Noncoding RNA and Polycomb recruitment

Abstract

A plethora of noncoding (nc) RNAs has been revealed through the application of high-throughput analysis of the transcriptome, and this has led to an intensive search for possible biological functions attributable to these transcripts. A major category of functional ncRNAs that has emerged is for those that are implicated in coordinate gene silencing, either in cis or in trans. The archetype for this class is the well-studied long ncRNA Xist which functions in cis to bring about transcriptional silencing of an entire X chromosome in female mammals. An important step in X chromosome inactivation is the recruitment of the Polycomb repressive complex PRC2 that mediates histone H3 lysine 27 methylation, a hallmark of the inactive X chromosome, and recent studies have suggested that this occurs as a consequence of PRC2 interacting directly with Xist RNA. Accordingly, other ncRNAs have been linked to PRC2 targeting either in cis or in trans, and here also the mechanism has been proposed to involve direct interaction between PRC2 proteins and the different ncRNAs. In this review, I discuss the evidence for and against this hypothesis, in the process highlighting alternative models and discussing experiments that, in the future, will help to resolve existing discrepancies.

FIGURE 1.

The A-repeat of Xist RNA. (A) Schematic representing the Xist gene locus and indicating the location of different tandemly repeated regions, labeled A–F (blue shading), and a short RNA, RepA, proposed to arise from within Xist exon 1 and to be important in PRC recruitment in X inactivation (see text). The A-repeat is essential for Xist-mediated chromosome silencing and is present in both full-length Xist RNA and RepA RNA. The consensus sequence of the A-repeat monomer with GC rich core (boxed) and U-rich spacer is shown below. (B) Proposed secondary structure of an A-repeat monomer, as determined by thermodynamic folding predictions and biophysical assays (see text). (C) Proposed secondary structure of a monomer of a tandem repeat located at the 5′ end of the marsupial ncRNA Rsx thought to function equivalently to Xist RNA A-repeat. This hypothetical structure is based solely on thermodynamic folding predictions.

FIGURE 2.

Major Polycomb complexes in mammals and proposed ncRNA interactions. (A) Representation of Polycomb repressive complex 2 (PRC2) illustrating core components with putative RNA binding domains (RBD) and catalytic histone methyltransferase domain (SET). Several ncRNAs, including Xist, Kcnq1ot1, and HOTAIR, and short RNAs transcribed from CpG island promoters of PcG target genes, have been proposed to interact with the putative RBDs of either EZH2 or SUZ12 (see text). Additional subunits that are either weakly associated or substoichiometric PRC2 components are linked by double-headed arrows. (B) Representation of the Arabidopsis thaliana PRC2 complex linked to regulation of vernalization. Color-coding indicates subunit homology with mammalian PRC2. The CXC domain in the CLF subunit is a putative RBD interacting with the ncRNA COLDAIR (see text). VRN5 and VIN3 are weakly associated or substoichiometric components potentially homologous to PCL1/2/3 in mammals and required to fully activate PRC2 at the FLC locus. (C) The classical PRC1 complex exists in two forms in which either CBX and MPH or RYBP subunits associate with the catalytic core subunits RING1A/B and PCGF2/4. The weakly associated/substoichiometric PRC1 component SCMLH1/2/3 is linked with a double-headed arrow. The chromodomain (CD) of the CBX7 protein has been shown to bind the ncRNA ANRIL (see text). The RanBP-ZF domain present in RYBP/YAF2 has an RNA binding function in some related proteins but apparently not in RYBP/YAF2. (D) Representation of three additional PRC1-related complexes in which the subunit composition results from incorporation of different homologs of the PCGF subunit, specifically PCGF1, PCGF6, and PCGF3/5. These complexes include the RYBP/YAF2 subunit but not PcG CBX or MPH proteins. A non-PcG CBX homolog, CBX3, that binds H3K9me3 as opposed to H3K27me3, is found in PCGF6 complexes. To date, there is no evidence that these complexes bind to ncRNA.

FIGURE 3.

Polycomb recruitment to the inactive X chromosome in response to A-repeat-deficient Xist transgene expression. Schematic illustrates the outcome of experiments inducing A-repeat-deficient Xist RNA transgenes in differentiating ES cells (Kohlmaier et al. 2004; and see text). (A) Induction of Xist RNA and chromosome coating in undifferentiated ES cells, with maintenance throughout subsequent differentiation, results in continual chromosomal localization of PRC2 and associated H3K27me3. (B) Cessation of Xist induction after ∼3 d results in rapid loss of PRC2/H3K27me3. (C) Xist induction and chromosome coating in late-stage differentiated cells does not recruit PRC2 unless (D) there was a transient expression of Xist with associated PRC2 recruitment in early differentiating cells. Collectively, these results demonstrate that early expression of the Xist transgenes confers an epigenetic memory to the chromosome that is required in order for differentiated cells to recruit PRC2 proteins in response to Xist RNA coating.

FIGURE 4.

Direct vs. indirect models for PRC2 recruitment in response to ncRNA expression. In the direct model (A), PRC2 complex binds to the ncRNA via an RBD domain and then, together with other RNA-bound chromatin modifiers (?), remodels the chromatin at sites where the ncRNA localizes. PRC2-mediated H3K27me3 is a component part of the resultant compacted chromatin configuration and could potentially have a causative role in establishing the modified state. In the indirect model (B), the ncRNA first modifies the underlying chromatin structure, for example, by erasing active chromatin marks, and the resultant compacted/modified chromatin then functions as a signal to recruit PRC2. In this scenario, the primary function of PRC2/H3K27me3 may be in facilitating stable propagation of modified/compacted chromatin states that were established by primary silencing factors.