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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.12.031 Byline: Samantha A. Beck, Ester Falconer, Amanda Catching, Jacob W. Hodgson, Hugh W. Brock Keywords: Syncytial embryo; DNA damage response; S phase; Polycomb group Abstract: Polycomb group (PcG) genes are required for heritable silencing of target genes. Many PcG mutants have chromatin bridges and other mitotic defects in early embryos. These phenotypes can arise from defects in S phase or mitosis, so the phenotype does not show when PcG proteins act in cell cycle regulation. We analyzed the cell cycle role of the proximal subunit of Polyhomeotic (PhP) in Drosophila. Time-lapse imaging reveals that chromatin bridges formed during mitosis are able to resolve but sometimes result in chromosome breakage. Chromosome bridging is also observed in canonical cell cycles occurring in larval brains and is therefore not unique to the rapid embryonic cycles. PhP colocalizes with chromatin in S phase but not in mitosis in early embryos, indicating a direct role in DNA synthesis. Time lapse imaging of ph.sup.p mutants reveals an acceleration of S phase, showing that ph.sup.p regulates S phase length. Like ph.sup.p mutations, mutations in DNA damage checkpoints result in S phase acceleration. Consistent with this model, mutations in ph do not affect DNA synthesis rates, but exhibit impaired ability to block cell cycle progression following exposure to gamma-rays. Our data show that the mitotic defects of ph.sup.p are caused by defects in the DNA damage response that occurs after DNA replication in S phase, and we propose that PhP has a direct role in DNA damage repair. Author Affiliation: Molecular Epigenetics Group, Department of Zoology, University of BC, Life Sciences Centre, 2350 Health Sciences Mall Vancouver, BC, Canada V6T 1Z3 Article History: Received 16 June 2009; Revised 30 October 2009; Accepted 19 December 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.11.015 Byline: Ester O'Dor, Samantha A. Beck, Hugh W. Brock Keywords: Mitosis; Polycomb; Chromatin bridges; Nuclear fallout; Mitotic delay Abstract: 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.sup.proximal but not polyhomeotic.sup.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. Author Affiliation: Department of Zoology, University of British Columbia, Life Sciences Centre, 2350 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3 Article History: Received 11 July 2005; Revised 4 November 2005; Accepted 9 November 2005

Polycomb group (PcG) proteins silence target loci in Drosophila. Although the mechanism of PcG-mediated silencing remains unknown, there is considerable evidence that PcG proteins act via multiple complexes. We have epitope-tagged Polyhomeotic Proximal, PHP, the major isoform of the proximal product of the polyhomeotic locus, at both termini (F-PHP-HA) and generated a stable Kc1 cell line in order to isolate F-PHP-HA-associated proteins. Using either column chromatography followed by immunoaffinity precipitation or a double immunoaffinity precipitation procedure, we observed multiple proteins that stably associate with F-PHP-HA. Sequencing the five major bands identified PHP-170 and PHP-140 isoforms, Polycomb, Heat shock cognate 4 (Hsc4), and a novel Drosophila J class chaperone we term Droj2. Mutations in both chaperone genes enhance homeotic transformations in PcG genes, suggesting that they have a role in silencing. We show by Western blotting that minor components of F-PHP-HA-associated proteins include TBP, TAFH42, TAFH85, and p55. However, unlike in PRC1, Psc, [TAF.sub.II]62, Modulo, dMI-2, or Rpd3/HDAC1 do not associate with F-PHP-HA. We discuss the role of chaperones and F-PHP-HA-associated proteins in PcG-mediated silencing and the evidence for different complexes containing Polyhomeotic in vivo. Keywords: Polyhomeotic; Chaperone; General transcription factor; Silencing; Polycomb group; Chromatin

The polyhomeotic (ph) gene is a member of the Polycomb group of genes (Pc-G), which are required for the maintenance of the spatial expression pattern of homeotic genes. In contrast to homeotic genes, ph is ubiquitously expressed and it is quantitatively regulated, ph is negatively regulated by the Pc-G genes, except Psc, and positively regulated by the antagonist trithorax group of genes (trx-G), suggesting that Pc-G and trx-G response elements (PREs and TREs) exist at the ph locus. In this study, we have functionally characterized PREs and TREs at the ph locus that function in transgenic constructs. We have identified a strong PRE and TRE in the ph proximal unit as well as a weak one in the ph distal unit. The PRE/TRE of both ph units appear atypical compared with the well-defined homeotic maintenance elements because the minimal ph proximal response element activity requires at least 2 kb of sequence and does not work at long range. We have used chromatin immunoprecipitation experiments on cultured cells and embryos to show that Pc-G proteins are located in restricted regions, close to the ph promoters that overlap functionally defined PRE/TREs. Our data suggest that ph PRE/TREs are cis-acting DNA elements that modulate rather than silence Pc-G- and trx-G-mediated regulation, enlarging the role of these two groups of genes in transcriptional regulation. Keywords: Drosophila; Polycomb; trithorax; polyhomeotic; PREs; TREs; Quantitative regulation

Polycomb group (PcG) genes of Drosophila are negative regulators of homeotic gene expression required for maintenance of determination. Sequence similarity between Polycomb and Su(var)205 led to the suggestion that PcG genes and modifiers of position-effect variegation (PEV) might function analogously in the establishment of chromatin structure. If PcG proteins participate directly in the same process that leads to PEV, PcG mutations should suppress PEV. We show that mutations in E(Pc), an unusual member of the PcG, suppress PEV of four variegating rearrangements: In(l)[w.sup.m4], [B.sub.SV], T(2;3)[Sb.sup.V], and In(2R)[bw.sup.VDe2]. Using reversion of a P element insertion, deficiency mapping, and recombination mapping as criteria, homeotic effects and suppression of PEV associated with E(Pc) co-map. Asx is an enhancer of PEV, whereas nine other PcG loci do not affect PEV. These results support the conclusion that there are fewer similarities between PcG genes and modifiers of PEV than previously supposed. However, E(Pc) appears to be an important link between the two groups. We discuss why Asx might act as an enhancer of PEV.

A study of the interaction of polyhomeotic (ph) with genes of the polycomb (Pc) group of Drosophila melangaster by crossing ph alleles to every Pc group member and to E(Pc) and Su(Z)2 reveals the presence of extragenic noncomplementation between ph 409 and Sce1, ScmD1 and E(Z)1 mutations in males and between ph503 and Pc, Psc1 and Su(Z)21 in females, confirming the direct interaction of the gene products with ph. Heterozygotes of extra sex combs, E(z), super sex combs or l(4)102EFc are not affected lethally by ph2, unlike the embryos that are homozygous for these genes. Homeotic phenotypes of esc heterozygotes from homozygous esc- mothers are not increased by ph503.

Gamma radiation was used to isolate new alleles of the Additional sex combs (Asx) locus of Drosophila melanogaster. The Asx region had a higher mutation rate than expected, hence it is either sensitive to radiation damage or is a large gene. Loss of the Asx gene resulted in segmentation defects and altered patterns of expression of the even-skipped gene, which suggests that Asx is a developmental requisite. Transposon tagging of the Asx locus was performed, allowing molecular studies to be started.

The Drosophila trithorax- and Polycomb-group (trxG and PcG) proteins maintain activated and repressed transcriptional states at specific target gene loci. The Additional sex combs (Asx) gene is of particular interest as it appears to function in both protein complexes and yet its effects on target genes are more restricted. A novel protein, Tantalus (TAN), was identified in a yeast two-hybrid screen for ASX-interacting proteins that might confer tissue-specific ASX functions. TAN contains consensus nuclear localization sites and binds DNA in vitro. However, its subcellular localization varies in a tissue-specific fashion. In salivary glands, TAN is predominantly nuclear and associates with 66 euchromatic sites on polytene chromosomes, more than half of which overlap with ASX. These loci do not include the homeotic genes of the ANT and BX complexes bound by other PcG and trxG proteins. Rather, tan mutant defects are restricted to sensory organs. We show that one of these defects, shared by Asx, is genetically enhanced by Asx. Taken together, the data suggest that TAN is a tissue-specific cofactor for ASX, and that its activity may be partially controlled by subcellular trafficking. Key Words: Additional sex combs; tantalus; tissue-specific; trithorax; Polycomb; Drosophila; sensory organs.

Background: Maintenance of cell fate determination requires the Polycomb group for repression; the trithorax group for gene activation; and the enhancer of trithorax and Polycomb (ETP) group for both repression and activation. Additional sex combs (Asx) is a genetically identified ETP for the Hox loci, but the molecular basis of its dual function is unclear. Results: We show that in vitro, Asx binds directly to the SET domains of the histone methyltransferases (HMT) enhancer of zeste [E(z)] (H3K27me3) and Trx (H3K4me3) through a bipartite interaction site separated by 846 amino acid residues. In Drosophila S2 cell nuclei, Asx interacts with E(z) and Trx in vivo. Drosophila Asx is required for repression of heat-shock gene hsp70 and is recruited downstream of the hsp70 promoter. Changes in the levels of H3K4me3 and H3K27me3 downstream of the hsp70 promoter in Asx mutants relative to wild type show that Asx regulates H3K4 and H3K27 trimethylation. Conclusions: We propose that during transcription Asx modulates the ratio of H3K4me3 to H3K27me3 by selectively recruiting the antagonistic HMTs, E(z) and Trx or other nucleosome-modifying enzymes to hsp70. [ABSTRACT FROM AUTHOR]

MLL (mixed-lineage leukemia) is a histone H3 Lys-4 specific methyltransferase that is a positive regulator of Hox expression. MLL rearrangements and amplification are common in acute lymphoid and myeloid leukemias and myelodysplastic disorders and are associated with abnormal up-regulation of Hox gene expression. Although Mu is expressed throughout hematopoiesis, Hox gene expression is sharply down-regulated during differentiation, suggesting that either the activity of MLL or its association with target promoters must be regulated. Here we show that MLL associates with actively transcribed genes but does not remain bound after transcriptional down-regulation. Surprisingly, MLL is associated not only with promoter regions but also is distributed across the entire coding regions of genes. Mu interacts with RNA polymerase Il (pol II) and colocalizes with RNA pol II at a subset of actively transcribed target in viva. Loss of function MLL results in defects in RNA pol II distribution. Together the results suggest that an intimate association between MLL and RNA pol Il occurs at MLL target genes in viva that is required for normal initiation and/or transcriptional elongation. [ABSTRACT FROM AUTHOR]

Immunological data, amino acid composition, and coordinate control during development suggest that the α, β and γ subunits of the major protein of Drosophila larval serum (LSP-1) are coded for by genes which evolved by replications of an ancestral gene followed by mutation. In order to test this hypothesis, and to study the relationship of these genes with that coding for the second major larval serum protein (LSP-2), we compared the one and two-dimensional peptide maps of all four larval serum protein subunits. One-dimensional maps generated by three different proteases showed many similarities among these proteins. Two-dimensional peptide mapping of the methionine-containing tryptic peptides showed that half of these peptides are common to all four larval serum protein subunits, and that about two-thirds are common to the three LSP-1 subunits. These observations show that the LSP-1 subunits are more closely related to each other than any is to LSP-2, and supported the initial suggestion that the proteins are homologous. Because the genes for the LSP-1 subunits are each located on a different chromosome, the LSP-I subunits are a suitable system for investigating the evolution and dispersal of related genes, and trans control in eukaryotes. [ABSTRACT FROM AUTHOR]