The role of chromatin structure in the regulation of gene switching
15 January 2010
Research on the mechanism of gene switching could pave the way for regenerative medicine and provide a phenomenological foundation for the emerging field of epigenetics
Laboratory for Chromatin Dynamics
RIKEN Center for Developmental Biology
The human body consists of about 60 trillion cells, each of which contains all the genes necessary to define a ‘human’. However, not all genes are active, and excellent genes may lie dormant and inactive without exhibiting their functions. The cells of individual organs, such as the skin, muscles and nerves, have certain genes switched on while other genes are suppressed. Recent research has revealed that the mechanism for regulating the switching of genetic expression is present in the chromatin structure formed by DNA winding around proteins.
Chromatin structure and gene switching
Figure 1: The structure of a gene.
DNA winds around histones to form the chromatin structure. A gene cannot be switched on unless the chromatin structure loosens to detach or shift the histones and unwind the DNA’s double helix, allowing the base sequence to be ‘read’ as RNA.
Our personalities and abilities are not solely dependent on the genome inherited from our parents — instead they can be varied by a process known as ‘gene switching’, which causes some genes to function and others to be deactivated. “It was around the mid-1990s when the mechanism for regulating the switching on and off of genes started to become clear,” says Jun-ichi Nakayama, Team Leader of the Laboratory for Chromatin Dynamics at the RIKEN Kobe Institute’s Center for Developmental Biology.
In 1996, when Nakayama was engaged in research into proteins related to aging and cancer and making the best use of the biochemical techniques available at graduate school, he stumbled upon a paper that would later lead him to dramatically change his research theme. “Dr David Allis, currently at the Rockefeller University, discovered an enzyme that catalyzes the attachment of an acetyl group to a particular site in a histone, and demonstrated its association with switching on genes.”
A gene is encoded by its base sequence, or the arrangement of the four bases in the DNA: adenine (A), thymine (T), guanine (G) and cytosine (C) (Fig. 1). DNA comprises two strands that are complementarily bound with each other between A and T and between G and C, forming its double helical structure. In this structure, a portion of the DNA contains genes bearing information for protein production. When one of these genes is switched on, the base sequence of the gene region of the DNA is read as RNA (transcription), and the unwanted portions are cut off. This process produces messenger RNA (mRNA), which subsequently becomes a protein.
What does it mean when a gene is switched ‘on’? The DNA contained in a single human cell measures about 1.8 m in length when drawn out. The DNA winds around the histone proteins, forming a structure known as chromatin. This chromatin is condensed and housed in the cell nucleus. Highly condensed chromatin is called heterochromatin (Fig. 2).
Figure 2: Heterochromatin.
When mammalian cell DNA is stained with fluorescent dye, heterochromatin, which is a densely condensed assembly of chromatin, becomes visible under bright light (left). The basic structure of heterochromatin is also found in fission yeast (right).
“For a gene to get switched on, the condensed chromatin structure must loosen to detach or shift the histones and unwind the DNA’s double helix. As the helix is unwound, the base sequence of the gene becomes available in a readable state as RNA. It was known that histones undergo modification by the attachment of various chemical entities such as acetyl groups and methyl groups. However, their role was elusive and did not attract significant attention. Dr Allis demonstrated that when an acetyl group attaches to a particular site in a histone, the chromatin structure loosens to allow the gene to be switched on.”
In 1999, Nakayama joined Cold Spring Harbor Laboratories as a postdoctoral researcher. “Dr Allis successfully linked the results of a biochemical study of tetrahymena, a model organism, to the genetics of yeast, thus clarifying the role of histone acetylation. I was deeply impressed by his approach, and it made me want to link my own biochemical skills to genetic investigations, and also to research that may lead to resolving the question of chromatin.”
In 2001, as a result of his study of fission yeast, Nakayama made the discovery that a methyl group is attached to a site in the heterochromatin histone H3K9 (Fig. 3). “Upon methylation of H3K9 by the action of methylase Clr4, a protein called HP1 that recognizes it assembles to condense the chromatin and form heterochromatin. Thus, the gene of interest remains off. Surprisingly, this mechanism was found to work the same way in humans.”
RNA interference and heterochromatin formation
Figure 3: Gene switching and chromatin structural change.
When an acetyl group (Ac) attaches to a particular site in a histone, the chromatin structure loosens to allow the gene to be switched on (left). Meanwhile, when methylase Clr4 acts to attach a methyl group (Me) to a site in histone H3K9, the HP1 protein gathers there with the methyl group as a marker, resulting in chromatin condensation and heterochromatin formation; the gene thus remains off (right).
In 2002, Nakayama established his own research unit, the Laboratory for Chromatin Dynamics, at the RIKEN Center for Developmental Biology. “One of the questions to be resolved concerns how the histones surrounding a particular gene are methylated to form heterochromatin in order to switch off the gene. A US study group pointed out that RNA interference is involved in the mechanism. Hence, we began studying the association in detail.”
RNA interference, a phenomenon that was observed for the first time in nematodes in 1998, marked a major breakthrough in life science, and its discovery was awarded the 2006 Nobel Prize in Physiology or Medicine. “Double-stranded RNA in a cell is cut into shorter fragments, which in turn bind to protein to form a complex. This complex complementarily binds to a particular mRNA to degrade it. This is RNA interference. Hence, RNA interference may be described as another mechanism for gene suppression.”
Several research groups around the world have engaged in experimental studies using fission yeast and have shown that methylation of a particular histone involves utilization of part of the RNA interference mechanism. The methylation is generally assumed to proceed as follows. First, the base sequence is read from a region of the DNA where the gene is otherwise suppressed, resulting in the formation of double-stranded RNA. The double-stranded RNA is then cut into shorter fragments through the same mechanism as RNA interference, thus forming a complex with the protein. Subsequently, the complex returns to the region where double-stranded RNA produced and complementarily binds to the RNA being transcribed. Triggered by this process, the complex attracts methylase Clr4 for attachment of a methyl group to the histone, and also the protein HP1, which recognizes the methyl group and condenses chromatin to form heterochromatin (Fig. 4). “We discovered an important protein that serves in this complex mechanism, and have been working to elucidate its functions.”
Figure 4: RNA interference and heterochromatin formation.
(1) The DNA base sequence is read, and double-stranded RNA is produced. (2) The double-stranded RNA is cleaved to produce shorter RNA, which in turn binds to protein to form a complex. (3) The complex returns to the region where double-stranded RNA is produced, and complementarily binds to the RNA being transcribed. The complex attracts methylase Clr4 and HP1 protein to form heterochromatin.
Traditionally, it had been thought that only as little as 2% of all DNA is read into RNA, the remainder being deemed ‘junk’ DNA. In 2005, however, Yoshihide Hayashizaki, director of the Omics Science Center at the RIKEN Yokohama Institute, and his colleagues upset this common belief. They found that more than 70% of DNA is read into RNA. More surprisingly, they revealed that much RNA lacks information for protein production. “It was found that some of this RNA is involved in the dynamic structural change of chromatin to regulate the switching on and off of genes. Hence, the ‘junk’ DNA proved to have a function,” says Nakayama.
Mechanism for flexible changes in the chromatin structure
In chromatin structure research, it is necessary to comprehensively clarify not only the mechanism for attaching a methyl group to a particular site in a histone, but also the process for recognizing the methyl group and forming heterochromatin. In 2008, Nakayama and his colleagues discovered that a complex mechanism is at work in this process. “There are two types of HP1 protein in fission yeast, which are distinguished by their slightly different shapes. One promotes heterochromatin formation, and the other suppresses heterochromatin formation. The promoting type of HP1 alone cannot form and maintain heterochromatin. We discovered that heterochromatin cannot be formed and maintained unless the two functionally opposite types of HP1 assemble in a certain balance.” Why is this complex mechanism required? “Probably to allow the chromatin structure to change flexibly according to varied circumstances.”
Three mechanisms involved in cell differentiation
In higher organisms like humans, a mechanism is also available in which genes are strongly suppressed by direct methylation of the DNA, not the histones. DNA methylation has also been found to be closely related to histone modification. “DNA methylation and RNA interference are thought to have originally emerged as a defensive system by which the DNA and RNA of cell-invading viruses are recognized and suppressed. Assuming that the defensive system has evolved to cause chromatin structural change by histone modification, it is easy to understand why these mechanisms are closely related to each other. Furthermore, this defensive system can be assumed to have changed in such a way that it is available in the process of differentiation, which produces a wide variety of cells in multicellular organisms.”
In the genesis of multicellular organisms, one fertilized egg proliferates and differentiates into a wide variety of cells, including muscle, skin and nerves, to give rise to an individual. Each cell has all the genes required for the entire organism. However, a fertilized egg cannot differentiate into skin cells, for example, unless only the genes required for the skin cells are switched on and the other genes are suppressed so that they do not work. This suppression is thought to be mediated by three closely associated mechanisms: DNA methylation, RNA interference and chromatin structural change by histone modification.
iPS cells and chromatin structure
Research into the regulatory mechanism for gene switching is also important in regenerative medicine. Induced pluripotent stem cells (iPS cells) are attracting attention as a key factor for major advances in regenerative medicine. When several genes are introduced into a somatic cell after differentiating into skin cells and the like, the somatic cell restores its ability to differentiate into all types of cell, like the cells in the early developmental stages. As such, iPS cells are versatile. Because iPS cells can be created from somatic cells, they offer great possibilities for application to regenerative medicine with minimal ethical concerns. “In the cells of the skin and muscles, for example, unnecessary genes are suppressed by the chromatin structure. In iPS cells, the system for gene suppression seems to be ‘reprogrammed’ to loosen the chromatin structure and allow all of the genes to be switched. However, the mechanism behind this change in the chromatin structure, which is induced merely by introducing several genes, remains unknown.”
The efficiency of iPS cell production remains low. “Studies on the chromatin structure are expected to contribute to research into the mechanism behind iPS cell genesis, leading to increases in the production efficiency and differentiation of iPS cells into selected cell types.”
Epigenetics, a new keyword in life science
There is another type of behavioral change in genes that cannot be explained solely by the DNA base sequence: epigenetics. “Epigenetics gives rise to variations such as personality differences between monozygotic twins. Although they share exactly the same genome, monozygotic twins have different characters and abilities. In some cases, the elder twin remains healthy, whereas the younger contracts lifestyle-related diseases or cancer. It is thought that environmental factors such as diet, exercise, learning and stress have differential effects and cause slightly different on-off states for various genes, and that the accumulation of these differences gives rise to the personality differences between monozygotic twins.” Are there excellent genes in our body that remain inactive and do not exhibit their functions? “Maybe so. I think a good living environment may lead to the accumulation of better on-off states in the genes. Conversely, a bad lifestyle and stress may lead to the accumulation of on-off states that herald the onset of disease.”
Epigenetic research is important. “Since the base sequences of the genomes of a wide variety of organisms have been decoded, a major challenge ahead is to elucidate the epigenetics.” This could lead to a better understanding of life phenomena, allow the causes of disease to be clarified and therapies to be developed, and ultimately form the basis for regenerative medicine. Epigenetic research is currently being most energetically conducted in Europe and the US. The Japanese Society for Epigenetics was formed in 2007. “Epigenetic research has been advanced through basic studies using various model organisms, including yeast, red bread mould, plants, nematodes and Drosophila. These seemingly modest investigations have come into the limelight. A strong point of research in Europe and the US is that there is a tradition to place importance on basic research.”
The EU and US are about to launch a major project called the Human Epigenome Project with the aim of exhaustively investigating all regions of the genome for DNA methylation and histone modification in a wide variety of cells. Histone modification includes not only acetylation and methylation, but also ubiquitination and phosphorylation, and even the same modification produces different modes in the regulation of gene switching depending on the portion of the histone that is modified. There is thus much to investigate. “The Epigenome Project is certainly important. However, I want to emphasize that the system for regulating the switching of genes cannot be clarified merely by examining their modifications. How do the modifications take place, and how are the modifications read to regulate switching? These are critical. I want to continue to work to elucidate the mechanism. To understand the regulatory system for the gene as a whole, basic research using a wide variety of model organisms must continue. We are determined to continue to work on elucidating this complex mechanism using fission yeast, and to conduct investigations to clarify how the mechanism found in that organism works in humans. We want our work to contribute to elucidating the mechanisms for genesis and to the development of regenerative medicine.”