Su(var)2-10 and the SUMO Pathway Link piRNA-Guided Target Recognition to Chromatin Silencing.

Regulation of transcription is the main mechanism responsible for precise control of gene expression. Whereas the majority of transcriptional regulation is mediated by DNA-binding transcription factors that bind to regulatory gene regions, an elegant alternative strategy employs small RNA guides, Piwi-interacting RNAs (piRNAs) to identify targets of transcriptional repression. Here, we show that in Drosophila the small ubiquitin-like protein SUMO and the SUMO E3 ligase Su(var)2-10 are required for piRNA-guided deposition of repressive chromatin marks and transcriptional silencing of piRNA targets. Su(var)2-10 links the piRNA-guided target recognition complex to the silencing effector by binding the piRNA/Piwi complex and inducing SUMO-dependent recruitment of the SetDB1/Wde histone methyltransferase effector. We propose that in Drosophila, the nuclear piRNA pathway has co-opted a conserved mechanism of SUMO-dependent recruitment of the SetDB1/Wde chromatin modifier to confer repression of genomic parasites.

The SUMO Ligase Su(var)2-10 Controls Hetero- and Euchromatic Gene Expression via Establishing H3K9 Trimethylation and Negative Feedback Regulation.

Сhromatin is critical for genome compaction and gene expression. On a coarse scale, the genome is divided into euchromatin, which harbors the majority of genes and is enriched in active chromatin marks, and heterochromatin, which is gene-poor but repeat-rich. The conserved molecular hallmark of heterochromatin is the H3K9me3 modification, which is associated with gene silencing. We found that in Drosophila, deposition of most of the H3K9me3 mark depends on SUMO and the SUMO ligase Su(var)2-10, which recruits the histone methyltransferase complex SetDB1/Wde. In addition to repressing repeats, H3K9me3 influences expression of both hetero- and euchromatic host genes. High H3K9me3 levels in heterochromatin are required to suppress spurious transcription and ensure proper gene expression. In euchromatin, a set of conserved genes is repressed by Su(var)2-10/SetDB1-induced H3K9 trimethylation, ensuring tissue-specific gene expression. Several components of heterochromatin are themselves repressed by this pathway, providing a negative feedback mechanism to ensure chromatin homeostasis.

Cutoff Suppresses RNA Polymerase II Termination to Ensure Expression of piRNA Precursors.

Small non-coding RNAs called piRNAs serve as guides for an adaptable immune system that represses transposable elements in germ cells of Metazoa. In Drosophila the RDC complex, composed of Rhino, Deadlock and Cutoff (Cuff) bind chromatin of dual-strand piRNA clusters, special genomic regions, which encode piRNA precursors. The RDC complex is required for transcription of piRNA precursors, though the mechanism by which it licenses transcription remained unknown. Here, we show that Cuff prevents premature termination of RNA polymerase II. Cuff prevents cleavage of nascent RNA at poly(A) sites by interfering with recruitment of the cleavage and polyadenylation specificity factor (CPSF) complex. Cuff also protects processed transcripts from degradation by the exonuclease Rat1. Our work reveals a conceptually different mechanism of transcriptional enhancement. In contrast to other factors that regulate termination by binding to specific signals on nascent RNA, the RDC complex inhibits termination in a chromatin-dependent and sequence-independent manner.

Disruption of IRE1α through its kinase domain attenuates multiple myeloma.

Multiple myeloma (MM) arises from malignant immunoglobulin (Ig)-secreting plasma cells and remains an incurable, often lethal disease despite therapeutic advances. The unfolded-protein response sensor IRE1α supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may co-opt the IRE1α-XBP1s pathway; however, the validity of IRE1α as a potential MM therapeutic target is controversial. Genetic disruption of IRE1α or XBP1s, or pharmacologic IRE1α kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice and augmented efficacy of two established frontline antimyeloma agents, bortezomib and lenalidomide. Mechanistically, IRE1α perturbation inhibited expression of key components of the endoplasmic reticulum-associated degradation machinery, as well as secretion of Ig light chains and of cytokines and chemokines known to promote MM growth. Selective IRE1α kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138- cells derived from bone marrows of newly diagnosed or posttreatment-relapsed MM patients, in both US- and European Union-based cohorts. Effective IRE1α inhibition preserved glucose-induced insulin secretion by pancreatic microislets and viability of primary hepatocytes in vitro, as well as normal tissue homeostasis in mice. These results establish a strong rationale for developing kinase-directed inhibitors of IRE1α for MM therapy.

Transgenerationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing.

Small noncoding RNAs that associate with Piwi proteins, called piRNAs, serve as guides for repression of diverse transposable elements in germ cells of metazoa. In Drosophila, the genomic regions that give rise to piRNAs, the so-called piRNA clusters, are transcribed to generate long precursor molecules that are processed into mature piRNAs. How genomic regions that give rise to piRNA precursor transcripts are differentiated from the rest of the genome and how these transcripts are specifically channeled into the piRNA biogenesis pathway are not known. We found that transgenerationally inherited piRNAs provide the critical trigger for piRNA production from homologous genomic regions in the next generation by two different mechanisms. First, inherited piRNAs enhance processing of homologous transcripts into mature piRNAs by initiating the ping-pong cycle in the cytoplasm. Second, inherited piRNAs induce installment of the histone 3 Lys9 trimethylation (H3K9me3) mark on genomic piRNA cluster sequences. The heterochromatin protein 1 (HP1) homolog Rhino binds to the H3K9me3 mark through its chromodomain and is enriched over piRNA clusters. Rhino recruits the piRNA biogenesis factor Cutoff to piRNA clusters and is required for efficient transcription of piRNA precursors. We propose that transgenerationally inherited piRNAs act as an epigenetic memory for identification of substrates for piRNA biogenesis on two levels: by inducing a permissive chromatin environment for piRNA precursor synthesis and by enhancing processing of these precursors.

Phytochemicals from Polyalthia Species: Potential and Implication on Anti-Oxidant, Anti-Inflammatory, Anti-Cancer, and Chemoprevention Activities.

Polyalthia belong to the Annonaceae family and are a type of evergreen tree distributed across many tropical and subtropical regions. Polyalthia species have been used long term as indigenous medicine to treat certain diseases, including fever, diabetes, infection, digestive disease, etc. Recent studies have demonstrated that not only crude extracts but also the isolated pure compounds exhibit various pharmacological activities, such as anti-oxidant, anti-microbial, anti-tumor, anti-cancer, etc. It is known that the initiation of cancer usually takes several years and is related to unhealthy lifestyle, as well as dietary and environmental factors, such as stress, toxins and smoking. In fact, natural or synthetic substances have been used as cancer chemoprevention to delay, impede, or even stop cancer growing. This review is an attempt to collect current available phytochemicals from Polyalthia species, which exhibit anti-cancer potentials for chemoprevention purposes, providing directions for further research on the interesting agents and possible clinical applications.

The Role of Autophagy in Anti-Cancer and Health Promoting Effects of Cordycepin.

Cordycepin is an adenosine derivative isolated from Cordyceps sinensis, which has been used as an herbal complementary and alternative medicine with various biological activities. The general anti-cancer mechanisms of cordycepin are regulated by the adenosine A3 receptor, epidermal growth factor receptor (EGFR), mitogen-activated protein kinases (MAPKs), and glycogen synthase kinase (GSK)-3ß, leading to cell cycle arrest or apoptosis. Notably, cordycepin also induces autophagy to trigger cell death, inhibits tumor metastasis, and modulates the immune system. Since the dysregulation of autophagy is associated with cancers and neuron, immune, and kidney diseases, cordycepin is considered an alternative treatment because of the involvement of cordycepin in autophagic signaling. However, the profound mechanism of autophagy induction by cordycepin has never been reviewed in detail. Therefore, in this article, we reviewed the anti-cancer and health-promoting effects of cordycepin in the neurons, kidneys, and the immune system through diverse mechanisms, including autophagy induction. We also suggest that formulation changes for cordycepin could enhance its bioactivity and bioavailability and lower its toxicity for future applications. A comprehensive understanding of the autophagy mechanism would provide novel mechanistic insight into the anti-cancer and health-promoting effects of cordycepin.

16-Hydroxycleroda-3,13-dien-15,16-olide Induces Apoptosis in Human Bladder Cancer Cells through Cell Cycle Arrest, Mitochondria ROS Overproduction, and Inactivation of EGFR-Related Signalling Pathways.

A clerodane diterpene compound 16-hydroxycleroda-3,13-dien-15,16-olide (CD) is considered a therapeutic agent with pharmacological activities. The present study investigated the mechanisms of CD-induced apoptosis in T24 human bladder cancer cells. CD inhibited cell proliferation in a concentration and time-dependent manner. CD-induced overproduction of reactive oxygen species and reduced mitochondrial membrane potential, associated with reduced expression of Bcl-2 and increased levels of cytosolic cytochrome c, cleaved PARP-1 and caspase-3. In addition, CD treatment led to cell cycle arrest at the G0/G1 phase and inhibited expression of cyclin D1 and cyclin-dependent kinases 2 and 4 and led to increased levels of p21, p27Kip1 and p53. All of these events were accompanied with a reduction of pEGFR, pMEK1/2, pERK1/2, pAkt, pmTOR, pP70S6K1, HIF-1α, c-Myc and VEGF. RNAseq-based analysis revealed that CD-induced cell death was characterised by an increased expression of stress and apoptotic-related genes as well as inhibition of the cell cycle-related genes. In summary, CD induces apoptosis in T24 bladder cancer cells through targeting multiple intracellular signaling pathways as a result of oxidative stress and cell cycle arrest.

Aqueous Extracts of Toona sinensis Leaves Inhibit Renal Carcinoma Cell Growth and Migration Through JAK2/stat3, Akt, MEK/ERK, and mTOR/HIF-2α Pathways.

Toona sinensis (TS) is a type of deciduous tree, which is distributed widely in Asia and used as a traditional herb medicine. Previously, we demonstrated that aqueous extracts of TS leaves (TSL-1) induce apoptosis in two clear types of human renal carcinoma cells (ccRCC) via mitochondria-dependent pathway. In this study, we further investigated the more detailed mechanism of TSL-1-induced antitumor effects on ccRCCs. TSL-1 treatment arrested ccRCC cells in G0/G1 phase through the decrease of cyclin D1, cyclin-dependent kinase (CDK)2, and CDK4 as well as induction of p53 and FOXO3a protein expressions. On the other hand, the inhibitory effects of TSL-1 on migration were also observed in 786-O and A-498 cells. Mechanically, we presented that TSL-1 could suppress cell cycle progression and motility via inhibiting the phosphorylation of JAK2/stat3, Akt, MEK/ERK, and mTOR in a concentration- and time-dependent manner. Moreover, we found that TSL-1 inhibited p21, HIF-2α, c-Myc, VEGF, and MMP9 protein expressions in both cell lines. In conclusion, these findings suggested that TS-induced apoptosis and its antimigration activity in ccRCC cells were accompanied by inactivation of several oncogenic pathways.

Pooled RNAi screen identifies ubiquitin ligase Itch as crucial for influenza A virus release from the endosome during virus entry.

Influenza viruses, like other viruses, rely on host factors to support their life cycle as viral proteins usually "hijack," or collaborate with, cellular proteins to execute their functions. Identification and understanding of these factors can increase the knowledge of molecular mechanisms manipulated by the viruses and facilitate development of antiviral drugs. To this end, we developed a unique genome-wide pooled shRNA screen to search for cellular factors important for influenza A virus (IAV) replication. We identified an E3 ubiquitin ligase, Itch, as an essential factor for an early step in the viral life cycle. In Itch knockdown cells, the incorporation of viral ribonucleoprotein complex into endosomes was normal, but its subsequent release from endosomes and transport to the nucleus was retarded. In addition, upon virus infection, Itch was phosphorylated and recruited to the endosomes, where virus particles were located. Furthermore, Itch interacted with viral M1 protein and ubiquitinated M1 protein. Collectively, our findings unravel a critical role of Itch in mediating IAV release from the endosome and offer insights into the mechanism for IAV uncoating during virus entry. These findings also highlight the feasibility of pooled RNAi screening for exploring the cellular cofactors of lytic viruses.

Antagonism of purinergic signalling improves recovery from traumatic brain injury.

The recent public awareness of the incidence and possible long-term consequences of traumatic brain injury only heightens the need to develop effective approaches for treating this neurological disease. In this report, we identify a new therapeutic target for traumatic brain injury by studying the role of astrocytes, rather than neurons, after neurotrauma. We use in vivo multiphoton imaging and show that mechanical forces during trauma trigger intercellular calcium waves throughout the astrocytes, and these waves are mediated by purinergic signalling. Subsequent in vitro screening shows that astrocyte signalling through the 'mechanical penumbra' affects the activity of neural circuits distant from the injury epicentre, and a reduction in the intercellular calcium waves within astrocytes restores neural activity after injury. In turn, the targeting of different purinergic receptor populations leads to a reduction in hippocampal cell death in mechanically injured organotypic slice cultures. Finally, the most promising therapeutic candidate from our in vitro screen (MRS 2179, a P2Y1 receptor antagonist) also improves histological and cognitive outcomes in a preclinical model of traumatic brain injury. This work shows the potential of studying astrocyte signalling after trauma to yield new and effective therapeutic targets for treating traumatic brain injury.

Significant head accelerations can influence immediate neurological impairments in a murine model of blast-induced traumatic brain injury.

Although blast-induced traumatic brain injury (bTBI) is well recognized for its significance in the military population, the unique mechanisms of primary bTBI remain undefined. Animate models of primary bTBI are critical for determining these potentially unique mechanisms, but the biomechanical characteristics of many bTBI models are poorly understood. In this study, we examine some common shock tube configurations used to study blast-induced brain injury in the laboratory and define the optimal configuration to minimize the effect of torso overpressure and blast-induced head accelerations. Pressure transducers indicated that a customized animal holder successfully reduced peak torso overpressures to safe levels across all tested configurations. However, high speed video imaging acquired during the blast showed significant head accelerations occurred when animals were oriented perpendicular to the shock tube axis. These findings of complex head motions during blast are similar to previous reports [Goldstein et al., 2012, "Chronic Traumatic Encephalopathy in Blast-Exposed Military Veterans and a Blast Neurotrauma Mouse Model," Sci. Transl. Med., 4(134), 134ra160; Sundaramurthy et al., 2012, "Blast-Induced Biomechanical Loading of the Rat: An Experimental and Anatomically Accurate Computational Blast Injury Model," J. Neurotrauma, 29(13), pp. 2352-2364; Svetlov et al., 2010, "Morphologic and Biochemical Characterization of Brain Injury in a Model of Controlled Blast Overpressure Exposure," J. Trauma, 69(4), pp. 795-804]. Under the same blast input conditions, minimizing head acceleration led to a corresponding elimination of righting time deficits. However, we could still achieve righting time deficits under minimal acceleration conditions by significantly increasing the peak blast overpressure. Together, these data show the importance of characterizing the effect of blast overpressure on head kinematics, with the goal of producing models focused on understanding the effects of blast overpressure on the brain without the complicating factor of superimposed head accelerations.

Proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2), a host membrane-deforming protein, is critical for membranous web formation in hepatitis C virus replication.

Hepatitis C virus (HCV) reorganizes intracellular membranes to establish sites of replication. How viral and cellular proteins target, bind, and rearrange specific membranes into the replication factory remains a mystery. We used a lentivirus-based RNA interference (RNAi) screening approach to identify the potential cellular factors that are involved in HCV replication. A protein with membrane-deforming activity, proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2), was identified as a potential factor. Knockdown of PSTPIP2 in HCV subgenomic replicon-harboring and HCV-infected cells was associated with the reduction of HCV protein and RNA expression. PSTPIP2 was localized predominantly in detergent-resistant membranes (DRMs), which contain the RNA replication complex. PSTPIP2 knockdown caused a significant reduction of the formation of HCV- and NS4B-induced membranous webs. A PSTPIP2 mutant defective in inducing membrane curvature failed to support HCV replication, confirming that the membrane-deforming ability of PSTPIP2 is essential for HCV replication. Taking these results together, we suggest that PSTPIP2 facilitates membrane alterations and is a key player in the formation of the membranous web, which is the site of the HCV replication complex.

Clerodane diterpene induces apoptosis/anoikis and suppresses migration and invasion of human bladder cancer cells through the histone deacetylases, integrin-focal adhesion kinase, and matrix metalloproteinase 9 signalling pathways.

Clerodane diterpene, a class of bicyclic diterpenoids, is found in hundreds of plant species. 16-hydroxycleroda-3,13-dien-15,16-olide (CD) can be isolated from the plant Polyalthia longifolia and has been applied against oral cancer and glioma by xenograft model. In this study, we aim to explore its antitumour action by examining its histone deacetylase (HDAC) activity and integrin-associated intracellular signalling pathway on T24 human bladder cancer (BC) cells. Our results revealed that CD-inhibited colony formation, HDAC activity, HDAC (1, 2 and 3) mRNA and cell spreading on fibronectin-coated surfaces in a concentration-dependent manner. Furthermore, decreased cFLIP and increased caspase-8 cleavage accompanied CD-induced cell death. At non-toxic concentrations, CD blocked the migration and invasion of T24 cells. CD hindered migration and invasion by the downregulation of fibronectin, integrin α5ß1, ß-catenin, FAK, vinculin and Rho A, as well as by reduction of phosphorylated glycogen synthase kinase 3ß (pGSK3ß), pSrc, pstat3 and pNFκB. We observed that the MMP9 gene was closely linked with prognosis of patients with bladder cancer. MMP9 protein levels and activity were largely attenuated by CD in a concentration-dependent manner. In conclusion, CD-induced caspase-8-dependent apoptosis and suppressed migration and invasion by blocking several intracellular signalling pathways, including downregulation of HDAC activity and integrin-FAK and MMP9 pathways.

Polo-like kinase 1 is involved in hepatitis C virus replication by hyperphosphorylating NS5A.

Hepatitis C virus (HCV) replication involves many viral and host factors. Here, we employed a lentivirus-based RNA interference (RNAi) screening approach to search for possible cellular factors. By using a kinase-phosphatase RNAi library and an HCV replicon reporter system, we identified a serine-threonine kinase, Polo-like kinase 1 (Plk1), as a potential host factor regulating HCV replication. Knockdown of Plk1 reduced both HCV RNA replication and nonstructural (NS) protein production in both HCV replicon cells and HCV-infected cells while it did not significantly affect host cellular growth or cell cycle. Overexpression of Plk1 in the knockdown cells rescued HCV replication. Interestingly, the ratio between the hyperphosphorylated form (p58) and the basal phosphorylated form (p56) of NS5A was lower in the Plk1 knockdown cells and Plk1 kinase inhibitor-treated cells than in the control groups. Further studies showed that Plk1 could be immunoprecipitated together with NS5A. Both proteins partially colocalized in the perinuclear region. Furthermore, Plk1 could phosphorylate NS5A to both the p58 and p56 forms in an in vitro assay system; the phosphorylation efficiency was comparable to that of the reported casein kinase. Taken together, this study shows that Plk1 is an NS5A phosphokinase and thereby indirectly regulates HCV RNA replication. Because of the differential effects of Plk1 on HCV replication and host cell growth, Plk1 could potentially serve as a target for anti-HCV therapy.

Schisandrin enhances dendrite outgrowth and synaptogenesis in primary cultured hippocampal neurons.

BACKGROUND: Schisandra chinensis, commonly used in Asia for tea material and traditional Chinese medicine, is presumed to enhance mental and intellectual functions. In this study, the effects and signalling mechanisms of a purified compound schisandrin, one of the lignan of Schisandra chinensis, on primary cultured hippocampal neurons were investigated. RESULTS: Schisandrin treatment enhanced total dendritic length and branching complexity, both of which were significantly suppressed in the presence of specific blockers for calmodulin-dependent kinase II (CaMKII), protein kinase C epsilon (PKCε), and mitogen activated protein kinase kinase (MEK). Moreover, schisandrin induced calcium influx, and phosphorylation of CaMKII, PKCε, and MEK. Inhibition of CAMKII and PKCε attenuated the schisandrin-induced phosphorylation of PKCε and MEK, and the phosphorylation of MEK, respectively. Moreover, schisandrin also stimulated the phosphorylation of cyclic AMP responsive-element binding protein (CREB) at Ser-133, an effect that was blocked by KN93. In addition to its neuritogenic effects, schisandrin increased the numbers of postsynaptic density-95-positive and FM1-43-positive puncta in dendrites and synaptic boutons, respectively. CONCLUSION: In hippocampal neurons, schisandrin exhibits neurotrophic properties that are mediated by the CaMKII-PKCε-MEK pathway.

Induction of mitochondrial-dependent apoptosis by essential oil of Toona sinensis root through Akt, mTOR and NF-κB signalling pathways in human renal cell carcinoma cells.

Natural products have long been considered as a kind of complementary medicine. In this study, we investigate the apoptotic effect of essential oils of Toona sinensis roots (TSR) on human clear cell renal cell carcinomas (ccRCC). The sesquiterpene content of TSR essential oil was determined via GC/MS analysis. TSR decreased ccRCC cell viabilities, inducing ROS generation and reduction of the mitochondrial membrane potential. Moreover, TSR inhibited Bcl-2 and Hsp90 expression but increased PARP-1 cleavage and cytochrome c release. Akt, mTOR and NF-κB phosphorylation and HIF-α expression were all inhibited, which likely contributed to the anti-proliferative and anti-adhesive effects of TSR.

Role of JNK activation in paclitaxel-induced apoptosis in human head and neck squamous cell carcinoma.

It has been reported that paclitaxel activates cell cycle arrest and increases caspase protein expression to induce apoptosis in head and neck squamous cell carcinoma (HNSCC) cell lines. However, the potential signaling pathway regulating this apoptotic phenomenon remains unclear. The present study used OEC-M1 cells to investigate the underlying molecular mechanism of paclitaxel-induced apoptosis. Following treatment with paclitaxel, cell viability was assessed via the MTT assay. Necrosis, apoptosis, cell cycle and mitochondrial membrane potential (∆Ψm) were analyzed via flow cytometric analyses, respectively. Western blot analysis was performed to detect the expression levels of proteins associated with the MAPK and caspase signaling pathways. The results demonstrated that low-dose paclitaxel (50 nM) induced apoptosis but not necrosis in HNSCC cells. In addition, paclitaxel activated the c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase or p38 mitogen-activated protein kinase. The paclitaxel-activated JNK contributed to paclitaxel-induced apoptosis, activation of caspase-3, -6, -7, -8 and -9, and reduction of ∆Ψm. In addition, caspase-8 and -9 inhibitors, respectively, significantly decreased paclitaxel-induced apoptosis. Notably, Bid was truncated following treatment with paclitaxel. Taken together, the results of the present study suggest that paclitaxel-activated JNK is required for caspase activation and loss of ∆Ψm, which results in apoptosis of HNSCC cells. These results may provide mechanistic basis for designing more effective paclitaxel-combining regimens to treat HNSCC.

Regulatory mechanisms of Cordyceps sinensis on steroidogenesis in MA-10 mouse Leydig tumor cells.

Cordyceps sinensis (CS) is an herbal medicine that increases steroidogenesis in Leydig cells and improves male reproductive dysfunction. We have found that CS stimulates Leydig cell steroidogenesis through the protein kinase A and protein kinase C signaling pathways. In the present study, we sought to determine the mechanisms of CS-stimulated steroidogenesis in MA-10 mouse Leydig tumor cells. Using pharmacological approaches, we found that de novo protein synthesis, protein transcription, a calcium signal, and a mitochondria electrochemical gradient were required for CS-stimulated steroidogenesis in MA-10 cells. mRNA expression of steroidogenic acute regulatory protein was activated by CS. However, CS had an adversary effect on P450 side-chain cleavage enzyme activity, but not in 3ß-hydroxysteroid dehydrogenase enzyme, in regulating MA-10 cell steroidogenesis. In conclusion, de novo protein synthesis, increased steroidogenic acute regulatory protein mRNA expression, and the mitochondria electrochemical gradient were involved in CS-stimulated steroidogenesis in MA-10 cells.

Repression of interrupted and intact rDNA by the SUMO pathway in Drosophila melanogaster.

Ribosomal RNAs (rRNAs) are essential components of the ribosome and are among the most abundant macromolecules in the cell. To ensure high rRNA level, eukaryotic genomes contain dozens to hundreds of rDNA genes, however, only a fraction of the rRNA genes seems to be active, while others are transcriptionally silent. We found that individual rDNA genes have high level of cell-to-cell heterogeneity in their expression in Drosophila melanogaster. Insertion of heterologous sequences into rDNA leads to repression associated with reduced expression in individual cells and decreased number of cells expressing rDNA with insertions. We found that SUMO (Small Ubiquitin-like Modifier) and SUMO ligase Ubc9 are required for efficient repression of interrupted rDNA units and variable expression of intact rDNA. Disruption of the SUMO pathway abolishes discrimination of interrupted and intact rDNAs and removes cell-to-cell heterogeneity leading to uniformly high expression of individual rDNA in single cells. Our results suggest that the SUMO pathway is responsible for both repression of interrupted units and control of intact rDNA expression.