The histone fold and formation of the nucleosome
We have seen how in the eubacterial chromosome, bending DNA serves to facilitate its compaction. A similar process occurs in eukaryotic cells in that DNA is bent and wrapped around a protein unit. In this case, the core unit is a protein–DNA complex termed a nucleosome. The nucleosome comprises the core histone proteins H2A, H2B, H3 and H4 arranged in a structure known as the core histone octamer, with an associated length of DNA. In order to understand how the nucleosome is assembled, we first need to study the structure of the individual histones that it contains.
Each histone protein folds to give the histone fold motif as shown in Figure 27. This folding involves the two small helices (domains I and III) crossing over the central helix (domain II). Note that the N-terminal ‘tail’ is not part of this fold but extends freely. The next stage is the dimerisation of two of these folded histones through the crossing-over of the two central helices (domains II). Dimerisation occurs between two molecules of H3 or H4, but histones H2A and H2B form H2A : H2B heterodimers. Finally, one each of H3 : H3 and H4 : H4 and two H2A: H2B dimers come together to form the core histone octamer. This structure resembles a small cylinder with the eight N-terminal tails protruding freely from it.
Between 146 and 180 bp of DNA can be wrapped in two turns around the histone octamer unit to form the nucleosome, the structure of which is shown in Figure 28. The DNA wraps around the histone octamer rather like a thread wrapped around a spool. Contact between the two components (DNA and protein) is between the DNA backbone and the surface of the octamer edges. The many interactions between the negatively charged DNA backbone and the positively charged histone proteins serve to stabilise the structure. Note that the interaction between the histone proteins and the DNA is not sequence-specific; thus the histone octamer is able to bind DNA of any sequence. As the DNA wraps around the octamer, torsional stress is introduced into it such that for every nucleosome, two left-handed superhelical turns are introduced into the DNA. If you look back at Figure 11b, you can see that one conformation adopted by supercoiled DNA is a solenoid. The DNA wrapped around an octamer adopts just such a conformation. Thus supercoiling of DNA is accommodated or maintained by the nucleosomes.
Along each long DNA chain within the eukaryotic nucleus it is estimated that, at any one time, over 80% of the DNA is packaged within nucleosomes. Between the nucleosomes are small lengths of ‘linker’ DNA. The location of a nucleosome along any particular stretch of duplex DNA, such as relative to regulatory elements in a gene's promoter, is called translational positioning. This positioning determines whether any segment of DNA lies within a nucleosome or within the linker region.
When isolated chromatin is partially denatured and examined under the EM, it is seen to have a ‘beads on a string’ structure known as the 10 nm fibre, being about 10 nm thick (Figure 29a and b). The DNA that is wrapped around the histone octamer is inaccessible to many chemicals and enzymes, fulfilling a primary objective of DNA packaging. This protection of DNA can be exploited as a means of determining which part of any one region of DNA is or isn't associated with the octamer. If chromatin is isolated from a eukaryotic nucleus and treated with a nuclease that cleaves the DNA double helix, the DNA helix will be cleaved where it is unprotected, i.e. in the ‘linker’ regions. An analysis of the DNA isolated from this preparation using agarose gel electrophoresis reveals a ladder of nucleosomal fragments (Figure 29c).
Figure 29: (a) Pictorial representation showing nucleosomes along a DNA strand (top) being treated with a nuclease that cleaves between nucleosomes (red arrows) to generate DNA chains carrying one, two or three nucleosomes. (b) Electron micrograph of partially denatured nucleosomal DNA showing the typical ‘beads on a string’ appearance. (c) Partial digestion of nucleosomal DNA linker regions with a micrococcal nuclease gives a mixture of fragments containing different numbers of nucleosomes, as in (a). The DNA from two chromatin samples treated in this way is shown, analysed by gel electrophoresis, revealing a ladder of fragments that correspond to different lengths of DNA. The fragments in successive bands differ in length by a stretch of DNA equivalent to one nucleosome repeat length (146 bp). Shown for comparison is the result of micrococcal nuclease digestion of naked DNA: a smear of fragments due to random cleavage.
The precise position of the octamer core along any one DNA strand can also influence which ‘face’ of the helix is exposed on the surface of the nucleosome. Recall that helical B-form DNA has minor and major grooves and that most sequence-specific DNA interactions with proteins occur in the major groove where the bases are ‘accessible’ for recognition. The positioning of the nucleosome relative to the rotation of the DNA helix is called rotational positioning. If the position of the helix on the octamer is such that the bases of a particular region are facing into the core unit, the recognition site will not be accessible (Figure 30).
Consider the Zif 268 protein (Figure 23). What would the effect be if the 9 bp recognition sequence was positioned as in Figure 30b?
The protein could not bind, as the major groove is inaccessible.
Thus the position of the nucleosome and rotation of the DNA upon it can influence accessibility of the DNA to proteins. Note that this is also the case for proteins that bind in the minor groove, such as TBP. The positioning of a nucleosome is dependent upon many factors including the presence of proteins bound to DNA, which serve to direct which face is accessible. Only very small rotations in DNA helix (5–6 base pairs) relative to the nucleosome core are required to shield or expose sites in the major groove. This provides an obvious point at which regulation can occur.