Nucleic acids and chromatin
Nucleic acids and chromatin

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Nucleic acids and chromatin

Core histone tail modification regulates DNA compaction

SAQ 34

What effect would neutralising the positive charges on the octamer N-terminal tails have upon the compaction of DNA by H1?

Answer

As the overall charge contribution from the octamer tails will be decreased, you would predict that compaction would be decreased.

As discussed earlier, the degree of compaction mediated by H1 is heavily influenced by the charge status of the N-terminal tails of the octamer proteins, as these assist in neutralising repulsive forces. Covalent modification of these histone tails can affect their net charge and thus their ability to mediate compaction of the DNA.

In addition, octamer tail modifications can create binding sites for proteins with specific recognition domains. Such proteins could alter the interactions between nucleosomes and influence compaction. The lysine residues of the core histone tails are frequently modified through the addition of methyl groups and acetyl groups or combinations of these. Similarly, the serine residues can also be modified by phosphorylation. Figure 32a shows four sites on the N-terminal region of histone H3 protein that are commonly modified. In many cases, such modifications are associated with transcriptional potential such as gene activation or silencing, or are correlated with cellular events such as mitosis (Figure 32b).

Figure 32
Figure 32 (a) Locations of residues in the N-terminal region of histone H3 that are subject to modification. (b) Examples of specific modifications in the histone H3 N-terminal tail region associated with various cellular events.

The most common modification of core histones is the addition of an acetyl group to lysine residues, which effectively removes the positive charge and therefore decreases the tails’ ability to interact with the DNA backbone. Thus regions where the N-terminal tails of histones are extensively acetylated are less able to form compacted 30 nm fibres. In such regions, the DNA is described as being in a more open conformation, and is more accessible to transcription factors and DNA polymerases. Modification of the histone protein tails by acetylation is regulated by a family of histone acetyltransferases (HAT) and histone deacetylases (HDAC). A family of proteins containing SET domains is responsible for the methylation of amino acid residues in the histone N-terminal tails. Many of these enzymes play key roles in the regulation of gene expression. Certain chemicals are known to inhibit histone deacetylase action and are commonly used to examine chromatin function. One example is trichostatin A. Treatment of cells with this drug results in the alteration of chromatin acetylation levels, with resulting alterations in gene expression.

As has already been described, it is possible to analyse how compact a region of chromatin is by examining its accessibility or sensitivity to nucleases. Analysis of chromatin sensitivity to DNAase I can be used to identify regulatory sequences in the genome. In regions of the genome protected in more compacted chromatin, DNAase I cannot access the DNA to cleave it. Regions where the chromatin fibre is less compacted have exposed stretches of DNA which are accessible to the DNAase I enzyme, so cleavage occurs. Analysis of chromatin susceptibility to DNAase I can be combined with restriction analysis and a Southern blot to reveal areas around genes that are accessible and hence most likely to be important sequences in gene regulation, as shown in Figure 33.

Figure 33
Figure 33 DNAase I hypersensitivity of chromatin.

Box 8

Figure 33: (a) Acetylation of the core histone tails causes the affected part of the chromatin structure to open up, making the DNA hypersensitive to cleavage by DNAase I. (b) Example of a DNAase hypersensitivity assay in the human FMR1 gene promoter region. Here, chromatin isolated from the nuclei of white blood cells was incubated with increasing amounts of DNAase I for 10 minutes, after which the DNA was separated from the chromatin proteins and treated with the restriction enzyme Eco RI (E  =  site of Eco RI cleavage). This DNA was then resolved by agarose gel electrophoresis, the fragments transferred by Southern blot to a membrane, and the fragment containing the FMR1 promoter detected using a specific hybridization probe (A). Treatment with increasing amounts of DNAase I causes the Eco RI fragment E1–E2, detected by probe A, to be cleaved at a single site (red arrow). Cleavage at this site reduces the intensity of the E1–E2 fragment and generates a smaller fragment (E2–D) (very pale). Hybridization of the same filter with probe B on the adjacent Eco RI fragment (E2–E3) serves as a control. This site of DNAase I hypersensitivity corresponds to the transcription start site of the FMR1 gene.

In addition to variations in the compactness of chromatin across a gene, the level of histone tail acetylation can also show very specific patterns, as illustrated in Figure 34. This figure shows the pattern of both DNAase I hypersensitivity and acetylation of H3 at lysine residue 9 (abbreviated as H3-K9-Ac) across the chicken β-globin locus. At the top is a map of the genomic region, with the sites of hypersensitivity to DNAase I identified by arrows and the position of the  β-globin gene cluster transcription units highlighted. Hypersensitivity sites HS1–4 play critical roles in gene regulation. The lower part of the figure shows the level of H3-K9 acetylation across this same region, assayed using a technique called chromatin immunoprecipitation (ChIp). , which is described in Box 9 This example uses an antibody that recognises the H3-K9 modification. As you can see, the level of acetylation of the H3 N-terminal tail at lysine 9 changes dramatically across the region, being virtually absent over much of the locus, but reaching high levels in discrete positions, several of which correspond to HS sites (such as HS4 and HS2).

Figure 34
Figure 34 Chromatin analysis across the chicken β-globin gene cluster, which comprises four genes: adult (two β) and developmental (ρ and η). Top: map of the locus indicating six DNAase I hypersensitive sites (red arrows), shown in relation to the coding regions of the β-globin genes; four of these hypersensitive sites (HS1–4) are known to be important in gene regulation. Bottom: the distribution of H3-K9-Ac across the locus as detected by chromatin immunoprecipitation (ChIp).

Box 9: Chromatin immunoprecipitation

Chromatin immunoprecipitation (abbreviated to ChIp) is a sensitive and common technique for analysing which proteins or specific protein modifications are associated with a region of DNA in vivo. (Note that this is the same principle as that of co-immunoprecipitation.)

Consider the region of DNA shown in Figure 35a. The DNA is wrapped into nucleosomes and two specific regions contain acetylated H3 N-terminal tails. Chromatin is isolated and cleaved at the linker regions to yield single nucleosomes and small DNA fragments. Nucleosomes carrying H3-K9-Ac are precipitated from this mixture using an antibody that specifically recognises the acetylated H3. The precipitated nucleosomes are purified and the DNA extracted from them. PCR is then used to assay for the presence or absence of particular markers sequences – A, B, C and D in the example in Figure 35b. As a positive control, purified DNA is used without any immunoprecipitation. In this example, markers B and D are detected in the immunoprecipitated DNA, indicating that these regions of the chromosome were originally associated with nucleosomes containing H3-Ac. Using this approach it is possible to screen large regions of chromosomes in vivo for specific histone protein modifications.

Figure 35
Figure 35 ChIp analysis of histone modification in chromatin. (a) Amap of a hypothetical stretch of DNA containing four marker sequences, A–D. (b) A stretch of DNA spanning the markers A–D shown complexed with nucleosomes. When subjected to treatment that cleaves the DNA at linker regions, single nucleosomes are generated. An antibody against H3 acetylated at lysine 9 (anti-H3-K9-Ac) is used to precipitate nucleosomes that contain acetylated H3 (right). The DNA is purified from the protein and the presence or absence of marker DNA for the four loci is assessed by PCR. Positive testing for markers B and D indicates that these regions of DNA were originally associated with nucleosomes containing H3-Ac. As a control, this procedure is performed with no immunoprecipitation (left).

Histone tail methylation also varies across eukaryote genomes. Which residue is modified can have a profound effect on the processing of the associated nucleosomal DNA, as the examples below illustrate. Modification at one residue may facilitate expression of a gene, whilst similar modification of a different residue in the same histone tail may result in transcriptional inactivation. These effects may be attributed to alterations in the stability of the chromatin structure, either directly, by affecting the net charge associated with the histone tails, or indirectly, by recruiting specific regulatory proteins to the affected stretch of chromatin.

An extreme case of histone tail methylation in chromatin can be seen with the mammalian X chromosome. In females, one copy of the chromosome is subject to X-inactivation. This chromosome is highly compacted and transcriptionally silent (i.e. inactive). An analysis of the complete chromosome complement of a female cell (shown in Figure 36) shows that, in contrast to the rest of the chromosomes, most of the inactivated X chromosome (indicated by the arrow) is not stained when probed with an antibody that recognises H3 methylated at lysine 4 (H3-K4-Me). The only region of the inactivated X chromosome that is labelled with this antibody is a very small region at the tip of one of the chromosome arms, a region known to be the only portion of this chromosome that is highly transcriptionally active.

Figure 36
Figure 36 Detection of lysine 4 methylation of H3 (H3-K4-Me) in chromosomes (blue) in a female human cell in metaphase using a fluorescently labelled antibody that specifically recognises H3-K4-Me. Note that staining (red) is absent from the majority of the transcriptionally silent, inactivated X chromosome (indicated by arrow).

Methylation of lysine 9 in histone H3 has a very different effect on transcriptional activity in the fission yeast S. pombe. Figure 37 illustrates the results of a ChIp analysis of the centromere from one chromosome of S. pombe. At the top is a map of the centromere and adjacent upstream and downstream regions and below are the levels of histone H3 methylated at the lysine 9 residue (abbreviated as H3-K9-Me). The pattern of H3-K9-Me modification shows a very clear association with the region around the centromere which is transcriptionally inactive.

As these examples illustrate, whilst the basic unit of chromatin structure, the histone octamer, remains the same across most of the DNA in the eukaryotic genome, the histone tails serve as a platform for many modifications. The pattern of these modifications in relation to their effects is known as the histone code.

Figure 37
Figure 37 The distribution of histone H3 lysine 9 methylation (H3-K9-Me) across the S. pombe centromere and adjacent upstream and downstream regions, detected by ChIp analysis. The transcriptionally silent region spanning the centromere is shown. Outside this region, actively transcribed genes are indicated (boxes and arrows).

The modifications of the histone tails discussed here are only a few of the many that are found within the eukaryotic cell. Research to decipher the code in different organisms is still under way. What we can say is that the modifications serve as a signalling system that promotes further interactions and activity from other proteins. For example, many transcriptional activators exert their effect through the local acetylation of histone tails, which in turn establishes a local environment in which transcription can occur.

There are situations where core histone proteins are replaced by specialised components. For example, histone 2AX is found incorporated adjacent to DNA breaks and plays a role in their repair, and a histone H2A variant called macroH2A is associated with the inactivated mammalian X chromosome and is involved in maintaining transcriptional silencing.

Box 10: The archaeal chromosome and eukaryote evolution

Archaea lack a nuclear membrane and hence are classified as prokaryotes. However, these organisms exhibit many features in common with eukaryotic cells. Recent studies on Archaea have identified the presence of proteins that, when dimerized, contain structures very similar to the histone fold seen in all eukaryotes. However, unlike their eukaryotic counterparts, these proteins contain no ‘tails’ (Figure 38a).

The dimers are able to wrap the chromosomal DNA into nucleosome-like particles (Figure 38b). The core elements of these structures appear to be dimers of a histone-like protein, HMf, with 80 bp of DNA wrapped around a core dimer. The interface with the DNA is dominated by lysine and arginine residues, which serve to counteract the negative charges on the sugar-phosphate DNA backbone.

The exact evolutionary relationship between Archaea and eukaryotes is uncertain, but these and other data suggest that they could share several common features indicative of a common ancestor. The development of a DNA compaction system such as one based on proteins carrying a histone fold structure may have solved the problem of balancing DNA compaction whilst still allowing access to the information stored within the DNA as a transcription template. Interestingly, many of the features of transcription in Archaea are similar to those in eukaryotes, such as the conservation of a TATA box, TBP and sequence similarity between RNA polymerases.

Figure 38
Figure 38 (a) The structure of the archaeal HMf dimer (cyan and green) highlighting secondary structure (pdb file 1bfm). (b) Nucleosome-like particles from M. thermoautotrophicum, a member of the Archaea.
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