Frog (and histone) tails tell the tale

Rockefeller researcher proposes “death code” for cells

Using laboratory cultures of human leukemia cells and the tails of tadpoles, a Rockefeller University researcher has shown that specialized proteins in the cell nucleus contain chemical flags that provide a “code” that spells death.

The finding, published in the May 16 issue of the journal Cell, strongly suggests that these proteins, called histones, provide the code that directs cell death as well as cell growth and proliferation. This “death code” may provide scientists with a new approach for cancer therapy.

“We propose there may be a ‘death’ histone code as well as the ‘life’ histone code we previously described,” says C. David Allis, professor and head of the Laboratory of Chromatin Biology at Rockefeller. “We believe this ‘death’ code directs cells to die and may be manipulated to kill tumor cells.”

Allis joined Rockefeller in March 2003. The research described in Cell occurred while he was a professor at the University of Virginia, in collaboration with the Wellcome Trust Centre for Cell Biology, Edinburgh, Scotland; the University of Texas M.D. Anderson Cancer Center, Houston, Texas; and the Fox Chase Cancer Center, Philadelphia, Pa.

Tale of tails

The now-familiar double helix structure of DNA actually is tightly coiled in a Slinky-like manner around protein “spools” called histones. The coiled DNA and histones together form a largely protective and highly constrained structure called chromatin. Poking through this tightly folded complex are long flexible proteins — the histone tails.

The Slinky-like DNA coil must expand to allow gene-activating machinery access to genes, and scientists now believe that the histone tails are crucial to switching genes on or off. In 2000, Allis and Brian Strahl, Ph.D., a postdoctoral fellow in his University of Virginia lab, first proposed what has now been referred to as the “histone code.”

According to their hypothesis, chemical modifications of the histone tails play a vital role in determining which genes are turned on or off. These modifications, which attach certain chemical groups to specific amino acids (the building blocks of proteins) on the histone tails, act like flags to direct the docking of other important proteins, some of which open up the tightly wrapped DNA, providing access to genes.

In addition, many scientists, including Allis, believe these chemical modifications are responsible for passing on inherited traits without changing the structure of DNA, an emerging field called epigenetics.

C. David Allis, Ph.D.

For the Cell paper, Allis and his colleagues focused on histone H2B (histones are made of four subunits called H2A, H2B, H3 and H4) and the addition of the phosphate chemical flag to H2B’s tail in cells undergoing programmed cell death. Also called apoptosis, programmed cell death is a naturally occurring process that many cells go through during the course of development from embryo to adult. For example, the resorption of the tadpole tail at the time of its metamorphosis into a frog, the formation of the fingers and toes of a human fetus and the formation of the proper connections between neurons in the brain are among the developmental processes that require the elimination of surplus cells through apoptosis.

An integral component of the well-being of adult organisms, including humans, apoptosis controls the number of cells in the body and eliminates genetically or environmentally damaged cells. The tumor suppressor p53, for example, induces apoptosis to prevent tumor cells from spreading. Many diseases can be seen as a result of an imbalance of cells. Cancer and autoimmune diseases result in part from the survival of too many cells, while immunological diseases, neurodegenerative disorders and infertility occur when there is an excess of cell death.

Apoptosis and mitosis, although very different biological processes, share one similar feature: the chromatin in the nucleus condenses and compacts. But while the nucleus of a normally dividing cell appears round, the nucleus of an apoptotic cell becomes fragmented into grape-like clusters, which is a hallmark of cells that die via programmed cell death.

Clues to a code

A previous study of phosphorylation — the process that attaches a phosphate to an amino acid — of histones in apoptotic cells by Kozo Ajiro at the Aichi Cancer Center in Japan showed that only histone H2B was phosphorylated in dying mammalian cells, including laboratory cultures of human leukemia cells called HL-60.

Previously, Allis and collaborators had shown that in yeast, worms, flies and mammalian cells undergoing mitosis, histone H3 was phosphorylated at the serine 10 (S10) position, which was demonstrated when an antibody designed to bind to an amino acid sequence containing S10 stained mitotic chromatin. When the researchers turned their attention to histone H2B in laboratory cultures of human leukemia cells, Allis and his co-authors designed an antibody to determine which amino acid site on the histone tail was phosphorylated. They found that it was serine 14 (S14); however, the antibody did not bind to mitotic chromatin as expected, but bound to apoptotic, or “death,” chromatin instead.

The researchers then found that the antibody for S10 of histone H3 did not bind to S14 of histone H2B in apoptotic human leukemia cells, nor did the antibody for S14 of histone H2B bind to S10 on histone H3 in mitotic cells, which confirmed that two separate biological events — apoptosis and mitosis — are associated with histone phosphorylation.

To prove that H2B phosphorylation occurs in living organisms and not just in cell cultures, the researchers injected the antibody for S14 into tadpoles undergoing tail resorption. They found zones of dying cells in the tails stained by the antibody.

Importantly, the kinase responsible for the addition of S10 (and S28) in histone H3 during mitosis is aurora B kinase, a kinase amplified in many human cancers.

“It is curious that there exist two serines in the H3 tail — S10 and S28 — that are well-documented mitotic marks,” says Allis. “In contrast, there are two serines in the H2B tail (S14 and S32) that may well prove to be apoptotic marks.”

Allis points out that the spacing between these serines is 17 amino acids in both tails. “What this means is uncertain, but certainly intriguing,” he says.

Executioners’ song

Allis and co-workers also identified the specific kinase that phosphorylates S14 on histone H2B in humans: Mst1, or mammalian sterile 20-like kinase.

“Mst1 is known to trigger cell death, but its targets, especially those in the nucleus, are largely unknown,” says Allis.

During apoptosis, Mst1 is cleaved by enzymes called caspases, the primary executioners in programmed cell death. Scientists knew that the cleaved form of Mst1 entered the nucleus, but its precise destination and function was unknown until now.

“Our findings strongly suggest that one of Mst1’s targets in the nucleus is histone H2B, where it phosphorylates S14 and induces cell death,” says Allis.

The discovery of Mst1’s role in cell death “could have therapeutic value,” says Allis.

“There is a lot of research into developing inhibitors of caspases as anticancer therapies, but clinical results have been disappointing so far,” Allis continues. “Mst1, because it is a kinase, has a chemical enzymatic function that could be inhibited and may provide a new target, which in tandem with caspase inhibitors could provide an effective cancer therapy.”

Histone research at Rockefeller

The role of histones in gene activation first became known in the 1960s, when the late Vincent G. Allfrey, Ph.D., and colleagues at Rockefeller made seminal contributions to understanding how histones control gene activation in higher organisms. Allfrey’s experiments provided evidence that histones are modified by enzymes that attach acetyl, phosphoryl or methyl chemical groups to them.

“I think Allfrey, more than anybody else, set the stage for the importance of histone modifications,” says Allis. “And I think his insights will stand the test of time and prove, in the end, to be largely on the mark.”

For the next three decades, while scientists focused on chromatin and transcription factors, an essential group of over 200 proteins responsible for turning genes on or off, histones seemed to fall off the radar screen. This changed in 1996 when Allis, then a professor at the University of Rochester, and colleagues identified the enzyme responsible for attaching acetyl groups to histones — an enzyme that was already known to be a vital player in gene activation. A month after Allis’s discovery, a team led by Stuart Schreiber, Ph.D., at Harvard University identified the enzyme that removes acetyl from histones. Schreiber’s enzyme was an already well-known gene repressor.

“The field has not been the same since,” says Allis.

Even before Allis’s arrival, several Rockefeller labs have been carrying the Allfrey legacy and studying the role of histone modifications in gene activation.

Last year, Robert Roeder, Ph.D., and colleagues showed that when tails are removed from histones, gene activation does not occur, suggesting that the tails act more like “arms” to directly affect gene activity once the DNA is unpackaged. In addition to this unexpected finding, the researchers also developed a powerful new biochemical tool for deciphering a possible “histone code.” And in 2003, Rockefeller researchers led by Sasha Tarakhovsky, Ph.D., discovered that histone H3 is modified at an early stage of the B cell’s maturation process. This necessary change to the chromatin accounts for much of the immune cell’s repertoire of possible antibodies, and is a first in understanding the functional effects of chromatin changes.

Allis’s co-authors are Wang L. Cheung, Kozo Ajiro, Ph.D., Peter Cheung, Ph.D., and Craig A. Mizzen, Ph.D., at the University of Virginia Health System; Kumiko Samejima, William C. Earnshaw, Ph.D., at Wellcome Trust Center for Cell Biology; Malgorzata Kloc, Ph.D., and Laurence D. Etkin, Ph.D., at University of Texas M.D. Anderson Cancer Center; and Alexander Beaser, Ph.D., and Jonathan Chernoff, M.D., Ph.D., at Fox Chase Cancer Center.

This research was supported by the National Institutes of Health.

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