Transcription and RNA interference in the formation of heterochromatin

Abstract

Transcription in heterochromatin seems to be an oxymoron--surely the 'silenced' form of chromatin should not be transcribed. But there have been frequent reports of low-level transcription in heterochromatic regions, and several hundred genes are found in these regions in Drosophila. Most strikingly, recent investigations implicate RNA interference mechanisms in targeting and maintaining heterochromatin, and these mechanisms are inherently dependent on transcription. Silencing of chromatin might involve trans-acting sources of the crucial small RNAs that carry out RNA interference, but in some cases, transcription of the region to be silenced seems to be required--an apparent contradiction.

Figure 1

Changes in histone modification implicated in the switch from a euchromatic to a heterochromatic state. Active genes are frequently marked by H3K4me (but see Berger, this issue); this mark must presumably be removed by LSD1 (not yet characterized in Drosophila). H3K9 is normally acetylated in euchromatin, and this mark must be removed by a histone deacetylase, typically HDAC1. Phosphorylation of H3S10 can interfere with methylation of H3K9; dephosphorylation may involved a phosphatase targeted through the carboxyl terminus of the JIL1 kinase. These transitions set the stage for acquisition of the modifications associated with silencing, including methylation of H3K9 by SU(VAR)3-9 or another HMT, binding of HP1, and subsequent methylation of H4K20 by SUV4-20, an enzyme recruited by HP1. Other silencing marks, such as methylation of H3K27 by E(Z) (not shown), appear to be relevant in some domains, although this mark is more prominently used by the Polycomb system. Supporting data comes from genetic identification of modifiers of PEV, biochemical characterization of the activities of such modifiers, and tests of protein-protein interactions . (Adapted from10)

Figure 2

Variegating phenotypes. While alternative chromatin packaging states can be inherited, they do switch at a low frequency, resulting in a variegating phenotype within a clone of cells. Picture: on the left, a fly eye; on the right a sectored colony of yeast S. pombe

Figure 3

HP1 interacts with H3K9me2/3 through its chromo domain, and with SU(VAR)3-9 through its chromoshadow domain. (Adapted from ref .)

Figure 4

Model showing RNAi-mediated heterochromatin assembly and silencing in S. pombe. Centromeric repeat (dg and dh) transcripts produced by RNA polymerase II are processed by the RNAi machinery, including RITS and RDRC effector complexes that interact with each other and localize across heterochromatic domains. The slicer activity of Ago1 (a component of RITS) and the RNA-dependent RNA polymerase activity of Rdp1 (a subunit of RDRC) are required for processing the repeat transcripts into siRNAs. siRNA-guided cleavage of nascent transcripts by Ago1 might make them preferential substrates for Rdp1 to generate dsRNA, which in turn is processed into siRNAs by Dicer. siRNAs are believed to mediate targeting of histone modifying activities including the Clr4 complex. This process most likely involves siRNA base pairing with nascent transcripts, but the precise mechanism remains undefined. siRNAs produced by heterochromatin-bound RNAi “factories” might also prime assembly of RISC-like complexes capable of mounting a classic RNAi response. Methylation of H3K9 by Clr4 is necessary for stable association of RITS with heterochromatic loci, apparently via binding of the Chp1 chromodomain. This methylation event also recruits Swi6, which along with other factors mediates spreading of various effectors such as SHREC. SHREC may facilitate proper positioning of nucleosomes to organize the higher-order chromatin structure essential for diverse heterochromatin functions, including transcriptional gene silencing. Swi6 also recruits an antisilencing protein, Epe1, that modulates heterochromatin to facilitate repeat transcription in addition to other functions. The system as a whole maintains a balance that results in loss of expression from reporter genes inserted within the domain.