Fig. 1. Differential histone methylation and acetylation at Snrpn. (A) Schematic presentation of Snrpn showing exons (vertical bars) and DMRs (horizontal lines). Regions analysed by ChIP are indicated underneath (small horizontal bars). A summary of the maternal (M) and/or paternal (P) allele specificity of the precipitated chromatin is presented underneath for ChIP assays on H3 acetylation on lysines 9 and 14 (H3-AcK9+14), H4 acetylated at lysines 5, 8, 12 and 16 (H4-Ac), H3 methylated at K4 (H3-MeK4) and H3 methylated at K9 (H3-MeK9). (B) ChIP at the DMR1 in brain. Chromatin was extracted from brain tissue dissected from mice that were (C57BL/6 × M.spretus) F₁ for chromosome 7, and was precipitated with antisera to H3-AcK9+14, H4-Ac, H3-MeK4 and H3-MeK9. DNAs extracted from antibody-bound (B) and unbound (U) fractions were used for amplification at the DMR1. PCR products were denatured and run through an SSCP gel. Maternal (M) and paternal (P) bands are indicated. To the left, PCR products from control C57BL/6 (D) and M.spretus (S) genomic DNAs and from the input chromatin (I) used for ChIP. (C) The DMR2 in brain. The same bound and unbound fractions as in panel B were used for PCR amplification from the DMR2. (D) The DMR1 in liver. Liver chromatin was precipitated with different antisera, and extracted DNA samples were amplified with the primer pair from the DMR1. (E) The DMR2 in liver. The same extracted DNA samples as in panel D were used as template for amplification with primers from the DMR2.

Fig. 3. Allele-specific histone methylation at the Igf2r gene. (A) Schematic presentation of the Igf2r gene, showing the DMR1 (paternal DNA methyl ation), DMR2 (maternal DNA methylation), the transcription initiation sites for sense and antisense transcription (arrows), and the position of exons (filled boxes). Regions analysed by ChIP are indicated underneath (small horizontal bars). (B) The DMR1 in liver. Presentation of results is as in Figure 1. (C) The DMR2 in liver. The same samples were analysed by PCR amplification with the primers from the DMR2. (D) 3′-end of the gene. The same samples as in Figure 2B were analysed by PCR amplification with primers at exon 48. (E) The DMR1 in brain. Chromatin extracted from brain was immunoprecipitated and analysed with the primers from the DMR1. (F) The DMR2 in brain. The precipitated samples as in Figure 2C were analysed with the primers at the DMR2. (G) 3′-end of the gene. The same samples as in Figure 2B were analysed by PCR amplification with primers at exon 48.

Fig. 5. MBD proteins associate with the maternal allele of U2af1-rs1. (A) Analysis of H3-K9 methylation on crosslinked chromatin. Formaldehyde crosslinked chromatin was precipitated with the α-linear and α-branched antisera to H3-MeK9. DNA from the bound and unbound fractions was amplified with primers from the 5′-UTR. To the left, amplification from input chromatin (I). P:M ratios, corrected for P:M in the input chromatin, are indicated, as well as the percentage of chromatin precipitated at U2af1-rs1. (B) The newly derived antisera αMeCP2 N-t, αMBD1 C-t, αMBD2 N-t and αMBD3 C-t were tested on a western blot with total nuclear extract from a murine cell line. The density markers indicate a pre-stained standard (Kaleidoscope, Bio-Rad). Comparable results were obtained with a human cell line (data not shown). (C) Crosslinked chromatin was precipitated with antisera to MeCP2, MBD1 and MBD3. Corrected P:M ratios are indicated for the precipitated fractions.

Fig. 2. H3-K9 hypermethylation on chromatin associated with the DNA-methylated allele of DMRs. Liver chromatin was precipitated with antisera to H3-K4 dimethylation (H3-MeK4), to a linear peptide with K9 dimethylation (α-linear) and to a branched peptide with K9 dimethylation (α-branched). (A) Maternal K9 methylation at the Snrpn imprinting-control centre. DNAs extracted from bound and unbound fractions were amplified with primers from the Snrpn DMR1; to the left, amplification from the input chromatin (I). P:M ratios (corrected for P:M in the input chromatin) are indicated. Real-time PCR was applied on the bound fractions to determine the percentage of chromatin precipitated. (B) Maternal K9 methylation at U2af1-rs1. PCR was with the primers from the 5′-UTR. (C) Paternal K9 methylation at the Igf2r DMR1 region. PCR was with the primers from the DMR1. (D) Maternal K9 methylation at the DMR2 of Igf2r. Amplification was with the DMR2 primers.

Fig. 4. Paternal H3 acetylation and K4 methylation at U2af1-rs1. (A) Schematic presentation of the intronless U2af1-rs1 gene (filled box) with the DMR indicated above. Regions analysed by ChIP are indicated underneath (small horizontal bars). (B) The 5′-UTR. Chromatin extracted from liver was precipitated with different antisera, and after extraction of DNA from the bound and unbound fractions, amplification was with primers from the 5′-UTR. (C) The 3′-UTR. The same precipitation samples and control DNAs as in (B) were used for amplification with a primer pair from the 3′-UTR. (D) Chromatin upstream of U2af1-rs1. The same DNA samples as in (B) were used for amplification with a primer pair from a region at 2 kb upstream of the gene.

Fig. 6. A working model for the maintenance of opposite epigenetic states at DMRs. Methylation (M) of DNA is present on one of the parental alleles only (at many DMRs, this is the maternal allele). This methylation is recognized by MBD proteins, which recruit HDAC/chromatin-remodelling complexes to the chromatin. HDACs (filled circles) ensure deacetylation of histones and may be associated with DNMTs, which maintain the DNA methylated. MBD protein binding may also be mechanistically linked to the H3-K9 hypermethylation on this parental allele, and this could constitute a second element in the maintenance of its DNA methylation. The opposite parental allele has no DNA methylation and is not accessible to HDAC complexes and associated DNMT activities, possibly in part because of the H3-K4 methylation. H3-K4 methylation could thus prevent DNA methylation from becoming established. In contrast, HATs and K4 histone methyltransferase (HMT) have access to the chromatin and this maintains the acetylation and K4 methylation on this allele.