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. 2007 May 4;3(5):e65. doi: 10.1371/journal.pgen.0030065

Figure 3. Epigenetic Modifications of Murine Klf14 .

Figure 3

(A) The location and distribution of regions analyzed in Mest and Klf14. The CpG islands overlapping Mest exon 1 and Klf14 are depicted by grey bars (row 1). The regions examined in the methylation analysis and ChIP assay are indicated in rows 2 and 3, respectively. The restriction enzymes used in the ChIP assay and the polymorphisms identified in BL6 and JF1 strains are also shown.

(B) Analysis of histone modifications by ChIP in fibroblast cells of BL6 × JF1 and JF1 × BL6 hybrids. ChIP was performed using formaldehyde fixed chromatin. Antibodies against histone 3 acetylated at lysines 9 and 14 (H3K9acK14ac), histone 4 acetylated at lysines 5, 8, 12, and 16 (H4ac), and H3K4me2 were used in the ChIP assay. Precipitated DNAs were PCR amplified using primers specific to the CpG islands of Mest and Klf14 and subsequently digested as shown in (A). DNA before immunoprecipitation (input) and the product obtained with no antibody (N.C.) were also included in the analysis. The difference in band intensities between the precipitated products and input DNA reveals that there is preferential precipitation of H3K9acK14ac, H4ac, and H3K4me2 on the paternal allele of the Mest CpG island, but no allelic differences were detected at the Klf14 region.

(C) Analysis of histone modifications by native-ChIP in whole embryos of BL6 × JF1 hybrids. Chromatin was immunoprecipitated using antibodies against H3K9ac, H3K4me2, H3K9me3, and H4K20me3. Anti-chicken was used as a nonspecific antibody (mock). Input DNA is denoted by I. Antibody-bound and unbound fractions of the precipitate are denoted by B and U, respectively. Precipitated DNA was PCR amplified using the same primers as in (B). The amplified DNA was analyzed by single strand conformation polymorphism. The results show differences in histone modifications between the two parental alleles in the Mest CpG island, but allelic enrichments were not observed for Klf14.

(D) Expression of Klf14 in offspring of Dnmt3a conditional knockout mice. The expression of Mest and Klf14 was examined in two embryos (e1–2) and corresponding extra-embryonic tissues (e1-2ex) from the offspring of female Dnmt3a conditional knockout mice, as well as a wild-type (wt) embryo. Klf14 expression is lost in the knockout mice, suggesting that its expression is dependent upon a maternally methylated region.

(E) Model of Klf14 expression in wild-type (wt) and Dnmt3a conditional knockout mice. In wild-type mice (upper panel), the maternally methylated CpG island in Mest (black circle) silences the expression of the gene from the maternal allele (M), while Klf14 is actively transcribed on this allele. The opposite pattern of expression is seen on the paternal strand (P), because of the unmethylated CpG island (white circle). In Dnmt3a −/+ embryos (lower panel), maternal methylation of the Mest CpG island is lost, causing increased expression of Mest and loss of expression of Klf14.

(F) Bisulfite sequencing results from 12.5-dpc whole embryos of BL6 × JF1 hybrids. Each block corresponds to a separate region analyzed in the Klf14 CpG island, as shown in (A) (Mest methylation analysis is not shown). Hollow circles and black circles indicate unmethylated and methylated CpG dinucleotides, respectively (N, could not be determined). Each row of circles represents CpGs in an individual PCR product clone. In each block, the top section and bottom sections correspond to clones from the maternal allele (BL6) and paternal (JF1) alleles, respectively, as determined by use of polymorphisms. The three regions analyzed indicate that the Klf14 CpG island is hypomethylated on both alleles.