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M·BssHII is a new multispecific C5-DNA-methyltransferase recognizing five different targets. As the enzyme has been isolated from a thermophilic Bacillus, the protein should show enhanced intrinsic thermostability and therefore be a promising candidate for crystallizing a multispecific MTase.

The precise mapping and quantification of DNA methylation as an epigenetic parameter during development and in diseased tissues is of great importance for functional genomics. Here we describe a rapid, quantitative method to assess methylation levels at specific CpG sites using PCR products of bisulfite-treated genomic DNA. Using single nucleotide primer extension (SNuPE) assays in combination with ion pair reverse phase high performance liquid chromatography (IP RP HPLC) separation techniques, methylated and unmethylated CpGs can be discriminated and quantified based on the different masses and hydrophobicities of the extended primer products. The assay is linear, highly reproducible and several sites can be measured simultaneously in one reaction. It can be semi-automated and eliminates the need for cloning and sequencing of individual bisulfite PCR products.

In order to test the preference of growing axons for membrane-associated positional specificity a new in vitro assay was developed. In this assay, membrane fragments of two different sources are arranged as a carpet of very narrow alternating stripes. Axons growing on such striped carpets are simultaneously confronted with the two substrates at the stripe borders. If there is a preference of axons for one or the other substrate they become oriented by the stripes and grow within the lanes of the preferred substrate. Such preferential growth could, in principle, be due to affinity to attractive factors on the preferred stripes or avoidance of repulsive factors on the alternate stripes. This assay system was used to investigate growth of chick retinal axons on tectal membranes. Tissue strips cut from various areas of the retina were explanted and the extending axons were confronted with stripes of cell membranes from various areas within the optic tectum. Tectal cell membranes prove to be an excellent substrate for the growth of retinal axons. Nasal and temporal axons can grow well on membranes of both posterior and anterior tectal cells. If, however, temporal axons are given a choice and encounter the border between anterior and posterior membranes they show a marked preference for growth on membranes of the anterior tectum, their natural target area. Nasal axons do not show a preference in this assay system. The transition from nasal to temporal properties within the retina is abrupt. In contrast, the transition from anterior to posterior properties of the tectal cell membranes occurs as a smooth gradient. Significantly, the positional differences of tectal membrane properties are only seen during the period of development of the retinotectal projection and are independent of tectai innervation by retinal axons. These anterior-posterior differences disappear by embryonic day 14.

Supplemental Information includes Supplemental Experimental Procedures, six figures, and four tables and can be found with this article online at http:// dx.doi.org/10.1016/j.celrep.2016.08.087., Mouse embryonic stem cells are dynamic and heterogeneous. For example, rare cells cycle through a state characterized by decondensed chromatin and expression of transcripts, including the Zscan4 cluster and MERVL endogenous retrovirus, which are usually restricted to preimplantation embryos. Here, we further characterize the dynamics and consequences of this transient cell state. Single-cell transcriptomics identified the earliest upregulated transcripts as cells enter the MERVL/Zscan4 state. The MERVL/Zscan4 transcriptional network was also upregulated during induced pluripotent stem cell reprogramming. Genome-wide DNA methylation and chromatin analyses revealed global DNA hypomethylation accompanying increased chromatin accessibility. This transient DNA demethylation was driven by a loss of DNA methyltransferase proteins in the cells and occurred genome-wide. While methylation levels were restored once cells exit this state, genomic imprints remained hypomethylated, demonstrating a potential global and enduring influence of endogenous retroviral activation on the epigenome., EMBO Fellowship (ALTF938-2014) and Marie Sklodowska-Curie Individual Fellowship, DFG grant WA1029 within the SFB1027, BBSRC (grant BB/K010867/1), Wellcome Trust (grant 095645/Z/11/Z), EU EpiGeneSys, BLUEPRINT, The authors thank all members of the W.R. laboratory for helpful discussions. We also thank Ferdinand von Meyenn and Mario Iurlaro for serum-2i transcriptome data, Simon Andrews for bioinformatics support, Simon Walker for imaging support, Nathalie Smerdon for high-throughput sequencing assistance, David Oxley for mass spectrometry, Rachael Walker for flow cytometry assistance, Ruslan Strogantsev for genomic imprinting discussions, Minoru Ko for the Zscan4c::EGFP plasmid, Samuel Pfaff for the MERVL::tdTomato plasmid, and Haruhiko Koseki for Dnmt1fl/fl ESCs. M.A.E.-M. is supported by an EMBO Fellowship (ALTF938-2014) and Marie Sklodowska-Curie Individual Fellowship. P.G. was supported by DFG grant WA1029 within the SFB1027. Research in W.R.’s lab is supported by the BBSRC (grant BB/ K010867/1), Wellcome Trust (grant 095645/Z/11/Z), EU EpiGeneSys, and BLUEPRINT.

CD4+ regulatory T cells (Tregs) are key mediators of immunological tolerance and promising effector cells for immuno-suppressive adoptive cellular therapy to fight autoimmunity and chronic inflammation. Their functional stability is critical for their clinical utility and has been correlated to the demethylated state of the TSDR/CNS2 enhancer element in the Treg lineage transcription factor FOXP3. However, proof for a causal contribution of the TSDR de-methylation to FOXP3 stability and Treg induction is so far lacking. We here established a powerful transient-transfection CRISPR-Cas9-based epigenetic editing method for the selective de-methylation of the TSDR within the endogenous chromatin environment of a living cell. The induced de-methylated state was stable over weeks in clonal T cell proliferation cultures even after expression of the editing complex had ceased. Epigenetic editing of the TSDR resulted in FOXP3 expression, even in its physiological isoform distribution, proving a causal role for the de-methylated TSDR in FOXP3 regulation. However, successful FOXP3 induction was not associated with a switch towards a functional Treg phenotype, in contrast to what has been reported from FOXP3 overexpression approaches. Thus, TSDR de-methylation is required, but not sufficient for a stable Treg phenotype induction. Therefore, targeted demethylation of the TSDR may be a critical addition to published in vitro Treg induction protocols which so far lack FOXP3 stability.

DNA methylation is a widely investigated epigenetic mark with important roles in development and disease. High-throughput assays enable genome-scale DNA methylation analysis in large numbers of samples. Here, we describe a new version of our RnBeads software - an R/Bioconductor package that implements start-to-finish analysis workflows for Infinium microarrays and various types of bisulfite sequencing. RnBeads 2.0 (https://rnbeads.org/) provides additional data types and analysis methods, new functionality for interpreting DNA methylation differences, improved usability with a novel graphical user interface, and better use of computational resources. We demonstrate RnBeads 2.0 in four re-runnable use cases focusing on cell differentiation and cancer.

In human and mouse most imprinted genes arearranged in chromosomal clusters. This linkedorganization suggests coordinated mechanismscontrolling imprinted expression. We havesequenced 250 kb in the centre of the mouseimprinting cluster on distal chromosome 7 andcompared it with the orthologous Beckwith–Wiedemann gene cluster on human chromosome11p15.5. This first comparative imprinting clusteranalysis revealed a high structural and functionalconservation of the six orthologous genes identified.However, several striking differences were alsodiscovered. First, compared with the mouse thehuman sequence is ∼40% longer, mostly dueto inser-tions of two large repetitive clusters. One of theseclusters encompasses an additional gene coding fora homologue of the ribosomal protein L26. Second,pronounced blocks of unique direct repeats charac-teristic of imprinted genes were only found in thehuman sequence. Third, two of the orthologous genepairs Tssc4/TSSC4 and Ltrpc5/LTRPC5 showedapparent differences in imprinting between humanand mouse, whereas others like Tssc6/TSSC6werenot imprinted in either organism. Together theseresults suggest a significant functional and struc-tural variability in the centre of the imprinting cluster.Some genes escape imprinting in both organismswhereas others exhibit tissue- and species-specificimprinting. Hence the control of imprinting in thecluster appears to be a highly dynamic processunder fast evolutionary adaptation. Intriguingly,whereas imprinted genes within the cluster containCpG islands the non-imprinted Ltrpc5 and Tssc6/TSSC6do not. This and additional comparisons withother imprinted and non-imprinted regions suggestthat CpG islands are key features of imprinteddomains.INTRODUCTIONImprinted genes are those that are predominantly expressedfrom only one of the parental alleles (1–6). The imprintingmechanism involves epigenetic modifications such as DNAmethylation and altered chromatin structures (7). Imprintedgenes play important roles in development, in particular infetal growth, placental development, cell proliferation andbehaviour after birth. Deregulation of imprinting is implicatedin a number of human cancers and genetic diseases, such as thePrader–Willi/Angelman syndromes (8) and Beckwith–Wiedemann syndrome (BWS) (9), which is associated withfetal overgrowth and a predisposition to childhood tumours.A significant number of imprinted genes are organized inclusters that are characterized by epigenetic features such asdifferences in allele-specific DNA replication, chromosomepairing of the two parental copies and sex-specific meioticrecombination frequencies (10–13). This suggests an epigeneticco-regulation of neighbouring imprinted genes. Some aspectsof regulation of imprinting could affect the whole cluster,either by the chromatin structure itself or by cis-actingelements such as imprinting centres or cis-acting transcripts.One prominent imprinting cluster in the human is the BWSregion on chromosome 11p15.5. In the mouse the imprintingcluster on distal chromosome 7 is homologous to the humanBWS region. We and others have identified a number of mousegenes in this region at the same relative positions as theirhuman orthologues, indicating a syntenic organization in bothorganisms (14–19). In the mouse and in the human theimprinting clusters contain the paternally expressed Igf2/IGF2(20–22) and Ins2/INS genes (23), the maternally expressedH19 (24,25), Mash2/ASCL2 (HASH2) (26,27), Cdkn1c/CDKN1C (28–31), Tssc3/TSSC3 (14), Tssc5/TSSC5 (15,16)and Kcnq1/KCNQ1 (17–19,32) genes and Cd81/CD81,whosematernal expression has so far only been shown in mouse(19,33,34). Antisense transcripts have been identified for