ß1 integrin signaling governs necroptosis via the chromatin-remodeling factor CHD4.

Fibrosis, characterized by sustained activation of myofibroblasts and excessive extracellular matrix (ECM) deposition, is known to be associated with chronic inflammation. Receptor-interacting protein kinase 3 (RIPK3), the central kinase of necroptosis signaling, is upregulated in fibrosis and contributes to tumor necrosis factor (TNF)-mediated inflammation. In bile-duct-ligation-induced liver fibrosis, we found that myofibroblasts are the major cell type expressing RIPK3. Genetic ablation of ß1 integrin, the major profibrotic ECM receptor in fibroblasts, not only abolished ECM fibrillogenesis but also blunted RIPK3 expression via a mechanism mediated by the chromatin-remodeling factor chromodomain helicase DNA-binding protein 4 (CHD4). While the function of CHD4 has been conventionally linked to the nucleosome-remodeling deacetylase (NuRD) and CHD4-ADNP-HP1(ChAHP) complexes, we found that CHD4 potently repressed a set of genes, including Ripk3, with high locus specificity but independent of either the NuRD or the ChAHP complex. Thus, our data uncover that ß1 integrin intrinsically links fibrotic signaling to RIPK3-driven inflammation via a novel mode of action of CHD4.

PD-1 instructs a tumor-suppressive metabolic program that restricts glycolysis and restrains AP-1 activity in T cell lymphoma.

The PDCD1-encoded immune checkpoint receptor PD-1 is a key tumor suppressor in T cells that is recurrently inactivated in T cell non-Hodgkin lymphomas (T-NHLs). The highest frequencies of PDCD1 deletions are detected in advanced disease, predicting inferior prognosis. However, the tumor-suppressive mechanisms of PD-1 signaling remain unknown. Here, using tractable mouse models for T-NHL and primary patient samples, we demonstrate that PD-1 signaling suppresses T cell malignancy by restricting glycolytic energy and acetyl coenzyme A (CoA) production. In addition, PD-1 inactivation enforces ATP citrate lyase (ACLY) activity, which generates extramitochondrial acetyl-CoA for histone acetylation to enable hyperactivity of activating protein 1 (AP-1) transcription factors. Conversely, pharmacological ACLY inhibition impedes aberrant AP-1 signaling in PD-1-deficient T-NHLs and is toxic to these cancers. Our data uncover genotype-specific vulnerabilities in PDCD1-mutated T-NHL and identify PD-1 as regulator of AP-1 activity.

Chromatin Preparation and Chromatin Immunoprecipitation from Drosophila Heads.

Chromatin immunoprecipitation (ChIP) is a widely used method to map protein-DNA interactions in vivo. Formaldehyde cross-linked chromatin is fragmented, and the protein of interest is immunoprecipitated using a specific antibody. The co-immunoprecipitated DNA is then purified and analyzed by quantitative PCR (ChIP-qPCR) or next-generation sequencing (ChIP-seq). Therefore, from the amount of DNA recovered, it can be inferred the localization and abundance of the target protein at specific loci or throughout the entire genome. This protocol describes how to perform ChIP from Drosophila adult fly heads.

WDR5 is required for DUX4 expression and its pathological effects in FSHD muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent neuromuscular disorders. The disease is linked to copy number reduction and/or epigenetic alterations of the D4Z4 macrosatellite on chromosome 4q35 and associated with aberrant gain of expression of the transcription factor DUX4, which triggers a pro-apoptotic transcriptional program leading to muscle wasting. As today, no cure or therapeutic option is available to FSHD patients. Given its centrality in FSHD, blocking DUX4 expression with small molecule drugs is an attractive option. We previously showed that the long non protein-coding RNA DBE-T is required for aberrant DUX4 expression in FSHD. Using affinity purification followed by proteomics, here we identified the chromatin remodeling protein WDR5 as a novel DBE-T interactor and a key player required for the biological activity of the lncRNA. We found that WDR5 is required for the expression of DUX4 and its targets in primary FSHD muscle cells. Moreover, targeting WDR5 rescues both cell viability and myogenic differentiation of FSHD patient cells. Notably, comparable results were obtained by pharmacological inhibition of WDR5. Importantly, WDR5 targeting was safe to healthy donor muscle cells. Our results support a pivotal role of WDR5 in the activation of DUX4 expression identifying a druggable target for an innovative therapeutic approach for FSHD.

Dominant role of DNA methylation over H3K9me3 for IAP silencing in endoderm.

Silencing of endogenous retroviruses (ERVs) is largely mediated by repressive chromatin modifications H3K9me3 and DNA methylation. On ERVs, these modifications are mainly deposited by the histone methyltransferase Setdb1 and by the maintenance DNA methyltransferase Dnmt1. Knock-out of either Setdb1 or Dnmt1 leads to ERV de-repression in various cell types. However, it is currently not known if H3K9me3 and DNA methylation depend on each other for ERV silencing. Here we show that conditional knock-out of Setdb1 in mouse embryonic endoderm results in ERV de-repression in visceral endoderm (VE) descendants and does not occur in definitive endoderm (DE). Deletion of Setdb1 in VE progenitors results in loss of H3K9me3 and reduced DNA methylation of Intracisternal A-particle (IAP) elements, consistent with up-regulation of this ERV family. In DE, loss of Setdb1 does not affect H3K9me3 nor DNA methylation, suggesting Setdb1-independent pathways for maintaining these modifications. Importantly, Dnmt1 knock-out results in IAP de-repression in both visceral and definitive endoderm cells, while H3K9me3 is unaltered. Thus, our data suggest a dominant role of DNA methylation over H3K9me3 for IAP silencing in endoderm cells. Our findings suggest that Setdb1-meditated H3K9me3 is not sufficient for IAP silencing, but rather critical for maintaining high DNA methylation.

HDAC2 Facilitates Pancreatic Cancer Metastasis.

The mortality of patients with pancreatic ductal adenocarcinoma (PDAC) is strongly associated with metastasis, a multistep process that is incompletely understood in this disease. Although genetic drivers of PDAC metastasis have not been defined, transcriptional and epigenetic rewiring can contribute to the metastatic process. The epigenetic eraser histone deacetylase 2 (HDAC2) has been connected to less differentiated PDAC, but the function of HDAC2 in PDAC has not been comprehensively evaluated. Using genetically defined models, we show that HDAC2 is a cellular fitness factor that controls cell cycle in vitro and metastasis in vivo, particularly in undifferentiated, mesenchymal PDAC cells. Unbiased expression profiling detected a core set of HDAC2-regulated genes. HDAC2 controlled expression of several prosurvival receptor tyrosine kinases connected to mesenchymal PDAC, including PDGFRα, PDGFRß, and EGFR. The HDAC2-maintained program disabled the tumor-suppressive arm of the TGFß pathway, explaining impaired metastasis formation of HDAC2-deficient PDAC. These data identify HDAC2 as a tractable player in the PDAC metastatic cascade. The complexity of the function of epigenetic regulators like HDAC2 implicates that an increased understanding of these proteins is needed for implementation of effective epigenetic therapies. SIGNIFICANCE: HDAC2 has a context-specific role in undifferentiated PDAC and the capacity to disseminate systemically, implicating HDAC2 as targetable protein to prevent metastasis.

Promoter G-quadruplexes and transcription factors cooperate to shape the cell type-specific transcriptome.

Cell identity is maintained by activation of cell-specific gene programs, regulated by epigenetic marks, transcription factors and chromatin organization. DNA G-quadruplex (G4)-folded regions in cells were reported to be associated with either increased or decreased transcriptional activity. By G4-ChIP-seq/RNA-seq analysis on liposarcoma cells we confirmed that G4s in promoters are invariably associated with high transcription levels in open chromatin. Comparing G4 presence, location and transcript levels in liposarcoma cells to available data on keratinocytes, we showed that the same promoter sequences of the same genes in the two cell lines had different G4-folding state: high transcript levels consistently associated with G4-folding. Transcription factors AP-1 and SP1, whose binding sites were the most significantly represented in G4-folded sequences, coimmunoprecipitated with their G4-folded promoters. Thus, G4s and their associated transcription factors cooperate to determine cell-specific transcriptional programs, making G4s to strongly emerge as new epigenetic regulators of the transcription machinery.

Epithelial cell plasticity drives endoderm formation during gastrulation.

It is generally accepted that epiblast cells ingress into the primitive streak by epithelial-to-mesenchymal transition (EMT) to give rise to the mesoderm; however, it is less clear how the endoderm acquires an epithelial fate. Here, we used embryonic stem cell and mouse embryo knock-in reporter systems to combine time-resolved lineage labelling with high-resolution single-cell transcriptomics. This allowed us to resolve the morphogenetic programs that segregate the mesoderm from the endoderm germ layer. Strikingly, while the mesoderm is formed by classical EMT, the endoderm is formed independent of the key EMT transcription factor Snail1 by mechanisms of epithelial cell plasticity. Importantly, forkhead box transcription factor A2 (Foxa2) acts as an epithelial gatekeeper and EMT suppressor to shield the endoderm from undergoing a mesenchymal transition. Altogether, these results not only establish the morphogenetic details of germ layer formation, but also have broader implications for stem cell differentiation and cancer metastasis.

Epstein-Barr virus inactivates the transcriptome and disrupts the chromatin architecture of its host cell in the first phase of lytic reactivation.

Epstein-Barr virus (EBV), a herpes virus also termed HHV 4 and the first identified human tumor virus, establishes a stable, long-term latent infection in human B cells, its preferred host. Upon induction of EBV's lytic phase, the latently infected cells turn into a virus factory, a process that is governed by EBV. In the lytic, productive phase, all herpes viruses ensure the efficient induction of all lytic viral genes to produce progeny, but certain of these genes also repress the ensuing antiviral responses of the virally infected host cells, regulate their apoptotic death or control the cellular transcriptome. We now find that EBV causes previously unknown massive and global alterations in the chromatin of its host cell upon induction of the viral lytic phase and prior to the onset of viral DNA replication. The viral initiator protein of the lytic cycle, BZLF1, binds to >105 binding sites with different sequence motifs in cellular chromatin in a concentration dependent manner implementing a binary molar switch probably to prevent noise-induced erroneous induction of EBV's lytic phase. Concomitant with DNA binding of BZLF1, silent chromatin opens locally as shown by ATAC-seq experiments, while previously wide-open cellular chromatin becomes inaccessible on a global scale within hours. While viral transcripts increase drastically, the induction of the lytic phase results in a massive reduction of cellular transcripts and a loss of chromatin-chromatin interactions of cellular promoters with their distal regulatory elements as shown in Capture-C experiments. Our data document that EBV's lytic cycle induces discrete early processes that disrupt the architecture of host cellular chromatin and repress the cellular epigenome and transcriptome likely supporting the efficient de novo synthesis of this herpes virus.

The MDM2 inducible promoter folds into four-tetrad antiparallel G-quadruplexes targetable to fight malignant liposarcoma.

Well-differentiated liposarcoma (WDLPS) is a malignant neoplasia hard to diagnose and treat. Its main molecular signature is amplification of the MDM2-containing genomic region. The MDM2 oncogene is the master regulator of p53: its overexpression enhances p53 degradation and inhibits apoptosis, leading to the tumoral phenotype. Here, we show that the MDM2 inducible promoter G-rich region folds into stable G-quadruplexes both in vitro and in vivo and it is specifically recognized by cellular helicases. Cell treatment with G-quadruplex-ligands reduces MDM2 expression and p53 degradation, thus stimulating cancer cell cycle arrest and apoptosis. Structural characterization of the MDM2 G-quadruplex revealed an extraordinarily stable, unique four-tetrad antiparallel dynamic conformation, amenable to selective targeting. These data indicate the feasibility of an out-of-the-box G-quadruplex-targeting approach to defeat WDLPS and all tumours where restoration of wild-type p53 is sought. They also point to G-quadruplex-dependent genomic instability as possible cause of MDM2 expansion and WDLPS tumorigenesis.

The Kidney Contains Ontogenetically Distinct Dendritic Cell and Macrophage Subtypes throughout Development That Differ in Their Inflammatory Properties.

BACKGROUND: Mononuclear phagocytes (MPs), including macrophages, monocytes, and dendritic cells (DCs), are phagocytic cells with important roles in immunity. The developmental origin of kidney DCs has been highly debated because of the large phenotypic overlap between macrophages and DCs in this tissue. METHODS: We used fate mapping, RNA sequencing, flow cytometry, confocal microscopy, and histo-cytometry to assess the origin and phenotypic and functional properties of renal DCs in healthy kidney and of DCs after cisplatin and ischemia reperfusion-induced kidney injury. RESULTS: Adult kidney contains at least four subsets of MPs with prominent Clec9a-expression history indicating a DC origin. We demonstrate that these populations are phenotypically, functionally, and transcriptionally distinct from each other. We also show these kidney MPs exhibit unique age-dependent developmental heterogeneity. Kidneys from newborn mice contain a prominent population of embryonic-derived MHCIInegF4/80hiCD11blow macrophages that express T cell Ig and mucin domain containing 4 (TIM-4) and MER receptor tyrosine kinase (MERTK). These macrophages are replaced within a few weeks after birth by phenotypically similar cells that express MHCII but lack TIM-4 and MERTK. MHCII+F4/80hi cells exhibit prominent Clec9a-expression history in adulthood but not early life, indicating additional age-dependent developmental heterogeneity. In AKI, MHCIInegF4/80hi cells reappear in adult kidneys as a result of MHCII downregulation by resident MHCII+F4/80hi cells, possibly in response to prostaglandin E2 (PGE2). RNA sequencing further suggests MHCII+F4/80hi cells help coordinate the recruitment of inflammatory cells during renal injury. CONCLUSIONS: Distinct developmental programs contribute to renal DC and macrophage populations throughout life, which could have important implications for therapies targeting these cells.

Cystatin B Involvement in Synapse Physiology of Rodent Brains and Human Cerebral Organoids.

Cystatin B (CSTB) is a ubiquitous protein belonging to a superfamily of protease inhibitors. CSTB may play a critical role in brain physiology because its mutations cause progressive myoclonic epilepsy-1A (EPM1A), the most common form of progressive myoclonic epilepsy. However, the molecular mechanisms underlying the role of CSTB in the central nervous system (CNS) are largely unknown. To investigate the possible involvement of CSTB in the synaptic plasticity, we analyzed its expression in synaptosomes as a model system in studying the physiology of the synaptic regions of the CNS. We found that CSTB is not only present in the synaptosomes isolated from rat and mouse brain cortex, but also secreted into the medium in a depolarization-controlled manner. In addition, using biorthogonal noncanonical amino acid tagging (BONCAT) procedure, we demonstrated, for the first time, that CSTB is locally synthesized in the synaptosomes. The synaptic localization of CSTB was confirmed in a human 3D model of cortical development, namely cerebral organoids. Altogether, these results suggest that CSTB may play a role in the brain plasticity and open a new perspective in studying the involvement of CSTB deregulation in neurodegenerative and neuropsychiatric diseases.

Pre-marked chromatin and transcription factor co-binding shape the pioneering activity of Foxa2.

Pioneer transcription factors (PTF) can recognize their binding sites on nucleosomal DNA and trigger chromatin opening for recruitment of other non-pioneer transcription factors. However, critical properties of PTFs are still poorly understood, such as how these transcription factors selectively recognize cell type-specific binding sites and under which conditions they can initiate chromatin remodelling. Here we show that early endoderm binding sites of the paradigm PTF Foxa2 are epigenetically primed by low levels of active chromatin modifications in embryonic stem cells (ESC). Priming of these binding sites is supported by preferential recruitment of Foxa2 to endoderm binding sites compared to lineage-inappropriate binding sites, when ectopically expressed in ESCs. We further show that binding of Foxa2 is required for chromatin opening during endoderm differentiation. However, increased chromatin accessibility was only detected on binding sites which are synergistically bound with other endoderm transcription factors. Thus, our data suggest that binding site selection of PTFs is directed by the chromatin environment and that chromatin opening requires collaboration of PTFs with additional transcription factors.

Point mutations in the PDX1 transactivation domain impair human ß-cell development and function.

OBJECTIVE: Hundreds of missense mutations in the coding region of PDX1 exist; however, if these mutations predispose to diabetes mellitus is unknown. METHODS: In this study, we screened a large cohort of subjects with increased risk for diabetes and identified two subjects with impaired glucose tolerance carrying common, heterozygous, missense mutations in the PDX1 coding region leading to single amino acid exchanges (P33T, C18R) in its transactivation domain. We generated iPSCs from patients with heterozygous PDX1P33T/+, PDX1C18R/+ mutations and engineered isogenic cell lines carrying homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations and a heterozygous PDX1 loss-of-function mutation (PDX1+/-). RESULTS: Using an in vitro ß-cell differentiation protocol, we demonstrated that both, heterozygous PDX1P33T/+, PDX1C18R/+ and homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations impair ß-cell differentiation and function. Furthermore, PDX1+/- and PDX1P33T/P33T mutations reduced differentiation efficiency of pancreatic progenitors (PPs), due to downregulation of PDX1-bound genes, including transcription factors MNX1 and PDX1 as well as insulin resistance gene CES1. Additionally, both PDX1P33T/+ and PDX1P33T/P33T mutations in PPs reduced the expression of PDX1-bound genes including the long-noncoding RNA, MEG3 and the imprinted gene NNAT, both involved in insulin synthesis and secretion. CONCLUSIONS: Our results reveal mechanistic details of how common coding mutations in PDX1 impair human pancreatic endocrine lineage formation and ß-cell function and contribute to the predisposition for diabetes.

BZLF1 interacts with chromatin remodelers promoting escape from latent infections with EBV.

A hallmark of EBV infections is its latent phase, when all viral lytic genes are repressed. Repression results from a high nucleosome occupancy and epigenetic silencing by cellular factors such as the Polycomb repressive complex 2 (PRC2) and DNA methyltransferases that, respectively, introduce repressive histone marks and DNA methylation. The viral transcription factor BZLF1 acts as a molecular switch to induce transition from the latent to the lytic or productive phase of EBV's life cycle. It is unknown how BZLF1 can bind to the epigenetically silenced viral DNA and whether it directly reactivates the viral genome through chromatin remodeling. We addressed these fundamental questions and found that BZLF1 binds to nucleosomal DNA motifs both in vivo and in vitro. BZLF1 co-precipitates with cellular chromatin remodeler ATPases, and the knock-down of one of them, INO80, impaired lytic reactivation and virus synthesis. In Assay for Transposase-Accessible Chromatin-seq experiments, non-accessible chromatin opens up locally when BZLF1 binds to its cognate sequence motifs in viral DNA. We conclude that BZLF1 reactivates the EBV genome by directly binding to silenced chromatin and recruiting cellular chromatin-remodeling enzymes, which implement a permissive state for lytic viral transcription. BZLF1 shares this mode of action with a limited number of cellular pioneer factors, which are instrumental in transcriptional activation, differentiation, and reprogramming in all eukaryotic cells.

The Aryl Hydrocarbon Receptor Pathway Defines the Time Frame for Restorative Neurogenesis.

Zebrafish have a high capacity to replace lost neurons after brain injury. New neurons involved in repair are generated by a specific set of glial cells, known as ependymoglial cells. We analyze changes in the transcriptome of ependymoglial cells and their progeny after injury to infer the molecular pathways governing restorative neurogenesis. We identify the aryl hydrocarbon receptor (AhR) as a regulator of ependymoglia differentiation toward post-mitotic neurons. In vivo imaging shows that high AhR signaling promotes the direct conversion of a specific subset of ependymoglia into post-mitotic neurons, while low AhR signaling promotes ependymoglial proliferation. Interestingly, we observe the inactivation of AhR signaling shortly after injury followed by a return to the basal levels 7 days post injury. Interference with timely AhR regulation after injury leads to aberrant restorative neurogenesis. Taken together, we identify AhR signaling as a crucial regulator of restorative neurogenesis timing in the zebrafish brain.

Genome-wide analysis of PDX1 target genes in human pancreatic progenitors.

OBJECTIVE: Homozygous loss-of-function mutations in the gene coding for the homeobox transcription factor (TF) PDX1 leads to pancreatic agenesis, whereas heterozygous mutations can cause Maturity-Onset Diabetes of the Young 4 (MODY4). Although the function of Pdx1 is well studied in pre-clinical models during insulin-producing ß-cell development and homeostasis, it remains elusive how this TF controls human pancreas development by regulating a downstream transcriptional program. Also, comparative studies of PDX1 binding patterns in pancreatic progenitors and adult ß-cells have not been conducted so far. Furthermore, many studies reported the association between single nucleotide polymorphisms (SNPs) and T2DM, and it has been shown that islet enhancers are enriched in T2DM-associated SNPs. Whether regions, harboring T2DM-associated SNPs are PDX1 bound and active at the pancreatic progenitor stage has not been reported so far. METHODS: In this study, we have generated a novel induced pluripotent stem cell (iPSC) line that efficiently differentiates into human pancreatic progenitors (PPs). Furthermore, PDX1 and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify PDX1 transcriptional targets and active enhancer and promoter regions. To address potential differences in the function of PDX1 during development and adulthood, we compared PDX1 binding profiles from PPs and adult islets. Moreover, combining ChIP-seq and GWAS meta-analysis data we identified T2DM-associated SNPs in PDX1 binding sites and active chromatin regions. RESULTS: ChIP-seq for PDX1 revealed a total of 8088 PDX1-bound regions that map to 5664 genes in iPSC-derived PPs. The PDX1 target regions include important pancreatic TFs, such as PDX1 itself, RFX6, HNF1B, and MEIS1, which were activated during the differentiation process as revealed by the active chromatin mark H3K27ac and mRNA expression profiling, suggesting that auto-regulatory feedback regulation maintains PDX1 expression and initiates a pancreatic TF program. Remarkably, we identified several PDX1 target genes that have not been reported in the literature in human so far, including RFX3, required for ciliogenesis and endocrine differentiation in mouse, and the ligand of the Notch receptor DLL1, which is important for endocrine induction and tip-trunk patterning. The comparison of PDX1 profiles from PPs and adult human islets identified sets of stage-specific target genes, associated with early pancreas development and adult ß-cell function, respectively. Furthermore, we found an enrichment of T2DM-associated SNPs in active chromatin regions from iPSC-derived PPs. Two of these SNPs fall into PDX1 occupied sites that are located in the intronic regions of TCF7L2 and HNF1B. Both of these genes are key transcriptional regulators of endocrine induction and mutations in cis-regulatory regions predispose to diabetes. CONCLUSIONS: Our data provide stage-specific target genes of PDX1 during in vitro differentiation of stem cells into pancreatic progenitors that could be useful to identify pathways and molecular targets that predispose for diabetes. In addition, we show that T2DM-associated SNPs are enriched in active chromatin regions at the pancreatic progenitor stage, suggesting that the susceptibility to T2DM might originate from imperfect execution of a ß-cell developmental program.

Loss of Uhrf1 in neural stem cells leads to activation of retroviral elements and delayed neurodegeneration.

In order to understand whether early epigenetic mechanisms instruct the long-term behavior of neural stem cells (NSCs) and their progeny, we examined Uhrf1 (ubiquitin-like PHD ring finger-1; also known as Np95), as it is highly expressed in NSCs of the developing brain and rapidly down-regulated upon differentiation. Conditional deletion of Uhrf1 in the developing cerebral cortex resulted in rather normal proliferation and neurogenesis but severe postnatal neurodegeneration. During development, deletion of Uhrf1 lead to global DNA hypomethylation with a strong activation of the intracisternal A particle (IAP) family of endogenous retroviral elements, accompanied by an increase in 5-hydroxymethylcytosine. Down-regulation of Tet enzymes rescued the IAP activation in Uhrf1 conditional knockout (cKO) cells, suggesting an antagonistic interplay between Uhrf1 and Tet on IAP regulation. As IAP up-regulation persists into postnatal stages in the Uhrf1 cKO mice, our data show the lack of means to repress IAPs in differentiating neurons that normally never express Uhrf1 The high load of viral proteins and other transcriptional deregulation ultimately led to postnatal neurodegeneration. Taken together, these data show that early developmental NSC factors can have long-term effects in neuronal differentiation and survival. Moreover, they highlight how specific the consequences of widespread changes in DNA methylation are for certain classes of retroviral elements.

Production of Small Noncoding RNAs from the flamenco Locus Is Regulated by the gypsy Retrotransposon of Drosophila melanogaster.

Protective mechanisms based on RNA silencing directed against the propagation of transposable elements are highly conserved in eukaryotes. The control of transposable elements is mediated by small noncoding RNAs, which derive from transposon-rich heterochromatic regions that function as small RNA-generating loci. These clusters are transcribed and the precursor transcripts are processed to generate Piwi-interacting RNAs (piRNAs) and endogenous small interfering RNAs (endo-siRNAs), which silence transposable elements in gonads and somatic tissues. The flamenco locus is a Drosophila melanogaster small RNA cluster that controls gypsy and other transposable elements, and has played an important role in understanding how small noncoding RNAs repress transposable elements. In this study, we describe a cosuppression mechanism triggered by new euchromatic gypsy insertions in genetic backgrounds carrying flamenco alleles defective in gypsy suppression. We found that the silencing of gypsy is accompanied by the silencing of other transposons regulated by flamenco, and of specific flamenco sequences from which small RNAs against gypsy originate. This cosuppression mechanism seems to depend on a post-transcriptional regulation that involves both endo-siRNA and piRNA pathways and is associated with the occurrence of developmental defects. In conclusion, we propose that new gypsy euchromatic insertions trigger a post-transcriptional silencing of gypsy sense and antisense sequences, which modifies the flamenco activity. This cosuppression mechanism interferes with some developmental processes, presumably by influencing the expression of specific genes.