Regulation of the Drosophila engrailed gene by Polycomb repressor complex 2

Suppressor-of-zeste-12 (Su(z)12) is a core component of the Polycomb repressive complex 2 (PRC2), which has a methyltransferase activity directed towards lysine residues of histone 3. Mutations in Polycomb group (PcG) genes cause de-repression of homeotic genes and subsequent homeotic transformations. Another target for Polycomb silencing is the engrailed gene, which encodes a key regulator of segmentation in the early Drosophila embryo. In close proximity to the en gene is a Polycomb Response Element, but whether en is regulated by Su(z)12 is not known. In this report, we show that en is not de-repressed in Su(z)12 or Enhancer-of-zeste mutant clones in the anterior compartment of wing discs. Instead, we find that en expression is down-regulated in the posterior portion of wing discs, indicating that the PRC2 complex acts as an activator of en. Our results indicate that this is due to secondary effects, probably caused by ectopic expression of Ubx and Abd-B.     

Uncovering a tumor-suppressor function for Drosophila Polycomb group genes

Comment on: Martinez AM, et al. Nat Genet 2009; 41:1076-82.     

Drosophila enhancer of Zeste/ESC complexes have a histone H3 methyltransferase activity that marks chromosomal Polycomb sites

Enhancer of Zeste is a Polycomb Group protein essential for the establishment and maintenance of repression of homeotic and other genes. In the early embryo it is found in a complex that includes ESC and is recruited to Polycomb Response Elements. We show that this complex contains a methyltransferase activity that methylates lysine 9 and lysine 27 of histone H3, but the activity is lost when the E(Z) SET domain is mutated. The lysine 9 position is trimethylated and this mark is closely associated with Polycomb binding sites on polytene chromosomes but is also found in centric heterochromatin, chromosome 4, and telomeric sites. Histone H3 methylated in vitro by the E(Z)/ESC complex binds specifically to Polycomb protein.     

Polycomb Group蛋白复合体与植物春化作用

Polycomb Group(PcG)蛋白能形成Polycomb Repressive Complex 1(PRC1)和PRC2等复合体,通过一个保守且表观遗传的机制调节基因表达并控制动植物的发育。拟南芥中由VERNALIZATION2参与形成的PRC2复合体(VRN2-PRC2)在春化过程中能对主要开花抑制基因FLOWER LOCUS C(FLC)的染色质进行组蛋白甲基化修饰,形成H3K27me3(组蛋白H3第27位赖氨酸三甲基化)等转录抑制标记,从而抑制FLC转录,促进开花。虽然麦类作物的春化机理与拟南芥有较大差异,但最近的研究表明麦类作物春化过程也受PcG蛋白调控。文章对拟南芥PcG蛋白介导的春化调节机制进行综述,期望能对植物尤其是麦类作物的春化机理研究提供资料。     

Alternative ESC and ESC-like subunits of a polycomb group histone methyltransferase complex are differentially deployed during Drosophila development

The Extra sex combs (ESC) protein is a Polycomb group (PcG) repressor that is a key noncatalytic subunit in the ESC-Enhancer of zeste [E(Z)] histone methyltransferase complex. Survival of esc homozygotes to adulthood based solely on maternal product and peak ESC expression during embryonic stages indicate that ESC is most critical during early development. In contrast, two other PcG repressors in the same complex, E(Z) and Suppressor of zeste-12 [SU(Z)12], are required throughout development for viability and Hox gene repression. Here we describe a novel fly PcG repressor, called ESC-Like (ESCL), whose biochemical, molecular, and genetic properties can explain the long-standing paradox of ESC dispensability during postembryonic times. Developmental Western blots show that ESCL, which is 60% identical to ESC, is expressed with peak abundance during postembryonic stages. Recombinant complexes containing ESCL in place of ESC can methylate histone H3 with activity levels, and lysine specificity for K27, similar to that of the ESC-containing complex. Coimmunoprecipitations show that ESCL associates with E(Z) in postembryonic cells and chromatin immunoprecipitations show that ESCL tracks closely with E(Z) on Ubx regulatory DNA in wing discs. Furthermore, reduced escl+ dosage enhances esc loss-of-function phenotypes and double RNA interference knockdown of ESC/ESCL in wing disc-derived cells causes Ubx derepression. These results suggest that ESCL and ESC have similar functions in E(Z) methyltransferase complexes but are differentially deployed as development proceeds.     

Genetic interactions and dosage effects of Polycomb group genes of Drosophila

The Polycomb (Pc) group of genes are required for maintenance of cell determination in Drosophila melanogaster. At least 11 Pc group genes have been described and there may be up to 40; all are required for normal regulation of homeotic genes, but as a group, their phenotypes are rather diverse. It has been suggested that the products of Pc group genes might be members of a heteromeric complex that acts to regulate the chromatin structure of target loci. We examined the phenotypes of adult flies heterozygous for every pairwise combination of Pc group genes in an attempt to subdivide the Pc group functionally. The results support the idea that Additional sex combs (Asx), Pc, Polycomblike (Pcl), Posterior sex combs (Psc), Sex combs on midleg (Scm), and Sex combs extra (Sce) have similar functions in some imaginal tissues. We show genetic interactions among extra sex combs (esc) and Asx, Enhancer of Pc, Pcl, Enhancer of zeste E(z), and super sex combs and reassess the idea that most Pc group genes function independently of esc. Most duplications of Pc group genes neither exhibit anterior transformations nor suppress the extra sex comb phenotype of Pc group mutations, suggesting that not all Pc group genes behave as predicted by the mass-action model. Surprisingly, duplications of E(z) enhance homeotic phenotypes of esc mutants. Flies with increasing doses of esc ⁺ exhibit anterior transformations, but these are not enhanced by mutations in trithorax group genes. The results are discussed with respect to current models of Pc group function.     

Dominant alleles identify SET domain residues required for histone methyltransferase of Polycomb repressive complex 2

Polycomb gene silencing requires histone methyltransferase activity of Polycomb repressive complex 2 (PRC2), which methylates lysine 27 of histone H3. Information on how PRC2 works is limited by lack of structural data on the catalytic subunit, Enhancer of zeste (E(Z)), and the paucity of E(z) mutant alleles that alter its SET domain. Here we analyze missense alleles of Drosophila E(z), selected for molecular study because of their dominant genetic effects. Four missense alleles identify key E(Z) SET domain residues, and a fifth is located in the adjacent CXC domain. Analysis of mutant PRC2 complexes in vitro, and H3-K27 methylation in vivo, shows that each SET domain mutation disrupts PRC2 histone methyltransferase. Based on known SET domain structures, the mutations likely affect either the lysine-substrate binding pocket, the binding site for the adenosylmethionine methyl donor, or a critical tyrosine predicted to interact with the substrate lysine epsilon-amino group. In contrast, the CXC mutant retains catalytic activity, Lys-27 specificity, and trimethylation capacity. Deletion analysis also reveals a functional requirement for a conserved E(Z) domain N-terminal to CXC and SET. These results identify critical SET domain residues needed for PRC2 enzyme function, and they also emphasize functional inputs from outside the SET domain.     

Maintenance of the engrailed expression pattern by Polycomb group genes in Drosophila.

The stable maintenance of expression patterns of homeotic genes depends on the function of a number of negative trans-regulators, termed the Polycomb (Pc) group of genes. We have examined the pattern of expression of the Drosophila segment polarity gene, engrailed (en), in embryos mutant for several different members of the Pc group. Here we report that embryos mutant for two or more Pc group genes show strong ectopic en expression, while only weak derepression of en occurs in embryos mutant for a single Pc group gene. This derepression is independent of two known activators of en expression: en itself and wingless. Additionally, in contrast to the strong ectopic expression of homeotic genes observed in extra sex combs- (esc-) mutant embryos, the en expression pattern is nearly normal in esc- embryos. This suggests that the esc gene product functions in a pathway independent of the other genes in the group. The data indicate that the same group of genes is required for stable restriction of en expression to a striped pattern and for the restriction of expression of homeotic genes along the anterior-posterior axis, and support a global role for the Pc group genes in stable repression of activity of developmental selector genes.     

Polycomb group mutants exhibit mitotic defects in syncytial cell cycles of Drosophila embryos

The Polycomb Group (PcG) of epigenetic regulators maintains the repressed state of Hox genes during development of Drosophila, thereby maintaining the correct patterning of the anteroposterior axis. PcG-mediated inheritance of gene expression patterns must be stable to mitosis to ensure faithful transmission of repressed Hox states during cell division. Previously, two PcG mutants, polyhomeotic and Enhancer of zeste, were shown to exhibit mitotic segregation defects in embryos, and condensation defects in imaginal discs, respectively. We show that polyhomeotic(proximal) but not polyhomeotic(distal) is necessary for mitosis. To test if other PcG genes have roles in mitosis, we examined embryos derived from heterozygous PcG mutant females for mitotic defects. Severe defects in sister chromatid segregation and nuclear fallout, but not condensation are exhibited by Polycomb, Posterior sex combs and Additional sex combs. By contrast, mutations in Enhancer of zeste (which encodes the histone methyltransferase subunit of the Polycomb Repressive Complex 2) exhibit condensation but not segregation defects. We propose that these mitotic defects in PcG mutants delay cell cycle progression. We discuss possible mitotic roles for PcG proteins, and suggest that delays in cell cycle progression might lead to failure of maintenance.     

Polycomb repressive complex 1 initiates and maintains tailless repression in Drosophila embryo

Maternally-deposited morphogens specify the fates of embryonic cells via hierarchically regulating the expression of zygotic genes that encode various classes of developmental regulators. Once the cell fates are determined, Polycomb-group proteins frequently maintain the repressed state of the genes. This study investigates how Polycomb-group proteins repress the expression of tailless, which encodes a developmental regulator in Drosophila embryo. Previous studies have shown that maternal Tramtrack69 facilitates maternal GAGA-binding factor and Heat shock factor binding to the torso response element (tor-RE) to initiate tailless repression in the stage-4 embryo. Chromatin-immunoprecipitation and genetic-interaction studies exhibit that maternally-deposited Polycomb repressive complex 1 (PRC1) recruited by the tor-RE-associated Tramtrack69 represses tailless expression in the stage-4 embryo. A noncanonical Polycomb-group response element (PRE) is mapped to the tailless proximal region. High levels of Bric-a-brac, Tramtrack, and Broad (BTB)-domain proteins are fundamental for maintaining tailless repression in the stage-8 to -10 embryos. Trmtrack69 sporadically distributes in the linear BTB-domain oligomer, which recruits and retains a high level of PRC1 near the GCCAT cluster for repressing tll expression in the stage-14 embryos. Disrupting the retention of PRC1 decreases the levels of PRC1 and Pleiohomeotic protein substantially on the PRE and causes tailless derepression in the stage-14 embryo. Furthermore, the retained PRC1 potentially serves as a second foundation for assembling the well-characterized polymer of the Sterile alpha motif domain in Polyhomeotic protein, which compacts chromatin to maintain the repressed state of tailless in the embryos after stage 14.