packaging DNA into chromatin allows for mitosis and meiosis, prevents chromosome breakage and controls gene expression and DNA replication.
Figure 1. Chromatin formation. DNA wraps around the histone proteins to form nucleosomes; these in turn couple to become the chromatin fiber. 1) Unpackaged DNA. 2) DNA wrapped around histone octamers to form nucleosomes. 3) Nucleosomes compacted into a chromatin fiber.
Heterochromatin is a tightly packed form of chromatin that can silence gene transcription. Heterochromatin constitutes telomeres, pericentric regions and areas rich in repetitive sequences. Euchromatin is less condensed and contains most actively transcribed genes.
Certain proteins – including histones, chromatin interacting proteins such as transcription factors and the DNA repair machinery – play a role in shaping chromatin structure.
Chromatin remodeling complexes can change chromatin architecture by modulating the interaction between nucleosomes and DNA, often by adding post-translational modifications to histones.
Heterochromatin is difficult to analyze because of its condensed state and repetitive DNA sequence. However, euchromatin poses fewer challenges as it contains active genes and maintains an open and extended structure.
Chromatin immunoprecipitation (ChIP) is the technique of choice to study chromatin modifications. It can be used to dissect the spatial and temporal dynamics of chromatin interaction with its associated factors, including transcription factors and histone modifications. This technique allows mapping of minute-by-minute changes of chromatin associated proteins at a single promoter, or to determine their binding sites over the entire human genome.
Figure 2. Schematic overview of a ChIP experiment workflow. 1) Cross-linking of DNA and proteins (optional). 2) Chromatin fragmentation by sonication or enzymatic methods. 3) Immunoprecipitation of chromatin fragments that interact with the target protein or modification. 4) Reversal of cross-links (when necessary) and DNA purification. 5) Analysis of the immunoprecipitated fraction to determine abudance of target sequence(s) relative to input. Common methods include qPCR, ChIP-seq and ChIP-Chip.
The principle of ChIP is simple: the selective enrichment of a chromatin fraction containing a specific antigen. Antibodies that recognize a protein or protein modification of interest can be used to determine the relative abundance of that antigen at one or more locations (loci) in the genome.
After ChIP, end point PCR and/or quantitative PCR (qPCR) can be used to verify whether a particular DNA sequence is associated with the protein of interest. Using these approaches researchers can evaluate the interaction of a protein of interest with a limited number of target DNA sequences (Figure 3).
Figure 3. ChIP performed on HeLa cells using anti-histone H3K4me3 antibody (ab8580) together with ChIP Kit – One Step (ab117138). No antibody was added to the blank. The immunoprecipitated DNA was quantified by RT-PCR. Primers and probes are located in the first kilobase of the transcribed region.
It is now possible to analyze the whole genome distribution of a histone modification or chromatin associated protein by coupling ChIP with either microarray platforms (ChIP-chip) or next generation sequencing technologies (ChIP-seq; Figure 4).