ly regulates the paternally expressed imprinted genes with the Snrpn and Ndn promoters are unmethylated on the CpG islands and modified with active chromatin marks H3K4me3 and H3Ac . On the maternal chromosome, the PWS-IC represses expression of the paternally expressed imprinted genes with the Snrpn and Ndn promoters are methylated on the CpG islands and modified with a repressive chromatin mark H3K9me3. Furthermore, we demonstrated PWS phenotypic rescues by maternal inheritance of the PWS-IC deletion, in contrast to paternal inheritance of the PWS-IC deletion causing the PWS phenotypes. We identified a previously unrecognized and important role of the PWS-IC at the PWS/AS domain. Materials and Methods Ethics statement All of the mice were bred and maintained according to a protocol approved by the Baylor College of Medicine Animal Care and Use Committee at the institution’s specific pathogen-free mouse facility, which is approved by the American Association for Accreditation of Laboratory Animal Care and is operated in accordance with current regulations and standards of the US Department of Agriculture and the Department of Health and Human Services. Mouse models Mutant mice with a 4.8-kb deletion at Snrpn exon 1were generated as described. Mutant mice carrying a deletion from Snrpn exon 2 to Ube3a were previously described. Mutant mice with a deletion at Ndn were described. Mice with the D4.8 mutation and mice with the DS-U mutation are maintained on a C57BL6/J genetic background. Mice with the DNdn mutation are maintained on a hybrid C57BL6/J and 129/ SvEv genetic background. RT-PCR analysis PWS-IC Is Required for Maternal Imprinting 14 PWS-IC Is Required for Maternal Imprinting sample. In each set of experiments, the normalized level of gene expression from the wild-type mouse was always set as 1. Western blot analysis Mouse whole brain was dissected from pups at day 1 of age. Brain samples were homogenized and lysed in NP40/SDS buffer. Sixty micrograms of mouse brain protein were used for electrophoresis on 10% Tris-Cl ready gels. The proteins were transferred to nitrocellulose membrane. The membranes were then incubated with the appropriate antibodies as follows: rabbit antihuman E6-AP was diluted 1:1000 or goat anti-human actin PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22187495 IgG was diluted 1:500. The membranes were then incubated with either goat anti-rabbit IgG horseradish peroxidase or donkey anti-goat HRP. The signals of western blotting were detected by enhanced chemiluminesence exposed to X-ray films. For quantification experiments, the X-ray films were scanned and the intensity of the signals was quantified by densitometry. We used a least 3 sets of mice with every genotypes. In each group of mice from different genotypes, the purchase BMS-833923 levels of E6-AP were normalized against the levels of actin in each sample. In each set of experiments, the normalized level of E6-AP from the wild-type mouse was always set as 1. Millipore/Upstate Biotechnology. For chromatin modification analysis, chromatin extracted from mouse brain was immunoprecipitated with antitrimethyl H3K4 antibodies, anti-acetyl Histone 3, or antitrimethyl H3K9me3. Then, qPCR analyses were performed using the primer sets to amplify co-precipitated DNA from Snrpn and Ndn. The primers used are listed in Mouse genomic tiling array We designed a custom mouse genomic tiling array using the Agilent E-array platform. The array included 44,000 oligonucleotides covering sequences of 
the mouse imprinted gene clusters from ATP10a
