All access genome: New study explores packaging of DNA

September 22, 2011

While efforts to unlock the subtleties of DNA have produced remarkable insights into the code of life, researchers still grapple with fundamental questions. For example, the underlying mechanisms by which human genes are turned on and off – generating essential proteins, determining our physical traits, and sometimes causing disease – remain poorly understood. 

Biophysicists Marcia Levitus and Kaushik Gurunathan at the Biodesign Institute at Arizona State University along with their colleagues Hannah S. Tims, and Jonathan Widom of Northwestern University in Evanston, Illinois have been preoccupied with tiny, spool-like entities known as nucleosomes. Their latest insights into how these structures wrap and unwrap, permitting regulatory proteins to access, bind with and act on regions of DNA, recently appeared in the Journal of Molecular Biology. Download Full Image

Nucleosomes, Levitus explains,  are essential components of the genome, acting to regulate access to DNA and protect it from harm. Nucleosome structure permits the entire strand of human DNA, roughly 6 feet in length, to be densely packed into the nucleus of every cell – an area just 10 microns in diameter. This occurs after nucleosomes assemble and fold into higher order structures, culminating in the formation of chromosomes.

Each nucleosome (there are roughly 30 million per cell) consists of a 147 base pair segment of DNA. This length of DNA thread is wound 1.67 times around the spool-like protein units, known as histones. The histone complex, together with its windings of DNA, forms the nucleosome core particle.

A multitude of proteins must act on regions of the DNA strand, by binding with appropriate target sites. Essential functions rely on these operations, including gene expression, replication and repair of damaged regions of the DNA molecule. But in eukaryotic cells like those of humans, some 75-80 percent of the DNA strand is curled up and hidden in the nucleosomes – inaccessible to protein binding interactions.

In earlier work, the group was able to show that nucleosomes are dynamic structures,  quite different from the static pictures produced by X-ray crystallography.  Lengths of DNA make themselves available for protein interaction by unwrapping and rewrapping around the histone core. When nucleosomes unwrap, proteins present in sufficient concentration can find their DNA targets and bind with them.

In order to observe and characterize the dynamic behavior of nucleosomes, the team relied on a versatile imaging method known as Fluorescence Resonance Energy Transfer or FRET. The technique allows researchers to look at a pair of fluorescent molecules or fluorophores, one of which is attached to the end of the exposed DNA strand, the other, to one of the histones around which the DNA is coiled, (see figure 1).

Fluorescence resonance energy transfer (FRET): experimental design. Nucleosomes are constructed having a fluorescent donor (cyan) attached to one end of the DNA, and a fluorescent acceptor (magenta) attached nearby on the histone protein core. In the middle diagram, spontaneous partial unwrapping of the DNA thread exposes a hidden DNA target site (hatched area), which is site-specific for the DNA binding protein LexA. When LexA is added in sufficient concentration, nucleosomes are temporarily trapped in their unwrapped state. The distance between the two fluorescent molecules changes as the DNA unwraps and rewraps, allowing the process to be precisely measured.

As Levitus explains, spontaneous unwrapping and rewrapping of DNA changes the distance between fluorophores, signaling that the process has occurred and allowing the group to quantify the frequency and rate of DNA exposure and concealment.

“Although FRET has been used for decades to measure molecular distances in biological systems, dynamic biomolecules such as nucleosomes present particular challenges,” notes Levitus. Traditionally, FRET experiments are performed with protein solutions containing  many billions of particles. In the case of nucleosomes however, the dynamic behavior of each particle is crucial and bulk measurements using FRET are not effective.

“In simple terms, if one wanted to understand how humans clap, it would be useless to listen to the whole planet clapping at once. Instead, one would listen to a few individuals, and that is exactly what we did with nucleosomes,” Levitus says.

The results of initial studies were revealing. For base pair sequences along the nucleosomes’ outer rind, spontaneous DNA unwrapping occurs at a rapid rate— about 4 times per second. This corresponds to a period of only 250 milliseconds during which this region of DNA remains fully wrapped and occluded by the histone complex. Once unwrapped, the DNA remains exposed for 10-50 milliseconds.

These findings present a plausible mechanism to allow protein binding with unwrapped DNA in vivo, so long as the binding sites occur near the ends of wrapped nucleosomal DNA.

The new study also examines, for the first time, the condition of DNA sequences occurring further along the wound length of nucleosomal DNA, that is, closer to the nucleosome’s center. Here, rates of DNA unwrapping decreased by orders of magnitude, (see figure 2).

This graphic shows the time elapsed during DNA unwrapping (a) and re-wrapping (b) as measured by FRET analysis. FRET works by measuring the distance between a pair of fluorescent molecules of fluorophores—one attached to the end of the DNA and the other attached to the histone protein spool around which the DNA “thread” winds and unwinds.

To examine this phenomenon, the group used a site-specific binding protein of Escherichia coli (known as LexA) to identify binding site exposure caused by nucleosome unwrapping. The nucleosomes were labeled with a FRET dye, which allowed the binding process of LexA and its target to be visualized. In successive experiments, the team shifted the binding sites in 10 base pair increments from the end of the nucleosome toward the middle.

The changes in unwrapping rate observed as the binding site was successively moved further inside the nucleosome were dramatic. In one case, a change in position of just 10 base pairs could produce a 250-fold decrease in unwrapping rate of the binding region.

These results prompt the question of how DNA binding sites more deeply wound within the nucleosome are able to successfully interact with their respective protein binders in vivo. The team proposes several possible mechanisms that would permit rapid access to hidden DNA binding regions, even where intrinsic rates of nucleosome unwrapping are low.

One hypothesis is that two or more proteins with target sites on the same nucleosome can act cooperatively, with one protein holding the momentarily unwrapped DNA open as the other enters the nucleosome and invades more inward regions of the DNA sequence, in what the authors describe as a ratcheting process.

Jonathan Widom, Dr. Levitus’ collaborator and a co-author of the new study was responsible for much of the pathbreaking research into nucleosome complexity. Dr. Widom died unexpectedly this past month. He was honored for his generosity, prolific research and outstanding contributions to biology in the August 25th issue of the journal Nature.

“I consider myself tremendously fortunate to have had the chance to collaborate with Jon Widom,” Levitus says. “Jon has been, and will continue to be, an incredible role model. His generosity, humility, and scientific genius has touched my life in many ways, and his death will leave a void that will be felt for many years to come.”

Ongoing research into the subtleties of nucleosome behavior promises to yield rich dividends for genomic science in general and provide a deeper appreciation for foundational issues of health and disease.

Marcia Levitus was the 2007 recipient of the prestigious National Science Foundation’s CAREER Award and the 2010 Inter-American Photochemistry Society Young Investigator Award. In addition to her tenured appointment in the Center for Single-Molecule Biophysics at the Biodesign Institute, she is an associate professor of chemistry and biochemistry in Arizona State University’s College of Liberal Arts and Sciences.

Figures reprinted from: Journal of Molecular Biology, volume 411(2), Tims HS, Gurunathan K, Levitus M, Widom J, Dynamics of nucleosome invasion by DNA binding proteins, pgs 430-48, with permission from Elsevier.


Richard Harth

Science writer, Biodesign Institute at ASU


Soccer opens Pac-12 play at No. 17 Cal

September 22, 2011

The Arizona State women’s soccer team opens Pac-12 play on Sept. 23 (4 p.m. PT) when it travels to Berkeley, Calif., to take on No. 17 California.

The Sun Devils went .500 (4-4) during the non-conference portion of their schedule. ASU opened the season with a pair of home wins against Northern Arizona (7-0 on Aug. 19) and South Carolina (1-0 on Aug. 28). Download Full Image

The Sun Devils would then drop their first two road games of the season, losing to No. 4 Virginia (3-0 on Sept. 2) and Old Dominion (3-1 on Sept. 4).

ASU has come away with splits each of the last two weeks. In its home tournament ASU hosted a pair of Big 12 teams, defeating Oklahoma (3-0 on Sept. 9) before losing to Missouri (1-0 on Sept. 11). ASU then closed out the non-conference portion of its schedule last week in Tucson where it fell to Texas Tech (5-0) before rebounding to beat Pacific (1-0).

ASU traveled to Tucson for the Loews Ventana Canyon Resort Cats Classic hosted by the University of Arizona and came away with a split, losing to Texas Tech 5-0 on Friday and defeating Pacific 1-0 on Sunday.

In the opening contest of the weekend the Sun Devils played Texas Tech to a scoreless tie in the first half before the Red Raiders came back for five goals in the second half to take the match.

Already down two starters on the front line – sophomore Devin Marshall and redshirt sophomore Nicki Stone – and missing goalkeeper Vittoria Arnold, who split time with Alyssa Gillmore in the season’s first three games, the injury-plagued Sun Devils were down another starter entering Sunday’s game against Pacific as sophomore defender Kaitlyn Pavlovich was sidelined after being injured against Texas Tech.

With all that going against them the Sun Devils still found a way to win against a Pacific squad which came into Sunday’s contest on a four-match winning streak. In addition, the Tigers had not allowed a goal during that stretch.

It appeared ASU would be headed for its first overtime match of the season until freshman Alexandra Doller scored her team-leading fifth goal in the 86th minute to give the Sun Devils the eventual 1-0 win.

It was the fourth shutout of the season for the Sun Devils and the second for goalkeeper Alyssa Gillmore, who was credited with one save and was extremely active throughout the contest in preventing the Tigers from scoring.

Coming off back-to-back NCAA Tournament appearances, the Sun Devils are looking to make it three NCAA bids in a row for the first time in school history. In order to do that ASU will have to overcome the challenge of losing its top two scorers from last season (and two of the top 10 scorers in school history) – Karin Volpe (No. 6 all time – 65 points, 28 goals) and Alexandra Elston (No. 9 all time – 48 points, 18 goals) – to graduation.

One player who figured to help the Sun Devils fill the aforementioned offensive void is sophomore forward Devin Marshall, who was named to the Pac-10 All-Freshman Team last season after finishing third on the team in scoring (10 points) and tied for first in assists (4). Marshall scored in ASU’s season opening win over Northern Arizona (Aug. 19). Unfortunately ASU was dealt a serious blow when Marshall sustained an ankle injury while assisting on teammate Alexandra Doller’s goal in the first half of ASU’s 3-1 loss at Old Dominion (Sept. 4). Marshall has missed ASU’s last five contests and her availability is currently being evaluated on a week-to-week basis.  

Other returners who have shown the ability to find the back of the net on multiple occasions include redshirt junior forward Sierra Cook (8 career goals - 3 in 2011) and redshirt junior Courtney Tinnin (4 career goals). Tinnin, who led ASU in assists in 2009 with six, did not play in 2010 due to a knee injury sustained in the spring of 2010. Tinnin has yet to play this season as she has been continuing rehab on the knee injury. It’s probable that she’ll be seeing her first action of the season on Friday against Cal.

Doller has been ASU’s top offensive performer thus far as she currently leads the team with five goals.

Another player who was expected to have an impact on the offensive end is redshirt sophomore Nicki Stone, who has spent the better part of the last two seasons recovering from knee injuries. Stone began the 2011 season as a starter on ASU’s front line and scored the game-winning goal in ASU’s win over South Carolina on Aug. 28. However Stone has not played since because of injury, missing ASU’s last six games. She is currently being evaluated on a week-to-week basis.

The ASU midfield figures to be a strength of the team as it returns 2010 All-Pac-10 Second-Team performer Taylor McCarter (has started all 48 career games) and sophomore Holland Crook, who started 14 of 20 contests last season and had a strong offseason according to Sun Devil head coach Kevin Boyd.

The Sun Devils return the majority of their starting back line from last season in addition to junior goalkeeper Alyssa Gillmore, who came into the season with 30 consecutive starts. Last season Gillmore posted five shutouts, including one of eventual College Cup participant Ohio State. Gillmore split time with sophomore keeper Vittoria Arnold in the season’s first three contests before Arnold was injured in the first half of ASU’s loss at Virginia (Sept. 2). Gillmore has played every minute of ASU’s last four contests as Arnold remains sidelined. Gillmore earned her second shutout of the season in last Sunday’s win over Pacific.

The injury bug made its way into the Sun Devils’ back line last week as sophomore defender Kaitlyn Pavlovich (had started all 27 games in her career) sustained a back injury against Texas Tech and is sidelined indefinitely.

The seven goals ASU scored in its 7-0 win over Northern Arizona on August 19 tied the single-game school record for most goals.  The original record was set and then equaled in consecutive weeks by the 2000 squad, which defeated Pittsburgh 7-0 on Sept. 10, 2000 and then followed that up with a 7-0 win over Eastern Michigan on Sept. 17.

Both Sierra Cook and Alexandra Doller scored a pair of goals against NAU. Also finding the back of the net for the Sun Devils were Jasmine Roth, Devin Marshall and Sarah Van Horn. Holland Crook assisted on a pair of ASU’s goals.

• Cal has 7-4-3 record against ASU although two of the last four meetings ended in a tie and three of the four games went to double overtime. The Sun Devils last win over the Bears came in 2006 in Berkley.

One of the biggest factors in ASU’s success the last two-plus seasons has been its ability to get off to good starts to open the season. Including the first eight games of 2011, the Sun Devils are 17-7-4 in August and September games since 2009.
Last season the Sun Devils began with a 6-0-1 record, which represented the second-longest unbeaten streak to start the season in school history. The only ASU team to have a longer streak was the 2000 squad, which began the season 9-0-0.

As has been the case in recent years, the Sun Devils will play one of the toughest schedules in the country. The Sun Devils will face seven teams in 2011 who were ranked in the NSCAA Preseason Top 25 (five of those seven squads reside in the Pac-12 Conference).
Aug. 28 – vs. South Carolina (#21 preseason)
Sept. 2 – at Virginia (#10 preseason)
Oct. 7 – vs. Oregon State (#19 preseason)
Oct. 14 – vs. Stanford (#2 preseason)
Oct. 21 – at USC (#25 preseason)
Oct. 23 – at UCLA (#16 preseason)
Oct. 30 – vs. Washington (#20 preseason)

The Sun Devils have made the most of their opportunities to play in front of their home crowd the last two-plus seasons as they are a combined 15-4-3 at Sun Devil Soccer Stadium since 2009. In addition, the Sun Devils are 15-1-2 in their last 18 non-conference games at home, a stretch which goes back to the 2008 season.

ASU’s seven home wins in 2009 tied the second-highest number of home wins in program history. Last season the Sun Devils established a new single-game attendance record when 1,513 fans poured into Sun Devil Soccer Stadium for ASU’s 2-1 State Farm Territorial Cup Series win over Arizona on October 15. The old record was 1,505 which came against Notre Dame in 2004.

Alexandra Doller’s two goals vs. Northern Arizona tied the school record for the most goals by a freshman in a season opener. Also accomplishing the feat was former Sun Devil Brittney Doty, who scored a pair of goals in ASU’s season opening win at UC Irvine in 2006.

Doller currently leads the Sun Devils in goals after notching her fifth of the season in last Sunday’s win vs. Pacific.

On August 9, former Sun Devil Soccer player Stacey Tullock became the first ever soccer player to be named to the ASU Sports Hall of Fame. Tullock is Arizona State’s all-time leader in goals (44), game-winning goals (15), assists (19) and total points (107). In 1998, she became the school’s first All-American selection, awarded by Soccer Buzz.