The tumour suppressor L(3)mbt inhibits neuroepithelial proliferation and acts on insulator elements

In Drosophila, defects in asymmetric cell division often result in the formation of stem-cell-derived tumours. Here, we show that very similar terminal brain tumour phenotypes arise through a fundamentally different mechanism. We demonstrate that brain tumours in l(3)mbt mutants originate from overproliferation of neuroepithelial cells in the optic lobes caused by derepression of target genes in the Salvador-Warts-Hippo (SWH) pathway. We use ChIP-sequencing to identify L(3)mbt binding sites and show that L(3)mbt binds to chromatin insulator elements. Mutating l(3)mbt or inhibiting expression of the insulator protein gene mod(mdg4) results in upregulation of SWH pathway reporters. As l(3)mbt tumours are rescued by mutations in bantam or yorkie or by overexpression of Expanded, the deregulation of SWH pathway target genes is an essential step in brain tumour formation. Therefore, very different primary defects result in the formation of brain tumours, which behave quite similarly in their advanced stages.     

A novel member of murine Polycomb-group proteins, Sex comb on midleg homolog protein, is highly conserved, and interacts with RAE28/mph1 in vitro

The Polycomb group of (PcG) genes were originally described in Drosophila, but many PcG genes have mammalian homologs. Genetic studies in flies and mice show that mutations in PcG genes cause posterior transformations caused by failure to maintain repression of homeotic loci, suggesting that PcG proteins have conserved functions. The Drosophila gene Sex comb on midleg (Scm) encodes an unusual PcG protein that shares motifs with the PcG protein polyhomeotic, and with a Drosophila tumor suppressor, lethal(3)malignant brain tumor (l(3)mbt). Expressed sequence tag (EST) databases were searched to recover putative mammalian Scm homologs, which were used to screen murine cDNA libraries. The recovered cDNA encodes two mbt repeats and the SPM domain that characterize Scm, but lacks the cysteine clusters and the serine/threonine-rich region found at the amino terminus of Scm. Accordingly, we have named the gene Sex comb on midleg homolog 1 (Scmh1). Like their Drosophila counterparts, Scmh1 and the mammalian polyhomeotic homolog RAE28/mph1 interact in vitro via their SPM domains. We analyzed the expression of Scmh1 and rae28/mph1 using northern analysis of embryos and adult tissues, and in situ hybridization to embryos. The expression of Scmh1 and rae28/mph1 is well correlated in most tissues of embryos. However, in adults, Scmh1 expression was detected in most tissues, whereas mph1/rae28 expression was restricted to the gonads. Scmh1 is strongly induced by retinoic acid in F9 and P19 embryonal carcinoma cells. Scmh1 maps to 4D1-D2.1 in mice. These data suggest that Scmh1 will have an important role in regulation of homeotic genes in embryogenesis and that the interaction with RAE28/mph1 is important in vivo.     

The Set2 methyltransferase associates with Ssn6 yet Tup1-Ssn6 repression is independent of histone methylation

The Tup1-Ssn6 corepressor regulates the expression of diverse classes of genes in Saccharomyces cerevisiae. Chromatin is an important component of Tup1-Ssn6-mediated repression. Tup1 binds to underacetylated tails of histones H3 and H4, and requires multiple histone deacetylases for the repression. Here we examine if histone methylation, in addition to histone deacetylation, plays a role in Tup1-Ssn6 repression. We found that like other genes, Tup1-Ssn6 target genes exhibit increased levels of histone H3 lysine 4 trimethylation upon activation. However, deletion of individual or multiple histone methyltransferases and other SET-domain containing genes has no apparent effect on Tup1-Ssn6-mediated repression of a number of well-defined targets. Interestingly, we discovered that Ssn6 interacts with Set2. Although deletion of SET2 does not affect Tup1-Ssn6 repression of a number of target genes, Ssn6 may utilize Set2 in specific contexts to regulate gene repression.     

A WD40 domain cyclophilin interacts with histone H3 and functions in gene repression and organogenesis in Arabidopsis

Chromatin-based silencing provides a crucial mechanism for the regulation of gene expression. We have identified a WD40 domain cyclophilin, CYCLOPHILIN71 (CYP71), which functions in gene repression and organogenesis in Arabidopsis thaliana. Disruption of CYP71 resulted in ectopic activation of homeotic genes that regulate meristem development. The cyp71 mutant plants displayed dramatic defects, including reduced apical meristem activity, delayed and abnormal lateral organ formation, and arrested root growth. CYP71 was associated with the chromatin of target gene loci and physically interacted with histone H3. The cyp71 mutant showed reduced methylation of H3K27 at target loci, consistent with the derepression of these genes in the mutant. As CYP71 has close homologs in eukaryotes ranging from fission yeast to human, we propose that it serves as a highly conserved histone remodeling factor involved in chromatin-based gene silencing in eukaryotic organisms.     

Separation-of-function mutation in HPC2, a member of the HIR complex in S. cerevisiae, results in derepression of the histone genes but does not confer cryptic TATA phenotypes

The HIR complex, which is comprised of the four proteins Hir1, Hir2, Hir3 and Hpc2, was first characterized as a repressor of three of the four histone gene loci in Saccharomyces cerevisiae. Using a bioinformatical approach, previous studies have identified a region of Hpc2 that is conserved in Schizosaccharomyces pombe and humans. Using a similar approach, we identified two additional domains, CDI and CDII, of the Hpc2 protein that are conserved among yeast species related to S. cerevisiae. We showed that the N terminal CDI domain (spanning amino acids 63-79) is dispensable for HIR complex assembly, but plays an essential role in the repression of the histone genes by recruiting the HIR complex to the HIR-dependent histone gene loci. The second conserved domain, CDII (spanning amino acids 452-480), is required for the stability of the Hpc2 protein itself as well as for the assembly of the HIR complex. In addition, we report a novel separation-of-function mutation within CDI of Hpc2, which causes derepression of the histone genes but does not confer other reported hir/hpc- phenotypes (such as Spt phenotypes, heterochromatin silencing defects and repression of cryptic promoters). This is the first direct demonstration that a separation-of-function mutation exists within the HIR complex.     

Polycomb group genes are required for neural stem cell survival in postembryonic neurogenesis of Drosophila

Genes of the Polycomb group (PcG) are part of a cellular memory system that maintains appropriate inactive states of Hox gene expression in Drosophila. Here, we investigate the role of PcG genes in postembryonic development of the Drosophila CNS. We use mosaic-based MARCM techniques to analyze the role of these genes in the persistent larval neuroblasts and progeny of the central brain and thoracic ganglia. We find that proliferation in postembryonic neuroblast clones is dramatically reduced in the absence of Polycomb, Sex combs extra, Sex combs on midleg, Enhancer of zeste or Suppressor of zeste 12. The proliferation defects in these PcG mutants are due to the loss of neuroblasts by apoptosis in the mutant clones. Mutation of PcG genes in postembryonic lineages results in the ectopic expression of posterior Hox genes, and experimentally induced misexpression of posterior Hox genes, which in the wild type causes neuroblast death, mimics the PcG loss-of-function phenotype. Significantly, full restoration of wild-type-like properties in the PcG mutant lineages is achieved by blocking apoptosis in the neuroblast clones. These findings indicate that loss of PcG genes leads to aberrant derepression of posterior Hox gene expression in postembryonic neuroblasts, which causes neuroblast death and termination of proliferation in the mutant clones. Our findings demonstrate that PcG genes are essential for normal neuroblast survival in the postembryonic CNS of Drosophila. Moreover, together with data on mammalian PcG genes, they imply that repression of aberrant reactivation of Hox genes may be a general and evolutionarily conserved role for PcG genes in CNS development.     

A Suv39h-dependent mechanism for silencing S-phase genes in differentiating but not in cycling cells

The Rb/E2F complex represses S-phase genes both in cycling cells and in cells that have permanently exited from the cell cycle and entered a terminal differentiation pathway. Here we show that S-phase gene repression, which involves histone-modifying enzymes, occurs through distinct mechanisms in these two situations. We used chromatin immunoprecipitation to show that methylation of histone H3 lysine 9 (H3K9) occurs at several Rb/E2F target promoters in differentiating cells but not in cycling cells. Furthermore, phenotypic knock-down experiments using siRNAs showed that the histone methyltransferase Suv39h is required for histone H3K9 methylation and subsequent repression of S-phase gene promoters in differentiating cells, but not in cycling cells. These results indicate that the E2F target gene permanent silencing mechanism that is triggered upon terminal differentiation is distinct from the transient repression mechanism in cycling cells. Finally, Suv39h-depleted myoblasts were unable to express early or late muscle differentiation markers. Thus, appropriately timed H3K9 methylation by Suv39h seems to be part of the control switch for exiting the cell cycle and entering differentiation.