Interaction of Polycomb-group proteins controlling flowering in Arabidopsis

In Arabidopsis, the EMBYRONIC FLOWER2 (EMF2), VERNALISATION2 (VRN2) and FERTILISATION INDEPENDENT ENDOSPERM2 (FIS2) genes encode related Polycomb-group (Pc-G) proteins. Their homologues in animals act together with other Pc-G proteins as part of a multimeric complex, Polycomb Repressive Complex 2 (PRC2), which functions as a histone methyltransferase. Despite similarities between the fis2 mutant phenotype and those of some other plant Pc-G members, it has remained unclear how the FIS2/EMF2/VRN2 class Pc-G genes interact with the others. We have identified a weak emf2 allele that reveals a novel phenotype with striking similarity to that of severe mutations in another Pc-G gene, CURLY LEAF (CLF), suggesting that the two genes may act in a common pathway. Consistent with this, we demonstrate that EMF2 and CLF interact genetically and that this reflects interaction of their protein products through two conserved motifs, the VEFS domain and the C5 domain. We show that the full function of CLF is masked by partial redundancy with a closely related gene, SWINGER (SWN), so that null clf mutants have a much less severe phenotype than emf2 mutants. Analysis in yeast further indicates a potential for the CLF and SWN proteins to interact with the other VEFS domain proteins VRN2 and FIS2. The functions of individual Pc-G members may therefore be broader than single mutant phenotypes reveal. We suggest that plants have Pc-G protein complexes similar to the Polycomb Repressive Complex2 (PRC2) of animals, but the duplication and subsequent diversification of components has given rise to different complexes with partially discrete functions.     

RYBP represses endogenous retroviruses and preimplantation- and germ line-specific genes in mouse embryonic stem cells

Polycomb repressive complexes (PRCs) are important chromatin regulators of embryonic stem (ES) cell function. RYBP binds Polycomb H2A monoubiquitin ligases Ring1A and Ring1B and has been suggested to assist PRC localization to their targets. Moreover, constitutive inactivation of RYBP precludes ES cell formation. Using ES cells conditionally deficient in RYBP, we found that RYBP is not required for maintenance of the ES cell state, although mutant cells differentiate abnormally. Genome-wide chromatin association studies showed RYBP binding to promoters of Polycomb targets, although its presence is dispensable for gene repression. We discovered, using Eed-knockout (KO) ES cells, that RYBP binding to promoters was independent of H3K27me3. However, recruiting of PRC1 subunits Ring1B and Mel18 to their targets was not altered in the absence of RYBP. In contrast, we have found that RYBP efficiently represses endogenous retroviruses (murine endogenous retrovirus [MuERV] class) and preimplantation (including zygotic genome activation stage)- and germ line-specific genes. These observations support a selective repressor activity for RYBP that is dispensable for Polycomb function in the ES cell state. Also, they suggest a role for RYBP in epigenetic resetting during preimplantation development through repression of germ line genes and PcG targets before formation of pluripotent epiblast cells.     

A phosphorylated form of Mel-18 targets the Ring1B histone H2A ubiquitin ligase to chromatin

Recent studies have shown that PRC1-like Polycomb repressor complexes monoubiquity-late chromatin on histone H2A at lysine residue 119. Here we have analyzed the function of the polycomb protein Mel-18. Using affinity-tagged human MEL-18, we identify a polycomb-like complex, melPRC1, containing the core PRC1 proteins, RING1/2, HPH2, and CBX8. We show that, in ES cells, melPRC1 can functionally substitute for other PRC1-like complexes in Hox gene repression. A reconstituted subcomplex containing only Ring1B and Mel-18 functions as an efficient ubiquitin E3 ligase. This complex ubiquitylates free histone substrates nonspecifically but is highly specific for histone H2A lysine 119 in the context of nucleosomes. Mutational analysis demonstrates that while Ring1B is required for E3 function, Mel-18 directs this activity to H2A lysine 119 in chromatin. Moreover, this substrate-targeting function of Mel-18 is dependent on its prior phosphorylation at multiple residues, providing a direct link between chromatin modification and cell signaling pathways.     

The polycomb protein Ring1B generates self atypical mixed ubiquitin chains required for its in vitro histone H2A ligase activity

Polycomb complexes mediate gene silencing, in part by modifying histones. Ring1B and Bmi1 are RING finger proteins that are members of the Polycomb repressive complex 1 (PRC1). Ring1B is an E3 that mediates its own polyubiquitination and monoubiquitination of histone H2A. In contrast, Bmi1 has no self-ubiquitinating activity. We show that unlike other RING finger proteins that are believed to mediate their own ubiquitination and degradation, Ring1B and Bmi1 are degraded by an exogenous E3, independent of their RING domain. The RING domains of both proteins mediate their association and subsequent stabilization. Consistent with the nonproteolytic self-ligase activity of Ring1B, it generates atypical mixed K6-, K27-, and K48-based polyubiquitin chains, which require the presence of all these lysine residues on the same ubiquitin molecule. The modification is required for Ring1B ability to monoubiquitinate H2A in vitro, unraveling an as yet undescribed mechanism for ligase activation via noncanonical self-ubiquitination.     

Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation

In many higher organisms, 5%-15% of histone H2A is ubiquitylated at lysine 119 (uH2A). The function of this modification and the factors involved in its establishment, however, are unknown. Here we demonstrate that uH2A occurs on the inactive X chromosome in female mammals and that this correlates with recruitment of Polycomb group (PcG) proteins belonging to Polycomb repressor complex 1 (PRC1). Based on our observations, we tested the role of the PRC1 protein Ring1B and its closely related homolog Ring1A in H2A ubiquitylation. Analysis of Ring1B null embryonic stem (ES) cells revealed extensive depletion of global uH2A levels. On the inactive X chromosome, uH2A was maintained in Ring1A or Ring1B null cells, but not in double knockout cells, demonstrating an overlapping function for these proteins in development. These observations link H2A ubiquitylation, X inactivation, and PRC1 PcG function, suggesting an unanticipated and novel mechanism for chromatin-mediated heritable gene silencing.     

Dominant modifiers of the polyhomeotic extra-sex-combs phenotype induced by marked P element insertional mutagenesis in Drosophila.

Members of the Polycomb group (Pc-G) and trithorax group (trx-G) of genes, as well as the enhancers of trx-G and Pc-G (ETP), function together to maintain segment identity during Drosophila development. In order to obtain new marked P mutations in these genes, we screened for dominant modifiers of the extra-sex-combs phenotype displayed by males mutant for the polyhomeotic (ph) gene, a member of the Pc-G group. Five P(lacW) insertions in four different genes were found to stably suppress ph: two are allelic to trithorax, one is the first allele specific to the Minute(2)21C gene, and the remaining two define new trx-G genes, toutatis (tou) in 48A and taranis (tara) in 89B10-13. tou is predicted to encode a 3109 amino acid sequence protein (TOU), which contains a TAM DNA-binding domain, a WAKZ motif, two PHD zinc fingers and a C-terminal bromodomain, and as such is likely to be involved in regulation of chromatin structure as a subunit of a novel chromatin remodelling complex. In a previous study, we found that insertion of a P(ph) transposable element containing ph regulatory sequences creates a high frequency of mutations modifying ph homeotic phenotypes. One such insertion enhanced the ph phenotype and we show that it is a new allele of UbcD1/eff, a gene encoding a ubiquitin-conjugating enzyme that is involved in telomere association and potentially in chromatin remodelling.     

Cutting edge: polycomb gene expression patterns reflect distinct B cell differentiation stages in human germinal centers

Polycomb group (Pc-G) proteins regulate homeotic gene expression in Drosophila, mouse, and humans. Mouse Pc-G proteins are also essential for adult hematopoietic development and contribute to cell cycle regulation. We show that human Pc-G expression patterns correlate with different B cell differentiation stages and that they reflect germinal center (GC) architecture. The transition of resting mantle B cells to rapidly dividing Mib-1(Ki-67)+ follicular centroblasts coincides with loss of BMI-1 and RING1 Pc-G protein detection and appearance of ENX and EED Pc-G protein expression. By contrast, differentiation of centroblasts into centrocytes correlates with reappearance of BMI-1/RING1 and loss of ENX/EED and Mib-1 expression. The mutually exclusive expression of ENX/EED and BMI-1/RING1 reflects the differential composition of two distinct Pc-G complexes. The Pc-G expression profiles in various GC B cell differentiation stages suggest a role for Pc-G proteins in GC development.     

Altered retinoic acid sensitivity and temporal expression of Hox genes in polycomb-M33-deficient mice

The Polycomb group genes are required for the correct expression of the homeotic complex genes and segment specification during Drosophila embryogenesis and larval development. In mouse, inactivation studies of several Polycomb group genes indicate that they are also involved in Hox gene regulation. We have used our previously generated M33 mutants to study the function of M33, the mouse homologue of the Polycomb gene of Drosophila. In this paper, we show that in the absence of M33, the window of Hoxd4 retinoic acid (RA) responsiveness is opened earlier and that Hoxd11 gene expression is activated earlier in development This indicates that M33 antagonizes the RA pathway and has a function in the establishment of the early temporal sequence of activation of Hox genes. Despite the early activation, A-P boundaries are correct in later stages, indicating a separate control mechanism for early aspects of Hox regulation. This raises a number of interesting issues with respect to the roles of both Pc-G proteins and Hox regulatory mechanisms. We propose that a function of the M33 protein is to control the accessibility of retinoic acid response elements in the vicinity of Hox genes regulatory regions by direct or indirect mechanisms or both. This could provide a means for preventing ectopic transactivation early in development and be part of the molecular basis for temporal colinearity of Hox gene expression.     

Molecular and genetic analysis of the Polycomb group gene Sex combs extra/Ring in Drosophila

Polycomb group (PcG) proteins repress homeotic genes and other developmental regulatory genes in cells where these genes must remain inactive during development. In Drosophila and in vertebrates, PcG proteins exist in two distinct multiprotein complexes, the Esc/Eed-E(z) complex and PRC1. Drosophila PRC1 contains Polycomb, Posterior sexcombs and Polyhomeotic, the products of three PcG genes that are critically needed for PcG silencing. Formation of stable PRC1 requires Ring, the product of a gene for which no mutations have been described. Here, we show that Sex combs extra (Sce) encodes Ring and that Sce/Ring function is critically required for PcG silencing.