Figure 4–48 A method for determining the position of loops in interphase chromosomes. In this technique, known as the chromosome conformation capture (3C) method, cells are treated with formaldehyde to create the indicated covalent DNA–protein and DNA–DNA cross-links. The DNA is then treated with an enzyme (a restriction nuclease) that chops the DNA into many pieces, cutting at strictly defined nucleotide sequences and forming sets of identical “cohesive ends” (see Figure 8–28). The cohesive ends can be made to join through their complementary base-pairing. Importantly, prior to the ligation step shown, the DNA is diluted so that the fragments that have been kept in close proximity to each other (through cross-linking) are the ones most likely to join. Finally, the cross-links are reversed and the newly ligated fragments of DNA are identified and quantified by PCR (the polymerase chain reaction, described in Chapter 8). From the results, combined with DNA sequence information, one can derive models for the interphase conformation of chromosomes.
Figure 4–49 A model for the organization of an interphase chromosome. A section of an interphase chromosome is shown folded into a series of looped domains, each containing perhaps 50,000–200,000 or more nucleotide pairs of double-helical DNA condensed into a chromatin fiber. The chromatin in each individual loop is further condensed through poorly understood folding processes that are reversed when the cell requires direct access to the DNA packaged in the loop. Neither the composition of the postulated chromosomal axis nor how the folded chromatin fiber is anchored to it is clear. However, in mitotic chromosomes, the bases of the chromosomal loops are enriched both in condensins (discussed below) and in DNA topoisomerase II enzymes (discussed in Chapter 5), two proteins that may form much of the axis at metaphase.
normal mitotic right arm of chromosomes at chromosome 2 same scale region where two homologous chromosomes are separated left arm of chromosome 2 left arm of chromocenter chromosome 3 20 µm right arm of chromosome 3 of the genome (see Figure 4–40). Such regions, where all of the chromatin has a similar structure, are separated from neighboring domains by barrier proteins (see Figure 4–41). At low resolution, the interphase chromosome can therefore be considered as a mosaic of chromatin structures, each containing particular nucleosome modifications associated with a particular set of non-histone proteins. Polytene chromosomes allow us to see details of this mosaic of domains in the light microscope, as well as to observe some of the changes associated with gene expression.
There Are multiple Forms of Chromatin
By staining Drosophila polytene chromosomes with antibodies, or by using a more recent technique called ChIP (chromatin immunoprecipitation) analysis (see Chapter 8), the locations of the histone and non-histone proteins in chromatin can be mapped across the entire DNA sequence of an organism’s genome. Such an analysis in Drosophila has thus far localized more than 50 different chromatin proteins and histone modifications. The results suggest that three major types of repressive chromatin predominate in this organism, along with two major types of chromatin on actively transcribed genes, and that each type is associated with a different complex of non-histone proteins. Thus, classical heterochromatin contains more than six such proteins, including heterochromatin protein 1 (HP1),