Transcriptional silencing by polycomb-group proteins

Abstract

Polycomb-group (PcG) genes encode chromatin proteins involved in stable and heritable transcriptional silencing. PcG proteins participate in distinct multimeric complexes that deposit, or bind to, specific histone modifications (e.g., H3K27me3 and H2AK119ub1) to prevent gene activation and maintain repressed chromatin domains. PcG proteins are evolutionary conserved and play a role in processes ranging from vernalization and seed development in plants, over X-chromosome inactivation in mammals, to the maintenance of stem cell identity. PcG silencing is medically relevant as it is often observed in human disorders, including cancer, and tissue regeneration, which involve the reprogramming of PcG-controlled target genes.

Figure 1.

The concept of cellular memory. Schematic illustration of the involvement of PcG and TrxG complexes in the determination of active and repressed states of gene expression and, thereby, cellular differentiation, which is maintained over many cell divisions. TA, transcriptional activator; TR, transcriptional repressor.

Figure 2.

Homeotic transformations in PcG mutants of various species. (A–D) Drosophila melanogaster, (E,F) Mus musculus, (G,H) Arabidopsis thaliana. (A,B) Leg imaginal discs undergoing a transdetermination event as indicated by the expression of the wing-specific gene vestigial (marked by green fluorescent protein [GFP]). (C,D) Cuticles of a wild-type (C) and a Su(z)12 mutant embryo (D). In the Su(z)12 mutant embryo, all abdominal, thoracic, and several head segments (not all visible in this focal plane) are homeotically transformed into copies of the eighth abdominal segment because of misexpression of the Abd-B gene in every segment. (E,F) Axial skeleton of newborn wild-type (E) and Ring1A^-/- mice (F). Views of the thoracic regions of cleared skeletons show bone (red) and cartilage (blue). The mutant displays anterior transformation of the eighth thoracic vertebra as indicated by the presence of an eighth (1–8) vertebrosternal rib, instead of seven (1–7) as in the wild type. (G,H) Wild-type (G) and clf-2 mutant (H) flowers. The wild-type flower shows the normal arrangement of sepals, petals, stamens, and carpels. In the clf-2 flower, petals are absent or reduced in number. (A,B, Courtesy of N. Lee and R. Paro; C,D, reprinted, with permission, from Birve et al. 2001, © Company of Biologists Ltd; E,F, reprinted, with permission, from Lorente et al. 2000, © Company of Biologists Ltd; G,H, courtesy of J. Goodrich.)

Figure 3.

Conserved PRC2 core complexes. The conserved core proteins of PRC2 (A) and PRC1 (B) complexes in Drosophila melanogaster, Mus musculus, Arabidopsis thaliana, and Caenorhabditis elegans are shown. (A) In the mouse, PRC2 variants containing EZH1 or EZH2 have distinct functions, whereas in Arabidopsis the ancestral complex has diversified into at least three variants with discrete functions during development. In C. elegans, the PRC2 core complex contains only three proteins, with MES-3 not having homology with any other identified PRC2 protein. Apart from these core proteins, several other proteins, which are not shown here, interact with PRC2. For instance, mammalian complexes can contain the histone lysine demethylase JARID2, the Zn-finger protein AEBP2, and various homologs of the Drosophila PCL protein (PCL1/2/3). Proteins that share the plant homeodomain (PHD)-domain with PCL, but are otherwise not closely related, are also associated with the VRN-PRC2 complex in Arabidopsis. Homologous proteins are indicated by the same color. (B) The core proteins of PRC1 are less conserved than those of PRC2 across the four species. In mammals, all genes encoding the PRC1 core subunits have been expanded (see Table 1), such that a variety of complexes with different isoform composition can be formed. In addition to the core components, several additional proteins can be found in PRC1 that are, however, less well characterized and are not shown. In plants, only homologs of Drosophila PSC and SCE have been identified; these are encoded by small gene families. Homologous proteins are indicated by the same color. (Based on Reyes and Grossniklaus 2003, Chanvivattana et al. 2004, and Margueron and Reinberg 2011.)

Figure 4.

Involvement of distinct PRC2 complexes at various stages of plant development. During the plant life cycle, distinct variants of PRC2 (see Fig. 3) control developmental progression. (A) A cleared wild-type ovule harboring the female gametophyte in its center is represented. The FIS-PRC2 represses unknown target genes that control proliferation of the central cell; consequently, in all fis class mutants, this cell proliferates in the absence of fertilization. Around fertilization, MEA is also required to maintain expression of the maternal MEA allele (MEA^m) at a low level, but this activity is independent of other FIS-PRC2 components. (B) Section of a wild-type seed harboring embryo and endosperm, enclosed by the seed coat. After fertilization, the FIS-PRC2 is involved in the control of cell proliferation in embryo and endosperm. It maintains a low level of expression of the maternal PHE1^m allele and is involved in keeping the paternal MEA^p allele silent, although FIS-PRC2 only plays a minor part in its repression. Both parental alleles of FUS3 are repressed by the FIS-PRC2. (C) Wild-type plant before flowering. The EMF-PRC2 prevents flowering by repressing FT and directly represses the floral genes AG and STM. (D) Wild-type plant after bolting—that is, floral induction induced by appropriate photoperiod and/or vernalization. The former relieves repression by EMF-PRC2 of FT, a promoter of flowering, whereas the latter leads to repression of the floral repressor FLC, thus inducing flowering. The maintenance of FLC repression depends on the VRN-PRC2. (E) Wild-type Arabidopsis flower. During flower organogenesis, the EMF complex regulates floral homeotic genes, such as AG, which determine the identity of floral organs, and STM, which is involved in floral organ development. (A, Courtesy of J.M. Moore and U. Grossniklaus; B, courtesy of J.-P. Vielle-Calzada and U. Grossniklaus; C,D, courtesy of D. Weigel; E, reprinted, with permission, from Page and Grossniklaus 2002, © Macmillan.)

Figure 5.

Schematic representation of the core PcG and TrxG protein complexes and their functions at promoters. Drosophila PcG proteins are depicted as red ovals with selected mammalian orthologs indicated in gray text. (A) Components and function of the PRC2 and counteracting activities of TrxG proteins (light green). (B) Components and functions of PRC1 and dRING-associated factor (dRAF) and the counteracting activities of the BAP SWI/SNF, facilitates chromatin transcription (FACT) remodeling complexes, and SET-domain histone KMTs TRX and ASH1. The TrxG protein Kismet-L is a member of the chromatin-helicase-DNA-binding (CHD) subfamily of chromatin-remodeling factors, stimulating elongation of Pol II. (Adapted from Enderle 2011.)

Figure 6.

PRC1 at paused promoters and during cell division. (A) The PRC1 complex may repress target genes by stalling the elongation of RNA Pol II. This may be achieved by ubiquitination of histone H2A through the subunit SCE/dRING, compacting promoter proximal chromatin, or direct physical interaction with the transcriptional machinery (including the short RNAs produced by the paused RNA Pol II). (B) A possible model for how differential gene expression states can be inherited. The process of intergenic transcription places positive epigenetic marks (e.g., acetylated histone tails, histone variants) at PREs that control active genes (PRE 2). All other PREs are silenced by default (PRE 1). During DNA replication and mitosis, only the positive epigenetic signal needs to be transmitted to the daughter cells, ensuring that in the next interphase intergenic transcription is restarted at PRE 2 before default silencing is reestablished at all other PREs.

Figure 7.

Chromosomal targeting of PRC1. (A) Immunostaining of Drosophila polytene chromosomes to visualize the distribution of the PC protein. (B) Genomic region encompassing the Drosophila PcG gene Psc and the Su(z)2 gene. The genome browser section shows the result of a ChIP-Seq and RNA-Seq analysis of Drosophila S2 tissue culture cells. The distributions of PRC1 components (red) and the TRX protein (green) are shown (data from Enderle et al. 2011). (C) In Drosophila, the PhoRC is a key player in chromatin targeting of PRC1 and PRC2, but a number of other transcription factors also contribute to target gene specificity. (D) In mouse and human, several different anchoring factors have been proposed. These include the Pho ortholog Ying and Yang 1 (YY1), transcription factors like Jarid 2 and Oct4, long ncRNAs, and the CpG content of the target sequence. (Adapted from Enderle 2011.)

Figure 8.

Interplay of PcG-mediated repression and DNA methylation regulates genomic imprinting in plants and mammals. (A) Regulation of genomic imprinting at the Kcnq1 domain on distal chromosome 7. The imprinting control element (ICE) is maternally methylated and prevents the transcription of the lncRNA Kcnq1ot1 from the maternal chromosome. The paternally expressed Kcnq1ot1 associates with chromatin and recruits chromatin modifying complexes, such as PRC2, to mediate and maintain transcriptional silencing of several paternal, protein-coding alleles. (B) In Arabidopsis seeds, the paternally expressed PHE1 gene is maternally repressed by the action of PRC2. A cis-regulatory element (shaded pink) downstream of the PHE1 gene must be methylated for paternal expression, but demethylated for maternal repression.

Figure 9.

PRC2 regulates cell proliferation in mammals and plants. (A,B) Plant embryos derived from wild-type and mea mutant egg cells. MEA encodes a protein of the FIS-PRC2 and regulates cell proliferation. The mea embryo (B) is much larger than the corresponding wild-type embryo (A) at the same stage of development (late heart stage). Mutant embryos develop slower and have approximately twice the number of cell layers. (C,D) Normal and cancerous prostate epithelium of mice. In the cancerous epithelium, Ezh2 expression is highly increased (labeled with an anti-Ezh2 antibody). Thus, both loss of E(Z) function in plants and overexpression of E(Z) function in mice can lead to defects in cell proliferation. (E,F) Control and RING1 overexpressing rat 1a fibroblast cells. Overexpression of RING1 leads to anchorage-independent growth in soft agar, typical of neoplastically transformed cells. (A,B, Courtesy of J.-P. Vielle-Calzada and U. Grossniklaus; C,D, reprinted, with permission, from Kuzmichev et al. 2005, © National Academy of Sciences; E,F, reprinted, with permission, from Satijn and Otte 1999, © American Society for Microbiology.)

Figure 10.

Sonic Hedgehog signaling maintains proliferation/self-renewal of cerebellar progenitor cells. The Shh signaling cascade regulates both the Rb pathway (which can be bound by the PRC2 RbAp48 protein) as well as the p53 pathway via Bmi1 control of the p16/p19 proliferation checkpoint. Inhibition of Smoothened (Smoh) by the Shh receptor Patched (Ptch) results in downstream signaling in the nucleus. One part of the signal induces N-Myc, Cyclin D1, and Cyclin D2, whereas the other part activates Bmi1 via the Gli effectors. (Adapted, with permission, from Valk-Lingbeek et al. 2004, © Elsevier.)