the effect of maternal inheritance of the D4.8 mutation is on the maternal allele, we used mice with paternal inheritance of the DNdn mutation. In this assay, only the wild-type Ndn allele on the maternal chromosome could be detected because the primer pair designed for ChIP-qPCR is located at the region deleted in the DNdn mutation. Compared with wild-type mice, the m+pDNdn mice showed a dramatic reduction of H3K4me3. This result clearly indicates paternal-specific H3K4me3 at Ndn and paternal deletion contributes to the significant reduction of H3K4me3. This is similar to the human NDN promoter showing paternal-specific association with H3K4me3. When maternally inheriting the D4.8 mutation, an elevated level of H3K4me3 was detected in the mD4.8pDNdn mice compared with the m+pDNdn mice. Since the paternal copy of Ndn was deleted in these mice, the elevated H3K4me3 was derived from the remaining maternal copy of Ndn in the mD4.8pDNdn mice. We next examined acetylation of histone 3 as an additional marker of an active gene expression state. ChIP-qPCR analyses showed reductions of H3Ac at both Snrpn and Ndn in the m+pD4.8 mice. On the other hand, maternal inheritance of the D4.8 mutation did not affect H3Ac at the Snrpn promoter in the mD4.8p+ mice, but did increase H3Ac at Ndn. When the paternal Ndn was deleted in the m+pDNdn mice, a marked reduction of H3Ac was observed, suggesting the paternal copy is the one preferentially modified with H3Ac. This is similar to the human NDN promoter showing paternal bias with H3Ac. Similar to increased H3K4me3, mD4.8pDNdn mice showed an PWS-IC Is Required for Maternal Imprinting 8 PWS-IC Is Required for Maternal Imprinting increase of H3Ac on the maternal copy of Ndn compared to the m+pDNdn mice. Finally, we examined H3K9me3 which is a mark of a repressive chromatin state. ChIP-qPCR analyses showed that the m+pD4.8 mice had marked increases in H3K9me3 at both Snrpn and Ndn, whereas the mD4.8p+ mice showed reductions of H3K9me3 compared with wild-type mice. Furthermore, the mD4.8pDNdn mice showed the reduction of H3K9me3 on the maternal copy of Ndn when compared with the m+pDNdn mice. There was approximately 2-fold enrichment of H3K9me3 in the m+pDNdn mice compared with wild-type mice. It should be noted that in ChIP-qPCR analysis, the level of ChIP was normalized against the level of input in each sample: ChIP from the m+pDNdn mice was normalized against the input with only one copy of the maternal Ndn allele, while ChIP from the m+p+ mice was normalized against the input with two Ndn copies from both parents. It is possible that the maternal copy of Ndn could be preferentially modified with H3K9me3, which is similar to the human NDN promoter with H3K9me3 towards maternal bias. Therefore, after normalization with input, the ChIP-qPCR PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189542 result might show a 2-fold enrichment of H3K9me3 in the m+pDNdn mice compared with the m+p+ mice, even though both mice could have similar levels of H3K9me3 enrichment on their maternal wild-type copies of Ndn. MedChemExpress RU 58841 However, we can not rule out the possibility that paternal inheritance of the DNdn mutation acts in trans to increase H3K9me3 on the maternal chromosome. Maternal inheritance of the D4.8 mutation altered DNA modification at Ndn and Mkrn3 Next, we analyzed whether maternal inheritance of the D4.8 mutation affects the DNA methylation status at the PWS/AS domain. Silencing of the maternal alleles of Snrpn, Ndn, and Mkrn3 is associated with maternal-specific CpG methyl
