Transgenic mosquitoes are beneficial for the design and implementation of various pathogen control programs. However, low and variable expression of transgenes caused by position effects is a hindrance to the characterization and effective use of transgenes in mosquito species. The use of insulator sequences to flank transgenes may have the ability to overcome position effects caused by the genomic environment surrounding the insertion site. CTCF is a multifunctional protein, conserved from humans to Drosophila. Its role as an enhancer blocker in the Drosophila bithorax complex and its proximal binding to other insulator proteins on Drosophila chromosomes makes it a good candidate for identifying insulator sequences throughout the mosquito genome that may be used to improve mosquito transgenesis. Its multi-functionality as a transcription factor and genome organizer also makes CTCF worthy of investigation for an improved understanding of the regulation of the mosquito genome. This study uses chromatin immunoprecipitation with an An. gambiae CTCF antibody followed by Illumina deep sequencing (ChIP-Seq) to identify regions of CTCF binding throughout the An. gambiae genome. A subset of the CTCF binding site peaks was validated using ChIP-PCR. Another subset of this data set, including the ChIP-PCR validated peaks, was input into the motif finding tool, AlignACE, in order to identify a CTCF binding site consensus. Four motifs were identified, none of which were found in more than 11.9% of the ChIP-Seq data set. These results lead us to conclude that An. gambiae CTCF binds to a wider variety of sequences compared to Drosophila CTCF. This work also includes a comparison of the expression profiles of the dipteran insulator proteins, Su(Hw) and CP190, with that of CTCF across multiple life stages in Ae. aegypti. The results of this study suggest the possibility of genomic colocalization, as has been recently discovered in Drosophila. The identification of CTCF binding site peaks throughout the An. gambiae genome provides a large data set of potential insulator sequences that may be used to improve mosquito transgenesis, and provide a new model for the study of CTCF function in a species with medical significance.

Chromatin remodeling protein Smarca5 participates on many cellular processes, which are important for tissue development and tumorigenesis. Among these processes utilizing ATPase activity of Smarca5 belong also transcription, replication and DNA repair. We hypothesized that Smarca5 represents essential molecule for chromatin modulation primarily at early developmental stages at the level of fast-dividing progenitors of many origins, in whose the ATPase is highly expressed. To such tissues may belong also hematopoiesis, in which the Smarca5 has highest expression. The subject of my doctoral thesis is therefore analysis of the effect Smarca5 depletion on proliferation and differentiation of hematopoietic progenitors in vivo and a search for mechanisms behind the resulted developmental defects. We utilized conditionally knockout allele of Smarca5 in blood precursors to study in a mouse model how depletion of the ISWI ATPase causes accumulation of earliest progenitors inhibited from further maturation to erythroid and other myeloid lines. The proerythroblasts became dysplastic and the majority of basophilic erythroblasts ceased cycling around the G2/M stage. An expected mechanism for observed changes appeared the activation of stress pathway of protein p53 that is often associated with unrepaired DNA...

MLL/KMT2A amplifications and translocations are prevalent in infant, adult and therapy-induced leukemia. However, the molecular contributor(s) to these alterations are unclear. Here we demonstrate that histone H3 lysine 9 mono- and di-methylation (H3K9me1/2) balance at the MLL/KMT2A locus regulates these amplifications and rearrangements. This balance is controlled by the cross-talk between lysine demethylase KDM3B and methyltransferase G9a/EHMT2. KDM3B depletion increases H3K9me1/2 levels and reduces CTCF occupancy at the MLL/KMT2A locus, and in turn, promotes amplification and rearrangements. Depleting CTCF is also sufficient to generate these focal alterations. Furthermore, the chemotherapy Doxorubicin (Dox), which associates with therapy-induced leukemia and promotes MLL/KMT2A amplifications and rearrangements, suppresses KDM3B and CTCF protein levels. KDM3B and CTCF overexpression rescues Dox-induced MLL/KMT2A alterations. G9a inhibition in human cells or mice also suppresses MLL/KMT2A events accompanying Dox treatment. Therefore, MLL/KMT2A amplifications and rearrangements are controlled by epigenetic regulators that are tractable drug targets, which has clinical implications. The data presented in this thesis were published in Cell in 2023. / Biomedical Sciences

Os antígenos cancer-testis (CT) são proteínas imunogênicas expressas em tecido gametogênico e em diferentes tipos de tumor, sendo considerados candidatos promissores para a imunoterapia do câncer. Entretanto, pouco se sabe sobre a função desses antígenos na tumorigênese. Em 2006, identificamos CTSP-1 como um novo antígeno CT, frequentemente expresso em vários tumores. Nesse trabalho, investigamos a função de CTSP-1 por meio da identificação de proteínas expressas em tumores de próstata e que são capazes de interagir fisicamente com esse antígeno. Demonstramos que CTSP-1 interage com a proteína CTCF em ensaios de duplo-híbrido em leveduras, pulldown e de co-localização e, em seguida, analisamos o impacto da superexpressão de CTSP-1 no controle da expressão de genes CT mediada por CTCF e na progressão do ciclo celular. Utilizando o CT NY-ESO-1 como modelo, demonstramos que a superexpressão de CTSP-1 não altera os níveis endógenos de NY-ESO-1 na linhagem celular tumoral H1299. Por outro lado, observamos que a superexpressão de CTSP-1 48h após as transfecções em H1299 induz um bloqueio do ciclo em G0/G1, reduzindo a capacidade clonogênica dessas células por um mecanismo dependente dos níveis de expressão de CTSP-1. Resultados semelhantes não foram observados em ensaios com clones superexpressando CTSP-1 estavelmente, o que sugere que eles tenham se originado de células que conseguiram escapar do bloqueio em G0/G1. Resultados preliminares sugerem que a redução da capacidade clonogênica das células H1299 que superexpressam CTSP-1 48h após as tansfecções não está associada à ocorrência de morte por apoptose. / Cancer-testis (CT) antigens are immunogenic proteins expressed in gametogenic tissues and in different histological types of tumors, being considered promising candidates for cancer immunotherapy. However, little is known about their role in tumorigenesis. In 2006, we identified CTSP-1 as a novel CT antigen, frequently expressed in different types of tumors. In this work, we investigated the functional role of CTSP-1 through the identification of proteins expressed in prostate tumors and that physically interact with this tumor antigen. We demonstrate that CTSP-1 interacts with the CTCF protein using the yeast two-hybrid system, pulldown and co-localization assays and have further analyzed the impact of CTSP-1 overexpression on the expression of CT genes mediated by CTCF and on the cell cycle progression. Using the CT antigen NY-ESO-1 as a model, we showed that the CTSP-1 overexpression does not alter the endogenous levels of NY-ESO-1 in the tumor cell line H1299. On the other hand, we observed that the overexpression of CTSP-1 in H1299 cells 48h after the transfections induces a cell cycle arrest in G0/G1 and reduces the clonogenic capacity of these cells by a mechanism dependent on the CTSP-1 expression levels. Similar results were not observed for cell clones stably overexpressing CTSP-1, suggesting that these clones have arisen from cells that managed to escape cell cycle arrest in G0/G1. Preliminary results suggest that the reduced clonogenic capacity of H1299 cells expressing CTSP-1 and analyzed 48h after the transfections is not associated with cell death by apoptosis.

Os antígenos cancer-testis (CT) são proteínas imunogênicas expressas em tecido gametogênico e em diferentes tipos de tumor, sendo considerados candidatos promissores para a imunoterapia do câncer. Entretanto, pouco se sabe sobre a função desses antígenos na tumorigênese. Em 2006, identificamos CTSP-1 como um novo antígeno CT, frequentemente expresso em vários tumores. Nesse trabalho, investigamos a função de CTSP-1 por meio da identificação de proteínas expressas em tumores de próstata e que são capazes de interagir fisicamente com esse antígeno. Demonstramos que CTSP-1 interage com a proteína CTCF em ensaios de duplo-híbrido em leveduras, pulldown e de co-localização e, em seguida, analisamos o impacto da superexpressão de CTSP-1 no controle da expressão de genes CT mediada por CTCF e na progressão do ciclo celular. Utilizando o CT NY-ESO-1 como modelo, demonstramos que a superexpressão de CTSP-1 não altera os níveis endógenos de NY-ESO-1 na linhagem celular tumoral H1299. Por outro lado, observamos que a superexpressão de CTSP-1 48h após as transfecções em H1299 induz um bloqueio do ciclo em G0/G1, reduzindo a capacidade clonogênica dessas células por um mecanismo dependente dos níveis de expressão de CTSP-1. Resultados semelhantes não foram observados em ensaios com clones superexpressando CTSP-1 estavelmente, o que sugere que eles tenham se originado de células que conseguiram escapar do bloqueio em G0/G1. Resultados preliminares sugerem que a redução da capacidade clonogênica das células H1299 que superexpressam CTSP-1 48h após as tansfecções não está associada à ocorrência de morte por apoptose. / Cancer-testis (CT) antigens are immunogenic proteins expressed in gametogenic tissues and in different histological types of tumors, being considered promising candidates for cancer immunotherapy. However, little is known about their role in tumorigenesis. In 2006, we identified CTSP-1 as a novel CT antigen, frequently expressed in different types of tumors. In this work, we investigated the functional role of CTSP-1 through the identification of proteins expressed in prostate tumors and that physically interact with this tumor antigen. We demonstrate that CTSP-1 interacts with the CTCF protein using the yeast two-hybrid system, pulldown and co-localization assays and have further analyzed the impact of CTSP-1 overexpression on the expression of CT genes mediated by CTCF and on the cell cycle progression. Using the CT antigen NY-ESO-1 as a model, we showed that the CTSP-1 overexpression does not alter the endogenous levels of NY-ESO-1 in the tumor cell line H1299. On the other hand, we observed that the overexpression of CTSP-1 in H1299 cells 48h after the transfections induces a cell cycle arrest in G0/G1 and reduces the clonogenic capacity of these cells by a mechanism dependent on the CTSP-1 expression levels. Similar results were not observed for cell clones stably overexpressing CTSP-1, suggesting that these clones have arisen from cells that managed to escape cell cycle arrest in G0/G1. Preliminary results suggest that the reduced clonogenic capacity of H1299 cells expressing CTSP-1 and analyzed 48h after the transfections is not associated with cell death by apoptosis.

La dystrophie Facio-Scapulo-Humérale est caractérisée par une faiblesse musculaire progressive et asymétrique. Elle affecte principalement les muscles faciaux, scapulaires et huméraux. L’association de plusieurs évènements épigénétiques à trois facteurs génétiques de la région subtélomérique du chromosome 4 résulte en un changement dans l’organisation chromatinienne la rendant permissive à l’expression aberrante des gènes de la région 4q35. Les myoblastes DFSH présentent des défauts de différenciation in vitro et des dérégulations dans des voies majeures comme celle de la réponse cellulaire au stress oxydant et de la différenciation myogénique. L’enjeu génétique et épigénétique complexe dans la DFSH et les limitations de la thérapie cellulaire dans son contexte laissent la DFSH jusque-là incurable. Toutefois les avancées dans les thérapies cellulaires et génétiques des myopathies ouvrent des horizons pour de futures applications dans le cadre de la DFSH.Le travail de thèse s’articule autour de trois thématiques. Premièrement, nous démontrons la faisabilité de la correction phénotypique et fonctionnelle des myotubes DFSH in vitro par la fusion de 50% de myoblastes normaux avec des myoblastes DFSH. Ensuite, nous évaluons deux approches d’édition génomique. Dans la première approche, nous ciblons le site de rattachement du chromosome 4 à la matrice nucléaire, FR-MAR avec la protéine CTCF à l’aide du système CRISPR/dCas9 en vue du rétablissement de l’organisation chromatinienne et de la fonction isolatrice de FR-MAR. Dans la deuxième, nous échangeons par translocation les régions homologues 4q35 et 10q26 dans le but de corriger les myoblastes DFSH comme les trois facteurs génétiques du locus 4q35 ne sont pathogéniques que sur un fond génétique lié au chromosome 4. Finalement, nous étudions le rôle du stress oxydant dans la DFSH. / Facio-Scapulo-Humeral dystrophy is characterized by progressive and asymmetrical muscle weakness. It mainly affects the facial, scapular and humeral muscles. The association of several epigenetic events with three genetic factors of the subtelomeric region of chromosome 4 results in a chromatin organization modification making it permissive to the aberrant expression of genes in the 4q35 region. FSHD myoblasts exhibit differentiation defects in vitro and dysregulations in major pathways such as the cellular response to oxidative stress and myogenic differentiation. The limitations of cell therapy and the complex genetic and epigenetic interplay in FSHD leave it, till now, incurable. However advances in cellular and genetic therapies of myopathies open up new horizons for future applications in the FSHD context. The thesis work is structured around three themes. First, we demonstrate the feasibility of phenotypic and functional correction of FSHD myotubes in vitro by fusing 50% of normal myoblasts with FSHD myoblasts. Next, we evaluate two genomic editing approaches. In the first one, we target the site of attachment of chromosome 4 to the nuclear matrix, FR-MAR with the CTCF protein using the CRISPR / dCas9 system for the purpose of restoring the chromatin organization and the insulating function of FR-MAR. In the second one, we exchange the homologous regions 4q35 and 10q26 by translocation in order to correct the FSHD myoblasts as the three genetic factors of the 4q35 locus are pathogenic only on a genetic background linked to chromosome 4. Finally, we study the role of the oxidative stress in the FSHD.

Die Anordnung von Tiergenomen in topologisch assoziierten Domänen (TADs) spielt eine entscheidende Rolle bei der Regulation von Genen. Diese TADs sind Bereiche mit erhöhter Interaktion, die durch kontaktarme Zonen getrennt sind. In Wirbeltieren erfolgt die Bildung von TADs durch die Bindung von Kohäsin und CTCF (CCCTC-bindender Faktor) im Rahmen eines dynamischen Prozesses namens Loop-Extrusion. Dieser Prozess erzeugt Chromatinschleifen, die gestoppt werden, wenn sie auf CTCF-Proteine in einer spezifischen Ausrichtung treffen. Obwohl CTCF in den meisten Bilaterien stark konserviert ist, wurde seine globale architektonische Funktion in Fliegen bisher nicht erforscht. In dieser Studie wurde ein innovativer Ansatz entwickelt, um die evolutionären Aspekte der CTCF-vermittelten 3D-Chromatinorganisation zu untersuchen. Die Auswirkungen des Austauschs von CTCF-Orthologen innerhalb der Bilateriengruppe auf Lebensfähigkeit, Phänotypen, Genexpression, Genomarchitektur und genomweite Bindungsmuster wurden analysiert. Die Ergebnisse zeigen, dass die nicht-vertebraten Chordatiere C. robusta, unabhängig von der Anwesenheit von CTCF, keine herkömmlichen TAD-Strukturen aufweisen. Dennoch kann das Ciona-Ortholog als Transkriptionsfaktor fungieren, um die Expression bestimmter Gene und die Lebensfähigkeit wiederherzustellen, die bei vollständigem CTCF-Verlust in embryonalen Stammzellen der Maus dysreguliert sind. Dies deutet darauf hin, dass CTCF eine konservierte Rolle als Transkriptionsregulator hat, die über seine bekannte Funktion als architektonisches Protein in einigen Arten hinausgeht. Weitere Untersuchungen sind erforderlich, um festzustellen, ob CTCF in Ciona das Genom in seiner nativen Umgebung bindet und die Bindung von Kohäsin aufrechterhält. Die Unfähigkeit des CTCF-Orthologs der Maus, Chromatinschleifen im Genom der Fruchtfliege zu erzeugen, legt nahe, dass die Wirbeltier-Version von CTCF allein nicht für eine funktionelle Schleifen-Extrusion ausreicht. Es könnte notwendig sein, dass sie mit Fliegen-Kohäsin oder spezifischen Kofaktoren kompatibel ist. Die Studie zeigt auch subtile Unterschiede in den Bindungsmotiven von CTCF zwischen den Arten. Während die Orthologe der Chordatiere ähnliche Motivstrukturen aufweisen, zeigt das Fliegen-Ortholog eine abweichende Musterpräferenz. Diese Erkenntnisse verdeutlichen die evolutionären Verschiebungen in den Bindungsvorlieben von CTCF in pan-chordaten Linien. Zusammenfassend bietet diese Forschung wertvolle Einblicke in die evolutionäre Bewahrung und funktionelle Divergenz von CTCF-vermittelten Chromatin-Kontakten bei Bilaterien. Sie betont die Bedeutung artspezifischer Faktoren und koevolutionärer Dynamiken bei der Gestaltung der Chromatinorganisation und Genregulation. Weitere Untersuchungen an verschiedenen Arten sind entscheidend, um die Entstehung und Bewahrung der CTCF-vermittelten Chromatinarchitektur im Verlauf der Evolution genau zu verstehen. / The three-dimensional organization of animal genomes, known as topologically associating domains (TADs), is crucial for controlling gene activity. TADs are regions with increased genetic interactions, separated by zones with fewer contacts. In vertebrates, the formation of TADs involves a dynamic process called loop extrusion, where cohesin and CTCFs bind to the chromatin. This process creates chromatin loops, with cohesin complexes pausing when they encounter CTCF molecules in a specific orientation. However, although CTCF is highly conserved among bilaterian species, its vital role in organizing genomes spatially has not been observed in invertebrates like flies. This study investigates the chromatin structure in Ciona robusta, a chordate species situated evolutionarily between well-studied organisms like mice and fruit flies. A unique approach was developed to explore the evolution of CTCF as a mediator of three-dimensional chromatin organization. By swapping CTCF orthologs from representative species across the bilaterian group, the research examined their impact on viability, traits, gene expression, genome architecture, and binding patterns across the genome. The findings indicate that Ciona robusta, a non-vertebrate chordate, lacks typical TAD structures, even in the presence of CTCF. However, although the Ciona ortholog cannot create TADs in mouse embryonic stem cells, it can act as a transcription factor, restoring the expression of specific genes and viability in cases of complete CTCF loss. This suggests that CTCF serves a conserved role as a transcription regulator, beyond its recognized role as a structural component in some species. Furthermore, when the mouse ortholog of CTCF was introduced into the fruit fly genome, it failed to induce the formation of chromatin loops, suggesting that the vertebrate version of CTCF alone is insufficient for effective loop extrusion. Additionally, the study revealed subtle differences in CTCF's binding motif preferences between species. While chordate orthologs shared similar motif structures, the fly ortholog had a distinct pattern. These findings underscore the evolutionary changes in CTCF binding preferences among chordate lineages. In summary, this research offers valuable insights into the evolutionary preservation and functional differences in CTCF-mediated chromatin interactions in bilaterian species. It highlights the significance of species-specific factors and co-evolutionary dynamics in shaping chromatin organization and gene regulation.

A controlled and efficient coordination of gene expression is the key for normal development of an organism. In mammals, a subset of autosomal genes is expressed monoallelically depending on the sex of the transmitting parent, a phenomenon known as genomic imprinting. The imprinted state of the H19 and Igf2 genes is controlled by a short stretch of sequences upstream of H19 known as the imprinting control region (ICR). This region is differentially methylated and is responsible for the repression of the maternally inherited Igf2 allele. It harbors hypersensitive sites on the unmethylated maternal allele and functions as an insulator that binds a chromatin insulator protein CTCF. Hence the H19 ICR, which plays an important role in maintaining the imprinting status of H19 and Igf2, was shown to lose the insulator property upon CpG methylation. Another ICR in the Kcnq1 locus regulates long-range repression of p57Kip2 and Kcnq1 on the paternal allele, and is located on the neighboring subdomain of the imprinted gene cluster containing H19 and Igf2, on the distal end of mouse chromosome 7. Similarly to the H19 ICR, the Kcnq1 ICR appears to possess a unidirectional and methylation-sensitive chromatin insulator property in two different somatic cell types. Hence, methylation dependent insulator activity emerges as a common feature of imprinting control regions. The protein CTCF is required for the interpretation and propagation of the differentially methylated status of the H19 ICR. Work in this thesis shows that this feature applies genomewide. The mapping of CTCF target sites demonstrated not only a strong link between CTCF, formation of insulator complexes and maintaining methylation-free domains, but also a network of target sites that are involved in pivotal functions. The pattern of CTCF in vivo occupancy varies in a lineage-specific manner, although a small group of target sites show constitutive binding. In conclusion, the work of this thesis shows that epigenetic marks play an important role in regulating the insulator property. The studies also confirm the importance of CTCF in maintaining methylation-free domains and its role in insulator function. Our study unravels a new range of target sites for CTCF involved in divergent functions and their developmental control.

Distal enhancer elements regulate the tissue-specific expression of their target genes via the establishment of physical interactions with the gene promoter. In mice, a cluster of five enhancers, jointly classified as a super-enhancer, specifically upregulate α-globin gene expression during erythroid differentiation. Aside from the Nprl3 gene, whose promoter is located inside this enhancer region, expression-levels of other genes within a short distance (&lt,50kb) of the enhancer region are not affected by the activation of the enhancer in erythroid cells, despite being located within the same sub-TAD in erythroid cells. The CCCTC-binding factor (CTCF) is implicated in the organisation of chromosome topology through the formation of interactions between its binding sites in an orientation-dependent manner. In this thesis, I demonstrate that CTCF functions in vivo as a boundary to maintain α-globin enhancer-promoter specificity in erythroid cells. The study of the local chromatin architecture by next-generation Capture-C reveals that α-globin enhancer and promoter interactions are constrained to a compartment of roughly 70kb. The unidirectional interaction profiles of the α-globin enhancers are delimited by the interactions between two genomic domains flanking the α-globin cluster. Further investigation shows that each of these domains contains several CTCF binding sites orientated in tandem, such that CTCF binding orientation between domains is convergent. Although CTCF binding across the α-globin locus is identical between mouse embryonic stem (ES) cells and erythroid cells, interaction between these domains occurs only in erythroid cells suggesting it is dependent on the formation of tissue-specific α-globin enhancer-promoter interactions. By generating a series of mouse models, deleting CTCF binding sites at the α-globin enhancers singly and in combination, I show that the deletion of two CTCF binding sites directly flanking the enhancer cluster results in a shift in interactions between flanking domains, away from the enhancer region. This leads to an expansion of enhancer interactions to include two genes directly upstream of the α-globin enhancers: Rhbdf1 and Mpg. Despite the Rhbdf1 gene being subject to polycomb group protein-mediated gene repression in erythroid cells, ablation of CTCF binding results in increased interactions between both the Rhbdf1 and Mpg gene promoters and the α-globin enhancers and concurrent strong transcriptional upregulation of both genes. The Rhbdf1 gene promoter acquires the active histone mark H3K4me3, but doesn't lose Polycomb Repressive Complex 2 (PRC2) mark H3K27me3 or binding of its catalytic component Ezh2. Despite the presence of this repressive mark, robust levels of Rhbdf1 expression are detected at levels higher than those in ES cells where this gene is actively expressed under the influence of its own enhancer. I conclude that regulation of the direction of enhancer interactions by CTCF is required for the promoter specificity of enhancers and the maintenance of transcriptional states of nearby genes.

<p>Genomic imprinting marks a subset of autosomal loci expressed in parent of origin-dependent monoallelic expression in a non-Mendelian fashion. To restore totipotency and to reset the imprint according to the sex of the individual, the mark must be erased during germline development. The imprinted <i>Igf2-H19</i> loci located distally on chromosome 7 in mouse and 11p15.5 in human, share common regulatory elements that regulate differential expression. Where the <i>H19 </i>is silenced when paternally inherited, the <i>Igf2</i> is silenced when maternally inherited. </p><p>The differentially methylated 5'-flank of <i>H19</i> gene, termed imprinting control region (ICR), shown to display a unique chromatin organisation harbours hypersensitive sites in linker regions flanked by positioned nucleosomes on the maternal allele. This unique chromatin conformation functions as a methylation-sensitive and unidirectional chromatin insulator, which later was found to depend on the chromatin insulator protein CTCF. </p><p>The <i>H19</i> ICR exhibits default-silencing functions in promoter-proximal positions. The maximal distance between the <i>H19</i> ICR and the promoter of the reporter gene required for this effect was 1.2 ± 0.3kb which can be compared to the 1.9 kb distance between the endogenous <i>H19 </i>ICR and <i>H19</i> promoter. Results suggest that the <i>H19</i> ICR adopts a chromatin conformation that must be separated by a minimal distance from pivotal <i>cis</i>-regulatory elements to avoid adverse effects on neighbouring promoters. </p><p>Poly(ADP-ribosy)lation represents a novel post-translational epigenetic mark that segregates with exclusively the maternal derived <i>H19</i> ICR and associated with factors that interact with the CTCF target sites. CTCF is itself poly(ADP-ribosy)lated and the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide relieves the insulator function of the <i>H19</i> ICR. </p><p>Designed zinc finger proteins were applied to examine if epigenetic marks provided an obstacle for targeted activation and silencing. The zinc finger protein ZFP809 with activator/repressor domain able to efficiently activate/silence the <i>IGF2</i> target. Murine hybrid cell lines of human chromosome 11, demonstrated that the ZFP809 overcame the epigenetic marks that repressed maternal <i>IGF2</i> and paternal <i>H19</i> allele, respectively. Results suggested that imprinted genes are not normally exposed to strong <i>cis</i>-regulatory elements and that the designed ZFPs can be exploited to develop a therapeutic method for rectifying epigenetic lesions.</p>