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Polycomb group (PcG) proteins are concentrated in nuclear foci called PcG bodies. Although some of these foci are due to the tendency of PcG binding sites in the genome to occur in linear clusters, distant PcG sites can contact one another and in some cases congregate in the same PcG body when they are repressed. Experiments using transgenes containing PcG binding sites reveal that co-localization depends on the presence of insulator elements rather than of Polycomb Response Elements (PREs) and that it can occur also when the transgenes are in the active state. A model is proposed according to which insulator proteins mediate shuttling of PcG target genes between PcG bodies when repressed to transcription factories when transcriptionally active., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Certain Polycomb group (PcG) genes are themselves targets of PcG complexes. Two of these constitute the Drosophila Psc-Su(z)2 locus, a region whose chromatin is enriched for H3K27me3 and contains several putative Polycomb response elements (PREs) that bind PcG proteins. To understand how PcG mechanisms regulate this region, the repressive function of the PcG protein binding sites was analyzed using reporter gene constructs. We find that at least two of these are functional PREs that can silence a reporter gene in a PcG-dependent manner. One of these two can also display anti-silencing activity, dependent on the context. A PcG protein binding site near the Psc promoter behaves not as a silencer but as a down-regulation module that is actually stimulated by the Pc gene product but not by other PcG products. Deletion of one of the PREs increases the expression level of Psc and Su(z)2 by twofold at late embryonic stages. We present evidence suggesting that the Psc-Su(z)2 locus is flanked by insulator elements that may protect neighboring genes from inappropriate silencing. Deletion of one of these regions results in extension of the domain of H3K27me3 into a region containing other genes, whose expression becomes silenced in the early embryo., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at "entry sites" that contain a consensus sequence motif ("MSL recognition element" or MRE). However, this motif is only ∼2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex) and female Kc cells (which lack the complex), we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome., Competing Interests: The authors have declared that no competing interests exist.

Chromatin is composed of DNA and a variety of modified histones and non-histone proteins, which have an impact on cell differentiation, gene regulation and other key cellular processes. Here we present a genome-wide chromatin landscape for Drosophila melanogaster based on eighteen histone modifications, summarized by nine prevalent combinatorial patterns. Integrative analysis with other data (non-histone chromatin proteins, DNase I hypersensitivity, GRO-Seq reads produced by engaged polymerase, short/long RNA products) reveals discrete characteristics of chromosomes, genes, regulatory elements and other functional domains. We find that active genes display distinct chromatin signatures that are correlated with disparate gene lengths, exon patterns, regulatory functions and genomic contexts. We also demonstrate a diversity of signatures among Polycomb targets that include a subset with paused polymerase. This systematic profiling and integrative analysis of chromatin signatures provides insights into how genomic elements are regulated, and will serve as a resource for future experimental investigations of genome structure and function.

Eukaryotic genomes are packaged in two basic forms, euchromatin and heterochromatin. We have examined the composition and organization of Drosophila melanogaster heterochromatin in different cell types using ChIP-array analysis of histone modifications and chromosomal proteins. As anticipated, the pericentric heterochromatin and chromosome 4 are on average enriched for the "silencing" marks H3K9me2, H3K9me3, HP1a, and SU(VAR)3-9, and are generally depleted for marks associated with active transcription. The locations of the euchromatin-heterochromatin borders identified by these marks are similar in animal tissues and most cell lines, although the amount of heterochromatin is variable in some cell lines. Combinatorial analysis of chromatin patterns reveals distinct profiles for euchromatin, pericentric heterochromatin, and the 4th chromosome. Both silent and active protein-coding genes in heterochromatin display complex patterns of chromosomal proteins and histone modifications; a majority of the active genes exhibit both "activation" marks (e.g., H3K4me3 and H3K36me3) and "silencing" marks (e.g., H3K9me2 and HP1a). The hallmark of active genes in heterochromatic domains appears to be a loss of H3K9 methylation at the transcription start site. We also observe complex epigenomic profiles of intergenic regions, repeated transposable element (TE) sequences, and genes in the heterochromatic extensions. An unexpectedly large fraction of sequences in the euchromatic chromosome arms exhibits a heterochromatic chromatin signature, which differs in size, position, and impact on gene expression among cell types. We conclude that patterns of heterochromatin/euchromatin packaging show greater complexity and plasticity than anticipated. This comprehensive analysis provides a foundation for future studies of gene activity and chromosomal functions that are influenced by or dependent upon heterochromatin.

The genomic binding sites of Polycomb group (PcG) complexes have been found to cluster, forming Polycomb "bodies" or foci in mammalian or fly nuclei. These associations are thought to be driven by interactions between PcG complexes and result in enhanced repression. Here, we show that a Polycomb response element (PRE) with strong PcG binding and repressive activity cannot mediate trans interactions. In the case of the two best-studied interacting PcG targets in Drosophila, the Mcp and the Fab-7 regulatory elements, we find that these associations are not dependent on or caused by the Polycomb response elements they contain. Using functional assays and physical colocalization by in vivo fluorescence imaging or chromosome conformation capture (3C) methods, we show that the interactions between remote copies of Mcp or Fab-7 elements are dependent on the insulator activities present in these elements and not on their PREs. We conclude that insulator binding proteins rather than PcG complexes are likely to be the major determinants of the long-range higher-order organization of PcG targets in the nucleus.

We have tested the specificity and utility of more than 200 antibodies raised against 57 different histone modifications in Drosophila melanogaster, Caenorhabditis elegans and human cells. Although most antibodies performed well, more than 25% failed specificity tests by dot blot or western blot. Among specific antibodies, more than 20% failed in chromatin immunoprecipitation experiments. We advise rigorous testing of histone-modification antibodies before use, and we provide a website for posting new test results (http://compbio.med.harvard.edu/antibodies/).

To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.

The Polycomb (PcG) and Trithorax (TrxG) group proteins are essential for development in all multicellular organisms. Mutations of the PcG and TrxG genes act as early embryonic lethals, while their overexpression correlates with malignancies. Comparative genome analysis showed that PcG and TrxG form a binary regulatory system that functions as an epigenetic rheostat to determine the threshold levels of extracellular signals affecting the expression levels of key developmental genes.

The dosage of X and Y chromosome genes varies systematically in males and females. In mammals, X inactivation largely compensates for this, but in this issue of Developmental Cell, Wijchers et al. show that the expression of hundreds of autosomal genes is still influenced by sex chromosome dosage, independently of physiological sex., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Polycomb (PcG) regulation has been thought to produce stable long-term gene silencing. Genomic analyses in Drosophila and mammals, however, have shown that it targets many genes, which can switch state during development. Genetic evidence indicates that critical for the active state of PcG target genes are the histone methyltransferases Trithorax (TRX) and ASH1. Here we analyze the repertoire of alternative states in which PcG target genes are found in different Drosophila cell lines and the role of PcG proteins TRX and ASH1 in controlling these states. Using extensive genome-wide chromatin immunoprecipitation analysis, RNAi knockdowns, and quantitative RT-PCR, we show that, in addition to the known repressed state, PcG targets can reside in a transcriptionally active state characterized by formation of an extended domain enriched in ASH1, the N-terminal, but not C-terminal moiety of TRX and H3K27ac. ASH1/TRX N-ter domains and transcription are not incompatible with repressive marks, sometimes resulting in a "balanced" state modulated by both repressors and activators. Often however, loss of PcG repression results instead in a "void" state, lacking transcription, H3K27ac, or binding of TRX or ASH1. We conclude that PcG repression is dynamic, not static, and that the propensity of a target gene to switch states depends on relative levels of PcG, TRX, and activators. N-ter TRX plays a remarkable role that antagonizes PcG repression and preempts H3K27 methylation by acetylation. This role is distinct from that usually attributed to TRX/MLL proteins at the promoter. These results have important implications for Polycomb gene regulation, the "bivalent" chromatin state of embryonic stem cells, and gene expression in development., Competing Interests: The authors have declared that no competing interests exist.

Polycomb group proteins have an essential role in the epigenetic maintenance of repressive chromatin states. The gene-silencing activity of the Polycomb repressive complex 2 (PRC2) depends on its ability to trimethylate lysine 27 of histone H3 (H3K27) by the catalytic SET domain of the EZH2 subunit, and at least two other subunits of the complex: SUZ12 and EED. Here we show that the carboxy-terminal domain of EED specifically binds to histone tails carrying trimethyl-lysine residues associated with repressive chromatin marks, and that this leads to the allosteric activation of the methyltransferase activity of PRC2. Mutations in EED that prevent it from recognizing repressive trimethyl-lysine marks abolish the activation of PRC2 in vitro and, in Drosophila, reduce global methylation and disrupt development. These findings suggest a model for the propagation of the H3K27me3 mark that accounts for the maintenance of repressive chromatin domains and for the transmission of a histone modification from mother to daughter cells.

The cohesin protein complex was first recognized for holding sister chromatids together and ensuring proper chromosome segregation. Cohesin also regulates gene expression, but the mechanisms are unknown. Cohesin associates preferentially with active genes, and is generally absent from regions in which histone H3 is methylated by the Enhancer of zeste [E(z)] Polycomb group silencing protein. Here we show that transcription is hypersensitive to cohesin levels in two exceptional cases where cohesin and the E(z)-mediated histone methylation simultaneously coat the entire Enhancer of split and invected-engrailed gene complexes in cells derived from Drosophila central nervous system. These gene complexes are modestly transcribed, and produce seven of the twelve transcripts that increase the most with cohesin knockdown genome-wide. Cohesin mutations alter eye development in the same manner as increased Enhancer of split activity, suggesting that similar regulation occurs in vivo. We propose that cohesin helps restrain transcription of these gene complexes, and that deregulation of similarly cohesin-hypersensitive genes may underlie developmental deficits in Cornelia de Lange syndrome.

Polycomb group proteins control a hierarchy of gene expression during the differentiation of stem cells. In this issue, Ezhkova et al. (2009) show that repression mediated by Polycomb proteins controls the timing of differentiation of precursor cells of the epidermal lineage.

Important advances in the study of Polycomb Group (PcG) complexes in the past two years have focused on the role of this repressive system in programing the genome. Genome-wide analyses have shown that PcG mechanisms control a large number of genes regulating many cellular functions and all developmental pathways. Current evidence shows that, contrary to the classical picture of their role, PcG complexes do not set a repressed chromatin state that is maintained throughout development but have a much more dynamic role. PcG target genes can become repressed or be reactivated or exist in intermediate states. What controls the balance between repression and derepression is a crucial question in understanding development and differentiation in higher organisms.

The Drosophila esc gene is a Polycomb Group (PcG) gene whose product is essential for histone H3 K27 methylation and PcG silencing yet genetic analysis indicated that its product was needed only in the very early embryo. We know now that escl, a close homologue of esc exists in the Drosophila genome. In contrast with earlier studies, we find that both esc and escl are expressed at all stages of development. We show that three major differences between the two genes are in the transcriptional control, which allows esc to make a much stronger maternal contribution; in the splicing efficiency, which makes a major difference in the early escl function; and in the lower participation of ESCL in the PRC2 complex and lower enzymatic activity of the resulting complex. Both genes can sustain normal development in the absence of the other except for the critical role provided by maternal esc product in early embryonic development. Finally, using zygotic mutations in both genes, we show that the gradual loss of function of PRC2 activity leads first to a loss of histone H3 K27 methylation and only at a later stage to a gradual loss of PRC1 binding to chromatin.

The mammalian Polycomblike protein PHF1 was previously shown to interact with the Polycomb group (PcG) protein Ezh2, a histone methyltransferase whose activity is pivotal in sustaining gene repression during development and in adulthood. As Ezh2 is active only when part of the Polycomb Repressive Complexes (PRC2-PRC4), we examined the functional role of its interaction with PHF1. Chromatin immunoprecipitation experiments revealed that PHF1 resides along with Ezh2 at Ezh2-regulated genes such as the HoxA loci and the non-Hox MYT1 and WNT1 genes. Knockdown of PHF1 or of Ezh2 led to up-regulated HoxA gene expression. Interestingly, depletion of PHF1 did correlate with reduced occupancy of Bmi-1, a PRC1 component. As expected, knockdown of Ezh2 led to reduced levels of its catalytic products H3K27me2/H3K27me3. However, reduced levels of PHF1 also led to decreased global levels of H3K27me3. Notably, the levels of H3K27me3 decreased while those of H3K27me2 increased at the up-regulated HoxA loci tested. Consistent with this, the addition of PHF1 specifically stimulated the ability of Ezh2 to catalyze H3K27me3 but not H3K27me1/H3K27me2 in vitro. We conclude that PHF1 modulates the activity of Ezh2 in favor of the repressive H3K27me3 mark. Thus, we propose that PHF1 is a determinant in PcG-mediated gene repression.

The cohesin complex is a chromosomal component required for sister chromatid cohesion that is conserved from yeast to man. The similarly conserved Nipped-B protein is needed for cohesin to bind to chromosomes. In higher organisms, Nipped-B and cohesin regulate gene expression and development by unknown mechanisms. Using chromatin immunoprecipitation, we find that Nipped-B and cohesin bind to the same sites throughout the entire non-repetitive Drosophila genome. They preferentially bind transcribed regions and overlap with RNA polymerase II. This contrasts sharply with yeast, where cohesin binds almost exclusively between genes. Differences in cohesin and Nipped-B binding between Drosophila cell lines often correlate with differences in gene expression. For example, cohesin and Nipped-B bind the Abd-B homeobox gene in cells in which it is transcribed, but not in cells in which it is silenced. They bind to the Abd-B transcription unit and downstream regulatory region and thus could regulate both transcriptional elongation and activation. We posit that transcription facilitates cohesin binding, perhaps by unfolding chromatin, and that Nipped-B then regulates gene expression by controlling cohesin dynamics. These mechanisms are likely involved in the etiology of Cornelia de Lange syndrome, in which mutation of one copy of the NIPBL gene encoding the human Nipped-B ortholog causes diverse structural and mental birth defects.

Intra- and interchromosomal interactions have been implicated in a number of genetic phenomena in diverse organisms, suggesting that the higher-order structural organization of chromosomes in the nucleus can have a profound impact on gene regulation. In Drosophila, homologous chromosomes remain paired in somatic tissues, allowing for trans interactions between genes and regulatory elements on the two homologs. One consequence of homolog pairing is the phenomenon of transvection, in which regulatory elements on one homolog can affect the expression of a gene in trans. We report a new instance of transvection at the Drosophila apterous (ap) locus. Two different insertions of boundary elements in the ap regulatory region were identified. The boundaries are inserted between the ap wing enhancer and the ap promoter and have highly penetrant wing defects typical of mutants in ap. When crossed to an ap promoter deletion, both boundary inserts exhibit the interallelic complementation characteristic of transvection. To confirm that transvection occurs at ap, we generated a deletion of the ap wing enhancer by FRT-mediated recombination. When the wing-enhancer deletion is crossed to the ap promoter deletion, strong transvection is observed. Interestingly, the two boundary elements, which are inserted approximately 10 kb apart, fail to block enhancer action when they are present in trans to one another. We demonstrate that this is unlikely to be due to insulator bypass. The transvection effects described here may provide insight into the role that boundary element pairing plays in enhancer blocking both in cis and in trans.

The organization of chromatin affects all aspects of nuclear DNA metabolism in eukaryotes. H3.3 is an evolutionarily conserved histone variant and a key substrate for replication-independent chromatin assembly. Elimination of chromatin remodeling factor CHD1 in Drosophila embryos abolishes incorporation of H3.3 into the male pronucleus, renders the paternal genome unable to participate in zygotic mitoses, and leads to the development of haploid embryos. Furthermore, CHD1, but not ISWI, interacts with HIRA in cytoplasmic extracts. Our findings establish CHD1 as a major factor in replacement histone metabolism in the nucleus and reveal a critical role for CHD1 in the earliest developmental instances of genome-scale, replication-independent nucleosome assembly. Furthermore, our results point to the general requirement of adenosine triphosphate (ATP)-utilizing motor proteins for histone deposition in vivo.

The Drosophila gypsy insulator contains binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein. Enhancer and silencer blocking require Su(Hw) recruitment of Mod(mdg4)-67.2, a BTB/POZ domain protein that interacts with Su(Hw) through a carboxyl-terminal acidic domain. Here we conducted mutational analyses of the Mod(mdg4)-67.2 BTB domain. We demonstrate that this domain is essential for insulator function, in part through direction of protein dimerization. Our studies revealed the presence of a second domain (DD) that contributes to Mod(mdg4)-67.2 dimerization when the function of the BTB domain is compromised. Additionally, we demonstrate that mutations in amino acids of the charged pocket in the BTB domain that retain dimerization of the mutated protein cause a loss of insulator function. In these cases, the mutant proteins failed to localize to chromosomes, suggesting a role for the BTB domain in chromosome association. Interestingly, replacement of the Mod(mdg4)-67.2 BTB domain with the GAF BTB domain produced a nonfunctional protein. Taken together, these data suggest that the Mod(mdg4)-67.2 BTB domain confers novel activities to gypsy insulator function.

Polycomb group complexes, which are known to regulate homeotic genes, have now been found to control hundreds of other genes in mammals and insects. First believed to progressively assemble and package chromatin, they are now thought to be localized, but induce a methylation mark on histone H3 over a broad chromatin domain. Recent progress has changed our view of how these complexes are recruited, and how they affect chromatin and repress gene activity. Polycomb complexes function as global enforcers of epigenetically repressed states, balanced by an antagonistic state that is mediated by Trithorax. These epigenetic states must be reprogrammed when cells become committed to differentiation.

Polycomb group proteins are transcriptional repressors that control many developmental genes. The Polycomb group protein Enhancer of Zeste has been shown in vitro to methylate specifically lysine 27 and lysine 9 of histone H3 but the role of this modification in Polycomb silencing is unknown. We show that H3 trimethylated at lysine 27 is found on the entire Ubx gene silenced by Polycomb. However, Enhancer of Zeste and other Polycomb group proteins stay primarily localized at their response elements, which appear to be the least methylated parts of the silenced gene. Our results suggest that, contrary to the prevailing view, the Polycomb group proteins and methyltransferase complexes are recruited to the Polycomb response elements independently of histone methylation and then loop over to scan the entire region, methylating all accessible nucleosomes. We propose that the Polycomb chromodomain is required for the looping mechanism that spreads methylation over a broad domain, which in turn is required for the stability of the Polycomb group protein complex. Both the spread of methylation from the Polycomb response elements, and the silencing effect can be blocked by the gypsy insulator.

Polycomb group (PcG) complexes are multiprotein assemblages that bind to chromatin and establish chromatin states leading to epigenetic silencing. PcG proteins regulate homeotic genes in flies and vertebrates, but little is known about other PcG targets and the role of the PcG in development, differentiation and disease. Here, we determined the distribution of the PcG proteins PC, E(Z) and PSC and of trimethylation of histone H3 Lys27 (me3K27) in the D. melanogaster genome. At more than 200 PcG target genes, binding sites for the three PcG proteins colocalize to presumptive Polycomb response elements (PREs). In contrast, H3 me3K27 forms broad domains including the entire transcription unit and regulatory regions. PcG targets are highly enriched in genes encoding transcription factors, but they also include genes coding for receptors, signaling proteins, morphogens and regulators representing all major developmental pathways.

Chromosome organization inside the nucleus is not random but rather is determined by a variety of factors, including interactions between chromosomes and nuclear components such as the nuclear envelope or nuclear matrix. Such interactions may be critical for proper nuclear organization, chromosome partitioning during cell division, and gene regulation. An important, but poorly documented subset, includes interactions between specific chromosomal regions. Interactions of this type are thought to be involved in long-range promoter regulation by distant enhancers or locus control regions and may underlie phenomena such as transvection. Here, we used an in vivo microscopy assay based on Lac Repressor/operator recognition to show that Mcp, a polycomb response element from the Drosophila bithorax complex, is able to mediate physical interaction between remote chromosomal regions. These interactions are tissue specific, can take place between multiple Mcp elements, and seem to be stable once established. We speculate that this ability to interact may be part of the mechanism through which Mcp mediates its regulatory function in the bithorax complex.

Transcriptional silencing in budding yeast and fruit fly is mediated by fundamentally unrelated proteins that assemble very different chromatin structures. Surprisingly, the repressive mechanisms evolved from these very different materials have similar features, including an epigenetic mode of inheritance and a block to transcription based on interference with the assembly or function of the promoter complex rather than with the binding of gene-specific activators.

Chromatin cross-linking is widely used for mapping the distribution of chromosomal proteins by immunoprecipitation, but our knowledge of the physical properties of chromatin complexes remains rudimentary. Density gradients have been long used to separate fragments of cross-linked chromatin with their bound proteins from free protein or free DNA. We find that the association of DNA fragments with very-high-molecular-weight protein complexes shifts their buoyant density to values much lower then that of bulk chromatin. We show that in a CsCl gradient, Polycomb response elements, promoters of active genes, and insulator or boundary elements are found at buoyant densities similar to those of free protein and are depleted from the bulk chromatin fractions. In these regions, the low density is associated with the presence of large protein complexes and with high sensitivity to sonication. Our results suggest that separation of different chromatin regions according to their buoyant density may bias chromatin immunoprecipitation results. Density centrifugation of cross-linked chromatin may provide a simple approach to investigate the properties of large chromatin complexes in vivo.

Two papers in this issue of Cell describe two roles of poly(ADP-ribose) polymerase (PARP) in modulating chromatin structure: as a structural component replacing linker histone (Kim et al., 2004) and as a constituent of a corepressor complex poised to dismiss repression upon receipt of an activating signal (Ju et al., 2004).

Polycomb (PcG) complexes maintain the silent state of target genes. The mechanism of silencing is not known but has been inferred to involve chromatin packaging to block the access of transcription factors. We have studied the effect of PcG silencing on the hsp26 heat shock promoter. While silencing does decrease the accessibility of some restriction enzyme sites to some extent, it does not prevent the binding of TBP, RNA polymerase, or the heat shock factor to the hsp26 promoter, as shown by chromatin immunoprecipitation. However, we find that in the repressed state, the RNA polymerase cannot initiate transcription. We conclude that, rather than altering chromatin structure to block accessibility, PcG silencing in this construct targets directly the activity of the transcriptional machinery at the promoter.

So far, two thioredoxin proteins, DHD and Trx-2, have been biochemically characterized in Drosophila melanogaster. Here, with the cloning and characterization of TrxT we describe an additional thioredoxin with testis-specific expression. TrxT and dhd are arranged as a gene pair, transcribed in opposite directions and sharing a 471 bp regulatory region. We show that this regulatory region is sufficient for correct expression of the two genes. This gene pair makes a good model for unraveling how closely spaced promoters are differentially regulated by a short common control region. Both TrxT and DHD proteins are localized within the nuclei in testes and ovaries, respectively. Use of a transgenic construct expressing TrxT fused to Enhanced Yellow Fluorescent Protein reveals a clear association of TrxT with the Y chromosome lampbrush loops ks-1 and kl-5 in primary spermatocytes. The association is lost in the absence of the Y chromosome. Our results suggest that nuclear thioredoxins may have regulatory functions in the germline.

The best characterized chromatin insulator in Drosophila is the Suppressor of Hairy wing binding region contained within the gypsy retrotransposon. Although cellular functions have been suggested, no role has been found yet for the multitude of endogenous Suppressor of Hairy wing binding sites. Here we show that two Suppressor of Hairy wing binding sites in the intergenic region between the yellow gene and the Achaete-scute gene complex form a functional insulator. Genetic analysis shows that at least two proteins, Suppressor of Hairy wing and Modifier of MDG4, required for the activity of this insulator, are involved in the transcriptional regulation of Achaete-scute.

Polycomb complexes assemble at their target sites and silence neighboring genes when these are not actively transcribed. The action of these complexes and of Trithorax complexes bound to the Polycomb Response Element establish alternative silent or derepressed states that are remembered through cell division and maintained for the rest of development. Recent results that may help explain the properties of these states are reviewed.

The morphological characteristics of intercalary heterochromatin (IH) are compared with those of other types of silenced chromatin in the Drosophila melanogaster genome: pericentric heterochromatin (PH) and regions subject to position effect variegation (PEV). We conclude that IH regions in polytene chromosomes are binding sites of silencing complexes such as PcG complexes and of SuUR protein. Binding of these proteins results in the appearance of condensed chromatin and late replication of DNA, which in turn may result in DNA underreplication. IH and PH as well as regions subject to PEV have in common the condensed chromatin appearance, the localization of specific proteins, late replication, underreplication in polytene chromosomes, and ectopic pairing.

Salivary gland polytene chromosomes of Drosophila melanogaster have a reproducible set of intercalary heterochromatin (IH) sites, characterized by late DNA replication, underreplicated DNA, breaks and frequent ectopic contacts. The SuUR mutation has been shown to suppress underreplication, and wild-type SuUR protein is found at late-replicating IH sites and in pericentric heterochromatin. Here we show that the SuUR gene influences all four IH features. The SuUR mutation leads to earlier completion of DNA replication. Using transgenic strains with two, four or six additional SuUR(+) doses (4-8xSuUR(+)) we show that wild-type SuUR is an enhancer of DNA underreplication, causing many late-replicating sites to become underreplicated. We map the underreplication sites and show that their number increases from 58 in normal strains (2xSuUR(+)) to 161 in 4-8xSuUR(+) strains. In one of these new sites (1AB) DNA polytenization decreases from 100% in the wild type to 51%-85% in the 4xSuUR (+) strain. In the 4xSuUR(+) strain, 60% of the weak points coincide with the localization of Polycomb group (PcG) proteins. At the IH region 89E1-4 (the Bithorax complex), a typical underreplication site, the degree of underreplication increases with four doses of SuUR(+) but the extent of the underreplicated region is the same as in wild type and corresponds to the region containing PcG binding sites. We conclude that the polytene chromosome regions known as IH are binding sites for SuUR protein and in many cases PcG silencing proteins. We propose that these stable silenced regions are late replicated and, in the presence of SuUR protein, become underreplicated.

Splicing is an efficient and precise mechanism that removes noncoding regions from a single primary RNA transcript. Cutting and rejoining of the segments occurs on nascent RNA. Trans-splicing between small specialized RNAs and a primary transcript has been known in some organisms but recent papers show that trans-splicing between two RNA molecules containing different coding regions is the normal mode in a Drosophila gene. The mod(mdg4) gene produces 26 different mRNAs encoding as many protein isoforms. The differences lie in alternative 3' exons encoded by different transcriptional units and spliced to the 5' common region by a surprising trans-splicing mechanism., (Copyright 2002 Wiley-Periodicals, Inc.)

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.

Gene expression in C. elegans germline cells is subject to strict controls. A set of MES proteins, including SET domain proteins and two homologs of Polycomb group proteins, establish an epigenetically transmitted silenced state that affects X chromosome gene expression.

Polycomb group (PcG) and Trithorax (TRX) complexes assemble at Polycomb response elements (PREs) and maintain respectively the repressed and active state of homeotic genes. Although PcG and TRX complexes are distinct, their binding to some PRE fragments in vitro depends on GAGA motifs. GAGA factor immunoprecipitates with both complexes. In presence of a PRE, TRX stimulates expression and prevents the return of repression at later stages. When TRX levels are reduced, repression is re-established in inappropriate regions of imaginal discs, suggesting that TRX insufficiency impairs the epigenetic memory of the active state. Targeting a GAL-TRX fusion shows that TRX is a coactivator that stimulates expression of an active gene but cannot initiate expression by itself. Targeting a histone acetylase to a PRE does not affect embryonic silencing but causes a loss of memory in imaginal discs, suggesting that deacetylation is required to establish the memory of the repressed state.

In many late-replicating euchromatic regions of salivary gland polytene chromosomes, DNA is underrepresented. A mutation in the SuUR gene suppresses underreplication and leads to normal levels of DNA polytenization in these regions. We identified the SuUR gene and determined its structure. In the SuUR mutant stock a 6-kb insertion was found in the fourth exon of the gene. A single SuUR transcript is present at all stages of Drosophila development and is most abundant in adult females and embryos. The SuUR gene encodes a protein of 962 amino acids whose putative sequence is similar to the N-terminal part of SNF2/SWI2 proteins. Staining of salivary gland polytene chromosomes with antibodies directed against the SuUR protein shows that the protein is localized mainly in late-replicating regions and in regions of intercalary and pericentric heterochromatin.

The Polycomb Response Element (PRE) is the nucleation site for the Polycomb silencing complexes. The sequences responsible for the recruitment of the components of the Polycomb complex are not well understood. A comparison of the bxd PRE sequences from several different Drosophila species shows that some changes have occurred during phylogeny but large blocks of sequence are conserved after a divergence of some 60 million years. We compare the PRE sequences, the sites of some known PRE binding proteins, the conservation of DNasel hypersensitive sites and relate them to the sequence of the Ultrabithorax promoter which these PREs regulate.

Chromosome-specific gene regulation is known thus far only as a mechanism to equalize the transcriptional activity of the single male X chromosome with that of the two female X chromosomes. In Drosophila melanogaster, a complex including the five Male-Specific Lethal (MSL) proteins, "paints" the male X chromosome, mediating its hypertranscription. Here, with the molecular cloning of Painting of fourth (Pof), we describe a previously uncharacterized gene encoding a chromosome-specific protein in Drosophila. Unlike the MSL proteins, POF paints an autosome, the fourth chromosome of Drosophila melanogaster. Chromosome translocation analysis shows that the binding depends on an initiation site in the proximal region of chromosome 4 and spreads in cis to involve the entire chromosome. The spreading depends on sequences or structures specific to chromosome 4 and cannot extend to parts of other chromosomes translocated to the fourth. Spreading can also occur in trans to a paired homologue that lacks the initiation region. In the related species Drosophila busckii, POF paints the entire X chromosome exclusively in males, suggesting relationships between the fourth chromosome and the X and between POF complexes and dosage-compensation complexes.

Chromatin insulators are regulatory elements that block the action of transcriptional enhancers when interposed between enhancer and promoter. The Drosophila Suppressor of Hairy wing [Su(Hw)] protein binds the Su(Hw) insulator and prevents enhancer-promoter interaction by a mechanism that is not understood. We show that when two copies of the Su(Hw) insulator element, instead of a single one, are inserted between enhancer and promoter, insulator activity is neutralized and the enhancer-promoter interaction may instead be facilitated. This paradoxical phenomenon could be explained by interactions between protein complexes bound at the insulators.

Polytene chromosomes of salivary glands of various Drosophila melanogaster strains containing two doses of the normal Su(UR)ES allele have a constant set of intercalary heterochromatin (IHC) sites. Their DNA is underreplicated, which leads to breaks and ectopic contacts emerging at a certain rate. Almost no underreplication, breaks, or ectopic conjugation are present in mutants lacking the normal Su(UR)ES gene product. It could be expected that an increase in the number of the Su(UR)ES+ gene doses would, in turn, drastically increase ectopic conjugation and breakage. To test this hypothesis, a strain of D. melanogaster was obtained with two additional doses of Su(UR)ES+ introduced into its genome. The flies with four gene doses exhibited a considerable increase in ectopic conjugation: both the proportion of regions participating in conjugation and the number of chromosomes with numerous contact nodes were increased. As a result, chromosomes that were straight and well-stretched in homozygotes for the mutation in Su(UR)ES became twisted and wound and contained many loops or nodes. Many chromosomes were wound too tightly for cytological analysis. Four doses of Su(UR)ES+ considerably increased the number of weak "points." For example, the 2R chromosome has only 3 weak points in strains with two doses of Su(UR)ES+ and as many as 22 weak points in the strain with four doses. In the transgenic strain, the frequency of breaks in previously known weak points increased, and new breaks appeared in 19 additional sites. All new break points appeared in the regions that were earlier described as regions of late replication in the S phase.

Polycomb response elements (PREs) are regulatory sites that mediate the silencing of homeotic and other genes. The bxd PRE region from the Drosophila Ultrabithorax gene can be subdivided into subfragments of 100 to 200 bp that retain different degrees of PRE activity in vivo. In vitro, embryonic nuclear extracts form complexes containing Polycomb group (PcG) proteins with these fragments. PcG binding to some fragments is dependent on consensus sequences for the GAGA factor. Other fragments lack GAGA binding sites but can still bind PcG complexes in vitro. We show that the GAGA factor is a component of at least some types of PcG complexes and may participate in the assembly of PcG complexes at PREs.

In the studies reported here, we have examined the properties of the Mcp element from the Drosophila melanogaster bithorax complex (BX-C). We have found that sequences from the Mcp region of BX-C have properties characteristic of Polycomb response elements (PREs), and that they silence adjacent reporters by a mechanism that requires trans-interactions between two copies of the transgene. However, Mcp trans-regulatory interactions have several novel features. In contrast to classical transvection, homolog pairing does not seem to be required. Thus, trans-regulatory interactions can be observed not only between Mcp transgenes inserted at the same site, but also between Mcp transgenes inserted at distant sites on the same chromosomal arm, or even on different arms. Trans-regulation can even be observed between transgenes inserted on different chromosomes. A small 800-bp Mcp sequence is sufficient to mediate these long-distance trans-regulatory interactions. This small fragment has little silencing activity on its own and must be combined with other Polycomb-Group-responsive elements to function as a "pairing-sensitive" silencer. Finally, this pairing element can also mediate long-distance interactions between enhancers and promoters, activating mini-white expression.

Purpose: Multiple studies have emphasized the higher yield of detection of metastatic neuroblastoma (MNb) by bone marrow biopsy (BMB) than by bone marrow aspiration (BMA). Because the need for BMA has been questioned, the yield of both procedures was investigated at diagnosis and during the course of disease., Methods: For morphologic and immunohistochemical detection of MNb, 289 specimens obtained by BMA and BMB from 57 children with neuroblastoma were reviewed., Results: In 34% of cases, MNb was present in both the aspirate and biopsy specimen. MNb was present in only the biopsy specimen in 8% and in only the aspirate in 6%. In 52%, neither BMA nor BMB detected MNb. In 15 of 18 cases in which MNb was present in the aspirate only, protein gene product 9.5 (PGP) stain was performed on the biopsy specimen. In one case, this helped to identify MNb that was not evident by routine hematoxylin and eosin stain. Of the 24 cases in which only the BMB was positive, 3 were identified only by means of PGP stain., Conclusions: Even with the additional use of immunohistochemistry, both BMA and BMB should be performed to have the highest yield of detection of MNb in bone marrow.