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.     

The polycomb group protein EED interacts with YY1, and both proteins induce neural tissue in Xenopus embryos

Polycomb group (PcG) proteins form multimeric protein complexes which are involved in the heritable stable repression of genes. Previously, we identified two distinct human PcG protein complexes. The EED-EZH protein complex contains the EED and EZH2 PcG proteins, and the HPC-HPH PcG complex contains the HPC, HPH, BMI1, and RING1 PcG proteins. Here we show that YY1, a homolog of the Drosophila PcG protein pleiohomeotic (Pho), interacts specificially with the human PcG protein EED but not with proteins of the HPC-HPH PcG complex. Since YY1 and Pho are DNA-binding proteins, the interaction between YY1 and EED provides a direct link between the chromatin-associated EED-EZH PcG complex and the DNA of target genes. To study the functional significance of the interaction, we expressed the Xenopus homologs of EED and YY1 in Xenopus embryos. Both Xeed and XYY1 induce an ectopic neural axis but do not induce mesodermal tissues. In contrast, members of the HPC-HPH PcG complex do not induce neural tissue. The exclusive, direct neuralizing activity of both the Xeed and XYY1 proteins underlines the significance of the interaction between the two proteins. Our data also indicate a role for chromatin-associated proteins, such as PcG proteins, in Xenopus neural induction.     

The Drosophila Polycomb group gene Sex combs extra encodes the ortholog of mammalian Ring1 proteins

In Drosophila, the Polycomb group (PcG) of genes is required for the maintenance of homeotic gene repression during development. Here, we have characterized the Drosophila ortholog of the products of the mammalian Ring1/Ring1A and Rnf2/Ring1B genes. We show that Drosophila Ring corresponds to the Sex combs extra (Sce), a previously described PcG gene. We find that Ring/Sce is expressed and required throughout development and that the extreme Pc embryonic phenotype due to the lack of maternal and zygotic Sce can be rescued by ectopic expression of Ring/Sce. This phenotypic rescue is also obtained by ectopic expression of the murine Ring1/Ring1A, suggesting a functional conservation of the proteins during evolution. In addition, we find that Ring/Sce binds to about 100 sites on polytene chromosomes, 70% of which overlap those of other PcG products such as Polycomb, Posterior sex combs and Polyhomeotic, and 30% of which are unique. We also show that Ring/Sce interacts directly with PcG proteins, as it occurs in mammals.     

YY1 controls Igκ repertoire and B‐cell development,and localizes with condensin on the Igκ locus

Conditional knock‐out (KO) of Polycomb Group (PcG) protein YY1 results in pro‐B cell arrest and reduced immunoglobulin locus contraction needed for distal variable gene rearrangement. The mechanisms that control these crucial functions are unknown. We deleted the 25 amino‐acid YY1 REPO domain necessary for YY1 PcG function, and used this mutant (YY1ΔREPO), to transduce bone marrow from YY1 conditional KO mice. While wild‐type YY1 rescued B‐cell development, YY1ΔREPO failed to rescue the B‐cell lineage yielding reduced numbers of B lineage cells. Although the IgH rearrangement pattern was normal, there was a selective impact at the Igκ locus that showed a dramatic skewing of the expressed Igκ repertoire. We found that the REPO domain interacts with proteins from the condensin and cohesin complexes, and that YY1, EZH2 and condensin proteins co‐localize at numerous sites across the Ig kappa locus. Knock‐down of a condensin subunit protein or YY1 reduced rearrangement of Igκ Vκ genes suggesting a direct role for YY1‐condensin complexes in Igκ locus structure and rearrangement.     

Association of Rex-1 to target genes supports its interaction with Polycomb function

Rex-1/Zfp42 displays a remarkably restricted pattern of expression in preimplantation embryos, primary spermatocytes, and undifferentiated mouse embryonic stem (ES) cells and is frequently used as a marker gene for pluripotent stem cells. To understand the role of Rex-1 in selfrenewal and pluripotency, we used Rex-1 association as a measure to identify potential target genes, and carried out chromatin-immunoprecipitation assays in combination with gene specific primers to identify genomic targets Rex-1 associates with. We find association of Rex-1 to several genes described previously as bivalently marked regulators of differentiation and development, whose repression in mouse embryonic stem (ES) cells is Polycomb Group-mediated, and controlled directly by Ring1A/B. To substantiate the hypothesis that Rex-1 contributes to gene regulation by PcG, we demonstrate interactions of Rex-1 and YY2 (a close relative of YY1) with Ring1 proteins and the PcG-associated proteins RYBP and YAF2, in line with interactions reported previously for YY1. We also demonstrate the presence of Rex-1 protein in both trophectoderm and Inner Cell Mass of the mouse blastocyst and in both ES and in trophectoderm stem (TS) cells. In TS cells, we were unable to demonstrate association of Rex-1 to the genes it associates with in ES cells, suggesting that association may be cell-type specific. Rex-1 might fine-tune pluripotency in ES cells by modulating Polycomb-mediated gene regulation.     

Ring1a/b polycomb proteins regulate the mesenchymal stem cell niche in continuously growing incisors

Rodent incisors are capable of growing continuously and the renewal of dental epithelium giving rise to enamel-forming ameloblasts and dental mesenchyme giving rise to dentin-forming odontoblasts and pulp cells is achieved by stem cells residing at their proximal ends. Although the dental epithelial stem cell niche (cervical loop) is well characterized, little is known about the dental mesenchymal stem cell niche. Ring1a/b are the core Polycomb repressive complex1 (PRC1) components that have recently also been found in a protein complex with BcoR (Bcl-6 interacting corepressor) and Fbxl10. During mouse incisor development, we found that genes encoding members of the PRC1 complex are strongly expressed in the incisor apical mesenchyme in an area that contains the cells with the highest proliferation rate in the tooth pulp, consistent with a location for transit amplifying cells. Analysis of Ring1a(-/-);Ring1b(cko/cko) mice showed that loss of Ring1a/b postnatally results in defective cervical loops and disturbances of enamel and dentin formation in continuously growing incisors. To further characterize the defect found in Ring1a(-/-);Ring1b(cko/cko) mice, we demonstrated that cell proliferation is dramatically reduced in the apical mesenchyme and cervical loop epithelium of Ring1a(-/-);Ring1b(cko/cko) incisors in comparison to Ring1a(-/-);Ring1b(fl/fl)cre- incisors. Fgf signaling and downstream targets that have been previously shown to be important in the maintenance of the dental epithelial stem cell compartment in the cervical loop are downregulated in Ring1a(-/-);Ring1b(cko/cko) incisors. In addition, expression of other genes of the PRC1 complex is also altered. We also identified an essential postnatal requirement for Ring1 proteins in molar root formation. These results show that the PRC1 complex regulates the transit amplifying cell compartment of the dental mesenchymal stem cell niche and cell differentiation in developing mouse incisors and is required for molar root formation.