Chapter 20 Experimental Systems and Methods

Analysis of Chromatin Structure

Three major approaches to the question of how DNA is folded within its native chromatin structure and which proteins interact with it at various developmental stages have been extensively used by immunologists in the last decade or so, and their application to the study of TCR and BCR gene structure was described in Chapter 6. The first, 3-D FISH, was described above in the section on immunofluorescence-based imaging techniques. Here, we briefly explain two other methods that have allowed investigators to understand how DNA and chromatin structure alters during recombination events in intact cells.

Chromatin Immunoprecipitation Experiments Characterize Protein-DNA Interactions

In a chromatin immunoprecipitation (ChIP) experiment, chromatin is treated with formaldehyde, which covalently cross-links DNA to any bound proteins. The chromatin is then isolated and the bound DNA is sheared into small pieces. Antibodies to a protein that is hypothesized to be interacting with DNA are used to immunoprecipitate that protein along with any bound DNA. After protein digestion, the released DNA fragment is amplified by PCR and sequenced, leading to knowledge of which proteins specifically bind to which DNA sequences.

Chromosome Conformation Capture Technologies Analyze Long-Range Chromosomal DNA Interactions

The technique of chromosome conformation capture (3C) was first described in 2002. The object of this process is to analyze long-range interactions between DNA sequences within a chromosomal context. One of the most recent variants of the 3C technique is called Hi-C (see Figure 20-30). In this form of the assay, cells are fixed with formaldehyde to cross-link macromolecules that are in geographical proximity to one another. Chromosomal DNA is then isolated and treated with a restriction enzyme. The overhangs left by the restriction endonuclease are filled in with nucleotide residues labeled with biotin, which marks the end of each fragment. Blunt-end ligation of fragments cross-linked by the same proteins then creates a genome-wide library of ligation products, with the demarcation between individual products indicated by the biotin residues. The DNA is then sheared, the biotin-containing sequences are purified, and those DNA sequences that are close to one another in the native chromatin structure can be identified by high-throughput sequencing and analysis. The eventual goal is to generate an interaction matrix of DNA sequences. Such matrices have been invaluable in revealing features of 3-D chromosomal organization.

An illustration describes the DNA Hi-C chromosomal capture process. — FIGURE 20-30 DNA Hi-C detects regions of DNA that interact in three-dimensional space in situ. Cells are fixed with formaldehyde, and chromosomal DNA is isolated and treated with a restriction enzyme. The overhangs left by the restriction endonuclease are filled in using biotinylated nucleotide residues. Fragments that were initially close in the chromosome are now close enough to be ligated, creating a genome-wide library of ligation products. The DNA is sheared, and the residual biotin-containing sequences are purified and sequenced in order to create a map of sequences that were associated in the original chromatin.

An illustration shows a cross-link DNA with Hind III restriction sites marked by a black line. A region of the cross link DNA with restriction site is zoomed out to show the nucleotide sequence in the two strands. The first strand reads, A A G C T T and the second strand reads, T T C G A A. A line marks the break in the two strands forming two fragments. The first fragment shows A of the first strand and T T C G A of the second strand. The second fragment shows A G C T T of the first fragment and A of the second fragment. In the second step, the DNA strand is cut with restriction enzyme. The illustration shows two strands with a cross mark between the two strands. The instruction over the third step reads, Fill ends and mark with biotin. The illustration shows spheres representing biotin bonded to the ends of the strands. The illustration in the fourth step shows the two strands joined to form a circular strand. Biotin ligands are seen at the joining points. A zoomed out image of the top joining region shows the nucleotide sequence of the two strands. The sequence of the first strand reads, A A G C T A G C T T and the sequence of the second strand reads, T T C G A T C G A A. A line marks the break in the two strands forming two fragments. The first fragment shows A A G of the first strand and T T C G A T C of the second strand. The region is labeled as Ligate. The illustration in the fifth step shows four fragments of DNA and two fragments with ligands. The corresponding text reads, Purify and shear DNA; pull down biotin. The illustration in the sixth step shows two pairs of DNA strands with the top strand in forward direction and the bottom strand in reverse direction, in each pair. The joined region with bonded ligand is at the center of each strand.