Epigenetic inheritance uncoupled from sequence-specific recruitment

Inheritance of a covalent histone modification Genomic DNA is the repository of all genetic information and is packaged into chromatin. Chromatin is also a repository of regulatory information in the form of covalent marks added to the histones that package the DNA. These marks can determine tissue- and organ-specific gene expression patterns, which must be transmitted to daughter cells to maintain their identity. Ragunathan et al. and Audergon et al. show that in fission yeast, a chromatin mark, like genetic information, can be inherited across many cell generations. The mark can be inherited independently of DNA sequence, DNA methylation, or RNA interference. Thus, histone marks constitute true epigenetic information. Science, this issue 10.1126/science.1258699; see also p. 132 Covalent modifications of histones are inherited epigenetically across many generations. INTRODUCTION Changes in histone posttranslational modifications are associated with epigenetic states that define distinct patterns of gene expression. Whereas sequence-specific DNA binding proteins play essential roles in establishing an epigenetic state, their contributions to maintenance remain unclear. Previous attempts to separate the inheritance of epigenetic states from sequence-specific establishment suggest that specific DNA sequences and DNA binding proteins are continuously required for epigenetic inheritance. Moreover, in addition to DNA binding proteins, the establishment and maintenance of epigenetic states involves self-reinforcing interactions between histone modifications and RNA interference (RNAi) or DNA methylation. Therefore, whether histone-based mechanisms can transmit epigenetic memory independently of specific DNA sequences remains unknown. RATIONALE The fission yeast Schizosaccharomyces pombe contains chromosomal domains that share many features with heterochromatin in multicellular eukaryotes, such as methylation of histone H3 lysine 9 (H3K9), catalysis by the human Suv39h homolog Clr4, association with HP1 proteins (Swi6 and Chp2), and histone hypoacetylation. We developed an inducible system for heterochromatin establishment in S. pombe by fusion of the Clr4 methyltransferase catalytic domain to the bacterial tetracycline repressor (TetR) protein. To generate a reporter locus, we introduced 10 tetracycline operators upstream of the normally expressed ade6+ gene (10XtetO-ade6+). The silencing of ade6+ results in the formation of red or pink colonies upon growth on medium with limiting adenine concentrations. This system allowed us to determine whether heterochromatin, once established, could be maintained after tetracycline-mediated release of the TetR-Clr4 initiator (TetR-Clr4-I) from DNA. RESULTS Cells containing the reporter gene in combination with the expression of TetR-Clr4-I formed pink colonies on low-adenine medium lacking tetracycline, indicating ade6+ silencing. The establishment of heterochromatin resulted in high levels of H3K9 methylation (H3K9me), which was subsequently lost upon tetracycline-induced release of TetR-Clr4-I within ~10 cell divisions, resulting in the appearance of white colonies. Whereas perturbations to pathways that altered the rate of histone exchange or eliminating competition from endogenous heterochromatic loci had subtle effects on epigenetic inheritance of ade6+ silencing, deletion of the putative JmjC domain–containing demethylase Epe1 resulted in cells that retained ade6+ silencing for >50 generations after tetracycline-induced release of TetR-Clr4-I or deletion of the TetR module. Furthermore, the chromodomain of Clr4, which is involved in recognition of the H3K9me mark, was indispensable for maintenance, suggesting that a direct “read-write” mechanism mediated by Clr4 propagates histone modifications and allows histones to act as carriers of epigenetic information. This mechanism allows epigenetic states to be inherited during mitosis and meiosis and is also critical for maintaining low levels of H3K9me at native pericentromeric repeats. CONCLUSION Our findings indicate that even in the absence of any coupling to other positive-feedback loops, or in the absence of sequence-dependent initiation signals, H3K9me defines a silent state that can be epigenetically inherited. Maintenance of the OFF state is determined by the balance between the rate of H3K9me by the Clr4 reader-writer module and the loss rate due to demethylation by an Epe1-dependent mechanism, transcription-coupled nucleosome exchange, and dilution of histones during DNA replication. The regulation of histone demethylation activity may play a broad role in determining the reversibility of epigenetic states. H3K9me defines a silent state that can be epigenetically inherited. A direct read-write mechanism involving the Clr4 H3K9 methyltransferase propagates histone modifications and allows histones to act as carriers of epigenetic information in the absence of any input from the DNA sequence, DNA methylation, or RNAi. Epe1, a putative demethylase, and other transcription-associated histone turnover pathways modulate the rate of decay of the epigenetic state. Changes in histone posttranslational modifications are associated with epigenetic states that define distinct patterns of gene expression. It remains unclear whether epigenetic information can be transmitted through histone modifications independently of specific DNA sequence, DNA methylation, or RNA interference. Here we show that, in the fission yeast Schizosaccharomyces pombe, ectopically induced domains of histone H3 lysine 9 methylation (H3K9me), a conserved marker of heterochromatin, are inherited through several mitotic and meiotic cell divisions after removal of the sequence-specific initiator. The putative JmjC domain H3K9 demethylase, Epe1, and the chromodomain of the H3K9 methyltransferase, Clr4/Suv39h, play opposing roles in maintaining silent H3K9me domains. These results demonstrate how a direct “read-write” mechanism involving Clr4 propagates histone modifications and allows histones to act as carriers of epigenetic information.