New & Noteworthy

New Twist on INO80 Chromatin Remodeling

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 H2AH2B histone dimer to form smaller hexasomes, which are slid and repositioned differently from nucleosomes.

From Hsieh et al., 2022

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. 

From Hsieh et al., 2022

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.

From Hsieh et al., model of Arp5p binding the hexasome in a different conformation due to the absence of the dimer (right)

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

Tags: chromatin, chromatin remodeling, hexasomes, INO80 complex, nucleosomes, Saccharomyces cerevisiae, transcription

Unlocking Chromatin

February 10, 2016

Transcription factors need to break through a number of locks in the right order to get to their prize. Image from Petar Milošević via Creative Commons.

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).

These authors set out to provide direct proof that nucleosome positioning over URS2-L is the key to the second lock. They did this by making a set of chimeric promoters between HO and CLN2.

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.

Given that it switches a yeast cell’s mating type, it isn’t surprising that the HO gene is under such lock and key. Image from Wikimedia Commons.

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

Tags: chromatin remodeling, HO endonuclease, nucleosomes, transcription regulation