12. Domains define regions that contain active genes

19.12 Domains define regions that contain active genes

Key terms defined in this section

Domain of a chromosome may refer either to a discrete structural entity defined as a region within which supercoiling is independent of other domains; or to an extensive region including an expressed gene that has heightened sensitivity to degradation by the enzyme DNAase I.

A region of the genome that contains an active gene may have an altered structure. The change in structure precedes, and is different from, the disruption of nucleosome structure that may be caused by the actual passage of RNA polymerase.

One indication of the change in structure of transcribed chromatin is provided by its increased susceptibility to degradation by DNAase I. DNAase I sensitivity defines a chromosomal domain, a region of altered structure including at least one active transcription unit, and sometimes extending farther. (Note that use of the term "domain" does not imply any necessary connection with the structural domains identified by the loops of chromatin or chromosomes.)

When chromatin is digested with DNAase I, it is eventually degraded into acid-soluble material (very small fragments of DNA). The progress of the overall reaction can be followed in terms of the proportion of DNA that is rendered acid soluble. When only 10% of the total DNA has become acid soluble, more than 50% of the DNA of an active gene has been lost. This suggests that active genes are preferentially degraded (Stalder et al., 1980).

Figure 19.42 Sensitivity to DNAase I can be measured by determining the rate of disappearance of the material hybridizing with a particular probe.

The fate of individual genes can be followed by quantitating the amount of DNA that survives to react with a specific probe. The protocol is outlined in Figure 19.42. The principle is that the loss of a particular band indicates that the corresponding region of DNA has been degraded by the enzyme.

Figure 19.43 In adult erythroid cells, the adult b-globin gene is highly sensitive to DNAase I digestion, the embryonic b-globin gene is partially sensitive (probably due tIn adult erythroid cells, the adult b-globin gene is highly sensitive to DNAase I digestion, the embryonic b-globin gene is partially sensitive (probably due to spreading effects), but ovalbumin is not sensitive. Data kindly provided by Harold Weintraub.

Figure 19.43 shows what happens to β-globin genes and an ovalbumin gene in chromatin extracted from chicken red blood cells (in which globin genes are expressed and the ovalbumin gene is inactive). The restriction fragments representing the β-globin genes are rapidly lost, while those representing the ovalbumin gene show little degradation. (The ovalbumin gene in fact is digested at the same rate as the bulk of DNA.)

So the bulk of chromatin is relatively resistant to DNAase I and contains nonexpressed genes (as well as other sequences). A gene becomes relatively susceptible to the enzyme specifically in the tissue(s) in which it is expressed.

Is preferential susceptibility a characteristic only of rather actively expressed genes, such as globin, or of all active genes? Experiments using probes representing the entire cellular mRNA population suggest that all active genes, whether coding for abundant or for rare mRNAs, are preferentially susceptible to DNAase I. (However, there are variations in the degree of susceptibility.) Since the rarely expressed genes are likely to have very few RNA polymerase molecules actually engaged in transcription at any moment, this implies that the sensitivity to DNAase I does not result from the act of transcription, but is a feature of genes that are able to be transcribed.

What is the extent of the preferentially sensitive region? This can be determined by using a series of probes representing the flanking regions as well as the transcription unit itself. The sensitive region always extends over the entire transcribed region; an additional region of several kb on either side may show an intermediate level of sensitivity (probably as the result of spreading effects).

The critical concept implicit in the description of the domain is that a region of high sensitivity to DNAase I extends over a considerable distance. Often we think of regulation as residing in events that occur at a discrete site in DNA Xfor example, in the ability to initiate transcription at the promoter. Even if this is true, such regulation must determine, or must be accompanied by, a more wide-ranging change in structure. This is a difference between eukaryotes and prokaryotes.

Research

Stalder, J. et al. (1980). Tissue-specific DNA cleavage in the globin chromatin domain introduced by DNAase I. Cell 20, 451-460.