RIKEN Center for Developmental Biology

2003 Annual Report

Modifications to the nuclear DNA-protein chromatin complex are central to the epigenetic regulation of gene transcription, an activity that must be maintained and propagated across mitotic cycles and throughout the development of the organism proper in order for cells to establish and maintain their identities. Chromatin occurs in highly-condensed and less spatially concentrated states, known as heterochromatin and euchromatin, respectively. Heterochromatin regions have fewer genes overall than euchromatic stretches of the genome, and many of the genes that are found there remain unexpressed. However, heterochromatin functions as more than a locked closet to store stretches of unused DNA; in fact, there appear to be a number of functionally distinct types of heterochromatin serving in a spectrum of developmentally important capacities, from the transcriptional regulation of cell-type specific genes to genomic self-defense by compartmentalizing and neutralizing foreign mobile genetic elements that might otherwise interfere with proper gene function. Heterochromatin also functions in two genetically silent chromosomal regions: telomeres, which play key roles in replicative senescence and cancer, and centromeres, the linchpins of mitosis.

Using the fission yeast Saccharomyces pombe as a model system, Jun-ichi Nakayama focuses on investigations of heterochromatin dynamics, modifications to the DNA-packing proteins called 'histones' in particular, and the molecular mechanisms that allow such chemical states to be heritably transmitted. Histones are the primary protein constituents of the nucleosome, the most basic unit of chromosomal organization, which provides highly compact but readily accessible packaging for a cell's gene-encoding DNA. The amino-terminal tails of histones protrude from the nucleosome and are subject to covalent modifications including phosphorylation, acetylation, and methylation. These histone modifications affect higher-order chromatin structure and influence gene expression.

In previous studies, Nakayama demonstrated that heterochromatin protein binding states play a role in the regulation of gene silencing. Nakayama performed detailed analyses of the binding states of Swi6, a homolog of the mammalian HP-1 heterochromatin protein at the silent mating-type (mat) locus of the fission yeast. The results of that study showed that Swi6 protein is a dosage-critical component involved in imprinting the mat locus. This binding of Swi6 is maintained both across mitotic cell cycles and intergenerationally, as it is propagated through meiosis as well. Nakayama's study also showed that the deacetylation and subsequent methylation of a specific histone H3 lysine residue are essential to this process.  Chemical modifications to histones are fundamental epigenetic processes that can act to switch the expression of a target gene on or off.

Nakayama has now linked Swi6 function to a number of other chromodomain proteins in fission yeast that seem to act in a stepwise and context-sensitive fashion to silence genes in the process of heterochromatin assembly. The number of species of these proteins, which possess characteristic SET or chromodomain sequence motifs, is much smaller in yeast than it is in human, making S. pombe an apt model for studying the basic means by which these molecules respond to the methyl modifications to histone residues. The methylation of specific sites on the histone H3 catalyzed by a SET domain-containing methyltransferase provides epigenetic markers that allow chromodomain proteins to bind the histone and direct it toward eu- or heterochromatin assembly or to initiate developmentally regulated gene silencing.

The Nakayama lab has shown the histone methyltransferase Clr4 to be essential for heterochromatin assembly as an upstream element that helps Swi6 to localize correctly. Two other chromodomain proteins, Chp1 and Chp2, have also been implicated in heterochromatin formation and function. Disruption of the gene encoding Chp1causes defects in centromeric silencing and higher mitotic loss rates in mini-chromosomes, while Chp2 mutations result in weak silencing defects in three heterochromatic regions: centromeres, telomeres and the mat locus.

The Nakayama’s group has demonstrated that Swi6, Chp1 and Chp2 localize at three heterochromatic regions (centromere, telomere and mat locus), and that this localization is clearly dependent on H3-lys9 methylation mediated by Clr4. Following on a series of experiments in which the gene for each protein was disrupted, Nakayama has developed a model in which Chp1 function is specific to the establishment and spreading of heterochromatin, while Chp2 and Swi6 form a dimer that supports the stable maintenance of heterochromatin. They also found that, among three heterochromatic regions, centromeres are more dynamic and require establishment steps.

Nakayama is also interested in how chromodomain proteins function in higher eukaryotic cells. In previous study, Nakayama found an evolutionally-conserved chromodomain protein is a stable component of histone deacetylase complex in fission yeast. Using mammalian cells, he is now investigating the function of a chromodomain protein which has been linked to cell senescence and development.

Through his work on heterochromatin histone modifications, Nakayama has uncovered potentially important new roles for proteins in the establishment, maintenance and transmission of epigenetic information. These findings show that the definition of a gene as a simple string of DNA nucleotides needs to be expanded to include the action of proteins in the functional genetic unit. In the future, Nakayama plans to perform more detailed analyses of the molecular mechanisms that underlie epigenetic function, as well as studies in higher organisms and epigenetic gene expression in developmental processes.