Extent of methyl modifications of H3 around SNRPN exon 1. DNA extracted from chromatin immunoprecipitated with methyl Lys9 H3 and methyl Lys4 H3 antibodies was amplified using primer pairs as shown. Primer pairs that showed preferential amplification of the maternal chromosome after immunoprecipitation with methyl H3 Lys9 antibody are shown as blackened squares; primer pairs that showed preferential amplification of the paternal chromosome after immunoprecipitation are shown as checkered squares; and primer pairs that showed no parent-specific preferential amplification are shown as unblackened squares. Primer sequences are as follows: UF1 (5′-CTAGAGGCCCCCTCTCATTGCAAC-3′), UR1 (5′-CTTCGCACACATCCCCGCCTGAGC-3′), UF2 (5′-TAGGAAGACCTGAGGGTGAG-3′), UR2 (5′-CACAGCACTGTTGCAATGAG-3′), UF3 (5′-TTTTCACAATTTACCCCCTC-3′), UR3 (5′-GGTCTTCCTATGTGCGGTAC-3′), UF4 (5′-ATTATCTCCCCCAAAATCAC-3′), UR4 (5′-TGTATCCAATTCTAATAGGC-3′), UF5 (5′-GTCTTTTCCCAAGCTACATC-3′), UR5 (5′-TGCCTATTAGAATTGGATAC-3′), UF6 (5′-GATCATTGGGAACTGAGCAG-3′), UR6 (5′-GATGTAGCTTGGGAAAAGAC-3′), UF7 (5′-TCCATCAGCCATATTGCATG-3′), UR7 (5′-TGCTCAGTTCCCAATGATCC-3′), DF1 (5′-CTCTCAAGAGACAGCCTGGGGAGC-3′), DR1 (5′-CCCCAATGCGAGCGGACAGGATAC-3′), DF2 (5′-ATCCTGTCCGCTCGCATTGG-3′), DR2 (5′-ACACGGAACTGCAATCACCC-3′), DF3 (5′-TCAGGGTGATTGCAGTTCCG-3′), and DR3 (5′-TACCCTGCTCCACCACGCAG-3′).

A, PCR analysis of chromatin prepared from lymphocytes from controls, from individuals with PWS, and from individuals with AS, immunoprecipitated with methyl H3 Lys9 antibody. Primary lymphocytes from controls (Control), from individuals with PWS deletion (Del), from individuals with PWS imprinting defect (Imp), and from individuals with AS deletion (Del) were stimulated with phytohemagglutinin and IL-2; then, chromatin was prepared, was sonicated to average size ∼0.5 kb, and was immunoprecipitated, as described by Kuo and Allis (1999), with rabbit antibody to a keyhole-limpet-hemocyanin–conjugated peptide containing amino acids 6–13 of H3 with dimethyl modification of Lys9 (Nakayama et al. 2001). DNA from the immunoprecipitated material was amplified by PCR with the following primers: SNRPN 5′ region (SNA-F, 5′-GATGCTCAGGCGGGGATGTGTGCG-3′; and SNA-R, 5′-GCTCCCCAGGCTGTCTCTTGAGAG-3′) (Saitoh and Wada 2000) and CEN16 (16CEN-F, 5′-GTCTCTTTCTTGTTTTTAAGCTGGG-5′; and 16CEN-R, 5′-TGAGCTCATTGAGACATTTGG-3′) (sequence from GenBank [accession number AC002307]). CEN16 was used as the control PCR for DNA immunoprecipitated with methyl H3 Lys9 antibody. Sample volumes were adjusted so that, for a given antibody, all samples yielded equal amounts of product with the control PCR primers. CEN16 PCR cycling conditions were 94°C for 3 min; 30 cycles at 94°C for 1 min, 54°C for 1 min, and 72°C for 1 min; and 1 cycle at 94°C for 1 min, 54°C for 1 min, and 72°C for 5 min. PCR products were separated on agarose gels and were visualized by ethidium bromide staining. Product sizes were 173 bp, for SNRPN 5′ region, and 198 bp, for CEN16. B, PCR analysis of chromatin prepared from lymphocytes from controls, from individuals with PWS, and from individuals with AS, immunoprecipitated with methyl H3 Lys4 antibody. The same chromatin preparations used in panel A were immunoprecipitated with rabbit antibody to a BSA-conjugated peptide containing amino acids 1–8 of H3 with dimethyl modification of Lys4. DNA from the immunoprecipitated material was amplified by PCR with primers used in panel A, as well as primers for the NDN promoter and for the positive control sequence GAPDH: GAPDH (GAPDH-F, 5′-GCATCACCCGGAGGAGAAAATCGG-3′; and GAPDH-R, 5′-GTCACGTGTCGCAGAGGAGC-3′) (Saitoh and Wada 2000) and NDN 5′ region (NCD-A, 5′-ATGGCGAGGCTTCACCTG-3′; and NCD-B, 5′-AACTGGCCCCTTCTCCAGTA-3′) (sequence from GenBank [accession number AF001013]). NDN PCR was performed in the presence of 10% dimethyl sulfoxide; cycling conditions were 94°C for 3 min; 30 cycles at 94°C for 1 min, 59°C for 1 min, and 72°C for 1 min; and 1 cycle at 94°C for 1 min, 59°C for 1 min, and 72°C for 5 min. Product sizes were 111 bp, for NDN 5′ region, and 269 bp, for GAPDH.

Hypothetical model for histone-modification–based gametic imprint. Blackened circles represent histone, and striped triangles represent protamine; “M” denotes maternal chromosome, and “P” denotes paternal chromosome. Both in male somatic cells and in female somatic cells, maternal chromosome histone is modified (represented by unblackened diamonds), whereas paternal chromosome histone is unmodified. In female germ cell, histones on both chromosomes become modified; in male germ cell, histones are replaced by protamines (Braun 2001). After fertilization, protamine associated with paternal chromosome is replaced by unmodified histone. Replacement of histones by protamines, during spermatogenesis, would provide a mechanism to reverse the chemically stable histone-methylation mark.