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The human epidermal growth factor receptor-2 (HER2/neu/ERBB2) is overexpressed in several cancer types. Although therapies targeting the HER2/neu protein result in inhibition of cell proliferation, the anticancer effect might be further optimized by limiting HER2/neu expression at the DNA level. Towards this aim, epigenetic editing was performed to suppress HER2/neu expression by inducing epigenetic silencing marks on the HER2/neu promoter.HER2/neu expression and HER2/neu promoter epigenetic modification status were determined in a panel of ovarian and breast cancer cell lines. HER2/neu-overexpressing cancer cells were transduced to express a zinc finger protein (ZFP), targeting the HER2/neugene, fused to histone methyltransferases (G9a, SUV39-H1)/super KRAB domain (SKD). Epigenetic assessment of the HER2/neu promoter showed that HER2/neu-ZFP fused to G9a efficiently induced the intended silencing histone methylation mark (H3K9me2). Importantly, H3K9me2 induction was associated with a dramatic downregulation of HER2/neu expression in HER2/neu- overexpressing cells. Downregulation by SKD, traditionally considered transient in nature, was associated with removal of the histone acetylation mark (H3ac). The downregulation of HER2/neu by induced H3K9 methylation and/or reduced H3 acetylation was sufficient to effectively inhibit cellular metabolic activity and clonogenicity. Furthermore, genome-wide analysis indicated preferential binding of the ZFP to its target sequence. These results not only show that H3K9 methylation can be induced but also that this epigenetic mark was instructive in promoting downregulation of HER2/neu expression.Implications: Epigenetic editing provides a novel (synergistic) approach to modulate expression of oncogenes. Mol Cancer Res; 11(9); 1029–39. ©2013 AACR.

PDF file - 72K, Sorted cells maintain their profile of Her2/neu and GFP expression.

PDF file - 48K, Her2/neu downregulation status in MCF7 by pMX-E2C fused to histone methyltransferase domains.

PDF file - 56K, Her2/neu Zinc Finger Protein fused to VP64 does not have any effect on an irrelevant gene.

PDF file - 60K, Induction of H3K9me3 in region B of Her2/neu gene in SKBR3 cells.

PDF file - 77K, Specific binding of HER2/neu ZFP (E2C).

Maternal embryonic leucine zipper kinase (MELK) is frequently overexpressed in cancer, but the role of MELK in cancer is still poorly understood. MELK was shown to have roles in many cancer-associated processes including tumor growth, chemotherapy resistance, and tumor recurrence. To determine whether the frequent overexpression of MELK can be exploited in therapy, we performed a high-throughput screen using a library of Saccharomyces cerevisiae mutants to identify genes whose functions become essential when MELK is overexpressed. We identified two such genes: LAG2 and HDA3. LAG2 encodes an inhibitor of the Skp, Cullin, F-box containing (SCF) ubiquitin-ligase complex, while HDA3 encodes a subunit of the HDA1 histone deacetylase complex. We find that one of these synthetic lethal interactions is conserved in mammalian cells, as inhibition of a human homolog of HDA3 (Histone Deacetylase 4, HDAC4) is synthetically toxic in MELK overexpression cells. Altogether, our work identified a novel potential drug target for tumors that overexpress MELK.

Maternal embryonic leucine zipper kinase (MELK) is frequently overexpressed in cancer, but the role of MELK in cancer is still poorly understood. MELK was shown to have roles in many cancer-associated processes including tumor growth, chemotherapy resistance, and tumor recurrence. To determine whether the frequent overexpression of MELK can be exploited in therapy, we performed a high-throughput screen using a library of Saccharomyces cerevisiae mutants to identify genes whose functions become essential when MELK is overexpressed. We identified two such genes: LAG2 and HDA3. LAG2 encodes an inhibitor of the SCF ubiquitin-ligase complex, while HDA3 encodes a subunit of the HDA1 histone deacetylase complex. We find that one of these synthetic lethal interactions is conserved in mammalian cells, as inhibition of a human homolog of HDA3 (HDAC4) is synthetically toxic in MELK overexpression cells. Altogether, our work might provide a new angle of how to exploit MELK overexpression in cancers and might thus lead to novel intervention strategies.

Translation efficiency can be affected by mRNA stability and secondary structures, including G-quadruplex structures (G4s). The highly conserved DEAH-box helicase DHX36/RHAU resolves G4s on DNA and RNA in vitro, however a systems-wide analysis of DHX36 targets and function is lacking. We map globally DHX36 binding to RNA in human cell lines and find it preferentially interacting with G-rich and G4-forming sequences on more than 4500 mRNAs. While DHX36 knockout (KO) results in a significant increase in target mRNA abundance, ribosome occupancy and protein output from these targets decrease, suggesting that they were rendered translationally incompetent. Considering that DHX36 targets, harboring G4s, preferentially localize in stress granules, and that DHX36 KO results in increased SG formation and protein kinase R (PKR/EIF2AK2) phosphorylation, we speculate that DHX36 is involved in resolution of rG4 induced cellular stress., Translation efficiency can be affected by mRNA stability and secondary RNA structures. Here the authors reveal that loss of DHX36 helicase activity leads to an accumulation of translationally inactive target mRNAs with G-rich structures in untranslated regions.

Accurate DNA replication is essential for genomic stability and cancer prevention. Homologous recombination is important for high-fidelity DNA damage tolerance during replication. How the homologous recombination machinery is recruited to replication intermediates is unknown. Here, we provide evidence that a Rad51 paralog-containing complex, the budding yeast Shu complex, directly recognizes and enables tolerance of predominantly lagging strand abasic sites. We show that the Shu complex becomes chromatin associated when cells accumulate abasic sites during S phase. We also demonstrate that purified recombinant Shu complex recognizes an abasic analog on a double-flap substrate, which prevents AP endonuclease activity and endonuclease-induced double-strand break formation. Shu complex DNA binding mutants are sensitive to methyl methanesulfonate, are not chromatin enriched, and exhibit increased mutation rates. We propose a role for the Shu complex in recognizing abasic sites at replication intermediates, where it recruits the homologous recombination machinery to mediate strand specific damage tolerance., The homologous recombination machinery needs to be recruited at replication intermediates for accurate functioning. Here, the authors reveal that a Rad51 paralog-containing complex, called the Shu complex, recognizes and enables tolerance of predominantly lagging strand abasic sites.

To ensure proper transmission of genetic information, cells need to preserve and faithfully replicate their genome, and failure to do so leads to genome instability, a hallmark of both cancer and aging. Defects in genes involved in guarding genome stability cause several human progeroid syndromes, and an age‐dependent accumulation of mutations has been observed in different organisms, from yeast to mammals. However, it is unclear whether the spontaneous mutation rate changes during aging and whether specific pathways are important for genome maintenance in old cells. We developed a high‐throughput replica‐pinning approach to screen for genes important to suppress the accumulation of spontaneous mutations during yeast replicative aging. We found 13 known mutation suppression genes, and 31 genes that had no previous link to spontaneous mutagenesis, and all acted independently of age. Importantly, we identified PEX19, encoding an evolutionarily conserved peroxisome biogenesis factor, as an age‐specific mutation suppression gene. While wild‐type and pex19Δ young cells have similar spontaneous mutation rates, aged cells lacking PEX19 display an elevated mutation rate. This finding suggests that functional peroxisomes may be important to preserve genome integrity specifically in old cells., We developed a high‐throughput approach to screen for genes important to suppress the accumulation of spontaneous mutations during yeast replicative aging. We found 13 known and 32 novel mutation suppression genes. Remarkably, one of these genes, PEX19, encoding an evolutionarily conserved peroxisome biogenesis factor, acts in an age‐dependent manner; while wild‐type and pex19Δ young cells have similar spontaneous mutation rates, aged cells lacking PEX19 display an elevated mutation rate.

Translation efficiency can be affected by mRNA stability and secondary structures, including so-called G-quadruplex (G4) structures. The highly conserved and essential DEAH-box helicase DHX36/RHAU is able to resolve G4 structures on DNA and RNA in vitro, however a system-wide analysis of DHX36 targets and function is lacking. We globally mapped DHX36 occupancy in human cell lines and found that it preferentially binds to G-rich sequences in the coding sequences (CDS) and 5' and 3' untranslated regions (UTR) of more than 4,500 mRNAs. Functional analyses, including RNA sequencing, ribosome footprinting, and quantitative mass spectrometry revealed that DHX36 decreased target mRNA stability. However, target mRNA accumulation in DHX36 KO cells did not lead to a significant increase in ribosome footprints or protein output indicating that they were translationally incompetent. We hypothesize that DHX36 resolves G4 and other structures that interfere with efficient translation initiation.

Author(s): Cornish-Bowden, Athel; Cornish-Bowden, Athel; Rasnick, David; Heng, Henry H; Horne, Steven; Abdallah, Batoul; Liu, Guo; Ye, Christine J; Bloomfield, Mathew; Vincent, Mark D; Aldaz, C Marcelo; Karlsson, Jenny; Valind, Anders; Jansson, Caroline; Gisselsson, David; Graves, Jennifer A Marshall; Stepanenko, Aleksei A; Andreieva, Svitlana V; Korets, Kateryna V; Mykytenko, Dmytro O; Huleyuk, Nataliya L; Baklaushev, Vladimir P; Kovaleva, Oksana A; Chekhonin, Vladimir P; Vassetzky, Yegor S; Avdieiev, Stanislav S; Bakker, Bjorn; Taudt, Aaron S; Belderbos, Mirjam E; Porubsky, David; Spierings, Diana CJ; de Jong, Tristan V; Halsema, Nancy; Kazemier, Hinke G; Hoekstra-Wakker, Karina; Bradley, Allan; de Bont, Eveline SJM; van den Berg, Anke; Guryev, Victor; Lansdorp, Peter M; Tatche, Maria Colome; Foijer, Floris; Liehr, Thomas; Baudoin, Nicolaas C; Nicholson, Joshua M; Soto, Kimberly; Quintanilla, Isabel; Camps, Jordi; Cimini, Daniela; Durrbaum, M; Donnelly, N; Passerini, V; Kruse, C; Habermann, B; Storchova, Z; Mandrioli, Daniele; Belpoggi, Fiorella; Silbergeld, Ellen K; Perry, Melissa J; Skotheim, Rolf I; Lovf, Marthe; Johannessen, Bjarne; Hoff, Andreas M; Zhao, Sen; SveeStromme, Jonas M; Sveen, Anita; Lothe, Ragnhild A; Hehlmann, R; Voskanyan, A; Fabarius, A; Bocking, Alfred; Biesterfeld, Stefan; Berynskyy, Leonid; Borgermann, Christof; Engers, Rainer; Dietz, Josef; Fritz, A; Sehgal, N; Vecerova, J; Stojkovicz, B; Ding, H; Page, N; Tye, C; Bhattacharya, S; Xu, J

Iron complexes of N,N-bis(2-Pyridylmethyl)-N-bis(2-pyridyl)-methylamine (N4Py) have proven to be excellent synthetic mimics of the Bleomycins (BLMs), which are a family of natural antibiotics used clinically in the treatment of certain cancers. However, most investigations of DNA cleavage activity of these and related metal complexes were carried out in 0, cell-free systems using plasmid DNA as substrate. The present study evaluated nuclear DNA cleavage activity and cell cytotoxicity of BLM and its synthetic mimics based on the ligand N4Py. The N4Py-based reagents induced nuclear DNA cleavage in living cells as efficiently as BLM and Fe(II)-BLM. Treatment of 2 cancer cell lines and 1 noncancerous cell line indicated improved cytotoxicity of N4Py when compared to BLM. Moreover, some level of selectivity was observed for N4Py on cancerous versus noncancerous cells. It was demonstrated that N4Py-based reagents and BLM induce cell death via different mechanistic pathways. BLM was shown to induce cell cycle arrest, ultimately resulting in mitotic catastrophe. In contrast, N4Py-based reagents were shown to induce apoptosis effectively. To the best of our knowledge, the present study is the first demonstration of efficient nuclear DNA cleavage activity of a synthetic BLM mimic within cells. The results presented here show that it is possible to design synthetic bioinorganic model complexes that are at least as active as the parent natural product and thereby are potentially interesting alternatives for BLM to induce antitumor activity.

The human epidermal growth factor receptor-2 (HER2/neu/ERBB2) is overexpressed in several cancer types. Although therapies targeting the HER2/neu protein result in inhibition of cell proliferation, the anticancer effect might be further optimized by limiting HER2/neu expression at the DNA level. Towards this aim, epigenetic editing was performed to suppress HER2/neu expression by inducing epigenetic silencing marks on the HER2/neu promoter.HER2/neu expression and HER2/neu promoter epigenetic modification status were determined in a panel of ovarian and breast cancer cell lines. HER2/neu-overexpressing cancer cells were transduced to express a zinc finger protein (ZFP), targeting the HER2/neugene, fused to histone methyltransferases (G9a, SUV39-H1)/super KRAB domain (SKD). Epigenetic assessment of the HER2/neu promoter showed that HER2/neu-ZFP fused to G9a efficiently induced the intended silencing histone methylation mark (H3K9me2). Importantly, H3K9me2 induction was associated with a dramatic downregulation of HER2/neu expression in HER2/neu- overexpressing cells. Downregulation by SKD, traditionally considered transient in nature, was associated with removal of the histone acetylation mark (H3ac). The downregulation of HER2/neu by induced H3K9 methylation and/or reduced H3 acetylation was sufficient to effectively inhibit cellular metabolic activity and clonogenicity. Furthermore, genome-wide analysis indicated preferential binding of the ZFP to its target sequence. These results not only show that H3K9 methylation can be induced but also that this epigenetic mark was instructive in promoting downregulation of HER2/neu expression. Implications: Epigenetic editing provides a novel (synergistic) approach to modulate expression of oncogenes. Mol Cancer Res; 11(9); 1029–39. ©2013 AACR.

Ovarian cancer is a difficult-to-treat cancer with a 5-year survival rate of only ∼45%, due to late diagnosis and therapy resistance. In need of new therapeutic approaches, induction of intercellular adhesion molecule (ICAM)-1 expression might be of interest, since the expression of ICAM-1 is lower in ovarian cancer cells compared with healthy ovarian cells and correlated with decreased tumorigenicity. Whereas ICAM-1 expression on tumor cells is of importance for attracting immune cells, ICAM-1 might also induce tumorigenicity and chemoresistance. In ovarian cancer, such a role of ICAM-1 is unclear. Here, we investigated whether ICAM-1 has a cell-biological role by bidirectional modulation of ICAM-1 expression using ICAM-targeting artificial transcription factors. For a panel of ovarian cancer cells, tumor growth and cisplatin sensitivity were evaluated. Induction of ICAM-1 expression (ranging from 3- to 228-fold on mRNA level and 1.7- to 108-fold on protein level) resulted in indications of decreased ovarian cancer cell growth and reduced cisplatin sensitivity. Repression ranged from 48 to 94% on mRNA level and 47 to 91% on protein level. This study shows that, next to its established immunogenic role, ICAM-1 affects cell biological behavior of ovarian cancer cells and, importantly, that reexpression by artificial transcription factors represents a powerful approach for functional validation of genes epigenetically silenced in cancer, such as ICAM-1.

Background: The epithelial cell adhesion molecule (EpCAM) is overexpressed on most carcinomas. Dependent on the tumour type, its overexpression is either associated with improved or worse patient survival. For ovarian cancer, however, the role of EpCAM remains unclear.Methods: Cell survival of ovarian cancer cell lines was studied after induction or repression of endogenous EpCAM expression using siRNA/cDNA or artificial transcription factors (ATF) consisting of engineered zinc-fingers fused to either a transcriptional activator or repressor domain.Results: Two ATFs were selected as the most potent down- and upregulator, showing at least a two-fold alteration of EpCAM protein expression compared with control. Downregulation of EpCAM expression resulted in growth inhibition in breast cancer, but showed no effect on cell growth in ovarian cancer. Induction or further upregulation of EpCAM expression decreased ovarian cancer cell survival.Conclusion: The bidirectional ATF-based approach is uniquely suited to study cell-type-specific biological effects of EpCAM expression. Using this approach, the oncogenic function of EpCAM in breast cancer was confirmed. Despite its value as a diagnostic marker and for immunotherapy, EpCAM does not seem to represent a therapeutic target for gene expression silencing in ovarian cancer.

Background Chromosome instability leads to aneuploidy, a state in which cells have abnormal numbers of chromosomes, and is found in two out of three cancers. In a chromosomal instable p53 deficient mouse model with accelerated lymphomagenesis, we previously observed whole chromosome copy number changes affecting all lymphoma cells. This suggests that chromosome instability is somehow suppressed in the aneuploid lymphomas or that selection for frequently lost/gained chromosomes out-competes the CIN-imposed mis-segregation. Results To distinguish between these explanations and to examine karyotype dynamics in chromosome instable lymphoma, we use a newly developed single-cell whole genome sequencing (scWGS) platform that provides a complete and unbiased overview of copy number variations (CNV) in individual cells. To analyse these scWGS data, we develop AneuFinder, which allows annotation of copy number changes in a fully automated fashion and quantification of CNV heterogeneity between cells. Single-cell sequencing and AneuFinder analysis reveals high levels of copy number heterogeneity in chromosome instability-driven murine T-cell lymphoma samples, indicating ongoing chromosome instability. Application of this technology to human B cell leukaemias reveals different levels of karyotype heterogeneity in these cancers. Conclusion Our data show that even though aneuploid tumours select for particular and recurring chromosome combinations, single-cell analysis using AneuFinder reveals copy number heterogeneity. This suggests ongoing chromosome instability that other platforms fail to detect. As chromosome instability might drive tumour evolution, karyotype analysis using single-cell sequencing technology could become an essential tool for cancer treatment stratification. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-0971-7) contains supplementary material, which is available to authorized users.

The CCHC-type zinc finger nucleic acid-binding protein (CNBP/ZNF9) is conserved in eukaryotes and is essential for embryonic development in mammals. It has been implicated in transcriptional, as well as post-transcri ptional, gene regulation; however, its nucleic acid ligands and molecular function remain elusive. Here, we use multiple systems-wide approaches to identify CNBP targets and function. We used photoactivatable ribonucleo side-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to identify 8,420 CNBP binding sites on 4,178 mRNAs. CNBP preferentially bound G-rich elements in the target mRNA coding sequences, most of which were previously found to form G-quadruplex and other stable structures in vitro. Functional analyses, including RNA sequencing, ribosome profiling, and quantitative mass spectrometry, revealed that CNBP binding did not influence target mRNA abundance but rather increased their translational efficiency. Considering that CNBP binding prevented G-quadruplex structure formation in vitro, we hypothesize that CNBP is supporting translation by resolving stable structures on mRNAs.

C13ORF18 is frequently hypermethylated in cervical cancer but not in normal cervix and might serve as a biomarker for the early detection of cervical cancer in scrapings. As hypermethylation is often observed for silenced tumor suppressor genes (TSGs), hypermethylated biomarker genes might exhibit tumor suppressive activities upon re-expression. Epigenetic drugs are successfully exploited to reverse TSG silencing, but act genome-wide. Artificial Transcription Factors (ATFs) provide a gene-specific approach for re-expression of silenced genes. Here, we investigated the potential tumor suppressive role of C13ORF18 in cervical cancer by ATF-induced re-expression.Five zinc finger proteins were engineered to bind the C13ORF18 promoter and fused to a strong transcriptional activator. C13ORF18 expression could be induced in cervical cell lines: ranging from >40-fold in positive (C13ORF18-unmethylated) cells to >110-fold in negative (C13ORF18-methylated) cells. Re-activation of C13ORF18 resulted in significant cell growth inhibition and/or induction of apoptosis. Co-treatment of cell lines with ATFs and epigenetic drugs further enhanced the ATF-induced effects. Interestingly, reactivation of C13ORF18 led to partial demethylation of the C13ORF18 promoter and decreased repressive histone methylation. These data demonstrate the potency of ATFs to re-express and potentially demethylate hypermethylated silenced genes. Concluding, we show that C13ORF18 has a TSG function in cervical cancer and may serve as a therapeutic anti-cancer target. As the amount of epimutations in cancer exceeds the number of gene mutations, ATFs provide promising tools to validate hypermethylated marker genes as therapeutic targets. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Ovarian cancer is a difficult-to-treat cancer with a 5-year survival rate of only ∼45%, due to late diagnosis and therapy resistance. In need of new therapeutic approaches, induction of intercellular adhesion molecule (ICAM)-1 expression might be of interest, since the expression of ICAM-1 is lower in ovarian cancer cells compared with healthy ovarian cells and correlated with decreased tumorigenicity. Whereas ICAM-1 expression on tumor cells is of importance for attracting immune cells, ICAM-1 might also induce tumorigenicity and chemoresistance. In ovarian cancer, such a role of ICAM-1 is unclear. Here, we investigated whether ICAM-1 has a cell-biological role by bidirectional modulation of ICAM-1 expression using ICAM-targeting artificial transcription factors. For a panel of ovarian cancer cells, tumor growth and cisplatin sensitivity were evaluated. Induction of ICAM-1 expression (ranging from 3- to 228-fold on mRNA level and 1.7- to 108-fold on protein level) resulted in indications of decreased ovarian cancer cell growth and reduced cisplatin sensitivity. Repression ranged from 48 to 94% on mRNA level and 47 to 91% on protein level. This study shows that, next to its established immunogenic role, ICAM-1 affects cell biological behavior of ovarian cancer cells and, importantly, that reexpression by artificial transcription factors represents a powerful approach for functional validation of genes epigenetically silenced in cancer, such as ICAM-1.

BACKGROUND: The epithelial cell adhesion molecule (EpCAM) is overexpressed on carcinomas, and its downregulation inhibits the oncogenic potential of multiple tumour types. Here, we investigated underlying mechanisms of epcam overexpression in ovarian carcinoma.METHODS: Expression of EpCAM and DNA methylation (bisulphite sequencing) was determined for ovarian cancer cell lines. The association of histone modifications and 16 transcription factors with the epcam promoter was analysed by chromatin immunoprecipitation. Treatment with 5-Aza-2'-deoxycytidine (5-AZAC) was used to induce EpCAM expression.RESULTS: Expression of EpCAM was correlated with DNA methylation and histone modifications. Treatment with 5-AZAC induced EpCAM expression in negative cells. Ten transcription factors were associated with the epcam gene in EpCAM expressing cells, but not in EpCAM-negative cells. Methylation of an Sp1 probe inhibited the binding of nuclear extract proteins in electromobility shift assays; such DNA methylation sensitivity was not observed for an NF-kappa B probe.CONCLUSION: This study provides insights in transcriptional regulation of epcam in ovarian cancer. Epigenetic parameters associated with EpCAM overexpression are potentially reversible, allowing novel strategies for sustained silencing of EpCAM expression. British Journal of Cancer (2011) 105, 312-319. doi: 10.1038/bjc.2011.231 www.bjcancer.com Published online 21 June 2011 (C) 2011 Cancer Research UK

Recently, we reported diagnostic marker gene promoters that are hypermethylated in cervical cancer. As hypermethylation is often seen for tumor suppressor genes (TSGs), such marker genes might exhibit tumor suppressive activities upon re-expression. In this study, we aim to specifically upregulate candidate TSGs (C13ORF18, EPB41L3 and CCNA1) using self-designed Artificial Transcription Factors (ATFs). An advantage of this approach is that genes are re-expressed from their natural promoter allowing all splice variants to be expressed in natural ratios In cervical cancer cell lines, DNA methylation status (methylation-specific PCR, bisulfite sequencing) and gene (re)expression levels (qRT-PCR) were determined, also after treatment with a DNA demethylating agent (5-Aza-2′-Deoxycytidine (DAC)) and a histone deacetylase inhibitor (Trichostatin A (TSA)). Sequence specific DNA-binding domains were constructed by engineering zinc finger proteins and fused to a strong transcriptional activator. Functional effects of re-expression of the genes were studied using cell survival/apoptosis/cell growth assays. Histone marks were identified using chromatin IP, before and after treatment with ATFs. C13ORF18, EPB41L3 and CCNA1 silencing in cervical cancer was associated with DNA hypermethylation and these genes could be re-expressed in a dose dependent manner using DAC. Gene-specific re-activation of the genes could be achieved by the ATFs: Up to 300-fold in negative (methylated) cells and up to 80-fold in positive (unmethylated) cells. Co-treatment of ATFs with DAC or TSA further enhanced the effect of the ATFs. The hypermethylation status of the genes was associated with low levels of histone acetylation and repressive histone marks (H3K9me3, H3K27me3). Interestingly, preliminary data show that re-activation of the silenced genes increased the histone acetylation, while repressive histone marks decreased, indicating that ATF-induced re-expression was associated with changes in the epigenetic code. Furthermore, it was found that specific re-activation of the genes inhibited cell growth and induced apoptosis. These data confirm our hypothesis that hypermethylated cancer-specific genes can be re-activated, resulting in a strong anti-tumor effect. Furthermore, this is the first time that these genes are reported as TSGs in cervical cancer. The great advantage of gene-specific DNA targeting is that only the TSGs of interest are upregulated, while current genome-wide epigenetic drugs also target metastasis inducing genes, and can therefore enforce cancer growth. Currently, the DNA binding domains are fused to epigenetic effector domains to explore the ability of gene specific epigenetic editing. Financed by NWO-VIDI to MR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3146. doi:1538-7445.AM2012-3146

Increasing evidence indicates that active DNA demethylation is involved in several processes in mammals, resulting in developmental stage-specificity and cell lineage-specificity. The recently discovered Ten-Eleven Translocation (TET) dioxygenases are accepted to be involved in DNA demethylation by initiating 5-mC oxidation. Aberrant DNA methylation profiles are associated with many diseases. For example in cancer, hypermethylation results in silencing of tumor suppressor genes. Such silenced genes can be re-expressed by epigenetic drugs, but this approach has genome-wide effects. In this study, fusions of designer DNA binding domains to TET dioxygenase family members (TET1, -2 or -3) were engineered to target epigenetically silenced genes (ICAM-1, EpCAM). The effects on targeted CpGs' methylation and on expression levels of the target genes were assessed. The results indicated demethylation of targeted CpG sites in both promoters for targeted TET2 and to a lesser extent for TET1, but not for TET3. Interestingly, we observed re-activation of transcription of ICAM-1. Thus, our work suggests that we provided a mechanism to induce targeted DNA demethylation, which facilitates re-activation of expression of the target genes. Furthermore, this Epigenetic Editing approach is a powerful tool to investigate functions of epigenetic writers and erasers and to elucidate consequences of epigenetic marks.

Previously we reported the production and characterization of monoclonal antibody 1H6 raised against (T(4)G(4))(2) intermolecular guanine quadruplex (G4) DNA structures (Henderson A. et al. (2014) Nucleic Acids Res., 42, 860-869; Hoffmann R. F. et al. (2016) Nucleic Acids Res., 44, 152-163). It was shown that 1H6 strongly stains nuclei and has exquisite specificity for heterochromatin by immuno-electron microscopy. Here we extend our studies of 1H6 reactivity using enzyme-linked immunosorbent assay (ELISA) and microscale thermophoresis (MST). As previously reported, 1H6 was found to strongly bind intermolecularG4 structureswith a (T(4)G(4))(2) sequence motif. However, using both methods we did not detect significant binding to G4 structures without thymidines in their sequence motif or to G4 structures made with (T(2)G(4))(2) oligonucleotides. In addition, we observed strong, sequence-specific binding of 1H6 by ELISA to immobilized single stranded poly(T) DNA but not to immobilized poly(C) or poly(A) homo-polymers. Cross-reactivity of 1H6 to poly(T) was not measured in solution using MST. 1H6 was furthermore found to bind to selected areas on DNA fibers but only after DNA denaturation. Based on these observations we propose that 1H6 binds with high affinity to adjacent T's that are restricted in their movement in selected G4 structures and denatured DNA. Cross-reactivity of 1H6 to immobilized single stranded T-rich DNA next to its previously reported specificity for bona fide G4 structures needs to be taken into account in the interpretation of 1H6 binding to (sub-) cellular structures.

Background: Alzheimer’s disease (AD) is a neurodegenerative disease of the brain and the most common form of dementia in the elderly. Aneuploidy, a state in which cells have an abnormal number of chromosomes, has been proposed to play a role in neurodegeneration in AD patients. Several studies using fluorescence in situ hybridization have shown that the brains of AD patients contain an increased number of aneuploid cells. However, because the reported rate of aneuploidy in neurons ranges widely, a more sensitive method is needed to establish a possible role of aneuploidy in AD pathology. Results: In the current study, we used a novel single-cell whole genome sequencing (scWGS) approach to assess aneuploidy in isolated neurons from the frontal cortex of normal control individuals (n = 6) and patients with AD (n = 10). The sensitivity and specificity of our method was shown by the presence of three copies of chromosome 21 in all analyzed neuronal nuclei of a Down’s syndrome sample (n = 36). Very low levels of aneuploidy were found in the brains from control individuals (n = 589) and AD patients (n = 893). In contrast to other studies, we observe no selective gain of chromosomes 17 or 21 in neurons of AD patients. Conclusion: scWGS showed no evidence for common aneuploidy in normal and AD neurons. Therefore, our results do not support an important role for aneuploidy in neuronal cells in the pathogenesis of AD. This will need to be confirmed by future studies in larger cohorts.

Epidermal Growth Factor Receptor-2 (HER-2) and Estrogen receptor-alpha (ER-a) have been found to be dysregulated in several types of cancer. Their dysregulation is associated with aberrant epigenetic modifications. Additionally, expression of ER-a and HER-2 is inversely correlated; their crosstalk has been shown to be involved in endocrine therapy resistance mechanisms [1]. Therapies targeting either receptor result in cell proliferation inhibition for some cancer types; the anti-cancer effect might be further optimized to be more efficient and applicable for more cancer types by co-targeting the genes at the DNA level. As epigenetic modifications provide a way to modify expression of genes in a sustained manner we aim to downregulate HER-2 and ER-a by inducing epigenetic silencing marks specifically onto their promoters (Epigenetic Editing [2]). We also aim to discover the possible crosstalk of ER-a with HER-2 and other important genes in cancer. Methods and materials Towards downregulation of HER-2 and ER-a, expression and epigenetic modification status of promoter of these genes were assessed in a panel of cancer cell lines (bisulfite sequencing and ChIP). Designed zinc finger proteins (ZFPs) targeting genes promoters were fused to a transcription repressor domain [SKD, histone methyltransferases (G9a, SUV39H1), or a DNA methyltransferase domain (C141S [3])] and expressed in cancer cells through viral transduction. Results HER2-ZFP fused to G9a or SUV39H1 induced the intended silencing histone methylation marks (H3K9me2, H3K9me3) on the HER-2 promoter. Up to 4-fold induced H3K9me2 mark was associated with up to 54 ± 2.9% downregulation of HER-2 expression (P