Proteasome Recognized as Nuclear Player on Gene-transcription Team

Findings in Yeast May Also Apply to Other Organisms, Including Humans

The origin of the nucleus is one of the great turning points in evolution. With its emergence more than two billion years ago, the once democratic cell became divided into two distinct realms: the workaday world of the cytoplasm, where proteins are assembled and carry out the sundry needs of the cell, and the more esoteric compartment housing the genetic material. For years, the nucleus was regarded as a highly restricted zone, admitting only those molecules and proteins, such as RNA polymerase and transcription factors, that are specialized for transcribing the DNA’s message. It now appears that one of the most common and versatile cytoplasmic agents, the proteasome, plays a widespread and critical role in transcription—and it does so from inside the nucleus.

Photo by Graham Ramsay

The discovery that nuclear proteasomes interact with a large number of genes contributes to a larger endeavor. “We are building a computational model of what the inside of the nucleus looks like. We will incorporate these data into that model,” said Pamela Silver (left), shown with co-authors Kathryn Auld, Christopher Brown, and Suzanne Komili.

Researchers first spotted the proteasome, a barrel-shaped multiprotein complex, in the nucleus of yeast about a decade ago. Though a flurry of more recent studies suggested that these organelles play a role in regulating transcription, it was not clear how many genes they actually regulate. Nor was it clear how exactly they carry out their gene-regulating functions.

Kathryn Auld, Pamela Silver, and their colleagues took a genomewide, two-pronged approach to the puzzle, looking first at which yeast genes are bound by the proteasome and then at which of those are expressed only in the presence of a proteasome that is fully functioning. As they report in the March 17 Molecular Cell, the proteasome binds and critically regulates the transcription of some of the most highly expressed and important genes in the yeast genome, including those involved in lipid metabolism, mating behavior, and the making of ribosomal proteins.

Though the findings were made in yeast, the researchers believe that they likely apply to more complex organisms, including humans. “We found the proteasome to be very important in so many roles in yeast transcription that I cannot imagine it is not important in other organisms,” said Auld, an HMS graduate student in the Biological and Biomedical Sciences (BBS) program.

Up Close and Personal
What makes the discovery especially tantalizing is the way the nuclear proteasomes appear to be regulating transcription. In the cytoplasm, the organelle works by swallowing proteins and chopping them up into peptide bits. In this simple but effective manner, the proteasome regulates almost every aspect of the cell—growth, differentiation, the stress response, DNA repair. Even transcription may be regulated through the degradation of transcription factors in the cytoplasm. Though the nuclear proteasomes appear to work by cleaving certain transcription factors, the new findings suggest that they also play a more independent role, possibly associating directly with the chromatin at different stages of transcription. “We think that maybe the proteasome can act in each of the steps of transcription and in different ways,” Auld said.

Illustration by Rachel Eastwood, based on original courtesy of Kathryn Auld

Mission creep. In the nucleus, the proteasome could play a role at each of the three stages of transcription. During initiation, the organelle could cleave transcription factors (TFs), thereby activating them. It may also recruit and stabilize transcriptional complexes at the promoter. The proteasome might help the RNA polymerase complex move along the DNA during elongation and release the complex from the DNA during termination.

Catching the proteasome in the yeast nucleus was part calculation, part luck. In the 1990s, members of Silver’s lab had tried to chart the proteasome’s comings and goings in the nucleus. Auld, who came to the lab several years ago, wanted to expand this work. She approached Jason Casolari, then a BBS graduate student at HMS, who had been developing a method for mapping the interaction of proteins and genes in the nucleus, called genomewide location analysis. “Other labs were using the method to identify binding sites for specific transcription factors,” said Silver, HMS professor of systems biology.

Casolari, who is now a postdoctoral fellow at Stanford University, was taking a more systems biology approach, using the method to look broadly at how whole classes of proteins and protein complexes interact with the genome. Auld proposed that they use the approach to map where in the nucleus the proteasomes were found.

The proteasome is a famously bulky complex, consisting of a central barrel, the 20S core particle, flanked on each side by a funnel, the 19S regulatory subunit. Auld began by selecting three proteasomal proteins, two from the funnel and one from the core. She expected genomic localization would show that the three proteasomal proteins bound many of the same genes. As it turned out, two of the them displayed a high degree of overlap, binding to the same highly transcribed genes, while the third bound a somewhat different constellation.

Though the three proteasomal proteins bound to important genes, it was not at all clear that they were required for those genes’ transcription. To find out, Auld and her colleagues looked at a series of mutants, each one lacking one of the three proteins. Two large classes of genes, ribosomal genes and mating-type genes, were misregulated in all three mutants.

Still, it was not clear how the proteasomal proteins were working—by direct association with the genes or through a transcription factor? To get at this question, the researchers performed genomic localization on two transcription factors, Mga2 and Spt23. It was a lucky pick—the pair bound to many of the same genes that the three proteasomal proteins did.

She and her colleagues were now in a position to carry out a telling experiment. If the proteasomes work simply as protein-chopping machines, in this case cleaving Mga2 and Spt23, then already-cleaved Mga2 and Spt23 should turn on a gene even in a cell lacking a functioning proteasome. But when they looked at the three proteasomal mutants, they found that a particular lipid-metabolizing gene, OLE1, did not turn on. This showed that in addition to processing Mga2 and Spt23, the proteasome was doing something else to turn on the gene.

Flouting Convention
It is still not clear what else the proteasome might be doing. “It could be acting as a big mass of protein that helps open up the chromatin during activation,” said Silver. She and her colleagues believe it might also play a role in subsequent stages of transcription (see figure). “One idea we like is that the proteasome is involved in the release of the RNA transcription complex from the gene,” Silver said. “Some of these things may not be happening through its conventional actions as a proteasome.”

In fact, their findings—in particular, that one of the proteasomal proteins binds to different genes than the other two proteins do—could mean that the proteasome may not even exist as a coherent structure. “There has been a suggestion, a controversial one, that the different proteins in the proteasome may actually form different subcomplexes,” Silver said.

The likelihood that nuclear proteasomes exist in higher eukaryotes, including humans, has other paradigm-bending implications. It is possible that proteasome inhibitors, such as those used in the newly developed cancer drug Velcade, may actually work in the nucleus, turning genes on and off in a direct manner, rather than degrading transcription factors and other proteins in the cytoplasm. “This study, and others like it, could help expand our view of how these antitumor drugs work,” Silver said.

—Misia Landau

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