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.     

A genetic framework for floral patterning

The initial steps of flower development involve two classes of consecutively acting regulatory genes. Meristem-identity genes, which act early to control the initiation of flowers, are expressed throughout the incipient floral primordium. Homeotic genes, which act later to specify the identity of individual floral organs, are expressed in distinct domains within the flower. The link between the two classes of genes has remained unknown so far. Here we show that the meristem-identity gene LEAFY has a role in controlling homeotic genes that is separable from its role in specifying floral fate. On the basis of our observation that LEAFY activates different homeotic genes through distinct mechanisms, we propose a genetic framework for the control of floral patterning.     

The enhancer of polycomb gene of Drosophila encodes a chromatin protein conserved in yeast and mammals

The Polycomb group of genes in Drosophila are homeotic switch gene regulators that maintain homeotic gene repression through a possible chromatin regulatory mechanism. The Enhancer of Polycomb (E(Pc)) gene of Drosophila is an unusual member of the Polycomb group. Most PcG genes have homeotic phenotypes and are required for repression of homeotic loci, but mutations in E(Pc) exhibit no homeotic transformations and have only a very weak effect on expression of Abd-B. However, mutations in E(Pc) are strong enhancers of mutations in many Polycomb group genes and are also strong suppressors of position-effect variegation, suggesting that E(Pc) may have a wider role in chromatin formation or gene regulation than other Polycomb group genes. E(Pc) was cloned by transposon tagging, and encodes a novel 2023 amino acid protein with regions enriched in glutamine, alanine and asparagine. E(Pc) is expressed ubiquitously in Drosophila embryogenesis. E(Pc) is a chromatin protein, binding to polytene chromosomes at about 100 sites, including the Antennapedia but not the Bithorax complex, 29% of which are shared with Polycomb-binding sites. Surprisingly, E(Pc) was not detected in the heterochromatic chromocenter. This result suggests that E(Pc) has a functional rather than structural role in heterochromatin formation and argues against the heterochromatin model for PcG function. Using homology cloning techniques, we identified a mouse homologue of E(Pc), termed Epc1, a yeast protein that we name EPL1, and as well as additional ESTs from Caenorhabditis elegans, mice and humans. Epc1 shares a long, highly conserved domain in its amino terminus with E(Pc) that is also conserved in yeast, C. elegans and humans. The occurrence of E(Pc) across such divergent species is unusual for both PcG proteins and for suppressors of position-effect variegation, and suggests that E(Pc) has an important role in the regulation of chromatin structure in eukaryotes.     

The cramped gene of Drosophila is a member of the Polycomb-group, and interacts with mus209, the gene encoding Proliferating Cell Nuclear Antigen

We have isolated and molecularly characterized the cramped (crm) gene of Drosophila melanogaster, and show that it can be classified as a Polycomb-group (Pc-G) gene. crm mutants exhibit typical Pc-G mutant phenotypes, reminiscent of ectopic homeotic gene expression, with additional sex comb teeth found on mesothoracic and metathoracic legs, and proximodistal transformations of the tarsal segments. crm encodes an 693 amino acids protein, with no significant homology to known proteins. We used polyclonal antibodies raised against bacterially expressed truncated CRM protein to show that the crm gene product is localized to the nucleus during embryogenesis. This nuclear localization appears to be restricted to S-phase nuclei, as CRM immunostaining disappears at mitosis. We found that this cell-cycle-dependent staining pattern was identical to that of Proliferating Cell Nuclear Antigen (PCNA). Furthermore, we provide evidence for co-localization of CRM and PCNA proteins in salivary gland polytene nuclei, and for a genetic interaction between crm and mus209, the Drosophila gene encoding PCNA. Together, our data suggest that these two proteins are involved in a common regulatory pathway and highlight possible interactions between Pc-G-mediated silencing and DNA replication in Drosophila.     

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.     

Enhancer point mutation results in a homeotic transformation in Drosophila

In Drosophila, the misexpression or altered activity of genes from the bithorax complex results in homeotic transformations. One of these genes, abd-A, normally specifies the identity of the second through fourth abdominal segments (A2 to A4). In the dominant Hyperabdominal mutations (Hab), portions of the third thoracic segment (T3) are transformed toward A2 as the result of ectopic abd-A expression. Sequence analysis and deoxyribonuclease I footprinting demonstrate that the misexpression of abd-A in two independent Hab mutations results from the same single base change in a binding site for the gap gene Krüppel protein. These results establish that the spatial limits of the homeotic genes are directly regulated by gap gene products.     

The trithorax group gene osa encodes an ARID-domain protein that genetically interacts with the brahma chromatin-remodeling factor to regulate transcription

The trithorax group gene brahma (brm) encodes the ATPase subunit of a chromatin-remodeling complex involved in homeotic gene regulation. We report here that brm interacts with another trithorax group gene, osa, to regulate the expression of the Antennapedia P2 promoter. Regulation of Antennapedia by BRM and OSA proteins requires sequences 5' to the P2 promoter. Loss of maternal osa function causes severe segmentation defects, indicating that the function of osa is not limited to homeotic gene regulation. The OSA protein contains an ARID domain, a DNA-binding domain also present in the yeast SWI1 and Drosophila DRI proteins. We propose that the OSA protein may target the BRM complex to Antennapedia and other regulated genes.     

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.     

The trithorax group gene moira encodes a brahma-associated putative chromatin-remodeling factor in Drosophila melanogaster

The genes of the trithorax group (trxG) in Drosophila melanogaster are required to maintain the pattern of homeotic gene expression that is established early in embryogenesis by the transient expression of the segmentation genes. The precise role of each of the diverse trxG members and the functional relationships among them are not well understood. Here, we report on the isolation of the trxG gene moira (mor) and its molecular characterization. mor encodes a fruit fly homolog of the human and yeast chromatin-remodeling factors BAF170, BAF155, and SWI3. mor is widely expressed throughout development, and its 170-kDa protein product is present in many embryonic tissues. In vitro, MOR can bind to itself and it interacts with Brahma (BRM), an SWI2-SNF2 homolog, with which it is associated in embryonic nuclear extracts. The leucine zipper motif of MOR is likely to participate in self-oligomerization; the equally conserved SANT domain, for which no function is known, may be required for optimal binding to BRM. MOR thus joins BRM and Snf5-related 1 (SNR1), two known Drosophila SWI-SNF subunits that act as positive regulators of the homeotic genes. These observations provide a molecular explanation for the phenotypic and genetic relationships among several of the trxG genes by suggesting that they encode evolutionarily conserved components of a chromatin-remodeling complex.     

Homeotic genes autonomously specify one aspect of pattern in the Drosophila mesoderm

Transplantation and ablation experiments have led to the generalization that in insects the mesoderm is naive, and that pattern is imposed upon it by the ectoderm. This has been demonstrated directly by mosaic analysis for the case of one muscle in Drosophila. The unique character of this muscle depends on the activity of sex-determining and homeotic genes, not in the muscle itself, but in the nerve that innervates it. Indirect evidence suggests, however, that homeotic genes specify some aspects of mesoderm patterning autonomously. Homeotic genes are expressed in the mesoderm, and are regulated in a segment-specific pattern analogous to, but different from, that seen in the ectoderm. Moreover, the effects of homeotic mutations on the muscles do not always mirror transformations seen in the epidermis. Here we examine this problem directly, by expressing homeotic genes ectopically in the mesoderm without altering their expression in the overlying ectoderm. We find that the pattern of adult muscle precursor cells characteristic of the thorax can be converted to that seen in the abdomen by expressing the homeotic gene abdominal-A specifically in the mesoderm.