Mammalian Polycomb group genes are categorized as a new type of early response gene induced by B-cell receptor cross-linking

Polycomb group (PcG) genes were initially described in Drosophila melanogaster as regulators of the homeobox gene. Four mammalian homologues, mel-18, bmi-1, M33 and rae-28, are analyzed in this study. They not only regulate mammalian homeotic genes by analogy with their Drosophila counterparts, but also have some influence on the growth and differentiation of B lymphocytes. Here we report that these four mammalian PcG genes are rapidly induced after antigen-receptor cross-linking in B cells. Thus we would like to propose that mammalian PcG genes can be categorized as a new type of immediate early gene.     

Altered cellular proliferation and mesoderm patterning in Polycomb-M33-deficient mice

In Drosophila, the trithorax-group and the Polycomb-group genes are necessary to maintain the expression of the homeobox genes in the appropriate segments. Loss-of-function mutations in those groups of genes lead to misexpression of the homeotic genes resulting in segmental homeotic transformations. Recently, mouse homologues of the Polycomb-group genes were identified including M33, the murine counterpart of Polycomb. In this report, M33 was targeted in mice by homologous recombination in embryonic stem (ES) cells to assess its function during development. Homozygous M33 (-/-) mice show greatly retarded growth, homeotic transformations of the axial skeleton, sternal and limb malformations and a failure to expand in vitro of several cell types including lymphocytes and fibroblasts. In addition, M33 null mutant mice show an aggravation of the skeletal malformations when treated to RA at embryonic day 7.5, leading to the hypothesis that, during development, the M33 gene might play a role in defining access to retinoic acid response elements localised in the regulatory regions of several Hox genes.     

white+ transgene insertions presenting a dorsal/ventral pattern define a single cluster of homeobox genes that is silenced by the polycomb-group proteins in Drosophila melanogaster

We used the white gene as an enhancer trap and reporter of chromatin structure. We collected white+ transgene insertions presenting a peculiar pigmentation pattern in the eye: white expression is restricted to the dorsal half of the eye, with a clear-cut dorsal/ventral (D/V) border. This D/V pattern is stable and heritable, indicating that phenotypic expression of the white reporter reflects positional information in the developing eye. Localization of these transgenes led us to identify a unique genomic region encompassing 140 kb in 69D1-3 subject to this D/V effect. This region contains at least three closely related homeobox-containing genes that are constituents of the iroquois complex (IRO-C). IRO-C genes are coordinately regulated and implicated in similar developmental processes. Expression of these genes in the eye is regulated by the products of the Polycomb-group (Pc-G) and trithorax-group (trx-G) genes but is not modified by classical modifiers of position-effect variegation. Our results, together with the report of a Pc-G binding site in 69D, suggest that we have identified a novel cluster of target genes for the Pc-G and trx-G products. We thus propose that ventral silencing of the whole IRO-C in the eye occurs at the level of chromatin structure in a manner similar to that of the homeotic gene complexes, perhaps by local compaction of the region into a heterochromatin-like structure involving the Pc-G products.     

Male-to-female sex reversal in M33 mutant mice

Polycomb genes in Drosophila maintain the repressed state of homeotic and other developmentally regulated genes by mediating changes in higher-order chromatin structure. M33, a mouse homologue of Polycomb, was isolated by means of the structural similarity of its chromodomain. The fifth exon of M33 contains a region of homology shared by Drosophila and Xenopus. In Drosophila, its deletion results in the loss of Polycomb function. Here we have disrupted M33 in mice by inserting a poly(A) capture-type neo(r) targeting vector into its fifth exon. More than half of the resultant M33cterm/M33cterm mutant mice died before weaning, and survivors showed male-to-female sex reversal. Formation of genital ridges was retarded in both XX and XY M33cterm/M33cterm embryos. Gonadal growth defects appeared near the time of expression of the Y-chromosome-specific Sry gene, suggesting that M33 deficiency may cause sex reversal by interfering with steps upstream of Sry. M33cterm/M33cterm mice may be a valuable model in which to test opposing views regarding sex determination.     

MES-2, a maternal protein essential for viability of the germline in Caenorhabditis elegans, is homologous to a Drosophila Polycomb group protein

A unique and essential feature of germ cells is their immortality. In Caenorhabditis elegans, germline immortality requires the maternal contribution from four genes, mes-2, mes-3, mes-4 and mes-6. We report here that mes-2 encodes a protein similar to the Drosophila Polycomb group protein, Enhancer of zeste, and in the accompanying paper that mes-6 encodes another Polycomb group protein. The Polycomb group is responsible for maintaining proper patterns of expression of the homeotic and other genes in Drosophila. It is thought that Polycomb group proteins form heteromeric complexes and control gene expression by altering chromatin conformation of target genes. As predicted from its similarity to a Polycomb group protein, MES-2 localizes to nuclei. MES-2 is found in germline nuclei in larval and adult worms and in all nuclei in early embryos. By the end of embryogenesis, MES-2 is detected primarily in the two primordial germ cells. The correct distribution of MES-2 requires the wild-type functions of mes-3 and mes-6. We hypothesize that mes-2 encodes a maternal regulator of gene expression in the early germline; its function is essential for normal early development and viability of germ cells.     

Expression, purification, and functional characterization of the carboxyl-terminal domain fragment of bacteriophage 434 repressor

The repressor protein of bacteriophage 434 binds to DNA as a dimer of identical subunits. Its strong dimerization is mediated by the carboxyl-terminal domain. Cooperative interactions between the C-terminal domains of two repressor dimers bound at adjacent sites can stabilize protein-DNA complexes formed with low-affinity binding sites. We have constructed a plasmid, pCT1, which directs the overproduction of the carboxyl-terminal domain of 434 repressor. The protein encoded by this plasmid is called CT-1. Cells transformed with pCT1 are unable to be lysogenized by wild-type 434 phage, whereas control cells are lysogenized at an efficiency of 1 to 5%. The CT-1-mediated interference with lysogen formation presumably results from formation of heteromeric complexes between the phage-encoded repressor and the plasmid-encoded carboxyl-terminal domain fragment. These heteromers are unable to bind DNA and thereby inhibit the repressor's activity in promoting lysogen formation. Two lines of evidence support this conclusion. First, DNase I footprinting experiments show that at a 2:1 ratio of CT-1 to intact 434 repressor, purified CT-1 protein prevents the formation of complexes between 434 repressor and its OR1 binding site. Second, cross-linking experiments reveal that only a specific heterodimeric complex forms between CT-1 and intact 434 repressor. This latter observation indicates that CT-1 interferes with 434 repressor-operator complex formation by preventing dimerization and not by altering the conformation of the DNA-bound repressor dimer. Our other evidence is also consistent with this suggestion. We have used deletion analysis in an attempt to define the region which mediates the 434 repressor-CT-1 interaction. CT-1 proteins which have more than the last 14 amino acids removed are unable to interfere with 434 repressor action in vivo.