July 15, 2014
In the art of rock balancing, the artist positions large rocks with exquisite precision. If he or she succeeds, the rocks counterbalance each other and stay in seemingly impossible positions to make a surprising and beautiful sculpture. But a little uneven pressure is enough to make the whole thing collapse.
It turns out that the cellular acetylation state is just as precisely balanced. In a new GENETICS paper, Torres-Machorro and Pillus identify Esa1p, an acetyltransferase, as the balancing artist in Saccharomyces cerevisiae cells.
Acetylation is an important type of protein modification. Histones, the proteins that interact with DNA to provide structure to chromosomes, are acetylated by histone acetyltransferases (HATs) and deacetylated by histone deacetylases (HDACs). Some HATs and HDACs also act on non-histone proteins.
The acetylation state in a cell is a dynamic process. All those HATs are adding acetyl groups at the same time that HDACs are removing them. The final level of acetylation depends on the activities of each of these classes of proteins.
Acetylation of histones has been associated with increases in gene expression and deacetylation with decreases. So to keep gene expression levels in balance, it is very important to keep acetylation balanced as well. Throwing acetylation patterns just a bit out of whack can have profound consequences on global gene expression that can ultimately lead to cell death.
The authors focused on one particular HAT, Esa1p, that acetylates histones H4 and H2A and also has non-histone targets. They were intrigued by the fact that yeast cells cannot survive without Esa1p, since no other HAT or HDAC subunit is essential in yeast.
An obvious explanation for lethality is that losing this protein leads to too low a level of acetylation. They reasoned that if they also knocked out an HDAC, then the overall acetylation levels might increase and so rescue the esa1 null mutant. And they were right.
Using a plasmid-shuffling method, they created various double mutant strains of esa1 and HDAC genes, and found that a strain that was mutant in esa1 and also in either the SDS3 or DEP1 genes was viable. SDS3 and DEP1 both encode subunits of the Rpd3L HDAC complex.
Torres-Machorro and Pillus next characterized the esa1 sds3 double mutant further. They found that although the sds3 mutation suppressed the inviability of the esa1 mutant, it did not suppress other phenotypes such as sensitivity to high temperature and DNA damaging agents.
The authors found that the sds3 mutation subtly increased histone H4 acetylation, which was low in the absence of Esa1p. However, acetylation levels of a different histone, H3, remained high even in the absence of Esa1p. This suggested that the fundamental problem in the esa1 null mutant was an imbalance in the global state of histone acetylation.
To test this hypothesis, the researchers used a variety of different genetic methods to tweak the balance of cellular acetylation in the esa1 sds3 mutant. They created mutations in histones H3 and H4 that made it seem as if acetylation was low or high, and they also mutated other genes for HDAC subunits. It is as if they were passers-by who decided to poke at a balanced rock sculpture to see what it took to bring the whole thing down.
Although the details are too numerous to report here, the results showed that by using these genetic methods to tweak the overall acetylation state of the cell, the fitness of the esa1 sds3 strain could be improved: phenotypes such as slow growth, sensitivity to high temperature or DNA damaging agents, or cell cycle defects were suppressed to some extent by the various manipulations. This lends support to the hypothesis that Esa1p is the master balancer of acetylation levels in the cell and that this is its essential function.
This balancing act may happen in human cells too. Esa1p has a human ortholog, TIP60, that has been implicated in cancer and other diseases. Like Esa1p, TIP60 is essential and is involved in the DNA damage response.
So yeast teaches us that the acetylation of proteins is balanced on a knife’s edge. Even the slightest changes can lead to a collapse in global gene regulation, which can have catastrophic effects like cancer. All that we learn about Esa1p, the acetylation balancing artist, may have much broader implications for human health.
by Maria Costanzo, Ph.D., Senior Biocurator, SGD