Preimplantation expression of the somatic form of Dnmt1 suggests a role in the inheritance of genomic imprints

Abstract

Background: Identical DNA methylation differences between maternal and paternal alleles in gametes and adults suggest that the inheritance of genomic imprints is strictly due to the embryonic maintenance of DNA methylation. Such maintenance would occur in association with every cycle of DNA replication, including those of preimplantation embryos.

Results: The expression of the somatic form of the Dnmt1 cytosine methyltransferase (Dnmt1s) was examined in cleavage-stage preimplantation mouse embryos. Low concentrations of Dnmt1s are found in 1-, 2-, 4-, and 8-cell embryos, as well as in morulae and blastocysts. Dnmt1s is present in the cytoplasm at all stages, and in the nuclei of all stages except the 1-cell, pronuclear-stage embryo. The related oocyte-derived Dnmt1o protein is also present in nuclei of 8-cell embryos, along with embryo-synthesized Dnmt1s. Dnmt1s protein expressed in 1-cell and 2-cell embryos is derived from the oocyte, whereas the embryo synthesizes its own Dnmt1s from the 2-cell stage onward.

Conclusion: These observations suggest that Dnmt1s provides maintenance methyltransferase activity for the inheritance of methylation imprints in the early mouse embryo. Moreover, the ability of Dnmt1o and Dnmt1s proteins synthesized at the same time to substitute for one another's maintenance function, but the lack of functional interchange between oocyte- and embryo-synthesized Dnmt1 proteins, suggests that the developmental source is the critical determinant of Dnmt1 function during preimplantation development.

Figure 1

Dnmt1o and Dnmt1s forms of Dnmt1 protein and their expression in oocytes. A. Schematic representation of the mouse Dnmt1s and Dnmt1o proteins. Near the N-termini two regions are highlighted, the PCNA binding domain (PBD) and targeting sequence (TS) that have been implicated in targeting Dnmt1 to replication foci [18,29]. The black rectangles identify the positions of the mouse Dnmt1s amino acid sequences used to immunize a rabbit to produce the UPT82 antiserum or to immunize a chicken to produce the UPTC21 antiserum [6]. B. Immunoblots showing expression of Dnmt1s protein in oocytes from various mouse lines. The upper and lower blots were probed with the UPT82 antiserum. + refers to wild-type oocytes; 1s/1o refers to homozygous mutant oocytes from the Dnmt1^1s/1o strain [6]; Δ1o refers to homozygous mutant oocytes from the Dnmt1^Δ1o strain; TgEL/+ refers to oocytes from hemizygous TgEL transgenic mice. Sample sizes for the upper blot: 100 wild-type GV oocytes; 120 wild-type MII oocytes; 25 1s/1o oocytes; 60 Δ1o oocytes. Sample sizes for the lower blot: 105 wild-type MII oocytes; 40 TgEL/+ oocytes. C. Expression of Dnmt1o protein in wild-type, 1s/1o and Δ1o oocytes. The same blot shown in panel B was probed with UPTC21.

Figure 2

Dnmt1s protein expression during preimplantation development. A. Relative amount of Dnmt1s protein in fully grown wild-type MII oocytes, and in embryos at different stages of development. MII = MII oocytes; 1 = 1-cell; 2 = 2-cell; 4 = 4-cell; 8 = 8-cell; Bl = blastocyst. Sample sizes: 105 wild-type MII oocytes; 105 1-cell embryos; 110 2-cell embryos; 110 4-cell embryos; 110 8-cell embryos; 120 blastocysts. B. Relative stability of oocyte-derived Dnmt1o and Dnmt1s proteins from homozygous Dnmt1^1s/1o mice during preimplantation development to the 8-cell stage. Sample sizes: 22 MII oocytes; 21 8-cell embryos. The blot was first probed with UPT82, stripped and then reprobed with UPTC21. The assay was repeated, and nearly identical results were obtained (data not shown). C. Relative levels of maintenance methyltransferase activity (MA) per oocyte. MA is expressed as [³H]methyl group incorporation into the synthetic template poly(dIdC). Oocyte genotype is the genotype of the diploid precursor of the analyzed MII oocyte. The assay was performed on three separate occasions (1–3). The background measurement is the cpm in sample containing poly(dIdC) and S-adenosyl methionine, but no oocyte extract. Dnmt1o MA/Total MA was calculated as [(Dnmt1^+/+ – background) – (Dnmt1^Δ1o/Δ1o – background)]/(Dnmt1^+/+ – background), expressed as a percentage.

Figure 3

Immunolocalization of Dnmt1o and Dnmt1s proteins in mouse embryonic fibroblasts and 1-cell embryos. A. Immunostaining of primary embryonic fibroblasts from either heterozygous Dnmt1^V/+ (V/+) or homozygous Dnmt1^V/V (V/V) mice using the UPT82 antiserum. Intracellular DNA was detected with DAPI. B. Immunostaining of MII oocytes and pronuclear-stage embryos of different genotypes with the UPT82 antiserum. PN3 and PN4 refer to pronuclear stage 3 and 4 as defined by morphologic criteria [17]. Insets are bright-field images of immunostained embryos. C. Immunostaining of pronuclear-stage embryos with the UPTC21 antiserum to detect the abundant Dnmt1o protein. Genotype abbreviations are the same as in the legends to Figures 1 and 2. Bar, 20 μm.

Figure 4

Expression of maternal and zygotic Dnmt1s during preimplantation development. A. Time course of Dnmt1s expression at defined preimplantation cleavage stages determined by immunostaining with the UPT82 antiserum. Mo = morula. Bl = blastocyst. B. Time course of Dnmt1s expression during preimplantation development in embryos derived from crosses between wild-type (+/+) female mice and homozygous Dnmt1^V/V (V/V) male mice. Inset is bright-field image of immunostained 1-cell embryo. C. Time courses of Dnmt1s expression during preimplantation development in embryos derived from crosses between heterozygous Dnmt1^V/+ (V/+) female mice and homozygous Dnmt1^V/V (V/V) male mice. The 4-cell and 8-cell embryos in the top row show primarily nuclear Dnmt1s staining whereas 4-cell and 8-cell embryos in the botton row show little or no nuclear Dnmt1s. All seven 2-cell, three out of seven 4-cell and six out of 13 8-cell embryos showed nuclear staining. Bar, 20 μm.

Figure 5

The forced expression of maternal Dnmt1s protein prevents loss of imprinting due to the absence of maternal Dnmt1o protein. A. Schematic of construct used to force expression of an epitope-tagged version of Dnmt1s in fully grown mouse oocytes of the TgEL transgenic mouse line. ZP3 = 6.6-kb mouse zona pellucida 3 gene promoter [23]. Dnmt1s = full-length Dnmt1s cDNA containing coding sequence for N-terminal Anti-Xpress epitope tag. BGH = bovine growth hormone polyadenylation and transcription termination sequence. B. Summary of bisulfite genomic sequencing of two normally imprinted genes in mice of various genotypes. Dnmt1^Δ1o/+ alleles were analyzed in a DNA sample isolated from an E13.5 embryo derived from a cross between a homozygous Dnmt1^Δ1o/Δ1o female and a Cast-7 male. Dnmt1^Δ1o/+, TgEL alleles were analyzed in DNA samples isolated either from an E13.5 embryo (Embryo #4) or from an adult (Adult #1) derived from crosses between a Dnmt1^Δ1o/Δ1o, TgEL female and a Cast-7 male. C. Analysis of H19 and Snurf/Snrpn gene expression in mice of various genotypes. Allele-specific expression of H19 was determined by restriction endonuclease digestion of RT-PCR products. Allele-specific expression of Snurf/Snrpn was determined by SnuPE assay. Lanes 1–4: Dnmt1^Δ1o/+, TgEL E13.5 embryos, Embryo #1 through Embryo #4. Lane 5–6: Dnmt1^Δ1o/+ embryos. Lanes 7–10: Dnmt1^Δ1o/+, TgEL adults, Adult #1 through Adult #4. Lane 11: inbred Cast-7. Lane 12: inbred FVB/N. Lane 13: F1 hybrid from cross between an FVB/N female mouse and a Cast-7 male mouse.

Figure 6

Model of Dnmt1o and Dnmt1s protein expression during preimplantation development. A. The top row of schematics depicts the pattern of maternal Dnmt1o protein expression seen during preimplantation development. Dnmt1o is localized in the nucleus of blastomeres of 8-cell embryos. Dnmt1o is not found in the pronuclei. B. The middle row of schematics depicts the pattern of maternal Dnmt1s protein expression in the early stages of preimplantation development. Dnmt1s is not found in the pronuclei. C. The bottom row of schematics depicts the pattern of zygotic Dnmt1s protein expression during preimplantation development. Dnmt1s is evident in nuclei of many stages, including in blastomeres of 8-cell embryos.