Mice doubly deficient for the Polycomb Group genes Mel18 and Bmi1 reveal synergy and requirement for maintenance but not initiation of Hox gene expression

Polycomb group genes were identified as a conserved group of genes whose products are required in multimeric complexes to maintain spatially restricted expression of Hox cluster genes. Unlike in Drosophila, in mammals Polycomb group (PcG) genes are represented as highly related gene pairs, indicative of duplication during metazoan evolution. Mel18 and Bmi1 are mammalian homologs of Drosophila Posterior sex combs. Mice deficient for Mel18 or Bmi1 exhibit similar posterior transformations of the axial skeleton and display severe immune deficiency, suggesting that their gene products act on overlapping pathways/target genes. However unique phenotypes upon loss of either Mel18 or Bmi1 are also observed. We show using embryos doubly deficient for Mel18 and Bmi1 that Mel18 and Bmi1 act in synergy and in a dose-dependent and cell type-specific manner to repress Hox cluster genes and mediate cell survival of embryos during development. In addition, we demonstrate that Mel18 and Bmi1, although essential for maintenance of the appropriate expression domains of Hox cluster genes, are not required for the initial establishment of Hox gene expression. Furthermore, we show an unexpected requirement for Mel18 and Bmi1 gene products to maintain stable expression of Hox cluster genes in regions caudal to the prospective anterior expression boundaries during subsequent development.     

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.     

Regulation of Hoxb3 expression in the hindbrain and pharyngeal arches by rae28, a member of the mammalian Polycomb group of genes

During animal development, Hox genes are expressed in characteristic, spatially restricted patterns and specify regional identities along the anterior-posterior (A-P) axis. Polycomb group (PcG) proteins in Drosophila repress Hox expression and maintain the expression patterns during development. Mice deficient for homologues of the Drosophila PcG genes, such as M33, bmi1, mel18, rae28 and eed, show altered Hox expression patterns. In this study, we examined the time course of Hoxb3 expression during late gastrulation and early segmentation of rae28-deficient mice. Hoxb3 was expressed ectopically in pharyngeal arch and hindbrain from embryonic day (E) 9.5 and 10.5, respectively. The anterior boundary of ectopic expression in the hindbrain extended gradually in the rostral direction as development proceeded from E10.5 to E12.5. Expression of kreisler and Krox20, which function as positive regulators of Hoxb3 expression, was not affected in rae28-deficient embryos. Analysis of a neural crest marker, p75, in rae28-deficient mice revealed that the neural crest cells begin to ectopically express Hoxb3 after leaving the hindbrain. Our results suggest that rae28 is not required for the establishment but maintenance of Hoxb3 expression.     

Axial skeletal patterning in mice lacking all paralogous group 8 Hox genes

We present a detailed study of the genetic basis of mesodermal axial patterning by paralogous group 8 Hox genes in the mouse. The phenotype of Hoxd8 loss-of-function mutants is presented, and compared with that of Hoxb8- and Hoxc8-null mice. Our analysis of single mutants reveals common features for the Hoxc8 and Hoxd8 genes in patterning lower thoracic and lumbar vertebrae. In the Hoxb8 mutant, more anterior axial regions are affected. The three paralogous Hox genes are expressed up to similar rostral boundaries in the mesoderm, but at levels that strongly vary with the axial position. We find that the axial region affected in each of the single mutants mostly corresponds to the area with the highest level of gene expression. However, analysis of double and triple mutants reveals that lower expression of the other two paralogous genes also plays a patterning role when the mainly expressed gene is defective. We therefore conclude that paralogous group 8 Hox genes are involved in patterning quite an extensive anteroposterior (AP) axial region. Phenotypes of double and triple mutants reveal that Hoxb8, Hoxc8 and Hoxd8 have redundant functions at upper thoracic and sacral levels, including positioning of the hindlimbs. Interestingly, loss of functional Hoxb8 alleles partially rescues the phenotype of Hoxc8- and Hoxc8/Hoxd8-null mutants at lower thoracic and lumbar levels. This suggests that Hoxb8 affects patterning at these axial positions differently from the other paralogous gene products. We conclude that paralogous Hox genes can have a unique role in patterning specific axial regions in addition to their redundant function at other AP levels.     

Are polycomb group bodies gene silencing factories?

Polycomb group (PcG) proteins mediate long-range associations between Hox genes, which correlate with gene repression in vivo. Bantignies et al. (2011) identify a physiological role for the nuclear localization of Hox genes in PcG-mediated gene silencing, strengthening the evidence that nuclear positioning regulates gene expression.     

Role of Bmi-1 and Ring1A in H2A ubiquitylation and Hox gene silencing

Polycomb group (PcG) proteins exist in at least two biochemically distinct protein complexes, the EED-EZH2 complex and the PRC1 complex, that respectively possess H3-K27 methyltransferase and H2A-K119 ubiquitin E3 ligase activities. How the enzymatic activities are regulated and what their role is in Hox gene silencing are not clear. Here, we demonstrate that Bmi-1 and Ring1A, two components of the PRC1 complex, play important roles in H2A ubiquitylation and Hox gene silencing. We show that both proteins positively regulate H2A ubiquitylation. Chromatin immunoprecipitation (ChIP) assays demonstrate that Bmi-1 and other components of the two PcG complexes bind to the promoter of HoxC13. Knockout Bmi-1 results in significant loss of H2A ubiquitylation and upregulation of Hoxc13 expression, whereas EZH2-mediated H3-K27 methylation is not affected. Our results suggest that EZH2-mediated H3-K27 methylation functions upstream of PRC1 and establishes a critical role for Bmi-1 and Ring1A in H2A ubiquitylation and Hox gene silencing.     

Nuclear re-organisation of the Hoxb complex during mouse embryonic development

The spatial and temporal co-linear expression of Hox genes during development is an exquisite example of programmed gene expression. The precise mechanisms underpinning this are not known. Analysis of Hoxb chromatin structure and nuclear organisation, during the differentiation of murine ES cells, has lent support to the idea that there is a progressive 'opening' of chromatin structure propagated through Hox clusters from 3'to 5', which contributes to the sequential activation of gene expression. Here, we show that similar events occur in vivo in at least two stages of development. The first changes in chromatin structure and nuclear organisation were detected during gastrulation in the Hoxb1-expressing posterior primitive streak region: Hoxb chromatin was decondensed and the Hoxb1 locus looped out from its chromosome territory, in contrast to non-expressing Hoxb9, which remained within the chromosome territory. At E9.5, when differential Hox expression along the anteroposterior axis is being established, we found concomitant changes in the organisation of Hoxb. Hoxb organisation differed between regions of the neural tube that had never expressed Hoxb [rhombomeres (r) 1 and 2], strongly expressed Hoxb1 but not b9 (r4), had downregulated Hoxb1 (r5), expressed Hoxb9 but not Hoxb1 (spinal cord), and expressed both genes (tail bud). We conclude that Hoxb chromatin decondensation and nuclear re-organisation is regulated in different parts of the developing embryo, and at different developmental stages. The differential nuclear organisation of Hoxb along the anteroposterior axis of the developing neural tube is coherent with co-linear Hox gene expression. In early development nuclear re-organisation is coupled to Hoxb expression, but does not anticipate it.     

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.