Researchers Find Protein That Silences Genes

Studying the phenomenon of nucleolar dominance, in which one parental set of ribosomal genes in a hybrid is silenced, Craig Pikaard, Ph.D., Washington University professor of biology in Arts & Sciences and colleagues have identified the protein HDA6 as an important player in the silencing. Using the experimental plant genus Arabidopsis, they have shown that HDA6 is located in the nucleus of Arabidopsis cells, and they have imaged it, characterized it biochemically and defined its role in two cellular activities that help bring about gene silencing.

According to Pikaard, genes can be turned off when acetyl groups — little two-carbon entities — are removed from histones, the proteins that wrap the DNA, and when methylation — a chemical modification of cytosine, one of the four chemical subunits of DNA — occurs. The removal of acetyl groups is called deacetylation. He and his collaborators found that one of many predicted histone deacetylases in Arabidopsis, HDA6 is a key player in both histone deacetylation and DNA methylation of ribosomal RNA genes. Both types of modification are studied as part of a biological field known as epigenetics, the goal of which is to understand how the packaging of DNA and its associated proteins can affect gene expression. In plants, as well as animals, some epigenetic traits are stable and can be inherited when a cell divides or even into the next generation.

Pikaard explains that understanding how some genes are selectively silenced and how silenced alleles can be turned on again may someday have practical benefits. For instance, tumor suppressor genes that normally help keep cells from dividing uncontrollably are often silenced by DNA methylation and histone modifications in cancer cells, contributing to tumor growth. And certain blood disorders resulting from defective genes expressed in adults might be alleviated if versions of those same genes that are only expressed very early in development, but are then silenced in adults, could only be turned on again. Though only dreams, at present, these sorts of ideas add to the excitement surrounding the field of epigenetics.

The big turn-off

For many years biologists thought that gene silencing in nucleolar dominance was a result of one set of ribosomal RNA genes being selectively turned on. But in 1997, Pikaard and colleagues found that they could switch on the silent genes using chemicals that inhibit either DNA methylation or histone deacetylation, indicating that turning off one parental set of ribosomal genes was really the secret to nucleolar dominance. In other words, all the factors needed for expression of the genes were in place but somehow the silenced genes were denied access to them. Since that time, Pikaard and his colleagues have been on the hunt for the proteins responsible for keeping the silenced genes off.

In their current paper, published on-line on April 28, 2006, in Genes and Development, and the cover story for the print version of the journal due out May 15, Pikaard and his collaborators describe a systematic effort to examine the 16 predicted histone deacetylases in the genome to see if any play a role in nucleolar dominance. They made transgenic hybrids in which each of the deacetylases were knocked out one by one and then examined the plants to see if there were effects on nucleolar dominance. In this process they found that knocking down HDA6 eliminated nucleolar dominance, such that the normally silent genes were now turned on.

To find out where HDA6 is located in the cell, the group then genetically engineered the protein to include a fluorescent tag and found that much of the HDA6, seen as a glowing red signal under the microscope, shows up in the nucleolus, which is precisely the site where ribosomal RNA genes are regulated and where nucleolar dominance occurs. "We found HDA6 at the scene of the crime, which was reassuring," Pikaard said.

Ph.D. student Keith Earley in the group characterized HDA6 biochemically and demonstrated that it was, in fact, a histone deacetylase, as predicted, and that the protein would remove acetyl groups from several different histones. A collaboration with mass spectrometry expert Michael Gross, Ph.D., Washington University professor of chemistry, helped define the precise locations of the acetyl groups that HDA6 can remove, down to which acetylated amino acids are involved.

"The bottom line is that HDA6 has very broad specificity. It can remove the acetyl groups from multiple histones and from multiple lysines of those histones" said Pikaard.

When multiple acetyl groups are on the histones, the genes are turned on, Pikaard explained. When they are removed by HDA6, it contributes to gene silencing. Using antibodies that recognize specific histone modifications that occur on the genes when they switch from off to on, the group was able to confirm that the deacetylation specificities they observed for HDA6 in the test tube fit with the changes in acetylation that occur on ribosomal RNA genes in living cells.

They also found that the mechanism behind the silencing involves both modifications of histones and changes in DNA methylation, and that HDA6 affects both.

Circular pathway to silence

"Somehow these modifications are linked together," Pikaard said. "We know that they work together and that HDA6 is a key player. They are intimately linked in a circular, self-reinforcing pathway. Each specifies the other. For instance, in modifying the histones a pathway is set in motion to recruit enzymes to perform DNA methylation. Likewise, changing DNA methylation leads to changes in histone modification".

Pikaard's other collaborators, all experts in microscopy, are researchers from the Instituto Superior de Agronomia, Tapada da Ajuda, in Lisbon, Portugal, and the Universidade Nova de Lisboa, Monte da Caparica, Caparica, Portugal. The work was supported by the National Institutes of Health and the National Science Foundation as well as the Fundação para a Ciência e Tecnologia, Portugal.

Pikaard's ultimate direction is to find out what makes the cell decide which set of ribosomal genes to silence.

"We understand better how the silencing is happening, but we don't know how the choice is made," Pikaard said. "Another thing we want to know is how all these activities for histone modification and DNA methylation are working together. At some point the various proteins must be interacting. The long term goal, though, is finding the choice mechanism."