Nucleosomes bundle up the DNA in a cell's nucleus, wrapping it around a complex of histone proteins. Studies of histone modifications and the proteins that bind to them reveal a mechanism that may control this packing.
Crack open any cell nucleus and look inside: you'll see what look like beads on a string. The beads are nucleosomes, small protein complexes that help to package the DNA (the strings) into the cramped confines of the nucleus. In the past fifteen years, nucleosomes have graduated in our understanding from being passive spools for DNA to full partners in the control of genetic information in the cell. Diverse chemical modifications of the histone proteins that form the nucleosome core can alter the expression of the associated genes1. These modifications make up what is known as the histone code, and a major challenge in molecular biology is to decipher how they affect gene expression.
Two of the most common modifications are phosphorylation and methylation — respectively the addition of a phosphate or a methyl group to the amino acids of which the histones are composed. Allis and colleagues2 previously proposed that reversible phosphorylation of the amino acids serine or threonine in the ‘tail’ regions of histones could antagonize the binding of regulatory proteins to neighbouring methylated lysine amino acids, creating a binary control switch. In this issue, Fischle et al. (page 1116)3 and Hirota et al. (page 1176)4 present data that strongly support this model and advance our understanding of how histone modification can control chromosome function.
Among the many histone modifications that have been described, methylation of lysines and phosphorylation of serines and threonines have attracted much attention. Phosphorylation of the serine at the tenth position in the tails of histone H3 (H3S10) occurs during cell division in eukaryotic (higher) cells. Once the cells have replicated their DNA and begin to prepare for division, nearly all of the histone H3 in the nucleus seems to be phosphorylated at this site. Phosphorylation of H3S10 also occurs at other stages in the cell cycle, but only at discrete chromosomal sites that are associated with gene expression.
The methylation of lysines is more complex. Methylation at lysine 9 of histone H3 (H3K9) is found mainly in the heterochromatin — the dense, mostly inactive regions of the genome. Methylation at lysine 4 of histone H3 (H3K4), by contrast, is associated with active genes. The different outcomes of lysine methylation result from the fact that each modification creates a binding target for a distinct protein. Heterochromatin protein 1 (HP1), which promotes heterochromatin formation (and the consequent gene silencing), recognizes and binds to methylated H3K9 using a region called the chromodomain5,6. CHD1, an enzyme that may destabilize nucleosomes and expose the DNA for gene expression, recognizes and binds to methylated H3K4 through two tandem chromodomains7.
Fischle et al.3 and Hirota et al.4 examined cells progressing into the metaphase stage of cell division, where the chromosomes become very tightly packed, or ‘condensed’, to facilitate their separation into the future daughter cells. Both groups discovered that these cells accumulated histone H3 that is both methylated at lysine 9 and phosphorylated at the neighbouring serine 10. Using antibodies specific for the doubly modified H3, the authors found that this dual modification occurred specifically in heterochromatic regions of chromosomes.
During most of the cell cycle, HP1 is also concentrated in heterochromatin, but with the onset of chromosome condensation, much of the HP1 leaves the chromosomes. Both labs link the dissociation of HP1 with the accumulation of phosphorylated H3S10 by showing that inactivation of an enzyme that phosphorylates H3S10 (Aurora B kinase) results in the retention of HP1. These observations suggest that H3S10 phosphorylation causes displacement of HP1 from heterochromatin with the onset of metaphase. To test this, the ability of HP1 to bind to dually modified H3 tail peptides was measured in vitro, using either fluorescence polarization3 or binding to peptide-coated beads4. In both assays, the binding of HP1 to the methylated H3K9 was substantially impaired when the neighbouring H3S10 was simultaneously phosphorylated.
The results demonstrate the need to examine the impact of multiple modifications on a histone tail; using an antibody that recognizes a single modification is clearly not sufficient to infer the histone state. The findings also provide experimental support for the regulatory-switch hypothesis of Allis and colleagues2. Methylation at H3K9, and concomitant binding of HP1, is not only found in heterochromatin, but also contributes to inactivation of some genes in euchromatin (the less dense and more active regions of the genome)8. If HP1 can be evicted by phosphorylation of H3S10, such repression might be reversed. However, the genomic region where HP1 binds would still be tagged by the methyl groups at H3K9. So if the H3S10 phosphate group were removed (by a protein phosphatase), that would leave the unopposed H3K9 methyl mark available to restore HP1 binding and reconstitute heterochromatin. This model is consistent with genetic studies implicating a histone methylase9,10 and a protein phosphatase11 in the control of heterochromatin formation in the fruitfly. Whether it applies to regulation in euchromatic domains remains to be seen.
Another study reported in this issue (page 1181)7 suggests that the methyl–phospho switch mechanism has wider applicability. Flanagan et al. describe the crystal structure of the double chromodomains of the mammalian CHD1 protein bound to an H3 peptide containing methylated H3K4. The way in which the CHD1 chromodomains bind to methyl-lysine seems different from how HP1 binds. But binding of the CHD1 chromodomains to methylated H3K4 is antagonized in vitro by phosphorylation of a neighbouring threonine (H3T3). CHD1 resembles a helicase, an enzyme capable of loosening nucleosome–DNA contacts. So controlled interaction of CHD1 with H3K4 by phosphorylation of H3T3 might regulate the access of regulatory proteins to DNA to control gene expression.
Using a binary switch to eject proteins that bind to methylated histones essentially reverses the effects of histone methylation. So far, the cell has been found to use two other methods to accomplish this end. First, as RNA polymerase traverses genes to make the encoded messenger RNA, it displaces the nucleosomes from the DNA. Nucleosomes re-form once the polymerase has passed, and the process can replace methylated histones with unmethylated ones12. Second, histone demethylase enzymes can directly strip the methyl groups off specific lysines13,14.
Why use three mechanisms to achieve the same biochemical result? Each has a different overall impact. Eviction of nucleosomes by RNA polymerase can remove all nucleosome modification marks (Fig. 1b). Lysine demethylation can target specific nucleosomes, but will also erase methylation patterns (Fig. 1c). However, by leaving methyl marks intact and ejecting the methyl-lysine binding factors through phosphorylation of neighbouring amino acids, cells can rapidly effect a large-scale reorganization of chromosome structure while preserving the underlying methylation pattern (Fig. 1a); this allows the original structure to be restored on dephosphorylation.
Fischle et al.2 identified 16 instances of lysines flanked by either serine or threonine among the four histone proteins that form the nucleosome, suggesting the possibility of other such binary switches. The monotonous beads-on-a-string conceal a rich variety of ornaments that compete with one another to control gene expression.
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