Regulation of heterochromatin by histone methylation and small RNAs
Introduction
Heterochromatin was originally defined as the fraction of the genome that remained visibly condensed during interphase [1]. More recently, it has been defined as the genomic regions that are gene-poor and are inaccessible to DNA-modifying reagents 2., 3.. Because the DNA within heterochromatin is condensed, it is largely believed that these regions are transcriptionally silenced. Early studies of heterochromatin led to the discovery of a phenomenon known as position effect variegation (PEV) [4], where a euchromatic gene placed near or within heterochromatin becomes epigenetically silenced [5]. The implications of epigenetic silencing in normal developmental gene regulation, aging and cancer progression have made heterochromatin the focus of intense investigation over the past few decades, although insights into heterochromatin establishment and maintenance have been lacking.
Recent findings have reshaped the way we think about heterochromatin, especially how it is formed and epigenetically maintained. In this review, we discuss some of the most recent findings in heterochromatin research with particular emphasis on histone H3 lysine 9 (H3 Lys9) mono-, di- and trimethylation and RNAi-mediated transcriptional silencing. Additionally, we will discuss some of the important implications of these recent findings and predict what the next few years will bring.
Section snippets
Histone methylation and heterochromatin
Within the eukaryotic nucleus, DNA is packaged with chromosomal proteins to form chromatin. The most fundamental repeating unit of chromatin is the nucleosome, which consists of 146 bp of DNA wrapped around an octamer of histone proteins made up of two copies each of H2A, H2B, H3 and H4 [6]. These evolutionarily conserved proteins consist of a globular C-terminal domain critical to nucleosome formation and a flexible N-terminal tail that protrudes from the nucleosome. These tails are targets for
Three degrees of methylation
One possible answer to these questions is that specific HMTs differentially methylate H3 Lys9 to a certain degree. The ε-amino group of lysine can accept up to three methyl groups and hence can be mono-, di- or trimethylated [13]. Different mass spectrometric techniques have identified several human histone lysine residues, including H3 Lys9, that are mono-, di- or trimethylated in vivo 35., 36., 37.. Although the biological significance of these differences was unknown, recent reports indicate
Targeted for silence: transposons and repeats
In addition to understanding the role of histone-modifying activities and the factors that recognize differentially modified histone tails, it is important to comprehend the mechanisms that define specific chromosomal domains as sites of heterochromatin assembly. In higher eukaryotes, a significant amount of genomic DNA is assembled into heterochromatic structures. Small blocks of silent chromatin structures are interspersed throughout the chromosomes and are essential for the maintenance of
DNA-based targeting of heterochromatin
Studies from plants, animals and fungi suggest that multiple mechanisms may be responsible for the initial targeting of heterochromatin complexes in different chromosomal contexts. One of these mechanisms probably involves factors that target specific DNA sequences. Certain DNA-binding factors that recognize specific DNA elements or silencers can nucleate heterochromatin, one example being the Sir-mediated silencing in S. cerevisiae. These DNA binding factors often possess modular structures
RNAi-mediated nucleation of heterochromatin
In addition to the DNA-binding proteins, recent studies suggest that RNA interference (RNAi), a new and increasingly well-studied process, targets repressive chromosomal complexes to specific chromosomal loci. In this fundamentally novel process, RNA provides specificity for the precise targeting of silent chromatin complexes to particular genomic loci. RNAi is an evolutionarily conserved silencing mechanism that is triggered by double stranded RNA (dsRNA) and serves to regulate gene expression
RITS: an RNAi effector complex for heterochromatin assembly
Although they were known to be involved in this pathway, it was not clear exactly how siRNAs promoted targeted assembly of heterochromatin. It had been hypothesized that a RISC-like heterochromatin-targeting complex containing the Argonaute protein binds to siRNAs and promotes their pairing to either nascent transcripts or homologous DNA sequences at the target locus 80., 81.. A recent study provided the first direct evidence for the existence of an RNAi effector complex called RITS
Step-wise model for RNAi-mediated epigenetic gene silencing
Increasing evidence indicates that heterochromatin assembly is a multi-step process. Heterochromatin is believed to nucleate at specific regulatory sequences and then spreads into neighboring sequences, resulting in epigenetic gene silencing [80]. There seem to be distinct requirements for the nucleation, spreading and maintenance of heterochromatin structures [43] (Figure 2). For nucleation, dsRNAs generated from the repetitive sequences are processed into siRNAs by the Dicer ribonuclease.
Conclusions
Quite recently, significant findings have led to a redefinition of heterochromatin regulation in terms of histone methylation and small RNAs. New data suggests that different degrees of H3 Lys9 methylation define specific heterochromatin domains (facultative versus constitutive) (Figure 1). It is clear that the degree of methylation is dictated by the specific HMT and where it is localized in the genome. For example, Suv39h1 and Suv39h2 localize to constitutive heterochromatin and specifically
Update
In recent paper, Freitag et al. showed that the HP1 homologue in Neurospora crassa was required for DNA methylation at the relics of transposons [83]. This new result extends previous studies by the Selker laboratory demonstrating that the histone H3 Lys9 trimethyltransferase DIM-5 is required for all known DNA methylation in Neurospora crassa [84]. Also recently, Chan et al. showed that factors involved in the RNAi pathway, such as RNA-dependent RNA polymerase 2 (rdr2), dicer-like 3 (dcr3),
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
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of special interest
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of outstanding interest
Acknowledgements
Our apologies to colleagues whose work has not been cited in this article due to space limitations. We thank E Chen, S Jia and T Sugiyama for critical comments and members of the Grewal laboratory for helpful discussions. Research in SG’s laboratory is supported by the National Cancer Institute.
References (85)
Heterochromatin and gene regulation in Drosophila
Curr Opin Genet Dev
(1996)The phenomenon of position effect
Adv Genet
(1950)- et al.
Signaling to chromatin through histone modifications
Cell
(2000) Histone modifications in transcriptional regulation
Curr Opin Genet Dev
(2002)- et al.
Histone methylation versus histone acetylation: new insights into epigenetic regulation
Curr Opin Cell Biol
(2001) - et al.
Methylation of histone H3 at Lys-9 is an early mark on the X chromosome during X inactivation
Cell
(2001) - et al.
Set domain-containing protein, G9a, is a novel lysine-preferring mammalian histone methyltransferase with hyperactivity and specific selectivity to lysines 9 and 27 of histone H3
J Biol Chem
(2001) - et al.
Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability
Cell
(2001) - et al.
Identification of novel histone post-translational modifications by peptide mass fingerprinting
Chromosoma
(2003) - et al.
Histone methyltransferases direct different degrees of methylation to define distinct chromatin domains
Mol Cell
(2003)