Genes VII

We know that nucleosomes can be reconstituted in vitro without regard to DNA sequence, but this does not mean that their formation in vivo is independent of sequence. Does a particular DNA sequence always lie in a certain position in vivo with regard to the topography of the nucleosome? Or are nucleosomes arranged randomly on DNA, so that a particular sequence may occur at any location, for example, in the core region in one copy of the genome and in the linker region in another?

To investigate this question, it is necessary to use a defined sequence of DNA; more precisely, we need to determine the position relative to the nucleosome of a defined point in the DNA. Figure 19.29 illustrates the principle of a procedure used to achieve this.

Suppose that the DNA sequence is organized into nucleosomes in only one particular configuration, so that each site on the DNA always is located at a particular position on the nucleosome. This type of organization is called nucleosome positioning (or sometimes nucleosome phasing). In a series of positioned nucleosomes, the linker regions of DNA comprise unique sites.

Consider the consequences for just a single nucleosome. Cleavage with micrococcal nuclease generates a monomeric fragment that constitutes a specific sequence. If the DNA is isolated and cleaved with a restriction enzyme that has only one target site in this fragment, it should be cut at a unique point. This produces two fragments, each of unique size.

The products of the micrococcal/restriction double digest are separated by gel electrophoresis. A probe representing the sequence on one side of the restriction site is used to identify the corresponding fragment in the double digest. This technique is called indirect end labeling.

Reversing the argument, the identification of a single sharp band demonstrates that the position of the restriction site is uniquely defined with respect to the end of the nucleosomal DNA (as defined by the micrococcal nuclease cut). So the nucleosome has a unique sequence of DNA.

What happens if the nucleosomes do not lie at a single position? Now the linkers consist of different DNA sequences in each copy of the genome. So the restriction site lies at a different position each time; in fact, it lies at all possible locations relative to the ends of the monomeric nucleosomal DNA. Figure 19.30 shows that the double cleavage then generates a broad smear, ranging from the smallest detectable fragment (~20 bases) to the length of the monomeric DNA.

In discussing these experiments, we have treated micrococcal nuclease as an enzyme that cleaves DNA at the exposed linker regions without any sort of sequence specificity. However, the enzyme actually does have some sequence specificity (biased toward selection of A PT-rich sequences). So we cannot assume that the existence of a specific band in the indirect end-labeling technique represents the distance from a restriction cut to the linker region. It could instead represent the distance from the restriction cut to a preferred micrococcal nuclease cleavage site!

This possibility is controlled by treating the naked DNA in exactly the same way as the chromatin. If there are preferred sites for micrococcal nuclease in the particular region, specific bands are found. Then this pattern of bands can be compared with the pattern generated from chromatin.

A difference between the control DNA band pattern and the chromatin pattern provides evidence for nucleosome positioning. Some of the bands present in the control DNA digest may disappear from the nucleosome digest, indicating that preferentially cleaved positions are unavailable. New bands may appear in the nucleosome digest when new sites are rendered preferentially accessible by the nucleosomal organization.

Nucleosome positioning might be accomplished in either of two ways:

• It is intrinsic: every nucleosome is deposited specifically at a particular DNA sequence. This modifies our view of the nucleosome as a subunit able to form between any sequence of DNA and a histone octamer.
• It is extrinsic: the first nucleosome in a region is preferentially assembled at a particular site. A preferential starting point for nucleosome positioning results from the presence of a region from which nucleosomes are excluded. The excluded region provides a boundary that restricts the positions available to the adjacent nucleosome. Then a series of nucleosomes may be assembled sequentially, with a defined repeat length.

It is now clear that the deposition of histone octamers on DNA is not random with regard to sequence. The pattern is intrinsic in some cases, in which it is determined by structural features in DNA. It is extrinsic in other cases, in which it results from the interactions of other proteins with the DNA and/or histones.

Certain structural features of DNA affect placement of histone octamers. DNA has intrinsic tendencies to bend in one direction rather than another; thus A PT-rich stretches locate so that the minor groove faces in towards the octamer, whereas G PC-rich stretches place so that the minor groove points out. Long runs of dA PdT (>8 bp) avoid positioning in the central superhelical turn of the core. It is not yet possible to sum all of the relevant structural effects and thus entirely to predict the location of a particular DNA sequence with regard to the nucleosome. Sequences that cause DNA to take up more extreme structures may have effects such as the exclusion of nucleosomes, and thus could cause boundary effects.

Positioning of nucleosomes near boundaries is common. If there is some variability in the construction of nucleosomes Xfor example, if the length of the linker can vary by, say, 10 bp Xthe specificity of location would decline proceeding away from the first, defined nucleosome at the boundary. In this case, we might expect the positioning to be maintained rigorously only relatively near the boundary.

The location of DNA on nucleosomes can be described in two ways. Figure 19.31 shows that translational positioning describes the position of DNA with regard to the boundaries of the nucleosome. In particular, it determines which sequences are found in the linker regions. Shifting the DNA by 10 bp brings the next turn into a linker region. So translational positioning determines which regions are more accessible (at least as judged by sensitivity to micrococcal nuclease).

Because DNA lies on the outside of the histone octamer, one face of any particular sequence is obscured by the histones, but the other face is accessible. Depending upon its positioning with regard to the nucleosome, a site in DNA that must be recognized by a regulator protein could be inaccessible or available. The exact position of the histone octamer with respect to DNA sequence may therefore be important. Figure 19.32 shows the effect of rotational positioning of the double helix with regard to the octamer surface. If the DNA is moved by a partial number of turns (imagine the DNA as rotating relative to the protein surface), there is a change in the exposure of sequence to the outside.

Both translational and rotational positioning can be important in controlling access to DNA. The best characterized cases of positioning involve the specific placement of nucleosomes at promoters. Translational positioning and/or the exclusion of nucleosomes from a particular sequence may be necessary to allow a transcription complex to form. Some regulatory factors can bind to DNA only if a nucleosome is excluded to make the DNA freely accessible, and this creates a boundary for translational positioning. In other cases, regulatory factors can bind to DNA on the surface of the nucleosome, but rotational positioning is important to ensure that the face of DNA with the appropriate contact points is exposed. We discuss the connection between nucleosomal organization and transcription in 21 Regulation of transcription.