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Background: The KDM1A histone demethylase regulates the cellular balance between proliferation and differentiation, and is often deregulated in human cancers including the childhood tumor neuroblastoma. We previously showed that KDM1A is strongly expressed in undifferentiated neuroblastomas and correlates with poor patient prognosis, suggesting a possible clinical benefit from targeting KDM1A. Methods: Here, we tested the efficacy of NCL-1, a small molecule specifically inhibiting KDM1A in preclinical models for neuroblastoma. Results: NCL-1 mimicked the effects of siRNA-mediated KDM1A knockdown and effectively inhibited KDM1A activity in four neuroblastoma cell lines and a patient-representative cell model. KDM1A inhibition shifted the aggressive tumor cell phenotypes towards less aggressive phenotypes. The proliferation and cell viability was reduced, accompanied by the induction of markers of neuronal differentiation. Interventional NCL-1 treatment of nude mice harboring established neuroblastoma xenograft tumors reduced tumor growth and inhibited cell proliferation. Reduced vessel density and defects in blood vessel construction also resulted, and NCL-1 inhibited the growth and tube formation of HUVEC-C cells in vitro. Conclusions: Inhibiting KDM1A could attack aggressive neuroblastomas two-fold, by re-directing tumor cells toward a less aggressive, slower-growing phenotype and by preventing or reducing the vascular support of large tumors. [ABSTRACT FROM AUTHOR]

In mouse development, long-term silencing by CpG island DNA methylation is specifically targeted to germline genes; however, the molecular mechanisms of this specificity remain unclear. Here, we demonstrate that the transcription factor E2F6, a member of the polycomb repressive complex 1.6 (PRC1.6), is critical to target and initiate epigenetic silencing at germline genes in early embryogenesis. Genome-wide, E2F6 binds preferentially to CpG islands in embryonic cells. E2F6 cooperates with MGA to silence a subgroup of germline genes in mouse embryonic stem cells and in embryos, a function that critically depends on the E2F6 marked box domain. Inactivation of E2f6 leads to a failure to deposit CpG island DNA methylation at these genes during implantation. Furthermore, E2F6 is required to initiate epigenetic silencing in early embryonic cells but becomes dispensable for the maintenance in differentiated cells. Our findings elucidate the mechanisms of epigenetic targeting of germline genes and provide a paradigm for how transient repression signals by DNA-binding factors in early embryonic cells are translated into long-term epigenetic silencing during mouse development. DNA methylation targets CpG island promoters of germline genes to repress their expression in mouse somatic cells. Here the authors show that a transcription factor E2F6 is required to target CpG island DNA methylation and epigenetic silencing to germline genes during early mouse development. [ABSTRACT FROM AUTHOR]

Background: ChIP-nexus, an extension of the ChIP-exo protocol, can be used to map the borders of protein-bound DNA sequences at nucleotide resolution, requires less input DNA and enables selective PCR duplicate removal using random barcodes. However, the use of random barcodes requires additional preprocessing of the mapping data, which complicates the computational analysis. To date, only a very limited number of software packages are available for the analysis of ChIP-exo data, which have not yet been systematically tested and compared on ChIP-nexus data. Results: Here, we present a comprehensive software package for ChIP-nexus data that exploits the random barcodes for selective removal of PCR duplicates and for quality control. Furthermore, we developed bespoke methods to estimate the width of the protected region resulting from protein-DNA binding and to infer binding positions from ChIP-nexus data. Finally, we applied our peak calling method as well as the two other methods MACE and MACS2 to the available ChIP-nexus data. Conclusions: The Q-nexus software is efficient and easy to use. Novel statistics about duplication rates in consideration of random barcodes are calculated. Our method for the estimation of the width of the protected region yields unbiased signatures that are highly reproducible for biological replicates and at the same time very specific for the respective factors analyzed. As judged by the irreproducible discovery rate (IDR), our peak calling algorithm shows a substantially better reproducibility. An implementation of Q-nexus is available at http://charite.github.io/Q/. [ABSTRACT FROM AUTHOR]

After extensive research on radiochemotherapy, 5-year survival rates of children with high risk neuroblastoma still do not exceed 50%, owing to adverse side-effects exemplified by doxorubicin-induced cardiomyopathy. A promising new approach is the combination of conventional therapies with specific modulation of cell signaling pathways promoting therapeutic resistance, such as inhibition of aberrant kinase activity or re-expression of silenced tumor suppressor genes by means of chromatin remodeling. In this regard, we established a system that allows to identify potential drug targets as well as to validate respective candidate inhibitors in high-risk neuroblastoma model cell lines. Cell culture, drug exposure, shRNA-mediated knockdown and phenotype analysis are integrated into an efficient and versatile single well-based protocol. By utilizing this system, we assessed RG108, SGI-1027 and nanaomycin A, three novel DNA methyltransferase inhibitors that have not been tested in neuroblastoma cell lines so far, for their potential of synergistic anti-tumor activity in combination with doxorubicin. We found that, similarly to azacytidine, SGI-1027 and nanaomycin A mediate synergistic growth inhibition with doxorubicin independently of N-Myc status. However, they display high cytotoxicity but lack global DNA demethylation activity. Secondly, we conducted a lentiviral shRNA screen of F-box proteins, key regulators of protein stability, and identified Fbxw11/β-TrCP2 as well as Fbxo5/Emi1 as potential therapeutic targets in neuroblastoma. These results complement existing studies and underline the reliability and versatility of our single well-based protocol. [ABSTRACT FROM AUTHOR]

Ten eleven translocation (Tet) enzymes oxidize the epigenetically important DNA base 5-methylcytosine (mC) stepwise to 5-hydroxymethylcytosine (hmC), 5-formylcytosine and 5-carboxycytosine. It is currently unknown whether Tet-induced oxidation is limited to cytosine-derived nucleobases or whether other nucleobases are oxidized as well. We synthesized isotopologs of all major oxidized pyrimidine and purine bases and performed quantitative MS to show that Tet-induced oxidation is not limited to mC but that thymine is also a substrate that gives 5-hydroxymethyluracil (hmU) in mouse embryonic stem cells (mESCs). Using MS-based isotope tracing, we show that deamination of hmC does not contribute to the steady-state levels of hmU in mESCs. Protein pull-down experiments in combination with peptide tracing identifies hmU as a base that influences binding of chromatin remodeling proteins and transcription factors, suggesting that hmU has a specific function in stem cells besides triggering DNA repair. [ABSTRACT FROM AUTHOR]

The INhibitor of Growth tumor suppressors (ING1-ING5) affect aging, apoptosis, DNA repair and tumorigenesis. Plant homeodomains (PHD) of ING proteins bind histones in a methylation-sensitive manner to regulate chromatin structure. ING1 and ING2 contain a polybasic region (PBR) adjacent to their PHDs that binds stress-inducible phosphatidylinositol monophosphate (PtIn-MP) signaling lipids to activate these INGs. ING1 induces apoptosis independently of p53 but other studies suggest proapoptotic interdependence of ING1 and p53 leaving their functional relationship unclear. Here we identify a novel ubiquitin-binding domain (UBD) that overlaps with the PBR of ING1 and shows similarity to previously described UBDs involved in DNA damage responses. The ING1 UBD binds ubiquitin with high affinity (Kd,100 nM) and ubiquitin competes with PtIn-MPs for ING1 binding. ING1 expression stabilized wild-type, but not mutant p53 in an MDM2- independent manner and knockdown of endogenous ING1 depressed p53 levels in a transcription-independent manner. ING1 stabilized unmodified and six multimonoubiquitinated forms of wild-type p53 that were also seen upon DNA damage, but not p53 mutants lacking the six known sites of ubiquitination. We also find that ING1 physically interacts with herpesvirus-associated ubiquitin-specific protease (HAUSP), a p53 and MDM2 deubiquitinase (DUB), and knockdown of HAUSP blocks the ability of ING1 to stabilize p53. These data link lipid stress signaling to ubiquitin-mediated proteasomal degradation through the PBR/UBD of ING1 and further indicate that ING1 stabilizes p53 by inhibiting polyubiquitination of multimonoubiquitinated forms via interaction with and colocalization of the HAUSP-deubiquitinase with p53. [ABSTRACT FROM AUTHOR]