Gene silencing protein discovered

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Gene silencing protein discovered

http://www.news-medical.net/?id=17801

A team of researchers, including biologists at Washington University

in St. Louis, has discovered the key role one protein plays in a

major turn-off -in this case, the turning off of thousands of nearly

identical genes in a hybrid plant.

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.

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 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 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.

" 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 Fundagco para a Cijncia 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. "