Everyone carries some baggage they would like to lose. For the histone protein H3, that baggage is a chunk of its tail, which when clipped off affects the expression of genes with which the histone is associated.
Chromatin — the complex of DNA and histone proteins — is highly dynamic. The histone constituents of chromatin can be reversibly modified, replaced with related proteins, or cleaved by proteolytic enzymes. Two studies1,2, published in Cell and Nature Structural and Molecular Biology, now show that histone modification by regulated proteolytic cleavage is required during cell differentiation in both mammals and yeast to alter gene expression.
A histone molecule is divided into two distinct regions: a carboxy terminus that organizes DNA on the surface of each unit of the DNA–histone complex (the nucleosome), and a flexible amino terminus dubbed the N tail, which protrudes from the nucleosome. N tails make contact with DNA and with histones in adjacent nucleosomes, thereby enabling chromatin folding. Histone proteins are also typically decorated with a large number and variety of small chemical groups, such as methyl and acetyl groups, and these modifications have a central role in regulating gene transcription3. Why so many modifications, and how do they affect transcription?
The prevailing view holds that distinct patterns of N-tail modifications, or 'marks', provide signals for the recruitment of specific transcription factors to chromatin or stimulate the activity of proteins that either modify the chromatin further or disrupt it3,4. As a consequence, transcription is turned on or off, depending on whether activating or repressing marks are present. The corollary of this is that, when the transcriptional status has to be reversed, the N-tail marks must be removed to allow appropriate restructuring of chromatin.
Commonly, marks are actively removed using the opposite reaction to the one that added them; for example, histone deacetylase enzymes remove acetyl marks, which were added by histone acetyltransferases. But what happens if more than one mark must be removed simultaneously? Most enzymes engaged in histone modification show specificity — that is, they only attack single, or several, marks of the same kind. Using this strategy, each type of modification would have to be removed by a different enzyme. Duncan et al.1 and Santos-Rosa and colleagues2 describe a more dramatic way in which multiple marks are simultaneously removed from the N tail of histone H3 in mouse and yeast cells, respectively. This mechanism involves enzymatic cleavage of the first 21 amino-acid residues at the N terminus of H3, thus sweeping away en masse all modifications present in this region (Fig. 1).
Despite the evolutionary conservation of this mechanism, there seems to be a striking difference between mice and yeast in the endopeptidase enzyme each organism uses for N-tail cleavage. The 'clipper' in mouse is cathepsin L, which belongs to a class of protease enzymes called cysteine proteases. Cathepsins are found in the cellular organelles called lysosomes, but are also present in the nucleus5. The as-yet-unidentified yeast enzyme is likely to be a serine protease2. The feature these enzymes share, however, is that they are regulated: cathepsin L is induced during differentiation of embryonic stem cells in mice, whereas the activity of the yeast enzyme is triggered under conditions of nutrient deprivation that lead either to the stationary phase of growth or to sporulation.
The signals that promote the activity of the mouse and yeast proteases are unknown. Both groups1,2 report, however, that the two enzymes are sensitive to the modification state of the target H3 N tail. The presence of certain marks inhibited the activity of the endopeptidases, whereas other marks increased their activity (Fig. 1). So it could be that enzymatic cleavage of H3 is regulated by an interplay between the endopeptidases and other chromatin-modifying activities.
What are the biological consequences of H3-tail clipping? The role of H3-tail cleavage during differentiation of embryonic stem cells is unknown, although stem cells contain a signature mark of trimethylation on lysines 4 and 27 (H3K4me3/H3K27me3) in the H3 N tail that is altered upon differentiation6. By removing this and other modifications during differentiation, H3-tail clipping could set the transcriptional state of a particular cell lineage.
At the cellular level, H3-tail clipping could simply clear all repressive marks from chromatin, thereby allowing the binding of transcription-activator complexes to the affected DNA. Indeed, the results of Santos-Rosa and colleagues2 support a role for this modification in regulating the selective activation of transcription in yeast, as mutation of the H3 cleavage site impaired expression of several genes normally activated during sporulation or entry into the stationary phase. The authors' work2 also hints at an additional effect of H3-tail clipping in yeast, involving regulation of nucleosome displacement. Cleavage of the H3 tail precedes loss of nucleosomes at several promoter sequences in vivo — an event that exposes promoter DNA and thus enhances the binding of transcription-activator complexes to the promoter during gene activation7. Intriguingly, a trimethylation mark, K4me3, which prevents clipping by the yeast endopeptidase in vitro, is maintained in chromatin at promoters during gene activation8. This observation supports a mechanism in which nucleosomes that do not contain K4me3 are marked for H3 cleavage and subsequent displacement.
Apart from the unknown identity of the serine protease that clips H3 in yeast, and the mysterious way in which clipper activities in mouse and yeast are regulated, many other questions arise from these fascinating observations1,2. The first relates to the unexpected role of lysosomal proteases in chromatin activities. Do other cysteine proteases also have nuclear targets? Second, is N-tail clipping unique to H3, or are other histone proteins also cleaved at their N tails in a regulated manner? There are reports that other histone N tails are proteolytically removed, notably during development in the ciliate Tetrahymena, in which many striking observations relating to chromatin structure have been made9. Third, could the loss of the N tail affect gene expression not just by removing some marks but also by actively preventing formation of others on N tails? And finally, how is the cleaved H3 molecule replaced with an intact H3, and could this provide a mechanism for substituting one set of tail marks for another? Just when it seemed that we knew how chromatin structure is regulated, previously unknown pathways emerge to keep this field of research vital and interesting.