Amyotrophic lateral sclerosis (ALS) is a devastating adult onset neurodegenerative disorder characterized by the selective degeneration of upper and lower motor neurons. Patients typically die from respiratory failures within 2-5 years after diagnosis. One of the milestones in ALS research is the discovery Fused in Sarcoma (FUS), an ALS causative gene. FUS is an RNA/DNA-binding protein and predominantly resides in the nucleus. Majority of the FUS mutations are located in the C-terminus and causing aberrant misdistribution to the cytoplasm. Currently, only a few binding partners of FUS are known, which makes it difficult to speculate on the function and interaction of the protein. In this study, we conducted a kinome-wide RNAi screen to identify kinases that affect the localization of FUS. A dual specificity protein kinase named CDC2-like kinase (CLK1) from the screen was found to be responsible for in post-translational modification of FUS and affects the localization of FUS in the nucleus. The identification of CLK1 as FUSmodifying kinase is consistent with roles ascribed to both in the binding and regulation of RNA.

Hedgehog (Hh) est un morphogène conservé au cours de l’évolution, et qui est impliqué dans un grand nombre de processus développementaux. Ma thèse vise à comprendre comment la sécrétion, et la mise en place du gradient d’Hh sont régulées à partir de son site de production, en utilisant la drosophile comme modèle animal. Pour identifier les régulateurs positifs impliqués dans la maturation du signal Hh, nous avons conçu et réalisé un crible génétique couvrant l’ensemble du génome, par ARNs interférents (ARNi). Grâce à ce crible, nous avons identifié la petite protéine GTPase Rab8 qui serait impliquée spécifiquement dans le routage intracellulaire de Hh. Selon notre modèle proposé, la protéine Hh serait secrétée de 2 façons. Sa sécrétion du coté apical est nécessaire à l'activation de gènes cibles à longue distance, alors que sa sécrétion du coté baso-latéral permettrait l'activation de gènes cibles à courte distance. La façon par laquelle Hh est transportée de la membrane apicale à la membrane basale à l’intérieur des cellules productrices n’est pas connue. La perte de fonction de Rab8 dans les cellules productrices de Hh induit une augmentation de l’activation des gènes cibles à courtes distances, alors l’expression des gènes cibles activés à longues distances est réduite. De plus, en utilisant des expériences sur tissus vivant pour suivre la dynamique de l'internalisation de la protéine endogène d’Hh, nous avons constaté que la perte de Rab8 n'a pas d’effet sur sa sécrétion primaire, mais entraine des défauts dans l’endocytose de Hh, affectant, par la suite, la mise en place du gradient morphogénétique. / Hedgehog (Hh) is a conserved secreted morphogen involved in an array of developmental processes. Using Drosophila as a model, during my thesis we aimed to ask how the secretion, extraction and transport of Hh protein are regulated at the site of its production. To understand the positive regulators of Hh secretion and transport we designed and performed a genome-wide RNAi screen in Drosophila to identify new regulators of Hh transport and identified the small GTPase Rab8 as a novel component required for Hh trafficking. According to our proposed model, there are two pools of secreted Hh. The apical pool is needed for long range target gene activation, and basolateral pool for short range target gene activation. It is not clear how Hh is sorted apico-basally in the producing cells. Interfering with Rab8 function in the Hh producing cells extends Hh short range targets. Conversely, it reduces the long range Hh targets, suggesting that interfering with Rab8 function in the Hh producing cells impairs Hh trafficking, thus hampering the fine tuning between the two secreted pools of Hh. Moreover, using live assays to track the dynamics of endogenous Hh internalization, we observed that loss of Rab8 in Hh producing cells does not affect its primary secretion, but causes defects in Hh endocytosis, subsequently affecting its gradient activity. We hypothesize a model where Hh is targeted for primary secretion to the apical side of the wing disc, which then is internalized, and this internalized Hh is then directed for recycling which is essential for its long range activity.

Le réseau de signalisation qui répond à l’insuline et aux nutriments est conservé chez les métazoaires, où il joue un rôle central dans le contrôle du métabolisme et de la croissance. Les nutriments assimilés sont soit directement utilisés pour la croissance tissulaire, soit stockés principalement sous forme de triglycérides. Chez la drosophile, l’activation de ce réseau de signalisation dans le corps gras, un organe qui remplit à la fois les fonctions hépatiques et destockage, induit une augmentation du stockage de lipides sous forme de nombreuses gouttelettes lipidiques (LDs). A l’inverse, la carence alimentaire se traduit par une augmentation de la taille des LDs et une diminution de lipides stockés. La kinase TOR (TargetOf Rapamycine) et son substrat S6 Kinase (S6K) jouent un rôle central dans cette régulation.Chez la drosophile, ces 2 kinases (dTOR et dS6K) contrôlent les aspects autonome-cellulaireset hormonaux de la croissance. En dépit de nombreuses études sur divers organismes modèles,destinées à comprendre les mécanismes régulateurs de S6K, rien n’est connu à ce jour sur lecontrôle de sa dégradation.Nous avons utilisé une banque de lignées exprimant des ARN interférant (RNAi) contre unegrande quantité de gènes de la drosophile, pour réaliser 3 des cribles génétiques destinés à identifier de nouveaux régulateurs du métabolisme et de la croissance. Dans le premier crible,les RNAi ont été induits dans la glande prothoracique, siège de la production de l’hormonestéroïde ecdysone connue pour réguler la croissance et les étapes du développement, souscontrôle de la nutrition et de la signalisation dTOR. Sur 7000 gènes criblés, 620 ont étéidentifiés comme nécessaire à la production d’ecdysone. Dans le second crible, nous avonsexprimé les RNAi de 4000 gènes dans le corps gras pour rechercher ceux qui induisaient uneaugmentation de la taille des LDs. L’objectif était d’identifier des gènes impliqués dans la réponse à la carence alimentaire, et nous avons ainsi retenu 24 candidats intéressants. Le troisième crible représente la majeure partie du travail de thèse, où nous avons criblé les RNAi susceptibles de modifier un phénotype de croissance induit par dS6K. Sur 7000 gènes testés,nous en avons retenu 45 qui ont ensuite été utilisés pour générer un diagramme d’interaction en utilisant les informations disponibles dans les banques de données. Les candidats les plus intéressants ont ensuite été analysés en culture de cellules pour identifier ceux qui régulent l’activité de dS6K et ceux qui régulent sont niveau d’expression. Parmi ces derniers, nousavons identifié le gène codant pour Archipelago (Ago), connue pour contrôler la dégradationrégulée des protéines-cibles au niveau du protéasome. Nous avons réalisé de nombreusesexpériences qui montrent que ago et dS6K interagissent génétiquement. En outre, il est indiquédans les banques de données que ces protéines interagissent entre elles par la technique des 2-hybrides en levure. Tous ces résultats révèlent que Ago régule la dégradation de dS6K, etposent les premières pierres de ce niveau de régulation. / The evolutionary conserved insulin and nutrient signaling network regulates growth andmetabolism. Nutrients are directly utilized for growth or stored, mostly as triglycerides. InDrosophila, activation of insulin/nutrient signaling in the fat body (the fly equivalent of liverand adipose tissue), causes an increase in fat stores composed of several small-size lipiddroplets (LDs). Conversely, fasting produces an increase in LD size and a decrease in fatcontents. The TOR kinase and its substrate S6 kinase (S6K) play a central role in this response,and particularly in Drosophila, they have been shown to orchestrate cell-autonomous andhormone-controlled growth. However, despite extensive research studies on different modelorganisms (mouse, fly, worm) to decipher the molecular and physiological functions of S6K,nothing is known about how its degradation is regulated.Taking advantage of the inducible RNA interfering (RNAi) library from NIG (Japan), we haveperformed three genetic screens to identify novel regulators of steroidogenesis, lipidmetabolism and dS6K-dependent growth. First, RNAi lines were screened in the ring gland; anorgan that controls the progression of the developmental steps by producing the steroidhormone ecdysone. Out of 7,000 genes screened, 620 positive candidates were identified toproduce developmental arrest and/or overgrowth phenotypes. Then, we challenged 4,000 genesby RNAi screening able to recapitulate the larger sized LD phenotype as obtained uponstarvation, leading to the identification of 24 potential candidates. Finally, the RNAi lines werescreened for their ability to enhance a growth phenotype dependent of the Drosophila S6K(dS6K). Out of 7,000 genes screened, 45 genes were identified as potential negative regulatorsof dS6K. These genes were further used to design a novel protein-protein interaction networkcentered on dS6K through the available data from yeast-2-hybrid (Y2H) assay. The most potentinteractors were then analyzed by treatment of cultured S2 cells with the corresponding doublestrand RNA (dRNA). Western blotting thus, allowed us to discriminate between the geneproducts that regulate dS6K levels versus those that regulate its phosphorylation, as a hallmarkfor its kinase activity. Interestingly, archipelago (ago), which encodes a component of an SCFubiquitinligase known to regulate the degradation of dMyc, Cyclin E and Notch, was identifiedas a negative regulator of dS6K-dependent growth. Based on the Y2H available data showingthat Ago and dS6K interact each other and the presence of a putative Ago-interaction motif indS6K, we hypothesized that Ago causes an ubiquitin-mediated degradation of dS6K. Ourmolecular data showed that loss of ago caused an elevated level of dS6K, which confirms arole of Ago in controlling dS6K degradation. Altogether our findings emphasize the importanceof the saturating screening strategies in Drosophila to identify novel regulators of metabolicand signaling pathways.

No description available.

<p>In order to propagate a solid tumor, cancer cells must adapt to and survive under various tumor microenvironment (TME) stresses, such as hypoxia or lactic acidosis. Additionally, cancer cells exposed to these stresses are more resistant to therapies, more likely to metastasize and often are worse for patient prognosis. While the presence of these stresses is generally negative for cancer patients, since these stresses are mostly unique to the TME, they also offer an opportunity to develop more selective therapeutics. If we achieve a better understanding of the adaptive mechanisms cancer cells employ to survive the TME stresses, then hopefully we, as a scientific community, can devise more effective cancer therapeutics specifically targeting cancer cells under stress. To systematically identify genes that modulate cancer cell survival under stresses, we performed shRNA screens under hypoxia or lactic acidosis. From these screens, we discovered that genetic depletion of acetyl-CoA carboxylase alpha (ACACA or ACC1) or ATP citrate lyase (ACLY) protected cancer cells from hypoxia-induced apoptosis. Furthermore, the loss of ACLY or ACC1 reduced the levels and activities of the oncogenic transcription factor ETV4. Silencing ETV4 also protected cells from hypoxia-induced apoptosis and led to remarkably similar transcriptional responses as with silenced ACLY or ACC1, including an anti-apoptotic program. Metabolomic analysis found that while α-ketoglutarate levels decrease under hypoxia in control cells, α-ketoglutarate was paradoxically increased under hypoxia when ACC1 or ACLY were depleted. Supplementation with α-ketoglutarate rescued the hypoxia-induced apoptosis and recapitulated the decreased expression and activity of ETV4, likely via an epigenetic mechanism. Therefore, ACC1 and ACLY regulated the levels of ETV4 under hypoxia via increased α-ketoglutarate. These results reveal that the ACC1/ACLY-α-ketoglutarate-ETV4 axis is a novel means by which metabolic states regulate transcriptional output for life vs. death decisions under hypoxia. Since many lipogenic inhibitors are under investigation as cancer therapeutics, our findings suggest that the use of these inhibitors will need to be carefully considered with respect to oncogenic drivers, tumor hypoxia, progression and dormancy. More broadly, our screen provides a framework for studying additional tumor cell stress-adaption mechanisms in the future.</p> / Dissertation

The evolutionary conserved insulin and nutrient signaling network regulates growth andmetabolism. Nutrients are directly utilized for growth or stored, mostly as triglycerides. InDrosophila, activation of insulin/nutrient signaling in the fat body (the fly equivalent of liverand adipose tissue), causes an increase in fat stores composed of several small-size lipiddroplets (LDs). Conversely, fasting produces an increase in LD size and a decrease in fatcontents. The TOR kinase and its substrate S6 kinase (S6K) play a central role in this response,and particularly in Drosophila, they have been shown to orchestrate cell-autonomous andhormone-controlled growth. However, despite extensive research studies on different modelorganisms (mouse, fly, worm) to decipher the molecular and physiological functions of S6K,nothing is known about how its degradation is regulated.Taking advantage of the inducible RNA interfering (RNAi) library from NIG (Japan), we haveperformed three genetic screens to identify novel regulators of steroidogenesis, lipidmetabolism and dS6K-dependent growth. First, RNAi lines were screened in the ring gland; anorgan that controls the progression of the developmental steps by producing the steroidhormone ecdysone. Out of 7,000 genes screened, 620 positive candidates were identified toproduce developmental arrest and/or overgrowth phenotypes. Then, we challenged 4,000 genesby RNAi screening able to recapitulate the larger sized LD phenotype as obtained uponstarvation, leading to the identification of 24 potential candidates. Finally, the RNAi lines werescreened for their ability to enhance a growth phenotype dependent of the Drosophila S6K(dS6K). Out of 7,000 genes screened, 45 genes were identified as potential negative regulatorsof dS6K. These genes were further used to design a novel protein-protein interaction networkcentered on dS6K through the available data from yeast-2-hybrid (Y2H) assay. The most potentinteractors were then analyzed by treatment of cultured S2 cells with the corresponding doublestrand RNA (dRNA). Western blotting thus, allowed us to discriminate between the geneproducts that regulate dS6K levels versus those that regulate its phosphorylation, as a hallmarkfor its kinase activity. Interestingly, archipelago (ago), which encodes a component of an SCFubiquitinligase known to regulate the degradation of dMyc, Cyclin E and Notch, was identifiedas a negative regulator of dS6K-dependent growth. Based on the Y2H available data showingthat Ago and dS6K interact each other and the presence of a putative Ago-interaction motif indS6K, we hypothesized that Ago causes an ubiquitin-mediated degradation of dS6K. Ourmolecular data showed that loss of ago caused an elevated level of dS6K, which confirms arole of Ago in controlling dS6K degradation. Altogether our findings emphasize the importanceof the saturating screening strategies in Drosophila to identify novel regulators of metabolicand signaling pathways.

For all living systems, there is a requirement to recycle and regulate proteins. In eukaryotic organisms this is accomplished by the proteasome. The p97 ATPase is another highly conserved and essential complex present throughout the eukaryotic cell. In Paper I we utilized UFD fluorescent substrates to address the role of p97 and cofactors in soluble proteasome degradation. Results using RNAi and Drosophila p97 mutants propose p97 to function upstream of the proteasome on cytosolic proteasome targets as an important unfoldase together with its Ufd1/Npl4 cofactors. The results implicate p97 to be important for degradation of proteasome substrates lacking natural extended peptide regions. In Paper II we focused on identifying transcription factors essential for production of proteasomal subunits and associated proteins in Drosophila S2 cells. We utilized an RNA library targeting 993 known or candidate transcription factors and monitored RNAi depleted Drosophila S2 cells expressing the UFD reporter UbG76VGFP. We identified a range of potential candidates and focused on the bZIP transcription factor Cnc-C. RNAi and qrt-PCR experiments implicated Cnc-C to be involved in transcription of proteasomal subunits. In Paper III we applied our knowledge gained from Paper I about p97 dependent substrates and set up a high-throughput microscopy screening method to potentially find inhibitors specifically targeting the p97 proteasomal sub-pathway. Utilizing UFD substrates with and without C-terminal peptide tails we determined if compounds inhibited the core proteasomal machinery or the p97 pathway specifically. Through a primary and secondary round of screening we identified several new compounds inhibiting the ubiquitin-proteasome pathway though none from our initial screening had specificity for p97. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.

L’établissement et le maintien du patron d’expression génique sont d’une importance critique pour l’identité cellulaire. Les protéines du groupe Polycomb (PcG) agissent sur la chromatine afin de maintenir la répression génique de ses gènes cibles à travers les cycles cellulaires de façon épigénétique. Toutefois, durant la mitose, la structure de la chromatine est grandement altérée par la répression de la transcription, la condensation de la chromatine et le relâchement de nombreux facteurs de transcription. Une question se pose alors : comment les protéines PcG peuvent-elles maintenir leur fonction à travers la mitose ? En interphase, les protéines PcG sont liées à leurs cibles sur la chromatine. Durant la mitose, la majorité des protéines PcG se libèrent de la chromatine mais une petite fraction persiste. Selon l’hypothèse du mitotic bookmarking, cette fraction agirait comme un ensemble de marqueurs guidant le recrutement des protéines PcG en fin de mitose pour maintenir le profil d’expression génique de la cellule. Cependant, nous ne savons pas comment ce recrutement à lieu, ni comment une fraction de protéines PcG est retenue à la chromatine. Afin de répondre à ces questions, un crible à ARN interférent a été établi pour identifier des facteurs contrôlant la liaison des protéines PcG à la chromatine à travers le cycle cellulaire. Quoiqu’une confirmation soit nécessaire, les facteurs spécifiques à l’interphase sont enrichis en protéines co-purifiant avec la protéine PcG testée et en hélicases alors que ceux spécifiques à la mitose sont enrichis en candidats liés aux protéines du groupe Trithorax (TrxG). / A critical part of cell identity is the establishment and maintenance of gene expression patterns. Polycomb group proteins (PcG) act on chromatin to maintain gene repression through cell cycles (epigenetically). However, during mitosis, chromatin structure is greatly altered by transcription repression, chromatin condensation, and the release of many transcription factors. A question then arises: how can PcG proteins maintain their function through mitosis? During interphase, PcG proteins are bound to their chromatin targets. During mitosis, most PcG proteins are released from chromatin, but a small fraction remains bound to chromatin. According to the mitotic bookmarking hypothesis, this fraction acts as a set of markers to guide the recruitment of PcG proteins at the end of mitosis to maintain the gene expression profile. However, we do not know how this recruitment takes place, nor do we know how a fraction of PcG proteins is retained on chromatin. To address these questions, an RNAi screen was established to identify factors that control the binding of PcG proteins to chromatin across the cell cycle. Although a confirmation is necessary, factors identified from interphase cells were enriched in proteins co-purifying with the tested PcG protein and in helicases while mitosis specific factors were enriched in Trithorax group (TrxG) protein related candidates.

Le sarcome de Ewing est la seconde tumeur pédiatrique de l’os la plus fréquente. Elle est caractérisée par une translocation chromosomique résultant à la fusion de EWSR1 avec un membre de la famille ETS. Chez 85% des patients, cette fusion conduit à l’expression de la protéine chimérique EWS-FLI1 qui est l’oncogène majeur de ce sarcome. Ce dernier agit principalement par son action transcriptionelle sur des cibles qui lui sont propres. Au niveau thérapeutique, le sarcome d’Ewing est traité par chimiothérapie, chirurgie locale et par radiothérapie. La survie à long terme des patients est de l’ordre de 70%, mais beaucoup plus basse pour les patients métastatiques et quasi nulle lors d’une récidive. Parmi maintes caractéristiques, certains cancers présentent une dérégulation énergétique. L’influence d’EWS-FLI1 sur cet aspect n’a fait l’objet d’aucune étude dans le contexte du sarcome d’Ewing. Nous avons donc étudié par profilage métabolomique des cellules de sarcome d’Ewing en présence ou en absence d’EWS-FLI1. En comparant ces deux conditions, des modulations du profil énergétique relatif au cycle de Krebs, des précurseurs de le glycosylation ainsi que des métabolites de la voie de la méthionine et du tryptophane ont été observés. En parallèle, grâce à un crible de banque de shRNAs réalisé dans des conditions expérimentales similaires à l’étude métabolomique (lignée d’Ewing avec ou sans EWS-FLI1), nous avons pu identifier des gènes présentant des caractéristiques « synthétique létales », c'est-à-dire tuant uniquement les cellules du sarcome d’Ewing en présence de son oncogène. / Ewing sarcoma, the second most commonly occurring pediatric bone tumor, is most often characterized by a chromosomal translocation between EWSR1 and FLI1. The gene fusion EWS-FLI1 accounts for 85% of all Ewing sarcoma and is considered the major oncogene and master regulator of Ewing sarcoma. EWS-FLI1 is a transcriptional modulator of targets, both directly and indirectly. Ewing sarcoma is aggressively treated with chemotherapy, localized surgery and radiation and has an overall survival of about 70%, however, survival for metastasis or relapsed cases remains low. One of the cancer hallmarks, metabolic deregulation, is most likely partly dependent on EWS-FLI1 in Ewing sarcoma cells. In order to get a better understanding of Ewing sarcoma biology and oncogenesis, it might be of high interest to investigate the influence of EWS-FLI1 in Ewing sarcoma cells. We therefore performed a global metabolic profiling of Ewing sarcoma cells with or without inhibition of EWS-FLI1. Several changes in the energy metabolism were observed throughout this study; the observed changes were consistent with an energy profile that moved from a cancer cell energy metabolism towards the energy metabolism of a more normal cell upon EWS-FLI1 inhibition, primarily based on the TCA cycle. Levels of TCA intermediates, glycosylation precursors, methionine pathway metabolites and amino acids, especially changes in the tryptophan metabolic pathway, were altered upon EWS-FLI1 inhibition. Parallel to this study, we performed a high-throughput synthetic lethality screen, in order to not only identify essential genes for cell survival and proliferation, but also to identify new synthetic lethal targets that could specifically target Ewing sarcoma cells carrying the EWS-FLI1 fusion gene.

Der Gen-Knockdown mittels RNAi hat sich als essentiell erwiesen, um Inhibitoren der induzierten Transdifferenzierung in C. elegans zu identifizieren (Tursun et al., 2011). Bakterienstämme, die dsRNA exprimieren, das die Expression spezifischer Gene mindert, können dem Wurm direkt zugefüttert werden, um einen genomweiten RNAi-screen der insgesamt 20.000 Gene in C. elegans durchzuführen. Allerdings werden die meisten biologischen Prozese durch mehr als ein Gen reguliert, was den Bedarf nach einer Methode generiert, die es erlaubt, zwei oder mehr Gene gleichzeitig herunter zu regulieren, um die Steuerung biologischer Prozesse studieren zu können. Die derzeitig vorhandenen Methoden liefern entweder nicht reproduzierbare Ergebnisse oder sind nicht skalierbar. Wir nutzen baktierelle Konjugation, die es durch ein konjugatives Plasmid ermöglicht Bakterienzellen zu generieren, die zwei verschiedene RNAi-Plasmide enthalten. Das Ziel war es, modifizierte RNAi-Donor-Plasmide mittels bakterieller Konjugation an eine Vielzahl anderer Bakterienzellen zu übertragen, die bereits ein anderes RNAi-Plasmid enthalten und dies dann im Hochdurchsatzverfahren durchführen zu können. Um Enhancer induzierter Expression von unc-25::gfp in der Keimbahn, ermöglicht durch den Knockdown des Histonchaperons LIN-53 (RbAp46/48 in Menschen), zu finden, wurden RNAi-Klone generiert, die gleichzeitig lin-53 als auch eines von insgesamt 800 verschiedenen Chromatin-bezogenen Gene herunter regulieren. Dabei identifizierten wir RBBP-5, Mitglied des Set1/ MLL-Methyltransferase-Komplexes, als neuen Barrierefaktor der induzierten Transdifferenzierung. RBBP-5 agiert dabei mutmaßlich parallel zu LIN-53. Doppelte RNAi, ermöglicht durch bakterielle Konjugation, erlaubt den simultanen Knockdown zweier oder mehr Gene, um genetische Interaktionen studieren zu können und erweitert damit die Einsatzmöglichkeiten von RNAi-Screens, um untereinander verbundene biologische Prozesse zu studieren. / The knock down of genes by RNAi has been fundamental to identify inhibitors of induced cell transdifferentiation in C. elegans (Tursun et al., 2011). Bacteria strains expressing dsRNA that target specific genes can be fed to the worm allowing straightforward whole-genome RNAi screens of the 20,000 genes in theC. elegans genome. However, many biological processes are regulated by more than one gene raising the need for simultaneous knock down of two or more genes to more fully interrogate the regulation of complex biological processes. Two approaches are currently available for double RNAi knockdown, − two bacteria strains expressing specific dsRNA can be mixed and grown together and fed simultaneously, which gives highly variable results. Alternatively, a new bacterial clone can be generated carrying a plasmid on which two RNAi targets of interest are 'stitched' together, which is not scalable. To address this challenge, we have developed a protocol using bacterial conjugation mediated by the 'Fertility Factor' (F) Episome in order to combine two different RNAi plasmids in a single bacterium. The objective was to be able to transfer a single RNAi plasmid to a large number of bacterial cells carrying different RNAi clones in one step in a high-throughput manner for large scale 'double' or even 'triple' RNAi screens. To find enhancers of induced unc-25::gfp expression in the germ line enabled by the depletion of histone chaperone LIN-53 (RbAp46/48 in humans), double RNAi clones targeting lin-53 and a total of 800 chromatin-related genes were generated and screened. We identified the Set1/MLL methyltransferase complex member RBBP-5 as a novel reprogramming barrier that putatively acts in a parallel pathway to LIN-53. Double RNAi by conjugation permits to reliably knock down two genes simultaneously in order to study genetic interactions at a genome-wide level, thus further increasing the versatility of RNAi screens to investigate interconnected biological processes.