Key Step in Gene Activation Discovered

Scientists have cracked the code of an essential signal in the sequence of steps that controls the molecular choreography of gene regulation. The discovery is expected to aid development of therapies and prevention strategies for certain genetically triggered diseases such as breast cancers, pediatric cancers, and leukemia.

The research is published in a recent edition of the journal Cell by a Penn State team lead by Jerry L. Workman, the Paul Berg Professor of Biochemistry at Penn State and an associate investigator with the Howard Hughes Medical Institute, whose lab during the past few years has produced a series of discoveries that have dramatically altered the understanding of gene activation. The lab's latest achievement adds an important piece to the increasingly detailed picture of the individual feats performed by the menagerie of molecules that team up to turn genes on. The researchers have identified the communication code between two types of these molecules, which triggers them to begin the process of rearranging the shape of a gene, effectively unlocking its genetic code. One is a molecule nicknamed "SWI/SNF" and the other is a molecular family nicknamed the "HATs."

Like a pack of comicbook superheroes, each with a different power, the team of molecules that control the use of a gene's code has members with special skills and strange names. Inactive genes are locked in the "off" position by being densely coiled into structures called "Nucleosomes," which are built like a spool of thread, with the long strand of gene-containing DNA tightly wrapped around a spool-like core of powerful proteins called "Histones." The job of the Histones is to bind very strongly with the DNA strand, keeping it tightly coiled so its code can't be copied. "A normal cell turns on a particular gene only when it needs to produce a particular protein for a particular job at a particular time," Workman explains.

Electron microscope image of a SWI/SNF complex (large dark spot) attached to a DNA strand containing nucleosomes (small spots on the encircling string)

Photo Credit: Bazett-Jones et al., Molecular and Cell Biology, February 1999, p. 1474

Each of the organism's many genes is distinguishable from other material along the long DNA strand by its distinctive ordering of DNA building blocks, known as nucleotides. The particular order of nucleotides specifies the kind of protein the gene is destined to generate whenever it is turned on. "To unlock the code of a gene, you first have to find it and unwind it," Workman says.

Before the Workman group's most recent discovery, the molecular team that unlocks genes was known to include a protein called a "Transcription Activator," whose particular skill is the ability to find the gene that needs to be turned on among the hundreds of other genes and miscellaneous DNA material strung end-to-end along the whole length of the DNA molecule. The Transcription Activator then attaches itself to the very beginning of the targeted gene, known as the "Promoter" site, which has its own distinctive nucleotide sequence.

Workman's lab also recently had discovered that one area on the Transcription Activator meshes like a matching lock and key with an area on a very large protein group called the "Histone Acetyltransferase" complex, a.k.a. "HAT." Although researchers had known for over three decades that each member of the family of HAT complexes has the power to grab a chemical configuration called an "Acetate Group" from the circulating cellular material and attach it to the Histone proteins in the core of a nucleosome, no one had been able to figure out the usefulness of that bit of molecular remodeling. However, it was known that the attachment of the Transcription Activator to the beginning of a gene is the signal that attracts a HAT complex to join the action, and Workman's lab recently had shown that the HATs add Acetate Groups only to those Histones that are located near the Promoter found by the Transcription Activator.

Workman's lab also recently had discovered the most powerful molecular character involved in unwinding a gene, an enzyme called "SWI/SNF" (pronounced "switch-sniff"). SWI/SNF breaks the grip of the histone proteins in the core of the nucleosome spool, liberating the gene on that stretch of DNA so it can unwind far enough to be accessible to another molecule, the "Transcription Enzyme," whose job is to move along the gene's length coping its code. Other molecules then use this copy to make proteins, which do the work specified by the gene.

Using these previous discoveries as clues, Workman speculated that a collaboration between a HAT complex, the Transcription Activator, and the Acetate Groups somehow helps the Transcription Enzyme to copy a gene. His lab now has confirmed that hunch and has discovered how the process works.

Illustration of an association between (from top to bottom) a SWI/SNF protein complex, a Transcription Activator, and a string of DNA wound around Histone proteins, forming nucleosome spools

Illustration Credit: Ahmed H. Hassan, Penn State

"Our experiments show that when the HAT complex adds an Acetate Group onto a Histone protein near the Promoter site of a gene, it constructs a code that signals the SWI/SNF complex to attach to the Promoter and begin turning that gene on," says Ahmed H. Hassan, a member of the Workman team that made the new discovery along with fellow graduate student Kristen E. Neely. The team also learned that the Acetate Group functions not only as a coded marker but also as a mooring post for the SWI/SNF complex to hold onto, which helps it attach to the Promoter and also to stay close to the beginning of a gene, where it works to unwind the gene from its nucleosome spool and to keep it in the "on" position by forcing it to stay unwound. "The acetylation is the communication code used by the HAT complex to tell the SWI/SNF complex where to attach and where to start working, plus it helps SWI/SNF work more efficiently," Workman explains.

To perform the elegant experiments that produced these discoveries, Workman and his students created purified experimental materials that contained only the essential DNA sections and protein complexes they wished to study. "We needed to isolate the SWI/SNF and HAT complexes from other cellular materials so we could figure out their particular functions," Workman explains.

Using standard biochemistry techniques, the researchers first isolated the small section of DNA containing the gene's Promoter site, then they induced bacteria to grow the Promoter DNA in an uncoiled but otherwise identical form. "Bacterial DNA does not contain histones so it doesn't wind up into nucleosomes," Hassan explains. The team then mixed their Promoter-site DNA strands with Histone proteins they also had carefully prepared, and processed them under just the right conditions to get the two purified ingredients to assemble together into nucleosome spools. With this starting material, they performed a series of experiments, adding into test tubes various combinations of other gene-controlling molecules--including isolated or purified parts of the Transcription Activator, Acetate Groups, the HAT complexes, and the SWI/SNF complex--to learn their roles and their order of interaction.

"We added and removed these elements in a variety of orders in our experiments to find out in what possible order they could do their work within a living cell," Workman says. "Our experiments demonstrate that the HATs do their job first, followed by SWI/SNF."

The Workman team's research brings into sharper focus the step-by-step process of gene regulation at the level of transcription. "The picture we see now is that the Transcription Activator sticks up a little when it attaches to the Promoter site of a gene, creating a region that provides a connection point for the HAT complex, which helps it add the Acetate Group to the histones, which provides a connecting point for the SWI/SNF complex, which stabilizes it and holds it in place so it can start unwinding the gene," Workman explains.

Defects in proteins in the HAT and SWI/SNF complexes can cause a number of cancers in humans, presumably because they are part of this one pathway of turning genes on that must work normally in order for cells to grow normally. "The kinds of studies we are doing to learn exactly how a gene should function can allows us to think of ways to intervene biochemically when it malfunctions," Workman adds.

This work was supported by a grant from the National Institute of General Medical Sciences to Jerry L. Workman. In addition to Workman other members of the Penn State research team include graduate students Ahmed H. Hassan and Kristen E. Neely. Workman is a former Leukemia Society Scholar and is currently a Howard Hughes Medical Institute Investigator.

Contacts:

Jerry L. Workman: (+1) 814-863-8256 (office), (+1) 814-863-8554 (lab), JLW10@psu.edu (e-mail), http://www.bmb.psu.edu/workman/workman.htm (web)

Barbara K. Kennedy (PIO): (+1) 814-863-4682 (office), science@psu.edu (e-mail)

Illustrations :

A high-resolution electron-microscope image of SWI/SNF bound to nucleosomes, plus an illustration of the processes described in this press release, are available on the web at HTTP://http://www.science.psu.edu/alert/Workman5-2001.htm