Photo by Todd McNaught
Though the genes we inherit dictate much of who we are, scientists have long known that that their mere presence is no guarantee of our fate. Among the thousands of genes that make up the DNA blueprint of an animal or human, it's those that are active at any given time or place in the body that govern the length of a fly's wing, the color of a cat's fur and even a person's likelihood of developing certain diseases.
With the development of a new technique by researchers in the Basic Sciences Division, scientists can now map the assortment of genes that are switched on or off in different situations more accurately than ever before. Applied to the study of fruit-fly cells, the method has already offered new insight into how gene-activity patterns may persist for many cell generations — a question that has long puzzled geneticists. It also may eventually help scientists determine the genetic choreography that allows an embryo to develop into an adult animal or that drives healthy cells to become cancerous.
Although the method was tested in fruit flies, it is based on tracking a protein that has nearly identical counterparts in all organisms, said Dr. Steven Henikoff, whose lab conducted the study. "We think this technique, which lacks many of the problems associated with existing methods, can be applied to plants, animals and humans," he said.
The study, led by Yoshiko Mito, a graduate student in the Molecular and Cellular Biology Program, is published in the October 2005 issue of Nature Genetics. Jorja Henikoff was a co-author. The new method emerged from a discovery made in Henikoff's lab several years ago. His group has a longstanding interest in how traits — such as the eye color of a fly — are determined not just by the information contained in a gene's DNA code but by proteins and chemical groups that latch on to genes, causing them to be turned on or off. This field is known as epigenetics, which is the study of inheritance of information that is not encoded in DNA sequence. Though the active or inactive states can be "inherited" as one cell divides into two — an outcome that is critical for endowing specific tissues or body parts with uniform characteristics — scientists have puzzled over how this epigenetic memory occurs.
Key players in epigenetics are nucleosomes, assemblies of proteins around which the DNA of chromosomes is wound. The proteins that comprise nucleosomes are called histones. When chromosomes duplicate — a prerequisite for cell division — new nucleosomes must assemble on the newly made DNA strands. Several years ago, Dr. Kami Ahmad in Henikoff's group had found that a variant of one of the histones, called H3.3, behaves differently from its more abundant counterpart, called H3. Unlike H3, H3.3 was found to exist in nucleosomes that are loaded onto chromosomes during times in the cell cycle other than when DNA is duplicated. The researchers showed that one such time is when standard nucleosomes are bumped off DNA during gene activation.
In the new study, Mito and colleagues used a method adapted by postdoc Dr. Takehito Furuyama in the lab to mark the two forms of histone in a way that allowed them to determine which parts of the genome are wrapped around nucleosomes containing histone H3 or H3.3. This "epigenomic profiling" method involves using DNA microarrays, which are chips containing small bits of DNA representative of the entire fruit fly genome. The microarrays allowed the scientists to determine the specific gene or part of a gene associated with the different histone forms.
The researchers saw H3.3 in parts of the fly genome that contain genes known to be active. They also saw that the amounts of H3.3 are higher over genes with higher activity levels, and that nucleosomes containing H3.3 exist not only on the active genes, but for some distance beyond each end of an active gene.
In their paper, Mito and colleagues provide evidence that the assembly of H3.3-containing nucleosomes on active genes may be what provides the "memory" of the active state from one cell generation to the next. The presence of the "active" nucleosomes would make genes easier to activate, or be decoded into RNA. This gene activation allows for the replacement of the H3-containing nucleosomes that are deposited after the DNA duplication process with H3.3-containing nucleosomes. This replacement would reset the active state of the gene.
Henikoff said that the new profiling technique can provide a much more accurate assessment of a gene's activity level than existing methods. Typically, other techniques rely on measuring the amount of RNA produced from a given gene or use antibodies to detect specific forms of histones. Both methods can provide less than accurate results. One project that could potentially benefit from his lab's new method, he said, is the Human Cancer Genome Project, a National Institutes of Health-sponsored effort to catalogue all of the genetic and epigenetic changes that contribute to the development of cancer.