Epigenetic instability caused by absence of CIZ1 drives transformation during quiescence cycles.

BACKGROUND: Cip1-interacting zinc finger protein 1 (CIZ1) forms RNA-dependent protein assemblies that stabilise epigenetic state, notable at the inactive X chromosome in females. CIZ1 has been linked with a range of human cancers and in mice genetic deletion of CIZ1 manifests as hyperproliferative lymphoid lineages in females. This suggests that its role in maintenance of epigenetic stability is linked with disease. RESULTS: Here, we show that male and female CIZ1-null primary murine fibroblasts have reduced H4K20me1 and that this compromises nuclear condensation on entry to quiescence. Global transcriptional repression remains intact in condensation-deficient CIZ1-null cells; however, a subset of genes linked with chromatin condensation and homology-directed DNA repair are perturbed. Failure to condense is phenotypically mimicked by manipulation of the H4K20me1 methyltransferase, SET8, in WT cells and partially reverted in CIZ1-null cells upon re-expression of CIZ1. Crucially, during exit from quiescence, nuclear decondensation remains active, so that repeated entry and exit cycles give rise to expanded nuclei susceptible to mechanical stress, DNA damage checkpoint activation, and downstream emergence of transformed proliferative colonies. CONCLUSIONS: Our results demonstrate a role for CIZ1 in chromatin condensation on entry to quiescence and explore the consequences of this defect in CIZ1-null cells. Together, the data show that CIZ1's protection of the epigenome guards against genome instability during quiescence cycles. This identifies loss of CIZ1 as a potentially devastating vulnerability in cells that undergo cycles of quiescence entry and exit.

Orai1 Channel Inhibition Preserves Left Ventricular Systolic Function and Normal Ca2+ Handling After Pressure Overload.

BACKGROUND: Orai1 is a critical ion channel subunit, best recognized as a mediator of store-operated Ca2+ entry (SOCE) in nonexcitable cells. SOCE has recently emerged as a key contributor of cardiac hypertrophy and heart failure but the relevance of Orai1 is still unclear. METHODS: To test the role of these Orai1 channels in the cardiac pathophysiology, a transgenic mouse was generated with cardiomyocyte-specific expression of an ion pore-disruptive Orai1R91W mutant (C-dnO1). Synthetic chemistry and channel screening strategies were used to develop 4-(2,5-dimethoxyphenyl)-N-[(pyridin-4-yl)methyl]aniline (hereafter referred to as JPIII), a small-molecule Orai1 channel inhibitor suitable for in vivo delivery. RESULTS: Adult mice subjected to transverse aortic constriction (TAC) developed cardiac hypertrophy and reduced ventricular function associated with increased Orai1 expression and Orai1-dependent SOCE (assessed by Mn2+ influx). C-dnO1 mice displayed normal cardiac electromechanical function and cellular excitation-contraction coupling despite reduced Orai1-dependent SOCE. Five weeks after TAC, C-dnO1 mice were protected from systolic dysfunction (assessed by preserved left ventricular fractional shortening and ejection fraction) even if increased cardiac mass and prohypertrophic markers induction were observed. This is correlated with a protection from TAC-induced cellular Ca2+ signaling alterations (increased SOCE, decreased [Ca2+]i transients amplitude and decay rate, lower SR Ca2+ load and depressed cellular contractility) and SERCA2a downregulation in ventricular cardiomyocytes from C-dnO1 mice, associated with blunted Pyk2 signaling. There was also less fibrosis in heart sections from C-dnO1 mice after TAC. Moreover, 3 weeks treatment with JPIII following 5 weeks of TAC confirmed the translational relevance of an Orai1 inhibition strategy during hypertrophic insult. CONCLUSIONS: The findings suggest a key role of cardiac Orai1 channels and the potential for Orai1 channel inhibitors as inotropic therapies for maintaining contractility reserve after hypertrophic stress.

Maintenance of epigenetic landscape requires CIZ1 and is corrupted in differentiated fibroblasts in long-term culture.

The inactive X chromosome (Xi) serves as a model for establishment and maintenance of repressed chromatin and the function of polycomb repressive complexes (PRC1/2). Here we show that Xi transiently relocates from the nuclear periphery towards the interior during its replication, in a process dependent on CIZ1. Compromised relocation of Xi in CIZ1-null primary mouse embryonic fibroblasts is accompanied by loss of PRC-mediated H2AK119Ub1 and H3K27me3, increased solubility of PRC2 catalytic subunit EZH2, and genome-wide deregulation of polycomb-regulated genes. Xi position in S phase is also corrupted in cells adapted to long-term culture (WT or CIZ1-null), and also accompanied by specific changes in EZH2 and its targets. The data are consistent with the idea that chromatin relocation during S phase contributes to maintenance of epigenetic landscape in primary cells, and that elevated soluble EZH2 is part of an error-prone mechanism by which modifying enzyme meets template when chromatin relocation is compromised.

Cardiac fibroblast-specific p38α MAP kinase promotes cardiac hypertrophy via a putative paracrine interleukin-6 signaling mechanism.

Recent studies suggest that cardiac fibroblast-specific p38α MAPK contributes to the development of cardiac hypertrophy, but the underlying mechanism is unknown. Our study used a novel fibroblast-specific, tamoxifen-inducible p38α knockout (KO) mouse line to characterize the role of fibroblast p38α in modulating cardiac hypertrophy, and we elucidated the mechanism. Myocardial injury was induced in tamoxifen-treated Cre-positive p38α KO mice or control littermates via chronic infusion of the ß-adrenergic receptor agonist isoproterenol. Cardiac function was assessed by pressure-volume conductance catheter analysis and was evaluated for cardiac hypertrophy at tissue, cellular, and molecular levels. Isoproterenol infusion in control mice promoted overt cardiac hypertrophy and dysfunction (reduced ejection fraction, increased end systolic volume, increased cardiac weight index, increased cardiomyocyte area, increased fibrosis, and up-regulation of myocyte fetal genes and hypertrophy-associated microRNAs). Fibroblast-specific p38α KO mice exhibited marked protection against myocardial injury, with isoproterenol-induced alterations in cardiac function, histology, and molecular markers all being attenuated. In vitro mechanistic studies determined that cardiac fibroblasts responded to damaged myocardium by secreting several paracrine factors known to induce cardiomyocyte hypertrophy, including IL-6, whose secretion was dependent upon p38α activity. In conclusion, cardiac fibroblast p38α contributes to cardiomyocyte hypertrophy and cardiac dysfunction, potentially via a mechanism involving paracrine fibroblast-to-myocyte IL-6 signaling.-Bageghni, S. A., Hemmings, K. E., Zava, N., Denton, C. P., Porter, K. E., Ainscough, J. F. X., Drinkhill, M. J., Turner, N. A. Cardiac fibroblast-specific p38α MAP kinase promotes cardiac hypertrophy via a putative paracrine interleukin-6 signaling mechanism.

The nuclear matrix protein CIZ1 facilitates localization of Xist RNA to the inactive X-chromosome territory.

The nuclear matrix protein Cip1-interacting zinc finger protein 1 (CIZ1) promotes DNA replication in association with cyclins and has been linked to adult and pediatric cancers. Here we show that CIZ1 is highly enriched on the inactive X chromosome (Xi) in mouse and human female cells and is retained by interaction with the RNA-dependent nuclear matrix. CIZ1 is recruited to Xi in response to expression of X inactive-specific transcript (Xist) RNA during the earliest stages of X inactivation in embryonic stem cells and is dependent on the C-terminal nuclear matrix anchor domain of CIZ1 and the E repeats of Xist CIZ1-null mice, although viable, display fully penetrant female-specific lymphoproliferative disorder. Interestingly, in mouse embryonic fibroblast cells derived from CIZ1-null embryos, Xist RNA localization is disrupted, being highly dispersed through the nucleoplasm rather than focal. Focal localization is reinstated following re-expression of CIZ1. Focal localization of Xist RNA is also disrupted in activated B and T cells isolated from CIZ1-null animals, suggesting a possible explanation for female-specific lymphoproliferative disorder. Together, these findings suggest that CIZ1 has an essential role in anchoring Xist to the nuclear matrix in specific somatic lineages.

Cardiomyocyte--specific expression of the nuclear matrix protein, CIZ1, stimulates production of mono-nucleated cells with an extended window of proliferation in the postnatal mouse heart.

Myocardial injury in mammals leads to heart failure through pathological cardiac remodelling that includes hypertrophy, fibrosis and ventricular dilatation. Central to this is inability of the mammalian cardiomyocyte to self-renew due to entering a quiescent state after birth. Modulation of the cardiomyocyte cell-cycle after injury is therefore a target mechanism to limit damage and potentiate repair and regeneration. Here, we show that cardiomyocyte-specific over-expression of the nuclear-matrix--associated DNA replication protein, CIZ1, extends their window of proliferation during cardiac development, delaying onset of terminal differentiation without compromising function. CIZ1-expressing hearts are enlarged, but the cardiomyocytes are smaller with an overall increase in number, correlating with increased DNA replication after birth and retention of an increased proportion of mono-nucleated cardiomyocytes into adulthood. Furthermore, these CIZ1 induced changes in the heart reduce the impact of myocardial injury, identifying CIZ1 as a putative therapeutic target for cardiac repair.

A state of reversible compensated ventricular dysfunction precedes pathological remodelling in response to cardiomyocyte-specific activity of angiotensin II type-1 receptor in mice.

Cardiac dysfunction is commonly associated with high-blood-pressure-induced cardiomyocyte hypertrophy, in response to aberrant renin-angiotensin system (RAS) activity. Ensuing pathological remodelling promotes cardiomyocyte death and cardiac fibroblast activation, leading to cardiac fibrosis. The initiating cellular mechanisms that underlie this progressive disease are poorly understood. We previously reported a conditional mouse model in which a human angiotensin II type-I receptor transgene (HART) was expressed in differentiated cardiomyocytes after they had fully matured, but not during development. Twelve-month-old HART mice exhibited ventricular dysfunction and cardiomyocyte hypertrophy with interstitial fibrosis following full receptor stimulation, without affecting blood pressure. Here, we show that chronic HART activity in young adult mice causes ventricular dysfunction without hypertrophy, fibrosis or cardiomyocyte death. Dysfunction correlated with reduced expression of pro-hypertrophy markers and increased expression of pro-angiogenic markers in the cardiomyocytes experiencing increased receptor load. This stimulates responsive changes in closely associated non-myocyte cells, including the downregulation of pro-angiogenic genes, a dampened inflammatory response and upregulation of Tgfß. Importantly, this state of compensated dysfunction was reversible. Furthermore, increased stimulation of the receptors on the cardiomyocytes caused a switch in the secondary response from the non-myocyte cells. Progressive cardiac remodelling was stimulated through hypertrophy and death of individual cardiomyocytes, with infiltration, proliferation and activation of fibroblast and inflammatory cells, leading to increased angiogenic and inflammatory signalling. Together, these data demonstrate that a state of pre-hypertrophic compensated dysfunction can exist in affected individuals before common markers of heart disease are detectable. The data also suggest that there is an initial response from the housekeeping cells of the heart to signals emanating from distressed neighbouring cardiomyocytes to suppress those changes most commonly associated with progressive heart disease. We suggest that the reversible nature of this state of compensated dysfunction presents an ideal window of opportunity for personalised therapeutic intervention.

Variant Ciz1 is a circulating biomarker for early-stage lung cancer.

There is an unmet need for circulating biomarkers that can detect early-stage lung cancer. Here we show that a variant form of the nuclear matrix-associated DNA replication factor Ciz1 is present in 34/35 lung tumors but not in adjacent tissue, giving rise to stable protein quantifiable by Western blot in less than a microliter of plasma from lung cancer patients. In two independent sets, with 170 and 160 samples, respectively, variant Ciz1 correctly identified patients who had stage 1 lung cancer with clinically useful accuracy. For set 1, mean variant Ciz1 level in individuals without diagnosed tumors established a threshold that correctly classified 98% of small cell lung cancers (SCLC) and non-SCLC patients [receiver operator characteristic area under the curve (AUC) 0.958]. Within set 2, comparison of patients with stage 1 non-SCLC with asymptomatic age-matched smokers or individuals with benign lung nodules correctly classified 95% of patients (AUCs 0.913 and 0.905), with overall specificity of 76% and 71%, respectively. Moreover, using the mean of controls in set 1, we achieved 95% sensitivity among patients with stage 1 non-SCLC patients in set 2 with 74% specificity, demonstrating the robustness of the classification. RNAi-mediated selective depletion of variant Ciz1 is sufficient to restrain the growth of tumor cells that express it, identifying variant Ciz1 as a functionally relevant driver of cell proliferation in vitro and in vivo. The data show that variant Ciz1 is a strong candidate for a cancer-specific single marker capable of identifying early-stage lung cancer within at-risk groups without resort to invasive procedures.

Constitutively active TRPC channels of adipocytes confer a mechanism for sensing dietary fatty acids and regulating adiponectin.

RATIONALE: Calcium entry is pivotal in the heart and blood vessels, but its significance and mechanisms in adipose tissue are largely unknown. An important factor produced by adipocytes is adiponectin, which confers myocardial protection, insulin-sensitization, and antiatherosclerotic effects. OBJECTIVE: To investigate the relevance of calcium channels to adipocytes and the production of adiponectin. METHODS AND RESULTS: Microarray analysis led to identification of transient receptor potential canonical (TRPC)1 and TRPC5 as channel subunits that are induced when adipocytes mature. Both subunits were found in perivascular fat of patients with atherosclerosis. Intracellular calcium and patch-clamp measurements showed that adipocytes exhibit constitutively active calcium-permeable nonselective cationic channels that depend on TRPC1 and TRPC5. The activity could be enhanced by lanthanum or rosiglitazone, known stimulators of TRPC5 and TRPC5-containing channels. Screening identified lipid modulators of the channels that are relevant to adipose biology. Dietary ω-3 fatty acids (eg, α-linolenic acid) were inhibitory at concentrations that are achieved by ingestion. The adipocyte TRPC1/TRPC5-containing channel was functionally negative for the generation of adiponectin because channel blockade by antibodies, knock-down of TRPC1-TRPC5 in vitro, or conditional disruption of calcium permeability in TRPC5-incorporating channels in vivo increased the generation of adiponectin. The previously recognized capability of α-linolenic acid to stimulate the generation of adiponectin was lost when calcium permeability in the channels was disrupted. CONCLUSIONS: The data suggest that TRPC1 and TRPC5 contribute a constitutively active heteromultimeric channel of adipocytes that negatively regulates adiponectin and through which ω-3 fatty acids enhance the anti-inflammatory adipokine, adiponectin.

Cancer-associated variant expression and interaction of CIZ1 with cyclin A1 in differentiating male germ cells.

CIZ1 is a nuclear-matrix-associated DNA replication factor unique to higher eukaryotes, for which alternatively spliced isoforms have been associated with a range of disorders. In vitro, the CIZ1 N-terminus interacts with cyclin E and cyclin A at distinct sites, enabling functional cooperation with cyclin-A-Cdk2 to promote replication initiation. C-terminal sequences anchor CIZ1 to fixed sites on the nuclear matrix, imposing spatial constraint on cyclin-dependent kinase activity. Here we demonstrate that CIZ1 is predominantly expressed as a predicted full-length product throughout mouse development, consistent with a ubiquitous role in cell and tissue renewal. CIZ1 is expressed in proliferating stem cells of the testis, but is notably downregulated following commitment to differentiation. Significantly, CIZ1 is re-expressed at high levels in non-proliferative spermatocytes before meiotic division. Sequence analysis identifies at least seven alternatively spliced variants, including a dominant cancer-associated form and a set of novel isoforms. Furthermore, we show that in these post-replicative cells, CIZ1 interacts with germ-cell-specific cyclin A1, which has been implicated in the repair of DNA double-strand breaks. Consistent with this role, antibody depletion of CIZ1 reduces the capacity for testis extract to repair digested plasmid DNA in vitro. Together, the data imply post-replicative roles for CIZ1 in germ cell differentiation that might include meiotic recombination - a process intrinsic to genome stability and diversification.

Cyclin E is recruited to the nuclear matrix during differentiation, but is not recruited in cancer cells.

Cyclin E supports pre-replication complex (pre-RC) assembly, while cyclin A-associated kinase activates DNA synthesis. We show that cyclin E, but not A, is mounted upon the nuclear matrix in sub-nuclear foci in differentiated vertebrate cells, but not in undifferentiated cells or cancer cells. In murine embryonic stem cells, Xenopus embryos and human urothelial cells, cyclin E is recruited to the nuclear matrix as cells differentiate and this can be manipulated in vitro. This suggests that pre-RC assembly becomes spatially restricted as template usage is defined. Furthermore, failure to become restricted may contribute to the plasticity of cancer cells.

Ciz1 cooperates with cyclin-A-CDK2 to activate mammalian DNA replication in vitro.

Initiation of mammalian DNA replication can be reconstituted from isolated G1-phase nuclei and cell extracts, supplemented with cyclin-dependent protein kinases (CDKs). Under these conditions, cyclin E supports pre-replication complex assembly, whereas cyclin-A-associated kinase acts later to terminate assembly and activate DNA replication. The mechanism by which these events are coordinated is unknown. Here, we show that the replication factor Ciz1 interacts with cyclins E and A sequentially through distinct cyclin-binding motifs. Cyclin A displaces cyclin E from Ciz1 in a manner that is dependent on functional domains that are essential for its role in DNA replication. Furthermore, in cell-free assays, recombinant cyclin-A-CDK2 complexes and recombinant Ciz1 cooperate to promote initiation of DNA replication in late G1-phase nuclei. In addition, Ciz1 supports immobilization of cyclin A in isolated nuclei and depletion of Ciz1 by RNAi impairs immobilization, suggesting that Ciz1 promotes initiation by helping to target the kinase to a specific subnuclear compartment. We propose that Ciz1 acts to coordinate the functions of cyclins E and A in the nucleus, by delivering cyclin-A-associated kinase to sites that are specified by cyclin E, helping to ensure that they execute their functions in the same place and in the correct order.

H19 acts as a trans regulator of the imprinted gene network controlling growth in mice.

The imprinted H19 gene produces a non-coding RNA of unknown function. Mice lacking H19 show an overgrowth phenotype, due to a cis effect of the H19 locus on the adjacent Igf2 gene. To explore the function of the RNA itself, we produced transgenic mice overexpressing H19. We observed postnatal growth reduction in two independent transgenic lines and detected a decrease of Igf2 expression in embryos. An extensive analysis of several other genes from the newly described imprinted gene network (IGN) was performed in both loss- and gain-of-function animals. We found that H19 deletion leads to the upregulation of several genes of the IGN. This overexpression is restored to the wild-type level by transgenic expression of H19. We therefore propose that the H19 gene participates as a trans regulator in the fine-tuning of this IGN in the mouse embryo. This is the first in vivo evidence of a functional role for the H19 RNA. Our results also bring further experimental evidence for the existence of the IGN and open new perspectives in the comprehension of the role of genomic imprinting in embryonic growth and in human imprinting pathologies.

Angiotensin II type-1 receptor activation in the adult heart causes blood pressure-independent hypertrophy and cardiac dysfunction.

AIMS: Sustained hypertension leads to cardiac hypertrophy that can progress, through pathological remodelling, to heart failure. Abnormality of the renin-angiotensin system (RAS) has been strongly implicated in this process. Although hypertrophy in human is an established risk factor independent of blood pressure (BP), separation of remodelling in response to local cues within the differentiated myocardium from that related to pressure overload is unresolved. This study aimed to clarify the role of local RAS activity, specifically in the adult heart, in modulating cardiac hypertrophy and pathological remodelling. METHODS AND RESULTS: Transgenic mice with inducible cardiomyocyte-specific expression of a wild-type or N111G mutant form of the human angiotensin II (Ang II) type-1 receptor (hAT1R) were generated. The wild-type receptor is primarily stimulated by Ang II. In contrast, the N111G receptor can also be fully stimulated by the Ang II derivative, Ang IV, at levels that do not stimulate the wild-type receptor. The unique properties of these models were used to investigate the myocardial growth, remodelling and functional responses to hAT1R stimulation, specifically in adult cardiomyocytes, under normal conditions and following Ang IV infusion. Low-level expression of wild-type or N111G hAT1R at the cardiomyocyte membrane, from the onset of adolescence, induced enhanced myocyte growth and associated cardiac hypertrophy in the adult. This was not associated with change in resting BP or heart rate, measured by longitudinal telemetric analysis, and did not progress to pathological remodelling or heart failure. However, selective activation of cardiomyocyte-specific N111G receptors by Ang IV peptide infusion induced adverse ventricular remodelling within 4 weeks. This was characterized by increased interstitial fibrosis, dilatation of the left ventricle, and impaired cardiac function. CONCLUSION: Low-level local AT1R activity in differentiated myocardium causes compensated cardiac hypertrophy, that is, increased myocardial mass but with the retention of normal function, whereas short-term increased stimulation induces cardiac dysfunction with dilatation, reduced ejection fraction, and increased fibrosis in the absence of change in systemic BP.

Cancer-associated missplicing of exon 4 influences the subnuclear distribution of the DNA replication factor CIZ1.

Cip1-interacting zinc finger protein 1 (CIZ1, also known as CDKN1A-interacting zinc finger protein 1) stimulates initiation of mammalian DNA replication and is normally tethered to the nuclear matrix within DNA replication foci. Here, we show that an alternatively spliced human CIZ1 variant, lacking exon 4 (Delta E4), is misexpressed as a consequence of intronic mutation in Ewing tumor (ET) cell lines. In all ET lines tested, exon 4 is skipped and an upstream mononucleotide repeat element is expanded to contain up to 28 thymidines, compared to 16 in controls. In exon-trap experiments, a 24T variant produced three-fold more exon skipping than a 16T variant, demonstrating a direct effect on splicing. In functional assays, Delta E4 protein retains replication activity, but fails to form subnuclear foci. Furthermore, coexpression of mouse Delta E4 with Ciz1 prevents Ciz1 from localizing appropriately, having a dominant negative effect on foci formation. The data show that conditional exclusion of exon 4 influences the spatial distribution of the Ciz1 protein within the nucleus, and raise the possibility that CIZ1 alternative splicing could influence organized patterns of DNA replication.

C-terminal domains deliver the DNA replication factor Ciz1 to the nuclear matrix.

Cip1-interacting zinc finger protein 1 (Ciz1) stimulates DNA replication in vitro and is required for mammalian cells to enter S phase. Here, we show that a significant proportion of Ciz1 is retained in nuclear foci following extraction with nuclease and high salt. This suggests that Ciz1 is normally immobilized by interaction with non-chromatin nuclear structures, consistent with the nuclear matrix. Furthermore, matrix-associated Ciz1 foci strikingly colocalize with sites of newly synthesized DNA in S phase nuclei, suggesting that Ciz1 is present in DNA replication factories. Analysis of green fluorescent protein-tagged fragments indicates that nuclear immobilization of Ciz1 is mediated by sequences in its C-terminal third, encoded within amino acids 708-830. Immobilization occurs in a cell-cycle-dependent manner, most probably during late G1 or early S phase, to coincide with its reported point of action. Although C-terminal domains are sufficient for immobilization, N-terminal domains are also required to specify focal organization. Combined with previous work, which showed that the DNA replication activity of Ciz1 is encoded by N-terminal sequences, we suggest that Ciz1 is composed of two functionally distinct domains: an N-terminal replication domain and a C-terminal nuclear matrix anchor. This could contribute to the formation or function of DNA replication factories in mammalian cells.

Pluripotency of reprogrammed somatic genomes in embryonic stem hybrid cells.

Somatic nuclei can be epigenetically reprogrammed by factors present in undifferentiated embryonic stem (ES) cells. The acquisition of pluripotency by somatic genomes could render such cells a viable source of personalized cell type(s) for therapeutic application, avoiding the need for controversial therapeutic cloning. To investigate this possibility, we first determined the origin of transcripts in teratomas generated from mouse (ES x somatic cell) hybrid clones. Transcription of markers from the somatic genome demonstrated efficient in vivo differentiation down independent lineages. The induction of dopaminergic neurons by coculture with stromal PA6 feeder cells also demonstrated efficient capacity to differentiate in vitro. Hybrid clone-derived neurons expressed appropriate markers, and transcription of Pitx3 from the somatic genome was confirmed. When transplanted into mouse brains, the dopaminergic neurons were successfully integrated and expressed tyrosine hydroxylase. Thus, it should be possible to produce personalized ES-like cells with the reprogrammed somatic genomes.

Polycomb-group proteins are involved in silencing processes caused by a transgenic element from the murine imprinted H19/Igf2 region in Drosophila.

A subset of autosomal genes undergo genomic imprinting which results in expression from only the paternal or maternal chromosome. While this phenomenon is restricted to mammals and angiosperms, the underlying silencing mechanisms appear to be evolutionarily conserved. A biallelically unmethylated DNaseI hypersensitive region (A6-A4) between the imprinted Igf2 and H19 genes is conserved in humans and mice and functions as a tissue-specific maintenance element for the imprinted growth factor IGF2. In order to analyse A6-A4 for potentially conserved transcriptional maintenance properties, we have generated transgenic Drosophila harbouring the element in a reporter construct. These flies depicted silencing of the reporter genes lacZ and mini -white. The silenced state of the mini -white gene showed variegation and sensitivity to temperature changes. In addition, two members of the conserved Polycomb group, Enhancer of zeste and Posterior sex combs, were needed for repression. Polycomb group proteins are essential for gene silencing during development. Our results indicate that Polycomb group proteins may also be involved in the regulation of mammalian imprinted genes.