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

What Happens in Genes, Stays in Genes

September 06, 2012

Chromatin proteins, primarily histones, are a great way to control what parts of a cell’s DNA are accessible to its machinery.  These proteins coat the DNA and are marked up in certain ways to indicate how available a piece of DNA should be.  A methyl group here, an acetyl group there and a cell “knows” where the genes are that it is supposed to read!

Why doesn't RNA polymerase get a strike every time?

Of course this structure needs to be maintained or a cell might start to misread parts of its DNA as starting points of genes.  Then RNA polymerase II (RNAPII), the enzyme responsible for reading most protein-coding genes, would start making RNA from the wrong parts of the DNA, wreaking havoc in a cell.

One place where maintaining chromatin structure might be especially tricky is within the coding parts of genes.  It is easy to imagine RNAPII barreling down the DNA, knocking the proteins aside like pins in a bowling alley.  But it doesn’t.  For the most part the chromatin structure stays the same and survives the onslaught of an elongating RNAPII.

Two key marks for keeping histones in place are the trimethylation of lysine 36 of histone H3 (H3K36me3) that is mediated by Set2p, and a general deacetylation of histone H4 that is mediated by the Rpd3S histone deacetylase complex.  We know this because loss of either complex causes an increase in H4 acetylation and transcription starts from within genes.

In a recent study in Nature Structural & Molecular Biology, Smolle and coworkers identified two key components that help chromatin resist an elongating RNAPII in the yeast S. cerevisiae.  The first, called the Isw1b complex, binds H3K36me3 and the second, the Chd1 protein, binds RNAPII itself.  That these two were involved wasn’t surprising since previous work had suggested they helped prevent histone exchange at certain genes.

What makes this work unique is that the researchers showed the global importance of these proteins in the process and were able to tease out some of the fine details of what is going on at the molecular level. They used electrophoretic mobility shift assays to show that Isw1b bound the trimethylated form of H3 via its Ioc4p subunit and used chromosome immunoprecipitation coupled to microarrays (ChIP-chip) to show that Isw1b localized to the middle of genes in vivo. They also showed that when Set2p was removed, the localization disappeared (presumably because of the loss of the trimethylation of lysine 36).  They clearly demonstrated that Isw1b is found primarily in the middle of genes.

While these results indicate that the Ioc4p-containing Isw1b complex is moored to the middle of genes via its interaction with H3K36me3, it does not establish what it is doing there.  For this the researchers knocked out Isw1b and Chd1 and showed via genome tiling arrays a global increase in cryptic transcription starts.  The DNA in the middle of genes was now being used inappropriately by RNAPII as starting points for transcription.  Further investigation with Isw1b and Chd1 knockouts showed an increase in chromosome exchange and an increase in acetylated H4 in the middle of genes.

Whew.  So it appears that Isw1b and Chd1 inhibit inappropriate starts of transcription by keeping hypoacetylated histones in place over the parts of a gene that are read.   They are two of the key players in maintaining the right chromatin structure over genes.  They help keep RNAPII from railroading histones aside as it elongates, thus protecting the cell from inappropriate transcription starts.

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: RNA polymerase II, Isw1b, Chd1, chromatin, Saccharomyces cerevisiae