June 03, 2022
The INO80 chromatin remodeling complex has long been the subject of intense study. Despite this, a recent report by Hsieh et al. in Molecular Cell reveals a new and unexpected biological activity: the INO80 complex (as compared to the other classes of chromatin remodelers) has a unique ability to act not only on nucleosomes but to enable transient detachment of an H2A–H2B histone dimer to form smaller hexasomes, which are slid and repositioned differently from nucleosomes.
Intriguingly, the authors demonstrate that the INO80 complex not only has the ability to act on hexasomes, but prefers to remodel hexasomes. Using in vitro biochemistry, they show that hexasomes are better substrates for the enzyme complex, better stimulate the enzyme’s ATPase activity, and are remodeled faster than full-size nucleosomes.
To explore the mechanisms underlying these observations, the authors asked about the acidic patches on H2A-H2B dimers. Given how previous studies had shown the importance of these patches for remodeling activity, the loss of one dimer of the two might be expected to hamper remodeling—not improve it. Instead, the team used a clever experiment with asymmetric nucleosomes containing mixtures of wild-type versus acidic patch mutant (APM) dimers to show how INO80 requires only a single acidic patch to maintain remodeling rates.
Arp5p is the protein within the INO80 complex that interacts most directly with acidic patches on histone H2 dimers. Using another series of in vitro experiments on reconstituted chromatin with a restriction enzyme accessibility assay and INO80(Δarp) (i.e. the complex lacking Arp5p), the authors show how the acidic patch specifically promotes formation of a key intermediate that primes the nucleosome for sliding along DNA.
That these complex experiments are so informative relies on the long history of studying yeast genes and proteins. These newer studies build on the breadth of earlier examinations to look at the complex abilities of protein assemblies to perform both overlapping and unique biochemical actions. The study of how chromatin is opened to allow transcription in a regulated fashion remains a critical area of study, for which yeast is an ideal model.
Categories: Research Spotlight
March 05, 2022
The “language” of histone methylation has been a subject of intensive study due to numerous diseases and disorders linked to faulty methylation patterns. Methylation patterns are “written” by enzymes in response to signals and then “read” by effector proteins recognizing methyl residues on highly specific lysine residues, leading to either large- or small-scale alterations in the transcriptional state of chromatin. In response to the cellular environment, signals are sent for the opposing processes of “writing” and “erasing” methylation. Conserved methyltransferase enzymes are the “writers” and demethylase enzymes are the “erasers,” with the activity of each regulated by cellular signals in ways that are poorly understood.
Whereas humans have 35 writers and 23 erasers, yeast has only four of each. Given orthology within each class of writers and erasers (as defined by the particular lysines methylated or demethylated), this makes yeast a perfect model system for digging into the links that connect cellular signals to specific methylation patterns on chromatin.
In a recent study in the Journal of Molecular Biology, Separovich et al. describe a systematic phosphosite mutant library that allowed the identification of key phosphorylated residues transducing cellular signals onto a writer/eraser pair. In response to environmental stress, Set2p methylates lysine 36 on histone H3 while Jhd1p opposes this action by demethylation. Using AlphaFold, they modeled the relationship between the specific phosphorylated residues and showed the key regulator of methylation activity (T127 on Set2p) is spatially proximal to the target lysine residue in the histone.
Upon analysis of differential expression for the sets of phosphonull and phosphomimetic mutants, they showed the proteins most affected by histone methylation clustered into GO categories consistent with cellular response to stress, e.g. ion membrane transport, lipid biosynthesis, ergosterol biosynthesis, and protein mannosylation.
While the kinase(s) responsible for phosphorylating the writer/eraser pair have yet to be identified, there are good candidates to test in yeast. The identification of the yeast players in signal transduction from environment to chromatin will undoubtedly be of use to those studying the much more complex system in humans.
Categories: Research Spotlight
April 06, 2018
This Research Spotlight also comes as a video animation. Be sure to check it out!
In the Star Wars movie Attack of the Clones, Padme and Anakin end up fighting Genosians on an automated assembly line. Padme manages to survive because the assembly line is set up in an ordered and predictable way. She knows when to jump, when to pause and so on to survive. If there was more chaos in the line, she might have been killed and then there’d be no Luke or Leia!
If the Genosian assembly line weren’t predictable, Padme might’ve ended up a pancake.
Just like the assembly line on Genosis, the gene is set up in an ordered and predictable way. Nucleosomes (barrel-shaped clusters of proteins) are spaced along the gene’s DNA to help keep it from getting tangled up. But when Pol II comes hustling along, the nucleosomes need to be carefully removed in front and then replaced after it passes.
Similar to the plight of poor Padme getting hurt if the assembly line were not predictable, genes can likewise get damaged if the nucleosome removal/replacement process goes haywire. In Star Wars, the result of a defective assembly line is damaged droids and TIE fighters. In cells, the result is a changed assembly line—the gene can end up longer! This change can be harmful for both the gene and the cell.
In a new study in GENETICS, Koch and coworkers use our favorite beast, the yeast Saccharomyces cerevisiae, to show that the ISW1 gene is a key player in making sure that the nucleosomes get back on DNA after being taken off. When yeast lack the ISW1 gene, nucleosomes don’t always return after being removed to make way for Pol II. And for some genes, this spells even more trouble.
The genes that have the most problems are those that have something called triplet repeats. Basically, this means the same 3 bases (e.g. CAG) are repeated many times in a row in the gene.
Using PCR, Koch and coworkers showed that a gene with triplet repeats ended up with more of them if the ISW1 protein (Isw1p) wasn’t there to shepherd the nucleosomes properly. And too many repeats in a gene can be harmful–not just to the yeast, but to us as well.
In fact, Triplet Repeat Expansion Disorders happen when the number of repeats increases from one generation to the next. These diseases are some of the most devastating ones around.
They mostly cause slow but steady degeneration of nerve and brain cells, and the cruel symptoms (loss of body movement control, dementia) often only show up in mid-life. The most well-known is probably Huntington’s disease, which killed folk-singer Woody Guthrie.
So understanding how Isw1p helps keep the Pol II assembly line running smoothly in yeast might help us understand how triplet repeat expansion happens in humans, and may eventually give us ideas how to keep it from happening in the first place. This is especially likely because humans have proteins that are similar to Isw1p and probably do something similar.
To determine how Isw1p regulates nucleosome reassembly, Koch and coworkers used Southern blots to show that yeast that lacked Isw1p couldn’t replace their nucleosomes after Pol II had passed as efficiently as yeast that had the protein. This means that when cells are missing the ISW1 gene, a long stretch of the DNA is left bare after Pol II has passed by.
This stretch of nucleosome-free DNA can end up forming new structures called hairpins that can cause cells to send in DNA repair machinery to deal with it. Unfortunately this machinery isn’t always that great at fixing the DNA. Like the Three Stooges adding more pipe to try to fix a leak, the cell can end up adding more DNA to deal with the hairpin.
Like adding extra DNA, throwing extra pipes at a plumbing leak is a great way to make a bad problem worse.
But for the cell, the results are not as hilarious as they were for Moe, Larry, and Curly. That extra DNA can lead to deadly genetic diseases.
A yeast cell needs Isw1p to keep the cell from bringing in the Three Stooges to mess up its DNA and potentially cause devastating genetic diseases. If it turns out the same is true in people, then once again yeast will have shown us how human genetic diseases might happen. And perhaps provide a target for us to go after to prevent these diseases from happening. #APOYG!
by Barry Starr, Ph.D., Barbara Dunn, Ph.D., and Kevin MacPherson, M.S.
Categories: Research Spotlight
February 10, 2016
In Die Hard, Hans Gruber and associates need to break through seven locks in the right order on a safe to get to bearer bonds worth 640 million dollars. Of course the hero John McClane foils the plot and beats the villains.
Nothing so exciting in yeast, but some genes are nearly as hard to turn on as that safe was to open. One of the most stubborn is the HO gene. It requires three locks or gates be opened in the right order to start making the HO endonuclease.
A new study in GENETICS by Yarrington and coworkers shows that the second lock for HO is a set of nucleosomes that blocks the binding of the transcription activator SBF. When they rejiggered this promoter so that these nucleosomes were removed, the HO gene needed fewer steps to get activated.
It is as if Hans Gruber and his gang only had five or six locks to get through to open their safe. And the 7th, hardest one was removed.
The HO gene is usually turned on in three sequential steps. First the Swi5p activator binds to a region called URS1, which recruits coactivators that then remodel the chromatin at the left half of URS2 (URS2-L). This allows SBF to bind its previously hidden binding sites which then remodels the chromatin again. Now a second set of SBF sites is revealed in the right half of URS2 (URS2-R).
Both of these promoters are activated by SBF. A key difference between the two is that the CLN2 promoter, like 95% of yeast promoters, is in a nucleosome depleted region (NDR).
The idea then is to make an HO promoter in which the usual URS2-L is replaced with the NDR region of CLN2. If the nucleosomes matter over URS2-L, then this construct should be activated in two instead of three steps.
Or, to put it another way, Swi5p binding to URS1, the first lock, will no longer be needed to open the second lock. HO activation will now be Swi5p independent. This is what the authors found.
When they looked at their chimeric protein that lacked nucleosomes over URS2-L, they found that using a strain deleted for SWI5 had very little effect on activity. There was only around a 2-fold difference in activity with this construct in the wild type and SWI5-deleted strains. This is very different than the wild type HO promoter where there was around a 15-fold difference between the two strains.
The authors then did an additional experiment where they took their chimeric reporter and mutated the nucleosome depleted region such that nucleosomes could bind there. This construct was now more Swi5p-dependent: there was around a 5-fold difference in activity between the wild type and SWI5 deletion strains. They had at least partially rebuilt that second lock.
Yarrington and coworkers continued with ChIP experiments to confirm that their chimeric construct was indeed depleted for nucleosomes, as well as other experiments to tease out more subtle details about the regulation.
Given that it switches a yeast cell’s mating type, it isn’t surprising that the HO gene is under such lock and key. The yeast cell wants to make sure it only turns on when needed. Just as the Nakatomi Corporation wanted to make sure only the right people could get to that fortune in bearer bonds.
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight