Fig 1.
Genomic structure of CoKO and U1 mutant alleles of the Peg3 locus.
(A) Schematic representation of WT and CoKO allele of the mouse Peg3 locus. Exons of Peg3 are indicated with blue boxes. The 7.1-kb insertion cassette contains promoterless galactosidase (β-Gal) and human β-actin promoter-driven neomycin resistance gene (NeoR), which are indicated with white boxes. The Poly(A) tails associated with these genes are indicated with grey boxes. This expression cassette is flanked by brown oval-shaped FRT boxes. The Poly(A) tails are accountable for the truncation of Peg3 expression, thus the 0x dosage of Peg3. (B) Schematic representation of WT and U1 alleles. The blue color indicates the genes associated with the paternal allele and the orange color represents the genes associated with the maternal allele. Maternal deletion of an alternative promoter, U1, causes the expression of Peg3 and Usp29 from the maternal allele, subsequently responsible for the expression levels of 2x dosage of Peg3.
Fig 2.
Peg3 downstream genes involved in major metabolic pathways.
The schematic figure shows the combination of several metabolic pathways from glycolysis to lipogenesis (fatty acid synthesis and mevalonate pathway). Glucose-derived citrate is converted to acetyl-CoA by ATP-citrate lyase (ACLY), which is used as a precursor in fatty acid and the mevalonate synthesis pathways. This current study identified 1073 genes as the downstream genes of Peg3 from genome-wide scanning of ChIP-seq peaks. More than 30 pathways were examined to test if there were any overlapping genes between these pathways and the genes identified from ChIP-seq analysis using the Kegg pathway database (https://www.kegg.jp/kegg/). Initial analysis identified several key genes critical for lipogenesis, including Acly, Idh1, Fasn, and Hmgcr.
Fig 3.
In vivo binding of PEG3 to the promoter of Acly.
(A) ChIP-seq results from the 80-kb genomic intervals encompassing Acly. The top panel is from WT-MEF cells and the bottom panel is from KO-MEF cells. Statistical p-values are indicated on the Y-axis with the maximum value being 10, whereas relative genomic positions are on the X-axis. An arrow represents the transcriptional direction of Acly. (B) Individual ChIP experiment confirming the in vivo binding of PEG3 to the promoter regions of Acly. Chromatins were prepared from the 14.5-dpc embryos and neonatal brain of WT and CoKO. Template for PCR reaction was the immunoprecipitated DNA either from WT or KO-MEF cells with the anti-PEG3 antibody. In addition, the Input and Negative (Neg) DNA was also included in this PCR amplification. The Neg control contained the DNA from the ChIP experiment without the antibody. (C) Quantitative PCR results from the immunoprecipitated DNA with the anti-PEG3 antibody. The red and blue bars indicate the relative enrichment levels of PEG3 IP and Neg compared to the Input in the promoter regions of Acly. Relative enrichment values are indicated on the Y-axis for each sample. All the analyses were performed in triplicates. The statistical significance of the observed difference between PEG3 IP and Neg was tested with a student t-test (*, p-value <0.05 and ***, p-value <0.0001).
Fig 4.
Expression level changes of Acly against the 0x and 2x dosage of Peg3.
Expression levels of Acly were measured using a series of qRT-PCR. This set of analyses was performed using two biological replicates for both the embryo and the mammary gland set of males (M1 and M2) and females (F1 and F2). The total RNA was isolated from the embryos and the mammary glands with WT (1x), CoKO (0x), and U1 (2x) dosage of Peg3. The expression level differences in the 14.5-dpc embryos were summarized in the first column (A, B) and the adult mammary glands in the second column (C, D). Individual gene expression levels were first normalized to Gapdh levels and then normalized values were further compared between WT and the mutants (CoKO or U1). Subsequent relative levels are presented in a graph with the average values and standard deviations. The statistical significance of the observed difference between WT and mutants was tested with a student t-test (*, p-value <0.05 and ***, p-value <0.0001). The corresponding p-values are listed in the text.
Fig 5.
Expression level changes of Fasn, Idh1, and Hmgcr in adult mammary gland.
A series of qPCR assays were performed to measure the expression levels of Fasn, Idh1, and Hmgcr between WT and CoKO/U1. For these analyses, two males (M1 and M2) and two females (F1 and F2) were used. Total RNA was isolated from the adult mammary glands, which was subjected to cDNA synthesis. A qRT-PCR was then performed. Gapdh was used to normalize the expression levels of each gene before comparing between WT and U1/CoKO. The results sets were summarized with the average values and standard deviations. The statistical significance is indicated in the following student’s t-test (*, p-value <0.05 and ***, p-value <0.0001). The corresponding p-values are listed in the text.
Fig 6.
Peg3’s potential control on lipogenesis through Acly.
Schematic representation of the potential downstream genes of Peg3 and their associated metabolic and lipogenesis pathways. Red color genes, Fasn, Idh1, and Hmgcr, are potential downstream genes predicted from the ChIP-seq analysis. All of these genes are involved in either regulating Acly or fatty acid and mevalonate pathways. With the variable dosages of Peg3, changes were observed in the expression levels of these genes, which are indicated with vertical arrows. Yellow arrows indicate 0x dosage of Peg3 and green arrows represent the 2x dosages of Peg3. Sex-specific response in the expression levels of Acly, Fasn, and Idh1 in the adult mammary gland was also observed in CoKO (0x Peg3). This suggests that Peg3 may play a role in lipogenesis by regulating the expression levels of Acly and several key genes.