epi-2021-0168 Supplementary Figures

figure

posted on 2021-09-14, 13:27 authored by Tie-Bo Zeng, Nicholas Pierce, Ji Liao, Piroska E. Szabó

S1. A truncated EHMT2 is expressed in the Ehmt2 homozygous embryo.

Western blots of 9.5 dpc embryos are displayed with the anti-EHMT2 (A) or the anti-EHMT1 (B) antibody. The GAPDH antibody was used as loading control. Protein was prepared from two 16-somite Ehmt2^-/- embryos combined, or one each of 22-somite Ehmt2^+/- and Ehmt2^+/+ embryo, yielding 34 µg, 142 µg and 181 µg protein, respectively. 5 µg protein was electrophoresed from each sample. Molecular weight marker locations are indicated to the right. Note the truncated EHMT2 in the Ehmt2^-/- embryo sample. Also note the reduced level of GLP in the Ehmt2^+/- sample. GLP levels in the Ehmt2^-/- embryo may be overcompensating for complete loss of EHMT2.

S2. Examples of the RNA-seq results in replicate EPC samples.

Two female maternal mutant (MATMUT) and maternal wild type (MATWT) EPC sample replicates are shown in the 129 x JF1 cross. Ehmt2^+/+ wild type (WT) and Ehmt2^-/- homozygous (HOMO) EPC samples are displayed in two female 6-somite stage EPCs each in the reciprocal crosses (129 x JF1 and JF1 x 129). Genome browser views shown for the split RNA-seq tracks in the paternal (P) and maternal (M) alleles. The samples are described in Table S1. (A) Canonical imprinted gene H19 is expressed from the maternal allele. (B) Canonical imprinted gene Igf2 is expressed from the paternal alleles. (C) Xist is expressed from the paternal allele except in one MATMUT EPC, where the maternal allele is partially relaxed. (D) Genes subject to imprinted X chromosome inactivation (XCI) are expressed from the maternal alleles. (E) An escapee of XCI, Taf1, is shown. (F) Kdm6a is subject to imprinted X chromosome inactivation (XCI). It is expressed from the maternal allele in MATMUT, MATWT, and WT samples but shows relaxation of the paternal allele in HOMO samples. This is the only exception where EHMT2 affects imprinted XCI.

S3. Reproducibility of the RNA-seq results in replicate EPC samples (A) CONTROL EPC samples are displayed in four (two female and two male) 6-somite stage EPCs each in two reciprocal crosses (129 x JF1 and JF1 x 129). Split-alleles are shown of Gab1 as described in Figure 2. Green box shows the location of the ERVK promoter. The scale is set to visualize the derepression from the normally silent maternal (M) allele initiated from the ERVK promoter, while the active paternal (P) allele is saturated. It is fully visible at the scale of [0-27], as shown in the bottom panel in Figure 2A. The samples are described in Table S1. (B) Four each (two female and two male) maternal mutant (MATMUT) and maternal wild type (MATWT) EPC sample replicates are shown in the 129 x JF1 cross. (C) Wild Type (WT) EPC samples. Four (two female and two male) 6-somite stage 129 x JF1, four 12-somite stage JF1 x 129, and four 6-somite-stage JF1 x 129 samples are shown. (D) Homozygous zygotic mutant (HOMO) EPC samples. Four (two female and two male) 6-somite stage 129 x JF1, 12-somite stage JF1 x 129, and four 6-somite-stage JF1 x 129 samples are shown. Note the change from paternal to biallelic expression in the mutant samples.

Fig. S4. The ERVK promoters of non-canonical imprinted are unmethylated in sperm. Genome browser views of DNA methylation data [31] are displayed at the ERVK promoters, associated with non-canonical imprinted genes in the oocyte, sperm, 2-cell and 4-cell embryos,

E3.5 ICM, E6.5 and E7.5 embryos. Potential oocyte-derived methylation (rectangles) is not maintained during preimplantation development.

S5. EHMT2 has a minor role in maintaining canonical imprinting in the EPC. (A) Allelic expression ratio (Mat/Mat+Pat) of selected maternally expressed canonical imprinted genes located along distal chromosome 7 in the EPC samples as described in Fig. 1A. (B) Slight derepression of the paternal allele of canonical imprinted genes. Genome browser images are shown as described in Figure 2.