Cis elements and protein binding factors in AS-IC. (A) Names and positions of the cis elements and binding proteins in AS-IC (position 1 corresponds to nucleotide 22716614 of chr. 15, according to hg18 at http://genome.ucsc.edu/cgi-bin/hgGateway). (B) Representative band-shift assays used to characterize the binding specificity of each element. All assays were performed with nuclear extracts of mouse ES cells. Numbers above the lanes indicate the position of the mutation in the AS-IC sequence. Mutations were single base replacements G↔ T and C↔ A. The mutated bases are marked by an asterisk. Capital letters indicate bases that are part of the binding sequence. Lowercase letters represent the sequence flanking the element. The shifted bands are marked by arrowheads. Other mutated oligomers, as well as no-extract controls, were also used. (C) Competition band-shift experiments were used to determine binding cooperation between different cis elements. Labeled oligomers for each experiment are listed above each picture. The left lane of each gel represents a no-competition control. Lanes to the right represent the effect of various unlabeled competitors (vertical lettering) that were included in 100× molar excess. Loss of bands (arrowheads) indicates competition. DNS-AS and DNS-PWS represent the DNS elements on AS-IC and PWS-IC, respectively. A reciprocal experiment for OCTA competition was also performed, with results similar to that of SOX.

Methylation analysis of AS-IC and PWS-IC upon mutations in AS-IC. (A) Top: Southern blot analysis of PWS-IC DMR in adult brain transgenic tissues using the methylation sensitive enzyme NotI. M, methylated band. U, unmethylated band. Up to 10 individual samples representing 2 generations were examined from each transgenic line upon both parental transmissions. Representative data are shown. ♂- paternal ♀- maternal. The number of founder lines, and copy number per each mutated transgene are listed at the bottom of the gel. Bottom: Bisulfite sequencing of PWS-IC DMR in adult brain of the various transgenic lines (as described in Fig. 2B). Each horizontal line represents a unique clone. Empty circles, unmethylated CpGs, filled circles, methylated CpGs. All bisulfite data are of a representative line from each mutation. Lines not shown gave similar results. (B) Bisulfite sequencing of AS-IC and PWS-IC DMRs in oocytes of the various mutant transgenic lines (as described in Fig. 2B). At least 5 separate pools of approximately 30 oocytes each were used. Each line represents a unique clone. Empty circles, unmethylated CpGs, filled circles, methylated CpGs. The DMR mutations were not analyzed for AS-IC, as they lack all 4 CpGs. (C) Bisulfite sequencing of AS-IC and PWS-IC DMRs in sperm of the various mutant transgenic lines. For AS-IC, each horizontal line represents a unique clone. For PWS-IC, the single horizontal line represents 12 unique clones that were all completely unmethylated. At least 3 sperm samples per mutated line were used. Southern blot analysis for PWS-IC yielded similar results. Similar results for PWS-IC were also obtained with primers that encompass region II in positions (−137)–(+101).

A proposed model describing the role of trans-acting factors in the establishment of imprinting in the PWS/AS domain. Top, maternal chromosome: Multiple protein factors bind the AS-IC (red) in the oocyte and protect its DMR from undergoing methylation (empty lollipops). An interaction between AS-IC and PWS-IC (blue) is achieved through separate DNS elements located on each IC. The interaction protects PWS-IC from methylation as well. In parallel, PWS-IC is marked (curved arrow) with an epigenetic modification that is maintained throughout the preimplantation period (yellow star). By implantation, DNA methyltransferase (Dnmt in green box) is recruited to gradually methylate PWS-IC (curved arrow, filled gray and black lollipops). AS-IC becomes methylated by the global wave of post implantation methylation. Bottom, paternal chromosome. Trans-acting factors are either not present, or fail to bind the methylated AS-IC in sperm (filled lollipops). This can be postulated based on the observed absence of Dmrbp in androgenetic ES cells, that express paternally derived genes, and based on its inability to bind a methylated DMR sequence as was demonstrated by in vitro experiments (4). Thus, AS-IC is inactive and leaves PWS-IC, which is a CpG island, unmethylated and active throughout.

Methylation analysis of the human AS-25I (WT) transgene. (A) Schematic structure of the transgene: AS-IC (white, positions 22716614–22717869), PWS-IC (black, 22746472–22751856), and IUS (intervening upstream sequence, gray, 22741535–22746471). PWS-IC is based on the PWS-SRO sequence obtained from Prader-Willi patients (3). SNRPN transcription start site (arrow), restriction sites used for Southern blotting [BglII (B), EcoR I (E,) and NotI (N), positions relative to SNRPN start site], probe (solid line), Roman numerals denote regions analyzed by bisulfite sequencing (dotted lines). (B) DNA bisulfite methylation analysis throughout development of AS-IC DMR (region I - positions 322–340 of AS-IC) and PWS-IC DMR [region III - positions (+218)–(+298) relative to SNRPN transcription start site, bottom]. DNA treatment and PCR were carried out as described in Materials and Methods. Empty circles, unmethylated CpGs. Filled circles, methylated CpGs. ♂- paternal transmission, ♀- maternal transmission. Each horizontal line represents a unique clone. At least 5 separate samples of pooled oocytes and 3.5 dpc embryos were treated for each stage and lineage. For adult brain and 8.5 dpc embryos, results represent 3 separate experiments. Similar results for PWS-IC were obtained using primers that encompass region II in positions (−137)–(+101), which includes the NotI sites used in Southern blot.

Dynamic changes in the methylation levels of the PWS/AS ICs throughout 4 stages of development. Both 3.5 and 8.5 represent the respective dpc (days post coitum) (A) Methylation levels of PWS-IC for the maternal (dashed lines) and paternal (dotted lines) alleles in the AS25I WT transgene (left) and its mutant derivatives (right). Analysis was performed by bisulfite sequencing for all stages, and by Southern blot for PWS-IC in sperm, 8.5 dpc and adult brain, as described in Figs. 2 and 3. (B) Methylation levels of AS-IC for the maternal (dashed lines) and paternal (dotted lines) alleles in the AS25I WT transgene (left) and its mutant derivatives (right). The maternal DNS mutation is drawn separately from the other mutations (solid line).