Posttranslational modifications (PTMs) play a crucial role in a wide range of biological processes. Lysine crotonylation (Kcr) is a newly discovered histone PTM that is enriched at active gene promoters and potential enhancers in mammalian cell genomes. However, the cellular enzymes that regulate the addition and removal of Kcr are unknown, which has hindered further investigation of its cellular functions. Here we used a chemical proteomics approach to comprehensively profile ‘eraser’ enzymes that recognize a lysine-4 crotonylated histone H3 (H3K4Cr) mark. We found that Sirt1, Sirt2, and Sirt3 can catalyze the hydrolysis of lysine crotonylated histone peptides and proteins. More importantly, Sirt3 functions as a decrotonylase to regulate histone Kcr dynamics and gene transcription in living cells. This discovery not only opens opportunities for examining the physiological significance of histone Kcr, but also helps to unravel the unknown cellular mechanisms controlled by Sirt3, that have previously been considered solely as a deacetylase.
Most of the DNA in a cell is wound around histone proteins to form a compacted structure called chromatin. Enzymes can modify the histones by adding small chemical tags on to them, and these histone modifications can cause the chromatin to either become more tightly packed or more open. Opening up the chromatin makes the DNA more accessible to the cellular machinery involved in gene expression. Thus, cells can regulate which genes they express, and by how much, by modifying the histone proteins.
Like all other proteins, histones are made of smaller molecules called amino acids. Specific amino acids within histone proteins can be modified in a number of different ways, with different effects. For instance, adding a chemical tag called an acetyl group onto an amino acid in a histone weakens the interaction between the histone and the DNA, which opens up the chromatin and increases gene expression.
Another way that histones can be modified is by the addition of crotonyl groups. These chemical tags have not been examined much because the enzymes that add or remove them remain to be identified. However, it was recently suggested that enzymes called sirtuins—which are known to remove acetyl groups from histones—might also remove the crotonyl groups.
Finding histone-modifying enzymes is challenging because the interactions between these enzymes and the histones are both weak and brief. Bao, Wang, Li, Li et al. have now overcome this challenge by developing a method to firmly link any protein that interacts with a crotonylated histone to the histone. Three out of the seven sirtuin enzymes found in humans were revealed to bind to crotonylated histones. All three of these enzymes—called Sirt1, Sirt2 and Sirt3—could remove crotonyl groups from histones in a test-tube, and Sirt3 could also do the same in living cells. Further biochemical experiments suggested that the mechanism used by these enzymes to remove crotonyl groups is the same as the mechanism they use to remove acetyl groups.
Bao, Wang, Li, Li et al. then uncovered the three-dimensional structure of the Sirt3 enzyme bound to a crotonylated histone, and revealed that the enzyme recognizes the crotonyl group on the histone via a unique interaction between the crotonyl group and a specific amino acid in the binding pocket of Sirt3. This amino acid is also found in Sirt1 and Sirt2, but not in other sirtuins; this interaction can thus explain why decrotonylation activity was only detected for these three enzymes.
Moreover, the levels of crotonylated histones and gene expression were higher in cells that lacked Sirt3, but not in those lacking Sirt1 or Sirt2. By identifying Sirt3 as the main decrotonylation enzyme in living cells, the role of histone crotonylation can now be investigated in greater detail.