An imprinting element from the mouse H19 locus functions as a silencer in Drosophila.

Genomic imprinting as originally described in Sciara is displayed by many organisms. In mammals, X-inactivation and the parent-of-origin-specific silencing of imprinted genes are examples of this phenomenon. A heritable chromatin structural modification may be the critical mechanism in such instances of chromosome condensation and preferential gene inactivation. H19 is an imprinted gene in which the repressed paternal allele is hypermethylated and the compacted chromatin is relatively resistant to digestion by nucleases. In order to uncover underlying conserved epigenetic mechanisms we have introduced a mouse H19 transgene into Drosophila. We show here that a 1.2-kb H19 upstream sequence functions in cis as a parent-of-origin independent silencing element in Drosophila. Strikingly, this cis-acting element is located within an upstream region that is necessary for H19 imprinting in mice. These results suggest involvement of an evolutionary conserved mechanism in both genes silencing in Drosophila and imprinting in mice.

Long-term use of HU210 adversely affects spermatogenesis in rats by modulating the endocannabinoid system.

Recent societal acceptance of cannabinoids as recreational and therapeutic drugs has posed a potential hazard to male reproductive health. Mammals have a highly sophisticated endogenous cannabinoid (ECS) system that regulates male (and female) reproduction and exo-cannabinoids may influence it adversely. Therefore it is imperative to determine their effects on male reproduction so that men can make informed choices as to their use. Here, an animal model was used to administer HU210, a synthetic analogue of Δ9-tetrahydrocannabinol (THC) and potent cannabinoid receptor (CB) agonist to determine its effects on reproductive organ weights, spermatogenesis, testicular histology and sperm motility. Its effects on the physiological endocannabinoid system were also investigated. Spermatogenesis was markedly impaired with reductions in total sperm count after 2 weeks of exposure. Spermatogenic efficiency was depleted, and Sertoli cell number decreased as exposure time increased with seminiferous tubules showing germ cell depletion developing into atrophy in some cases. Sperm motility was also adversely affected with marked reductions from 2 weeks on. HU210 also acted on the sperm's endocannabinoid system. Long-term use of exo-cannabinoids has adverse effects on both spermatogenesis and sperm function. These findings highlight the urgent need for studies evaluating the fertility potential of male recreational drug users. HU210, a selective agonist for CB1 and CB2 cannabinoid receptors impairs spermatogenesis and sperm motility and deregulates the endocannabinoid system.

Mechanisms of heritable gene repression during development of Drosophila.

During development, patterns of differential gene expression, defining determined states of cells, need to be maintained over many cell generations. In Drosophila, genetic and molecular analyses led to the discovery of a set of proteins which seem to exert such a memory function by using epigenetic mechanisms. Recent experiments demonstrate that, in particular, the heritable inactivation of regulatory genes relies on stable changes in the higher-order constitution of chromatin.

Chromo-domain proteins: linking chromatin structure to epigenetic regulation.

Chromo-domain proteins appear to be a central component in the epigenetic regulation of heterochromatin function and euchromatic gene expression. The recent discovery of a variety of interacting partners of chromo-domain proteins is yielding new molecular insights into epigenetic regulatory processes acting at the level of higher order chromatin structure.

Epigenetic inheritance of active chromatin after removal of the main transactivator.

The Drosophila Polycomb and trithorax group proteins act through chromosomal elements such as Fab-7 to maintain repressed or active gene expression, respectively. A Fab-7 element is switched from a silenced to a mitotically heritable active state by an embryonic pulse of transcription. Here, histone H4 hyperacetylation was found to be associated with Fab-7 after activation, suggesting that H4 hyperacetylation may be a heritable epigenetic tag of the activated element. Activated Fab-7 enables transcription of a gene even after withdrawal of the primary transcription factor. This feature may allow epigenetic maintenance of active states of developmental genes after decay of their early embryonic regulators.

Chromatin multiprotein complexes involved in the maintenance of transcription patterns.

In Drosophila, the maintenance of active and inactive patterns of gene expression during development involves the activity of two genetically complex systems. Molecular analysis of the components, apparently acting in large multiprotein complexes, has allowed a substantial advancement in our understanding of the role of chromatin higher order structures in gene regulation and nuclear organization. The Polycomb-group factors induce heterochromatin-like structures on genes that need to be stably and heritably inactivated. The role of the trithorax-group factors is to counteract these repressed chromatin domains and thus to render the genes accessible to activating factors.

Imprinting a determined state into the chromatin of Drosophila.

The Polycomb gene of Drosophila melanogaster is a member of a class of genes involved in the clonal transmission of the repressed state of bomeotic regulatory genes through development. Genetic evidence, and the finding of a molecular similarity between the Polycomb protein and a heterochromatin-associated protein of Drosophila, suggest that this mechanism of repression might be imprinted in the structure of the chromatin, rather than being sustained through the action of diffusible regulatory factors.

Epigenetic regulation in Drosophila.

Epigenetic regulation of gene transcription relies on molecular marks like DNA methylation or histone modifications. Here we review recent advances in our understanding of epigenetic regulation in the fruit fly Drosophila melanogaster. In the past, DNA methylation research has primarily utilized mammalian model systems. However, several recent landmark discoveries have been made in other organisms. For example, the interaction between DNA methylation and histone methylation was first described in the filamentous fungus Neurospora crassa. Another example is provided by the interaction between epigenetic modifications and the RNA interference (RNAi) machinery that was first reported in the fission yeast Schizosaccharomyces pombe. Another organism with great experimental power is the fruit fly Drosophila. Epigenetic regulation by chromatin has been extensively analyzed in the fly and several of the key components have been discovered in this organism. In this chapter, we will focus on three aspects that represent the complexity of epigenetic gene regulation. (1) We will discuss the available data about the DNA methylation system, (2) we will illuminate the interaction between DNA methylation and chromatin regulation, and (3) we will provide an overview over the Polycomb system of epigenetic chromatin modifiers that has proved to be an important paradigm for a chromatin system regulating epigenetic programming.

The polycomb group protein complex of Drosophila melanogaster has different compositions at different target genes.

In Drosophila the Polycomb group genes are required for the long-term maintenance of the repressed state of many developmental regulatory genes. Their gene products are thought to function in a common multimeric complex that associates with Polycomb group response elements (PREs) in target genes and regulates higher-order chromatin structure. We show that the chromodomain of Polycomb is necessary for protein-protein interactions within a Polycomb-Polyhomeotic complex. In addition, Posterior Sex Combs protein coimmunoprecipitates Polycomb and Polyhomeotic, indicating that they are members of a common multimeric protein complex. Immunoprecipitation experiments using in vivo cross-linked chromatin indicate that these three Polycomb group proteins are associated with identical regulatory elements of the selector gene engrailed in tissue culture cells. Polycomb, Polyhomeotic, and Posterior Sex Combs are, however, differentially distributed on regulatory sequences of the engrailed-related gene invected. This suggests that there may be multiple different Polycomb group protein complexes which function at different target sites. Furthermore, Polyhomeotic and Posterior Sex Combs are also associated with expressed genes. Polyhomeotic and Posterior Sex Combs may participate in a more general transcriptional mechanism that causes modulated gene repression, whereas the inclusion of Polycomb protein in the complex at PREs leads to stable silencing.

The Drosophila polycomb protein interacts with nucleosomal core particles In vitro via its repression domain.

The proteins of the Polycomb group (PcG) are required for maintaining regulator genes, such as the homeotic selectors, stably and heritably repressed in appropriate developmental domains. It has been suggested that PcG proteins silence genes by creating higher-order chromatin structures at their chromosomal targets, thus preventing the interaction of components of the transcriptional machinery with their cis-regulatory elements. An unresolved issue is how higher order-structures are anchored at the chromatin base, the nucleosomal fiber. Here we show a direct biochemical interaction of a PcG protein-the Polycomb (PC) protein-with nucleosomal core particles in vitro. The main nucleosome-binding domain coincides with a region in the C-terminal part of PC previously identified as the repression domain. Our results suggest that PC, by binding to the core particle, recruits other PcG proteins to chromatin. This interaction could provide a key step in the establishment or regulation of higher-order chromatin structures.

A single ancestral gene of the human LIM domain oncogene family LMO in Drosophila: characterization of the Drosophila Dlmo gene.

Members of the human TTG/RBTN family, now renamed 'LMO' for LIM-only proteins, encode proteins with two tandem copies of a LIM motif. There are three members of this family, two have been isolated at the sites of chromosomal translocations in T-cell leukaemia. The function of the LIM motifs is at present unknown. We found that the LMO-2 gene is highly conserved between mammals, Drosophila and yeast. As a first step to obtain a model system for studying the function of the LIM motifs we have isolated the Drosophila homologue Dlmo. In contrast to mammals Drosophila appears to have only one lmo gene. A 2087 bp cDNA clone was isolated from a larval cDNA library, encoding a protein of 266 amino acids. A second transcript with an alternative 5' end was identified in RNA from embryos. The Drosophila lmo protein consists of two tandem copies of the conserved LIM domain characteristic of the human LMO family and an extended amino and carboxy terminus, which is not present in the human proteins. The amino acid sequence similarity with human LMO-1 and LMO-2 in LIM 1 is 79% and 69% and in LIM-2 90% and 60%, respectively. In addition a short stretch of 25 nucleotides with a homology of 83% between LMO-2 and Dlmo is found in the 3' UTR. Dlmo, like LMO-1, has an intron after the second LIM encoding region, which is not present in LMO-2. It is expressed maternally and at a high level in early embryogenesis as well as in adults. Interestingly we observed that the Dlmo protein is immunologically related to LMO-2 and can be detected by immunohistochemistry in early cellular blastoderm embryos. The gene was localised to a genetically well characterized region (17C on the X chromosome) opening the way for identification of mutations.

Epigenetics: unforeseen regulators in cancer.

The past several years have seen a tremendous advance in the understanding of the basic mechanisms of epigenetic regulation. A large number of studies have not only linked epigenetics with cell cycle regulation but also partially unravelled how epigenetics may regulate gene expression. The aim of this review is to provide an overview of the latest findings and current ideas on epigenetics with a focus on emphasizing the emerging influence epigenetics has on the onset and progression of cancer.

The Polycomb gene is differentially regulated during oogenesis and embryogenesis of Drosophila melanogaster.

Homeotic genes are responsible for determining the identity of body structures along the anterior-posterior axis. In Drosophila the early patterning system defines the differential expression pattern of the homeotic genes. In laterstages the Polycomb-group (Pc-G) genes were found to keep homeotic genes stably repressed in those domains where they have to be inactive. At the molecular level the Pc-G is supposed to exert its repressory role by influencing the higher order structure of chromatin. Here we show that during oogenesis the Polycomb (Pc) protein is localized in the polytene nuclei of the nurse cells. In addition, in late stages we observe overlapping gradients of expression in the somatic follicle cells, suggesting also an important function of Pc on the determinants involved in egg formation. During embryogenesis Pc is found in all tissues, though in later stages it preferentially accumulates in the CNS. Interestingly, we have identified a feedback-type regulation: the Ultrabithorax gene, a homeotic target gene of Pc, in its own domain of expression is down-regulating Pc.

Heritable chromatin states induced by the Polycomb and trithorax group genes.

In Drosophila the Polycomb group (PcG) and trithorax group (trxG) genes are required to maintain differential expression patterns of many important developmental regulatory genes. The PcG is responsible for heritable silencing throughout development. At target genes PcG response elements (PREs) attract PcG protein complexes and induce the formation of higher-order chromatin structures. We have mapped the distribution of Polycomb and other PcG members at various target genes by using an improved formaldehyde cross-linking and chromatin immunoprecipitation technique. We find that Polycomb spreads locally from PREs over several kilobases, thereby probably stabilizing the silencing complexes. Members of the trxG co-localize at PREs. GAGA factor was found to be constitutively bound to PREs independently of gene activity. PREs associated with active genes appear to have increased amounts of bound GAGA. We have developed a system capable of switching a PRE between the on/off modes. PREs and trxG-regulated elements are common chromosomal elements through which the proteins of the PcG/trxG exert their maintenance function on adjacent chromatin structures.

Imprinting and gene silencing in mice and Drosophila.

H19 and Igf2 are located within a large imprinting domain that confers monoallelic silencing of parental alleles. The silent paternal allele of H19 is hypermethylated and relatively resistant to nucleases. Using a 130 kb yeast artificial chromosome clone, appropriate imprinting of both H19 and Igf2 was observed at single insert loci in transgenic mice. Imprinting was also observed for H19-lacZ transgenes containing 4 kb of upstream sequence, but only at multicopy loci. The H19 RNA is therefore not essential for imprinting. When the H19-lacZ transgene was introduced into Drosophila, a 1.2 kb region was identified within the 4 kb upstream flank that functioned as a bi-directional silencer. This cis element is located within a region that is apparently necessary for imprinting in mice. These studies suggest an evolutionarily conserved mechanism for gene silencing in Drosophila and imprinting in mice. We propose a new model for imprinting of H19 and Igf2 in mice in which silencing of H19 is the default state, and activation of the maternal allele requires a specific activator element.

[The cloning and analysis of a single gene in Drosophila homologic with human oncogene TTG].

The human TTG/RBTN family is an oncogene family. There are three members in this family: TTG-1/RBTN-1, TTG-2/RBTN-2 and TTG-3/RBTN-3. Two of them, TTG-1/RBTN-1 and TTG-2/RBTN-2 have been isolated at the sites of chromosomal translocations in T-cell leukaemia. This gene family encodes cysteine-rich proteins with two tandem copies of a LIM motif. The function of the LIM motif is unknown. We found that the TTG-2 gene is highly conserved among mammals; Drosophila and yeast. As a first step to obtain a model system for studying the function of the LIM motifs, we isolated the Drosophila homologue dttg. In contrast to human, Drosophila appeared to have only one ttg/rbtn gene. A 2087bp cDNA clone was isolated, encoding a protein of 266 amino acids. A second transcript with an alternative 5' end was identified in RNA from embryos. The Drosophila ttg protein consisted of two tandem copies of the conserved LIM domain characteristic of the human TTG/RBTN family. The amino acid sequence similarity with human TTG-1 and TTG-2 is 79% and 62%, respectively. The dttg, like TTG-1, have an intron in the second LIM encoding region, which is not present in TTG-2.

Drosophila Polycomb-group regulated chromatin inhibits the accessibility of a trans-activator to its target DNA.

The genes of the Polycomb-group (Pc-G) are responsible for maintaining the inactive expression state of homeotic genes. They act through specific cis-regulatory DNA elements termed PREs (Pc-G Response Elements). Multimeric complexes containing the Pc-G proteins are thought to induce heterochromatin-like structures, which stably and heritably inactivate transcription. We have tested the functional role of the FAB fragment, a PRE of the bithorax complex. We find that this element behaves as an orientation dependent silencer, capable of inducing mosaic gene expression on neighboring genes. Transgenic fly lines were constructed containing a PRE adjacent to a reporter gene inducible by the yeast GAL4 trans-activator. The competition between the activator and Pc-G-containing chromatin was visualized on polytene chromosomes using immunocytochemistry. The Pc-G protein Polycomb and GAL4 have mutually exclusive binding patterns, supporting the notion that Pc-G-induced chromatin structures can prevent activators from binding to their target sequences. However, this antagonistic function can be overcome by high doses of GAL4, even in the absence of DNA replication.

Spreading the silence: epigenetic transcriptional regulation during Drosophila development.

In early Drosophila development a complex cascade of diffusible transcription factors generates an intricate expression pattern of developmental regulators such as the homeotic genes. The mechanism which subsequently maintains the pattern during the rest of development is mainly using epigenetic features for its function. Evidence comes from the analysis of the Polycomb-group (Pc-G), a class of genes which is responsible for maintaining the inactive state of expression. The Pc-G was found to share many parallels to genes involved in heterochromatin formation. Different members of the Pc-G interact in large multiprotein complexes, which apparently can cover and inactivate large chromosomal domains. Specific DNA elements have been identified that are used by the Pc-G proteins to nucleate these specialized domains of silent chromatin. Thus, the Pc-G proteins appear to permanently inactivate genes by generating heterochromatin-like structures which could then be inherited by the daughter cells in an epigenetic manner. Heritable gene silencing is an important but little understood mechanism in pattern formation. Phenomenologically related effects have been observed in many organisms. These range from the transcriptional silencing of the inactive mating type loci in yeast to parental imprinting phenomena and X-chromosome inactivation in mammals. Analysis of these functions in Drosophila provides an excellent model system for studying the molecular basis of such epigenetic mechanisms that use higher order chromatin structures for transcriptional repression.