Genes VII

Interphase chromatin is a tangled mass occupying a large part of the nuclear volume, in contrast with the highly organized and reproducible ultrastructure of mitotic chromosomes. What controls the distribution of interphase chromatin within the nucleus?

Some indirect evidence on its nature is provided by the isolation of the genome as a single, compact body. Using the same technique just described for isolating the bacterial nucleoid, nuclei can be lysed on top of a sucrose gradient. This releases the genome in a form that can be collected by centrifugation. As isolated from D. melanogaster, it can be visualized as a compactly folded fiber (10 nm in diameter), consisting of DNA bound to proteins.

Supercoiling measured by the response to ethidium bromide corresponds to about one negative supercoil / 200 bp. These supercoils can be removed by nicking with DNAase, although the DNA remains in the form of the 10 nm fiber. This suggests that the supercoiling is caused by the arrangement of the fiber in space, and represents the existing torsion.

Full relaxation of the supercoils requires one nick / 85 kb, identifying the average length of "closed" DNA. This region could comprise a loop or domain similar in nature to those identified in the bacterial genome. Loops can be seen directly when the majority of proteins are extracted from mitotic chromosomes. The resulting complex consists of the DNA associated with ~8% of the original protein content. As seen in Figure 18.7, the protein-depleted chromosomes take the form of a central scaffold surrounded by a halo of DNA.

The metaphase scaffold consists of a dense network of fibers. Threads of DNA emanate from the scaffold, apparently as loops of average length 10 V30 µm (30 V90 kb). The DNA can be digested without affecting the integrity of the scaffold, which consists of a set of specific proteins. This suggests a form of organization in which loops of DNA of ~60 kb are anchored in a central proteinaceous scaffold.

The appearance of the scaffold resembles a mitotic pair of sister chromatids. The sister scaffolds usually are tightly connected, but sometimes are separate, joined only by a few fibers. Could this be the structure responsible for maintaining the shape of the mitotic chromosomes? Could it be generated by bringing together the protein components that usually secure the bases of loops in interphase chromatin?

Interphase cells possess a nuclear matrix, a filamentous structure on the interior of the nuclear membrane. Chromatin often appears to be attached to the matrix, and there have been many suggestions that this attachment is necessary for transcription or replication. When nuclei are depleted of proteins, the DNA extrudes as loops from the residual nuclear matrix.

Is DNA attached to the matrix or scaffold via specific sequences? DNA sites attached to proteinaceous structures in interphase nuclei are called MAR (matrix attachment regions); it is confusing that they are sometimes also called SAR (scaffold attachment regions), although they concern the nuclear matrix.

How might we demonstrate that particular DNA regions are genuinely associated with the matrix? In vivo and in vitro approaches are summarized in Figure 18.8. Both start by isolating the matrix as a crude nuclear preparation containing chromatin and nuclear proteins. Different treatments can then be used to characterize DNA in the matrix or to identify DNA able to attach to it.

To analyze the existing MAR, the chromosomal loops can be decondensed by extracting the proteins. Removal of the DNA loops by treatment with restriction nucleases leaves only the (presumptive) in vivo MAR sequences attached to the matrix.

The complementary approach is to remove all the DNA from the matrix by treatment with DNAase; then isolated fragments of DNA can be tested for their ability to bind to the matrix in vitro.

The same sequences should be associated with the matrix in vivo or in vitro. Once a potential MAR has been identified, the size of the minimal region needed for association in vitro can be determined by deletions. We can also then identify proteins that bind to the MAR sequences.

A surprising feature is the lack of conservation of sequence in MAR fragments. They are usually ~70% A PT-rich, but otherwise lack any consensus sequences. However, other interesting sequences often are in the DNA stretch containing the MAR. Cis-acting sites that regulate transcription are common. And a recognition site for topoisomerase II is usually present in the MAR. It is therefore possible that an MAR serves more than one function, providing a site for attachment to the matrix, but also containing other sites at which topological changes in DNA are effected.

What is the relationship between the chromosome scaffold of dividing cells and the nuclear matrix of interphase cells; are the same DNA sequences attached to both structures? In several cases, the same DNA fragments that are found with the nuclear matrix in vivo can be retrieved from the metaphase scaffold. And fragments that contain MAR sequences can bind to a metaphase scaffold. It therefore seems likely that DNA contains a single type of attachment site, which in interphase cells is connected to the nuclear matrix, and in mitotic cells is connected to the chromosome scaffold.

The nuclear matrix and chromosome scaffold consist of different proteins, although there are some common components. Topoisomerase II is a prominent component of the chromosome scaffold, and is a constituent of the nuclear matrix, suggesting that the control of topology is important in both cases.