New & Noteworthy

Puzzling Out Gene Expression

February 21, 2013

Have you ever put together a million piece puzzle that was all blue? That is sort of what it sometimes feels like figuring out how genes are turned on or off, up or down.

jigsaw puzzle

There are hundreds or even thousands of proteins called transcription factors (TFs) controlling gene expression. And there is a seemingly simple but frustratingly opaque string of DNA letters dictating which TFs are involved at a particular gene. Figuring out which sets of proteins bind where to control a gene’s expression can be a baffling ordeal.

Up until now most of the ways of identifying which TFs are bound at which genes have been incredibly labor intensive to do on a large scale. With all of the current techniques, researchers need to construct sets of reagents before they even get started. For example, to be able to immunoprecipitate TFs along with the DNA sequences they bind, you need to insert epitope tags in all the TF genes so an antibody can pull them down. Other techniques are just as involved.

What the field needs is a quick and dirty way to find where TFs bind in the genome. And now they just might have one.

In a new study, Mirzaei and coworkers used a modification of the well-known technique mass spectrometry (mass spec) to identify TFs that bind to a specific piece of DNA. With this technique, called selected reaction monitoring, the mass spec looks only for specific peptide sequences. This not only makes it much more sensitive and reproducible than ordinary mass spec, but it should also be relatively straightforward to do if a lab has access to the right sort of mass spec. They haven’t worked out all the bugs and it is definitely still a work in progress, but the technique looks promising.

Mirzaei and coworkers set up assays to detect 464 yeast proteins that are known or suspected to be involved in regulating RNA polymerase II transcription. Then they tested their assay on a 642 base pair piece of DNA known to contain signals that affect the levels of FLO11 transcription. They found fifteen proteins (out of the 222 they searched) that bound this piece of DNA. Of these, only one, Msn1p, had been previously identified as regulating the FLO11 gene. The other fourteen had not been found in any previous assays.

The authors next showed that two of these fourteen proteins, Mot3p and Azf1p, represented real regulators of the FLO11 gene. For example, deletion of MOT3 led to a threefold increase in FLO11 expression under certain conditions. And when AZF1 was deleted, FLO11 could not be activated under a different set of conditions. So Mot3p looks like a repressor of FLO11 and Azf1p looks like an activator.

This was a great proof of principle experiment, but much more work needs to be done before this will become a standard assay in the toolkit of scientists studying gene expression. They need to figure out why some known regulators of FLO11 (Flo8p, Ste12p, and Gcn4p) were missed in the assay and whether the other twelve proteins they discovered play a role in the regulation of the FLO11 gene.

Having said this, it is still important to note that even this early stage model of the assay identified two proteins that scientists did not know controlled FLO11 gene expression. At the very least this is a quick and easy way to quickly identify candidates for gene expression. We may not be able to use it to see the whole picture on the puzzle, but it will at least get us a good start on it.

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

Categories: Research Spotlight

Tags: transcription , RNA polymerase II , Saccharomyces cerevisiae

When Polymerases Collide

October 25, 2012

Lots of recent studies are showing that transcription happens over way more DNA than anyone previously thought. For example, the ENCODE project has shown that most of a genome gets transcribed into RNA in humans, fruit flies and nematodes. This transcriptional exuberance was recently confirmed in the yeast S. cerevisiae as well.

There is also a whole lot of antisense transcription going on. Taken together, these two observations suggest that there are lots of opportunities for two polymerases to run headlong into each other. And this could be a big problem if polymerases can’t easily get past one another.

Goats butting heads

What happens when RNA polymerases meet head-on?

Imagine that the two polymerases clash in the middle of some essential gene. If they can’t somehow resolve this situation, the gene would effectively be shut off. Bye bye cell!

Of course this is all theoretical at this point. After all, smaller polymerases like those from T3 and T4 bacteriophages manage to sneak past one another. It looks like this isn’t the case for RNA polymerase II (RNAPII), though.

As a new study by Hobson and coworkers in Molecular Cell shows, when two yeast RNAPII molecules meet in a head on collision on the same piece of DNA, they have real trouble getting past each other. This is true both in vitro and in vivo.

For the in vivo experiments, the authors created a situation where they could easily monitor the amount of transcription close in and far away from a promoter in yeast. Basically they pointed two inducible promoters, from the GAL10 and GAL7 genes, at one another and eliminated any transcription terminators between them. They also included G-less cassettes (regions encoding guanine-free RNA) at different positions relative to the GAL10 promoter, so that they could use RNAse T1 (which cleaves RNA at G residues) to look at how much transcription starts out and how much makes it to the end.

When they just turned on the GAL10 promoter, they saw equal amounts of transcription from both the beginning and the end of the GAL10 transcript. But when they turned on both GAL10 and GAL7, they saw only 21% of the more distant G-less cassette compared to the one closer to the GAL10 promoter.

They interpret this result as meaning the two polymerases have run into each other and stalled between the two promoters. And their in vitro data backs this up.

Using purified elongation complexes, they showed that when two polymerases charge at each other on the same template, transcripts of intermediate length are generated. They again interpret this as the polymerases stopping dead in their tracks once they run into one another. Consistent with this, they showed that these stalled polymerases are rock stable using agarose gel electrophoresis.

Left unchecked, polymerases that can’t figure out how to get past one another would obviously be bad for a cell. Even if it were a relatively rare occurrence, eventually two polymerases would clash somewhere important, with the end result being a dead cell. So how do cells get around this thorny problem?

King Arthur and the Black Knight

To get past the Black Knight, Arthur had to destroy him. Hopefully the cell has more tricks up its sleeve than that!

One way is to get rid of the polymerases. The lab previously showed that if a polymerase is permanently stalled because of some irreparable DNA lesion, the cell ubiquitinates the polymerase and targets it for destruction. In this study they used ubiquitin mutants to show that the same system can work at these paused polymerases too. Ubiquitylation-compromised yeast took longer to clear the polymerases than did their wild type brethren.

The authors think that this isn’t the only mechanism by which polymerases break free though. They are actively seeking factors that can help resolve these crashed polymerases. It will be interesting to see what cool way the cell has devised to resolve this dilemma.

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

Categories: Research Spotlight

Tags: transcription , RNA polymerase II , ubiquitin-mediated degradation , 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: Chd1 , chromatin , Isw1b , RNA polymerase II , Saccharomyces cerevisiae

New data tracks added to GBrowse

April 23, 2012

SGD has added a new mix of data tracks to our GBrowse genome viewer from seven publications covering transcriptome exploration via tiling microarrays (David et al. 2006), genomic occupancy of RNA polymerase II and III and associated factors (Kim et al. 2010; Ghavi-Helm 2008), 3′ end processing (Johnson et al. 2011), histone H2BK123 monoubiquitination (Schulze et al. 2011) and high-resolution ChIP by a novel method called ChIP-exo (Rhee et al. 2011; Rhee et al. 2012). Download data tracks, metadata and supplementary data by clicking on the ‘?’ icon on each data track within GBrowse or directly from the SGD downloads page. We welcome new data submissions pre- or post-publication and invite authors to work with us to integrate their data into our GBrowse and PBrowse viewers. Please contact us if you are interested in participating or have questions and comments. Happy browsing!

Categories: New Data

Tags: histone modifications , RNA polymerase II , RNA polymerase III , transcriptome , ChIP-exo

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